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Volume 3 Issue 1 Jan-March, 2013 Editor-In-Chief JAYAPAL REDDY GANGADI M.Pharm, M.Phil.,FICCP, Ph.D

Associate Editors Mr.E.Venkateshwarlu Mr.K.Venu Mr.K.Naresh Mr.M.Satish

Department Of Pharmacology, India Department Of Pharmacology, India Department Of Pharmaceutical Chemistry, India Department Of Pharmaceutical Chemistry, India

Editorial Advisory Board Members Prof.Dr.J.Venkateshwar Rao Dr.J.Raju, Dr.A.Srinivas Dr.K.S.Nataraj Dr.G.K.Mallaiah Dr.G.Shyam Prasad Dr.B.Srinivas Dr.B.K.Prusty Dr.T.Sreekanth Dr.A.Shyam Sunder Dr. M.R.Jayapal Dr.Deepak Prashar Prof.Dr.Prakash.MMS.Kinthada Dr.Seshikala Durisetti Dr.Anshu Srivastava Dr.Arvind R.Umarkar Dr.M.Jagadishnaik Dr.Rajsekhar Paul Dr.Shidlingappa Shirol Dr. DSVGK Kaladhar Prof. Dr.G.Vidyasagar Dr.Surapaneni Krishna Mohan Dr.N.N.Rajendran Dr.Mohammed Rageeb Md Usman Dr.Thirumalai.T Dr.Pulak Majumder Dr. Mirza Rafiullah Baig Dr. Kammuluri Ratna Kumar Mr. Sai Krishna Mr.Vamshi Krishna

Department Of Pharmaceutical Chemistry, India Department Of Pharmaceutics, India Department Of Pharmaceutical Chemistry, India Department Of Pharmaceutical Analysis, India Department Of Pharmacognosy, India Department Of Microbiology, India Department Of Pharmaceutical Chemistry, India Department Of Pharmacology, India Department Of Pharmaceutical Chemistry, India Department Of Pharmacology, India Department Of Organic Chemistry, India Department Of Pharmacy, Kullu (H.P.),India Department Of Chemistry, India Kakatiya University College Of Engineering Department of Applied science & Humanities, India Department of Pharmaceutical Chemistry, India Department Of Zoology, India Principal Scientist, Novartis Pharma, SWITZERLAND Department of Plastic Surgery, K.L.E.S ,India Department of Bioinformatics, GITAM University, India Dean, Faculty Of Pharmaceutical Sciences, India Department of Biochemistry, India Director of PG Studies and Research, India Department of Pharmacognosy, India Post graduate and Research Department of Zoology, India Department of Pharmacognosy, India Department of Clinical Pharmacy, Malaysia Department of Pharmaceutics, USA Department of Pharmaceutics, UK Department of Pharmaceutics, USA

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Research Article Biological Sciences EFFECT OF MERCURY AND CADIMIUM ON THE ENZYME ACTIVITY OF HYDRILLA PLANT Goutam C. Das**, K. Bijay Kumar and B.K.Mohanty* * P.G.Department of Botany &Biotechnology, Khallikote Autonomous College, BERHAMPUR-760001, ODISHA **Lecturer in Botany, A.S. Collge, Balia-755005, Jajpur, ODISHA *Corresponding Author Email: mohantysir57@yahoo.com

ABSTRACT The study of toxicity of metals on the enzyme activity in the Hydrilla plant indicated that in general there is induction or inhibition in enzyme activity. The enzymes studied here in relation Hg and Cd toxicity like catalase, peroxidase and polyphenol oxidase found in the plant responded differently to the metal treatment. Catalase and polyphenol oxidase showed a decrease in the activity whereas peroxidase enzymes were stimulated by the metals. When the metals were treated along with growth hormone Kinetin, there was substantial recovery of the enzyme activity in the test plant. Other enzymes like acid phosphatase, acid/alkaline protease, acid /alkaline prophophatase and RNase were also studied to correlate the decrease in the molecular content in the test plant responded a decline in its activity with the metal treatment. However, the addition of Kinetin to the metals (HgCl2 and CdSo4) showed a recovery in the enzyme activity in the hydrilla plant .All these observations indicates that the aquatic plant hydrilla can be used as an indicator plant to assess the metal toxicity in the aquatic system.

KEY WORDS Mercury, Cadimium, Enzymes, Hydrilla

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INTRODUCTION Life depends on a complex network of chemical reactions brought about by a specific enzyme. The enzymes are the primary instruments for the expression of gene action. Enzymes are of biological importance in metabolic function, which have the most highly specialized class of proteins. The change in metabolism can well be correlated with the disturbance in behavior and action of the enzymes. The reduction in active metabolism has been correlated with the toxic effective different types of toxicants. The disturbance in enzymatic action can be related to the specific action of certain toxicants by inhibiting the enzymes to act, bind the active sites of the enzyme and enzymes become nonfunctional due to disintegration .Inhibition of enzymes by chemical agents may be reversible or

irreversible, competitive or noncompetitive. There are many different mechanisms through which thiol groups of enzymes can be acted upon by heavy metals. The importance of enzymatic studies in the system lies in the functional interpretation of the pollutant causing different types of disorders. Induction of catalase activity is brought about when toxic does of various metals are present (Van Assche and Clijsters, 1990). Taking into consideration the effect of the metal application, some workers have reported an induction of catalase in response to various heavy metals (Mukharji and Das Gupta, 1972; Subhadra et al., 1991). There are reports that cadmium decreased the activity of catalase, an antioxidant enzyme in germinating seedlings of mung bean phaseolus vilgaris. (Somasekharaiah et al., 1992). A decline

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www.ijpbs.com (or) www.ijpbsonline.com in catalase activity was also observed in rhy leaves when exposed to cadmium (Streh et al., 1993). Catalase activity increased significantly in Posidonia oceanica following 48h exposure to low concentrations of HgCl2 (0.01 Îźg L-1 and 0.1 Îźg L-1) as compared to controls (Ferrat et al., 2002b). Increase in Peroxidase has been observed in Glycine max (Lee et al., 1976) and Phaseolus Vulgaris (Van Assche et al., 1988) in response to cadmium. Reddy and Prasad (1992) observed an increased peroxidase Oryza sativa treated with different concentrations of cadmium. Karataglis et al., (1991) reported that increasing concentrations of copper, zinc, lead, nickel, chromium caused complex changes in the forms of isoperxidases in the seedlings of wheat. In unicellular green alga Scenedesmus subspicatus Reinhold et al., (1994) observed observed a significant rise in peroxidase activity in response to cadmium. Patro et al.,(2001), reported that all concentrations of the effluents found to have strong effect on the activity of peroxidase in the leaves of Oryza sativa L. In Ceratophyllum demersum mercury induced oxidative stress increased anti-oxidant enzymes like peroxidase. Rama Devi and Prasad, 1998, Sandalio et al., 2001 Metwally, 2003, 2004; Gallego et al., 1996; Balestrasse et al., 2003). Mukherji and Maitra (1976) have demonstrated the stimulated activities of peroxidase under conditions of lead toxicity of growth of rice seedlings. Very low cadmium levels in vitro have shown to stimulate the activities of certain enzymes like peroxidase, acid phosphatase etc. (Ernst 1980, Shah and Dubey, 1997).

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MATERIALS AND METHODS TEST MATERIAL: The submerged rooted macrophyte Hydrilla Verticillata casp is an aquatic weed belonging to family Hydrocharitaceae.It grows abundantly in lakes, ponds and ditches of tropical and

temperature climates. The coarsely serrated leaves of this plant occur in whorls of three to eight and have characteristic spines on the underside of the midrib. Hydrilla produces reproductive propagates called turions and tubers. Turions are compact dormant buds that are produced in leaf axils and fall from the plant, when they are mature. Tubers are formed terminally on rhizomes and can be found up to a foot deep in the sediment. A pond near A.S.College, Balia Dist: Jajpur (Orissa) was selected for the collection of Hydrilla verticillata plants. Samples of the plant were obtained from the site and grown in experimental ponds near the college premises. TEST CHEMICALS: The chemicals used in the present study were of the purest grade available and were obtained from renowned laboratories. Mercuric chloride [Hg Cl2] (M.W-272)] and cadmium sulphate [3 CdSO4, 8H2O (M.W.769.51)] were used at the test chemicals. Different concentration of the test chemicals were prepared by using double distilled water as the solvent. The selected concentration of the test chemicals used in the present study for both Mercury and cadmium were 1, 5, 10, 25mg-1 for experimentations. EXPERIMENTAL SETUP: Hydrilla growth in experimental tanks were collected in the form of sample and after acclimatization that would be subjected to tested in four different concentrations.(1,5,10.25mgL-1) of cd and Hg for 5 days and 10 days separately. Various physiological and biochemical parameters of the plant were measured in response to the metal treatment. After assessing the toxic effects of CD and Hg at various selected concentration on the test plant, an attempt would be made to study the amelioratine effects of growth regulators, Kintein (5mg-1) on toxic effects were assessed by adding 2ml in each concentrations of Cd and Hg.

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www.ijpbs.com (or) www.ijpbsonline.com The enzymes like Catalase, Peroxydase, Poly Phenyl oxidase (Kar and Mishra, 1976), Acid Phosphatase (Sacher and De Leo, 1977) Acid/Alkaline Protease (Moore and Stein, 1948) were estimated following standard procedures.

RESULTS The data pertaining to the effect of different concentrations of mercury and cadmium on the various enzyme activities and their percent increase/decrease was depicted in Table No. 1 and 2.The catalase enzyme activity was least effected and the highest percent decrease was only up to 5.28% in case of Hg and 7.69% in case of Cd. The peroxydase enzyme was elevated with the increase in Hg and Cd treatment and highest %increase was seen in case of 25 mg Hg treatment for 10 days. The polyphenol oxidase enzyme showed a decrease in activity with the increase in metal concentration and the maximum decrease was seen in case of 25 mg Cd treatment for 10 days. The results obtained with enzymes like Peroxydase and Poly phenyl oxidase was statistically significant. The Acid Phosphatease enzyme showed an increase in activity with the increase in metal concentration but the trend was not statistically significant.The Acid protease activity showed a decreasing trend with the increase in metal concentration and the highest decrease (71.25%) was seen in case of 25 mg Hg treatment for 5 days.

DISCUSSION

the signaling events that lead to the variation of the transcript levels of catalase in Pea plants under cadmium stress. The activity of several enzymes like RNase, catalase etc., also decrease due to toxic action of chromium compounds (Panda et al., 1997). In the present investigation catalase showed a decreased trend in response to increasing concentrations of mercury and cadmium. There was a â&#x20AC;&#x201C;ve correlation between various concentrations of both metals and the enzyme activity in Hydrilla plant after 5 and 10 days of interval. Similar decreasing trend also been observed when treated with metals and kinetin. Compared to metals the activity of catalase is more expressed in presence of kinetin. Peroxidase is present in grana and stroma lamellae (Henry, 1974, 1975.a.b. Henry et al., 1981.b) and thus may be closely associated with the changes in chloroplast including variation in chlorophyllII Pigment. Peroxidase is considered as a reliable indicator of various physiological processes. Some workers observed a marked change in peroxidase activity during root initiation and considered as an indicator of the process (Gasper et al., 1982, 1985; Moncousin 1986; Moncousin et al., (1987). There are also reports of other metals inducing peroxidase activity viz. mercury in Oryza sativa, mercury in Lemna minor (Subhadra et al.1991), choromium in Eichhornia crassipes (Satyakala and Jamil, 1993). In the present investigation there was induction of peroxidase activity in Hydrilla plant in response to both mercury and cadmium after 5 to 10 days of treatment, though there were some differences in degree of induction. There was a +ve correlation between induction of peroxidase and metal concentration both for mercury and cadmium.

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The catalase activity decreased following exposure to elevated cadmium concentrations (Shim et al., 2003; Dalurzo et al., 1997; 2001; Fornazier et al., 2002) Romero â&#x20AC;&#x201C; Puertas et al., (2004) studied the involvement of H2O2 and O2 in

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TABLE NO: 1: EFFECT OF DIFFERENT CONCENTRATIONS OF HgCl2 AND CdSO4 ON VARIOUS ENZYME ACTIVITIES OF HYDRILLA Sp. (EACH DATUM IS THE MEAN OF SIX REPLICATES) (Values in parentheses indicate the percent increase / decrease in content from control values) Treatment Metal CATALASE PEROXIDASE POLYPHENOL OXIDASE ACID PHOSPHATASE ACID PROTEASE Conc. In µ moles of H2O2 In absorbance (A) units In absorbance (A) units In absorbance (A) units In absorbance (A) units utilized / min-1 g-1 fresh weight / min-1 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days Control 119.25 119.18 1.88 1.89 1.24 1.25 1.18 1.18 1.6 1.6 1mg 118.23 117.89 2.02 2.01 1.09 1.08 1.03 1.01 1.04 1.03 (-0.855) (-2.591) (7.446) (6.349) (-12.096) (-13.6) (12.711) (-14.406) (-35) (-35.625) 5mg 116.16 115.07 3.04 3.01 1.02 0.96 2.09 2.06 1.01 0.98 Hg Cl2 (-2.591) (-3.448) (61.702) (59.259) (-17.741) (-23.2) (77.118) (74.576) (-36.875) (-38.75) 10mg 114.04 113.98 3.8 3.71 0.96 0.90 1.94 1.84 0.82 0.99 (-4.368) (-4.363) (106.914) (96.296) (-22.580) (-28) (64.406) (55.932) (-48.750) (-51.25) 25mg 113.05 112.88 4.12 4.08 0.80 0.76 2.48 2.46 0.46 0.68 (-5.199) (-5.286) (119.148) (115.873) (-35.483) (-39.2) (110.169) (108.474) (-71.250) (-57.5) r Value -0.893* -0.858* 0.868* 0.889* -0.924** -0.888* 0.849* 0.853* -0.868* -0.767 Ns Control 1mg 5mg Cd SO4 10mg 25mg r Value

119.24 119.19 1.89 1.88 1.24 1.25 1.18 1.18 117.18 117.18 1.92 1.89 1.11 1.09 1.06 1.02 (-1.727) (-1.686) (1.587) (0.531) (-10.483) (-12.8) (-10.169) (-13.559) 115.14 115.01 2.11 1.90 0.96 0.84 2.06 2.03 (-3.438) (-3.507) (11.640) (1.063) (-22.580) (-32.8) (74.576) (72.033) 111.98 111.08 3.29 3.08 0.78 0.72 1.05 1.03 (-6.088) (-6.804) (74.074) (63.829) (-37.096) (-42.4) (-11.016) (-12.711) 110.46 110.02 4.06 4.01 0.45 0.36 2.20 2.11 (-7.363) (-7.693) (114.814) (113.297) (-63.709) (-71.2) (86.440) (78.813) -0.900* -0.883* 0.958** 0.962** -0.977*** -0.957** 0.643 Ns 0.617 Ns ( *- Significant at P ≤ 0.05, **- Significant at P ≤ 0.01, ***- Significant at P ≤ 0.001, NS- Non-Significant.) International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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1.6 1.63 (1.875) 1.48 (-7.5) 0.98 (-38.75) 0.88 (-45) -0.897*

1.6 1.60 (0.00) 1.46 (-8.75) 0.96 (-46) 0.85 (-46.875) -0.902*

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TABLE NO: 2: EFFECT OF DIFFERENT CONCENTRATIONS OF HgCl2 WITH KINETIN AND CdSO4 WITH KINETIN ON VARIOUS ENZYME ACTIVITIES OF HYDRILLA Sp. (EACH DATUM IS THE MEAN OF SIX REPLICATES)(Values in parentheses indicate the percent increase / decrease in content from control values) Treatment Metal CATALASE PEROXIDASE POLYPHENOL OXIDASE ACID PHOSPHATASE ACID PROTEASE Conc. In µ moles of H2O2 In absorbance (A) units In absorbance (A) units In absorbance (A) units In absorbance (A) units utilized / min-1 g-1 fresh weight / min-1 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days 5 days 10 days Control 119.43 119.25 1.9 1.9 1.25 1.26 1.19 1.19 1.7 1.7 1mg 118.63 117.95 2.12 2.05 1.11 1.10 1.06 1.04 1.05 1.04 (-0.669) (-1.090) (11.578) (7.894) (-11.2) (-12.698) (-10.169) (12.605) (-38.235) (-380823) 5mg 116.56 115.87 3.14 3.11 1.06 1.02 2.11 2.09 1.02 1.01 Hg Cl2 (-2.403) (-2.834) (65.263) (63.684) (-15.2) (-19.047) (78.813) (75.630) (-40) (-40.588) Kinetin 10mg 117.34 114.02 3.96 3.92 0.98 0.87 1.98 1.88 0.89 0.86 (-1.749) (-4.385) (108.421) (106.315) (-21.6) (-30.952) (67.796) (57.983) (-47.647) (-49.411) 25mg 113.35 113.12 4.22 4.18 0.82 0.79 2.58 2.56 0.56 0.54 (-5.090) (-5.140) (122.105) (120) (-34.4) (-37.301) (118.644) (115.126) (-67.058) (-68.235) r Value -0.948** -0.878* 0.870* 0.869* -0.940** -0.880* 0.898* 0.870* -0.813* -0.813* Control 119.25 119.28 1.90 1.90 1.25 1.26 1.19 1.19 1.7 1.7 1mg 117.58 117.43 1.95 1.93 1.13 1.12 1.08 1.03 1.65 1.62 (-1.400) (-1.550) (2.631) (1.578) (-9.6) (-11.111) (-9.243) (-13.445) (-2.941) (-4.7.5) 5mg 115.34 115.11 2.21 2.19 0.98 0.88 2.09 2.06 1.58 1.56 Cd SO4 with (-3.278) (-3.495) (16.315) (15.263) (-21.6) (-30.158) (75.630) (73.109) (-7.058) (-8.235) Kinetin 10mg 112.63 111.98 2.12 3.28 0.82 0.76 1.11 1.09 1.08 1.06 (-5.551) (-6.120) (11.578) (72.631) (-934.4) (-39.682) (-6.722) (-8.403) (-36.470) (-37.647) 25mg 111.56 110.76 4.26 4.11 0.47 0.38 2.24 2.15 0.98 0.95 (-6.448) (-7.142) (124.210) (116.315) (-62.4) (-69.841) (88.235) (80.672) (-42.352) (-44.117) Ns Ns r Value -0.884* -0.888* 0.951** 0.966** -0.982*** -0.967** 0.660 0.636 -0.899* -0.902* (*- Significant at P ≤ 0.05, **- Significant at P ≤ 0.01, ***- Significant at P ≤ 0.001, NS- Non-Standard)

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www.ijpbs.com (or) www.ijpbsonline.com In the present study it is observed that polyphenol oxidase activity was reduced in Hydrilla plant in response to mercury and cadmium after 5 to 10 days of interval. There was a close-ve correlation between metal concentration applied and the decrease in the activity of the enzyme in response to both the metals. Our investigation is contradictory to the views of Garg et al., (1994) that an increase in polyphenol oxidase activity in Marsilea minuta under cadmium induced stress. Activity of polyphenol oxidase increased into a lot by the addition of kinetin with heavy metals and was observed in Hydrilla plant after 5 to 10 days of interval. As far as Hydrilla plant is concerned polyphenol oxidase can safely be used as an indicator of mercury and cadmium toxicity. A rise in acid phosphate activity was observed in Glycine max in response tyo cadmium (Lee et al., 1976). Medicago polymorpha in response to drought stress. (Ehsanpour et al., 2003). Trticum aestivum in response to chromium (Sharma and Sharma, 1996). On the other hand an inhibition of acid phosphatase activity was observed by Kong and Chen, (1995) in response to Al and Zn, and Rajni et al., (1991) in response to Cu, Zn, Hg in potato tubers are available. In Hydrilla at low concentrations of mercury and cadmium levels (5 mg L -1) stimulate the activity of acid phosphatase. Whereas at higher concentrations of both metals (10mg L-1) have inhibitory to thjis enzyme. Further increasing the concentrations of both metals (25mg L -1) the activity of acid phosphatase again increases after 5 to 10 days of interval. This observations was supported by Ernst, (1980); Shah and Dubey,(1997); Kumar and Banerjii, (1992). Similar trend was also observed when the test plant was both metals and kinetin. There was a +ve correlation between mercury and enzyme activity and mercury and kinetin and enzyme activity both after 5 to 10 days of treatment but a

non-significant correlation was observed between the enzyme activity with cadmium and enzyme activity with cadmium and kinetin. The trends in acid phosphatase activity in response to metals shows that the enzyme cannot be used as a general indicator of metal toxicity as its responses are dependent on species and organ. Sneh Lata(1989) on Phaseolus aureus had shown that cadmium influences the activity of protease which seem to inhibit the seedling growth. Cadmium inhibits protease activity in germinating pea seeds was reported by Bansal et al.,(2001). The protease enzyme inhibition with HgCl 2 was also observed in germinating mungbean seeds. (Yamaoka et al., 1990). Oats (Drivdhal and Thimann, 1978). Common bean (Rascusen and foote, 1970), Soybean (Ragster and Chrispeels, 1979) and Agave (Du Toit, 1976). In Hydrilla we report the presence of two sulfhydryl protease, one alkaline with pH optimum of 8.0 and the other a highly acidic one, with optimum pH of 3.0. Activity of acidic and alkaline protease were inhibited by the response of heavy metals in Kalachoe leaves. (Jasrai et al., 1992). In the present investigation an attempt is made to analyse the activity of both acidic protease and alkaline protease in Hydrilla plant. There was a significant decrease in the activity of both acidic and alkaline protease Hydrilla in response to both mercury (after 5 and 10 days) of interval. But a non-significant correlation was observed after 10 days of interval with mercury treatment. In the present investigation the acid pyrophsphatase showed decreasing trend in response to both the metal and enzyme activity after 5 and 10 days interval. Similar trend exists when treated with metals and kinetin and showed non-significant correlation between the enzyme and metal with kinetin. The alkaline pyrophosphatase enzyme also showed a decreased trend in response to both the

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www.ijpbs.com (or) www.ijpbsonline.com metals and there was a –ve correlation and enzyme activity after 5 days of treatment but after 10 days non-significant correlation exists between them. In response to metals and kinetin similar decreasing trend also observed in Hydrilla and showed a –ve correlation between enzyme activity and metal with kinetin after 5 days of treatment but the non-significant correlation was existing after 10 days of interval. Thus inorganic pyrophosphatase can be used as an indicator of metal toxicity response. Change in synthesis or alterations in levels of RNase may be involved in the regulation of RNA content. To explore the second possibility RNase was included in the present investigation. There was a decline in the RNase activity in Hydrilla in response to both mercury and cadmium after 5 and 10 days of interval. A non-significant correlation exists between mercury and enzyme activity after subsequent days but a close –ve significant correlation exists between cadmium and enzyme acivity. Induction of kinetin to the metals showed similar trend in Hydrilla plant and a non-significant correlation exists between enzyme activity and mercury with kinetin, but with cadmium and kinetin a significant close –ve correlation exist after 5 and 10 days of interval. Compared to only metal treatment RNase activity is more pronounced when treated with metal and kinetin Hydrilla after 5 and 10 days of interval. It may be concluded that RNase may be involved in the regulation of RNA content in Hydrilla in response to metals under study.

ACKNOWLEDGEMENT

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Authors are thankful to Principal, Khallikote autonomous College, Berhampur for providing necessary laboratory facilities.

REFERENCE

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and physiological roles in higher plants. Univ. of Geneva, Switzerland. 1982 Henry, E.W.; Ultrastructural localization- polyphenol oxidase activity in chloroplasts byNicotina tobacum chloroplast. Plant Physiol. I50 (Suppl.): 357.1974 Henry, E.W; Ployphenoloxidase activity in thylakooids and membrance bound granular components of Nicotina tobacum- Chloroplasts. J. Microsco. 22: 10116, 1975a Henry, E.W.; preoxidase in tobacco abscission zone. J. Ultrastruct. Res. 289-299, 1975b Henry, E.W.; De Poore, M.J.; Oâ&#x20AC;&#x2122; Conner, M.N. and De Morrow, J.M. Photobiochem. Photobiophys., Sorbitol disrupted spinach (Spinacea oleracea L.) chloroplasts. II. ATPsae, per oxidase and chlorophyll.3:145-151,1981b Jasrai, Y.T., Bhatt, P.N. and Mehta, A.R.; Highly acidic and alkaline proteases in Kalanchoe leaves and their activity during dormant epiphyllous bud reactivation. Plant physiol and Biochem. Vol. 19; 88-92, 1992 Karataglis, S.; Moustakes, M. and Symeonidis, L.; Effect of heavy metals on isoperoxidates of wheat. Biol. Plant. (Prague). 33(I): 3-9, 1991 Kumar, S., Banerjee, D.; effect of some heavy metals on in vitro activities of certain enzymes, Plant physiol. Biochem. 19: 33-35, 1992 Lee, K.C.; Cunningham, B.A.; Paulsen, G.M.; Liang, G.H. and Moore, R.B.; effects of cadmium on respiration rate and activities of several enzymes in soybean seedlings. Physiol. Plant. 36: 4-6, 1976a Lee, K.C.; Cunningham, B.A.; Paulsen, G.M.; Chung, K.H. and Liang, G.H.; Lead effects on several enzymes and nitrogenous compounds in soybean leaf. Journal of Environmental Quality. 5: 357-35, 1976b Maier, R.; Aktivitat und multiple Foremen der Peroxidase in unverbleiten und verbleiten Pflanzen von Zea mays und Medicago sativa. Phyton. 19: 83-96, 1978a Metwally, A.; Finkemeier, I.; Georgi, M. and Dietz, K.J.; salicylic acid alleviates the cadmium toxicity in barley seedlings. Plant physiol. 132: 272-281, 2003 Monocousin, C.; Preoxidase as a marker for rooting improvement of clones of Vitis cultured in vitro. In: Molecular and physiological aspects of plant peroxidases. Univ. of Geneva, Switzerland, 1986 Monocousin, C.; Fevre, J.M.; Gasper, T.; In; Physiology and Biochemistry of auxines in plants. (M.Kuttaceak, R.S. Bandurski and J.Krekuleeds) Academia, Praha 331337, 1987 Mukherji, S.; maitra, P.; Toxic efftecs of lead on growth and metabolism of germinating rice seeds and on mitosis of cells. Ind. J.Exp. boil. 14: 519-571, 1976

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Mukherji, S. and Dasgupta, B.; characterization of copper toxicity in lettuce seedlings. Physiologia Plantarum. 27: 126-129, 1972 Panda, S.K. and Patra, H.K.; Physiology of chromium toxicity in plants. A review plant physiol and Biochem, vol. 24(I): 10-17,1997 Patro, B.; Panigrahi, S.; Das, M. and tripathy, S.; the effectiveness of industrial effluents on chlorophyll degradation and hydrogen peroxide utilization in rice leaves during senescence. Asian J. of Microbiol. Biotech. And Env. Sc. Vol.3: 183-190,2001 Racusen, D.; Foote, M., An endopeptidase of bean leaves. Canadian J. Bot. 48:1017-1021,1970 Ragster, L. and Chrispeels, M.J.; Azocoll-digesting Proteinase in Soybean leaves. Plant physiol. 64: 857862, 1979 Rajni, K.; Malhotra, N.; and Dua, S.; Characteristics of acid phosphateses from Ambari (Hibiscus Cannabinus L.) seedlings. Plant physiol and Biochem. Vol. 18(I): 4753,1991 Rama Devi, S., Prasad, M.N.V. (1998) - Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte; response of antioxidant enzymes and antioxidants. Plant Sci. 138, 157-165,1998 Reinheld, D.; Hoefner, W. and Koehler, W.; Influence of Cu, Cd and altrazine on the metabolism of the unicellular green alga scenedesmus subspicatus. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde. 157 (2): 145-150 ,1994 Romero-Puertas, M.C.; Rodriguez-Serrano, M.; Corpas, F.J.; Gomez, M.; del Rio, L.A. and Sandalio, L.M. Cadmium induced subcellular accumulation of O2 and H2O2 in pea leaves. Plant cell Environ. 27:1122-1134, 2004 Sandalio, L.M.; Dalurzo, H.C.; Gomez, M.; RomeroPuertas, M.c. and del Ri; Cadmium induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52: 2115-2126, 2001 Satyakala, G. and Jamil, K.; Effect of trace metal (Cu and Cr) on the aquaticweed Eichornia crassipes. Int.J.Toxic Occup Envir. Health. (2):41 (abstr), 1993 Sharma, D.C. and Sharma, C.P.; Chromium uptake and toxicity effects on growth and metabolic activities in wheat, ev 2003. Indian J. Expt. Biol. 34: 689-691, 1996 Shim, I.S.; Monose, Y.; Yamamoto, A.; Kim, D.W. and Usui, K. ; Inhibition of catalase activity by Oxidative Stress and its relationship to salicyclic acid accumulation in plants. Plant Growth Regul. 39: 285-292, 2003 Sneh Lata ; Pretreatment effects of cadmium on seedlings growth, mobilization and activity of hydrolytic enzymes in phaselous aureus Cv.T-44. Mendel. 6, 69772, 1989

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Somashokaraiah, B.K.; Padmaja, K. and Prasad, A.R.K.; Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaselous vulgaris): involvement of lipid peroxides in chlorophyll degradation. Physiol. Plant. 85(I); 85-89, 1992 Streh, P.; Michael-knauf, A. and Feirabend, J.; Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms under various osmotic and chemical stress conditions. Physiol. Plant. 88(II): 590-598, 1993 Subhadra, A.V.; Nanda, A.K.; Behera, P.K. and Panda, B.B. ;Acceleration of catalase and preoxidase activities in Lemna minor L. and Allium cepa L. in response to low

levels of aquatic mercury. Environ. Pollut. 69; 169-197, 1991 Van Assche, F. and Clijsters, H.; Effects of metals on enzyme activity in plants. Plant, Cell and Environ. 13: 195-20, 1990. Van Assche, F.; Cardinales, C. and Clijsters, H.; Induction of enzyme capacity in plants as a result of heavy metal toxicity: dose-response relations in Phaseolus vulgaris L., treated with zinc and cadmium. Environmental Pollution. 52: 103-115, 1998 Yamadka, Y.; Takimura, O.; Fuse, H. and Kamimura, K.; Effect of magnetism on growth of Dunaliela salina. 51: 87-90, 1993

*Corresponding Author: Dr. B.K.Mohanty,

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Reader in Botany P.G.Department of Botany &Biotechnology, Khallikote Autonomous College, BERHAMPUR-760001, ODISHA, mohantysir57@yahoo.com, Mob: 09437216341

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Research Article Pharmaceutical Sciences DIELECTRIC STUDY OF DIMETHYL FORMAMIDE WITH ETHOXY ETHANOL USING TIME DOMAIN REFLECTOMETRY TECHNIQUE KRISHNA H. FATTEPUR*1, JAGADISH G. BARAGI 2 1

Department of Physics, National College, Basavanagudi, Bangalore-560 004, Karnataka, India 2 Department of Chemistry, J.S.S. College, Vidyagiri, Dharwad-580004, Karnataka, India *Corresponding Author Email: krishnafattepur@yahoo.com

ABSTRACT Dielectric relaxation studies of Dimethyl Formamide (DMF) with Ethoxy Ethanol (EE) mixture for different concentrations over the frequency range 10 MHz to 20 GHz in the temperature range 278K to 308K have been investigated using time domain reflectometry (TDR) technique. Static permittivity ( s), dielectric constant at high frequency () and relaxation time () were found through dielectric measurements. The excess dielectric parameters, Bruggeman factor, Kirk wood correlation factor and thermodynamic parameters like change in enthalpy (H) and change in entropy (S) have been determined and discussed to yield information on the molecular interactions and microdynamics of the mixture of the system.

KEY WORDS Dielectric Relaxation, Dimethyl Formamide, Ethoxy Ethanol, Bruggeman Factor, Cole Davidson model, Kirkwood Correlation factor.

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INTRODUCTION Studies on frequency dependent dielectric relaxation of binary mixture DMF and aqueous solutions have been reported 1-2 to understand the microdynamics and hydrogen bond structure in these systems. The hydrogen bonding causes a significant effect upon the structure, thermodynamic properties like change in enthalpy (H) and change in entropy (S). Referring work of Gestblom and J. Sjoblom 3-4 small - chain alcohols like methanol have faster relaxation time and low activation energy where as long chain molecules have slower relaxation  and high activation energy. Several researchers have studied the temperature dependent dielectric relaxation parameters, activation energy and Kirkwood correlation factor of various binary mixtures 5-7. In this paper an attempt has been made to report a

systematic investigation of dielectric properties and dielectric mechanism, thermodynamic properties, Bruggeman factor and Kirkwood correlation factor and the results are discussed over the seven different mole fractions at four different temperatures from 278K to 308K.

MATERIALS AND METHODS (i) Chemicals and Sample Preparation: Analytical reagent grade samples of Dimethyl Formamide (DMF) and Ethoxy Ethanol (EE)) were procured from S.D. fine-Chem. Pvt. Ltd., Mumbai, India. In the present study they are used without further purification. The solutions were prepared at seven different mole fractions of DMF and EE at room temperature. The mole fraction of components DMF (1) and EE (2) is calculated using the following Equation 1 7-8.

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VA  A MA X1  V A  A VB  B  MA MB VB  B MB X2  V A  A VB  B  M M B ---------- (1) A And Where MA, MB are the molecular weights VA, VB are the volumes and A, B are the density of liquid A and B respectively. The limiting high frequency  of pure ideal solvents and m of the binary mixtures were taken as the square of refractive index nD at the wavelength of sodium Dline.

empty cell wave-form is used as the reference wave-form. Both response wave-forms are the reflected wave-forms from the sample cell with open termination of transmission line. The data acquisition is carried out for 7 concentrations in the temperature range 278K to 308K with an accuracy of ±1 0C. At each time the response wave-forms without sample and with sample were recorded. The time-dependent response wave-form without sample is referred as R1 (t) and with sample is referred as Rx (t). In this process, the time-dependent data were processed to obtain complex reflection coefficient spectra *() using Fourier transformation 9-10 in the frequency range of 10 MHz to 10 GHz. (iii) Data Analysis: As explained earlier the reflection coefficient is related to dielectric response of a sample under study for the frequency range 10 MHz to 10 GHz in terms of complex permittivity spectra. The complex permittivity * can be written as ’ – i”, where ’ is real part, is called dielectric dispersion and it is proportional to stored energy. Where ” is imaginary part and is called dielectric loss. The value of  is not so sensitive to ε* and it is taken as a fixed value 3.0. A sample of complex permittivity dispersion spectra with ’ and ” is shown in Graph 1. This corresponds to 0.5438 mole fraction of EE at 278K.

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(ii) Apparatus: The Hewlett Packard HP54750A sampling oscilloscope with HP54754A TDR plug-in module has been used. After observing TDR response for the sample under study, the time window was kept to 5 ns. Also by observing TDR response for the sample under study, the SMA sample cell with 1.35 mm effective pin length has been used. To reduce noise, time-dependent response curve was averaged for 64 times and then stored in the memory of the oscilloscope with 1024 points per wave-form. First, the reflected pulse from the empty cell is acquired and stored in the memory and then, the reflected pulse from the cell with sample is acquired and stored in the memory. The

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Graph 1: Complex permittivity dispersion spectra with’ and ” corresponds to 0.5438 mole fraction of EE at 278K

The frequency dependent complex permittivity ε* = ε'-iε'' data was obtained by using dielectric parameters of raw data. The static dielectric constant (s) and relaxation time () for the mixture obtained by using Havriliak-Negami equation 2 11.

 * ( )    

s  

1  (it )   ------ (2) 1

Where s is the static dielectric constant,  is the high frequency dielectric constant,  is the angular frequency and  is the relaxation time,  and  are the experimental parameters for the distribution of relaxation time. While fitting the data, equation 2 includes Cole-Cole (=1) 12, Davidson–Cole (=0) 13 and Debye ((=0, =1) 14 relaxation models.

RESULTS The Physical properties of pure liquids DMF and EE used for calculation are listed in Table 1. Table 1: Literature values of Physical properties of DMF and EE Name Mol. Formula Mol. Wt. Density R I (nD) Dipole -3 moment µ  gcm

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DMF EE

C3H7NO C4H10O2

73.094 90.121

0.944255 0.925235

1.430520 1.405425

3.86 D 2.19 D

Source: Hand book of Chemistry and Physics David R Lide 84 th Edition CRC Press (i) Permittivity and Relaxation Time: The evaluated values of s,  as a function of mole fraction of EE at four different temperatures are given in Table 2. A graphical representation of s, and  is shown in Graphs 2 and 3 respectively.

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Table 2: Temperature dependent dielectric parameters for binary mixture of DMF+EE Mole Fraction of EE 278K 288K 298K 308K

0.0000 0.1658 0.3463 0.4428 0.5438 0.7607 1.0000

s

 (ps)

s

 (ps)

s

45.0 38.8 33.2 25.2 29.4 38.6 17.5

25.2 30.2 40.4 64.7 58.6 56.4 55.8

41.1 35.3 32.2 21.8 4.4 34.5 16.5

19.3 25.3 34.5 60.3 54.4 48.9 46.5

39.4 34.3 30.7 19.2 22.7 27.7 15.7

 (ps) 11.1 22.1 30.2 57.0 48.6 41.9 38.6

s 34.7 32.1 27.1 17.8 19.0 23.5 14.2

 (ps) 8.4 16.8 27.3 50.1 46.6 36.2 34.5

Graph 2: Variation of static dielectric constant (s) as a function of mole fraction of EE at different temperatures.

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Graph 3: Variation of static relaxation time (s) as a function of mole fraction of EE at different temperatures.

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(ii) Excess Permittivity and Excess Inverse Relaxation Time:

E

s The excess permittivity, at various concentrations is calculated using the equation 3.

 sE = ( ) – [( ) X + ( ) X ] -------(3) s m s A A s B B Where X is the mole fraction and subscripts m, A and B represent the binary mixtures and components DMF and EE of the binary mixture respectively. The excess permittivity provides qualitative information about structural aspects of the mixture.

E

If s = 0 indicates the liquids A and B do not interact at all.

E

If s < 0 indicates the liquids A and B interact in such a way that the total effective dipole gets reduced. This shows that liquids A and B may form multimers leading less effective dipoles or there is a tendency of dipoles to align in anti-parallel direction.

1    

The excess parameter at various concentrations is calculated using the Equation 4. (1/)E = (1/)m – [(1/)A XA + (1/)B XB] --------(4) Where X is the mole fraction and subscripts m, A and B represent the binary mixtures and components DMF and EE of the binary mixture respectively. It provides the qualitative information about the formation of multimers and monomers in the following way – (1/E= 0: Indicates there was no change in the dynamics of liquids A and B. (1/0: Indicates liquids A and B interaction produced a field such that the effective dipole rotated slowly. (1/0: Indicates liquids A and B interaction produced a field such that the effective dipoles rotated rapidly. E

E If s > 0 indicates the liquids A and B interact in such a way that the total effective dipole moment increases. There is a tendency of dipole aligned in parallel direction. Table 3: Excess permittivity Mole Fraction 278K of EE (1/)E  sE

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0 0.1658 0.3463 0.4428 0.5438 0.7607 1

0 -1.6419 -2.2760 -7.6227 -0.6451 14.5190 0

0.0000 -0.0030 -0.0074 -0.0136 -0.0108 -0.0054 0.0000

 sE

E

1   and    values of the DMF-

The evaluated s EE binary mixture are given in Table 3.

and excess inverse relaxation time (1/)E of DMF+EE 288K 298K 308K E E E E (1/) (1/) (1/)E s s  sE 0 -1.7225 -0.3803 -8.4068 -23.322 12.1133 0

0.0000 -0.0073 -0.0123 -0.0218 -0.0169 -0.0083 0.0000

0 -1.172 -0.492 -9.705 -3.812 6.3287 0

0.0000 -0.0342 -0.0347 -0.0441 -0.0346 -0.0174 0.0000

0 0.7979 -0.5 -7.822 -4.552 4.3944 0

0.0000 -0.0446 -0.0512 -0.0592 -0.0486 -0.0229 0.0000

A graphical representation of excess permittivity and excess inverse relaxation time against mole fraction of EE for four different temperatures are shown in Graphs 4 and 5 respectively.

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Graph 4: Variation of Excess permittivity SE as a function of mole fraction of EE at different temperatures

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Graph 5: Variation of excess inverse relaxation time (1/)E as a function of mole fraction of Ethoxy Ethanol at different temperatures.

(iii) Bruggeman Factor (fb): The Bruggeman mixture formula can be used as first evidence of molecular interactions in binary mixture A and B respectively 15. The equation for Bruggeman factor (fB) is given by

   0B f B  0m  0 A   0B

  0A    0m

  

 1  V ---------- (5)

According to equation (5) a linear relation is expected between Bruggeman factor and mole fraction of EE. Any deviation from this linear relation indicates existence of intermolecular

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www.ijpbs.com (or) www.ijpbsonline.com interactions. Many similar observations have been reported in 16-17. The evaluated values of Bruggeman factor fB for binary mixture DMF+EE

at four different temperatures are given in Table 4.

Table 4: Bruggeman Factor, fB of DMF+EE Mole Fraction fB of EE 278K 288K 298K 308K 0.0000 1 1 1 1 0.1658 0.81378 0.804 0.82192 0.8961 0.3463 0.63182 0.6923 0.6878 0.6833 0.4428 0.3397 0.2662 0.18767 0.2194 0.7607 0.80753 0.7757 0.56943 0.5166 1.0000 0 0 0 0

Ideal 1 0.8342 0.6537 0.5572 0.2393 0

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A graphical representation of fB against mole fraction is shown in Graph 6. Graph 6: Variation of Bruggeman Factor f B as a function of mole fraction of Ethoxy Ethanol at different temperatures.

(iv) Effective Kirkwood Correlation Factor (geff): The structural information about the liquids from the dielectric relaxation parameter may be obtained using the effective Kirkwood correlation factor, geff 17. This factor is also a parameter for obtaining information regarding orientation of electric dipoles in polar liquids. The Kirkwood equation for the binary mixture can be expressed as 18-19

    2 sm   m  2 4N   A2  A  X A  B B X B  g eff  sm m 2 KT  M A MB  sm  m  2  -- (6)

Where XA and XB are mole fractions liquids A and B respectively, s is static permittivity,  is permittivity at high frequency,  is dipole moment,  is density at temperature T, M is molecular weight, K is Boltzmann’s constant, N is Avogadro’s number. The values of geff are evaluated for different concentrations at

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www.ijpbs.com (or) www.ijpbsonline.com different temperatures using equation 6. The evaluated values of geff as a function of mole

fraction of EE at four different temperatures are given in Table 5.

Table 5: Kirkwood Correlation Factor geff of DMF + EE Mole geff fraction 278K 288K 298K 308K Of EE 0.0000 1.27 1.20 1.19 1.08 0.1658 1.25 1.18 1.18 1.14 0.3463 1.27 1.27 1.25 1.14 0.4428 1.06 0.94 0.85 0.81 0.5438 1.40 0.15 1.15 0.98 0.7607 2.57 2.37 1.95 1.70 1.0000 1.92 1.87 1.83 1.70 The graphical representation of variation of geff with mole fraction of EE are given in Graph 7. Graph 7: Variation of Kirkwood correlation factor geff as a function of mole fraction of Ethoxy Ethanol at different temperatures.

(v) Arrhenius Plot: The thermodynamic parameters like molar enthalpy of activation H* and molar entropy of activation S* are calculated by Eyring’s rate equation 7 using least square fit method. 20-21

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

G* h RT exp KT

--------- (7) Where G is molar free activation energy, *

G  H  TS ------ (8) *

Equation 7 can also be written as * *  h  H  TS ln T   ln    RT K

  h   S *   H *     ln(T )  ln        RT  K R        ------- (9) The slope of the linear plot between ln(T) and

 H    (1/T) gives  R  .

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www.ijpbs.com (or) www.ijpbsonline.com Using values of molar enthalpy H* and molar entropy S*, molar free activation energy, G* can be calculated using equation 8. The evaluated

T in K 278 288 298 308

1000/T K-1 3.597 3.472 3.356 3.247

values of ln(T) and (1/T) are tabulated in table 6 and the graphical representation of Arrhenius plot is shown in Graph 8.

Table 6: Arrhenius Plot for DMF + EE ln(T) Mole Fraction of EE 0 0.1658 0.3463 0.4428 0.5438 -18.777 -18.596 -18.305 -17.897 -17.933 -19.130 -18.737 -18.427 -17.869 -17.972 -19.420 -18.838 -18.526 -17.891 -18.050 -19.770 -19.080 -18.590 -17.99 -18.060

0.7607 -17.971 -18.078 -18.199 -18.310

1.000 -17.980 -18.130 -18.280 -18.360

Graph 8: Arrhenius plot to calculate thermodynamic parameters for different concentrations.

which shows the change in activation energy of the system. 6. Molar Enthalpy and Molar Entropy: The evaluated values of molar enthalpy and molar entropy from Arrhenius plot for different molar concentrations are tabulated in Table 7.

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From Graph 8 linear nature of Arrhenius plot shows that equivalent changes in values of molar enthalpy of activation H in the observed temperature range from 278 K to 308 K. The slope of Arrhenius plot changes with concentration,

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Table 7: Change in Enthalpy and Entropy of DMF + EE Mole Fraction of ΔH in KJ ΔS in KJ EE 0.0000 24.941395 0.047927 0.1658 11.000624 -0.003533 0.3463 6.908211 -0.020428 0.4428 2.0059208 -0.041773 0.5438 3.2762430 -0.036662 0.7607 8.1292074 -0.018940 1.0000 9.1884923 -0.014922 The graphical representation of molar enthalpy and molar entropy are shown in Graph 9. Graph 9: Variation of molar enthalpy and molar entropy as a function of mole fractions (EE).

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DISCUSSION From Graph 2, the nonlinear behavior of these parameters confirms the H-Bond complex function over the entire concentration range of DMF-EE mixture. The maximum decrease in s value is observed when mole fraction of EE is 0.4428 reveals that dipole interaction is minimum for all observed temperatures 278, 288, 298 and 308K.This suggests that weak intermolecular interaction due to shielded charge distribution in DMF. The

maximum value of s observed for temperatures 278, 298 and 308K when mole fraction of EE is 0.7607 reveals the dipole interaction between DMF-EE binary mixtures is maximum. From graph 3, Relaxation time is found to be maximum for all temperatures when mole fraction of EE is 0.4428. The Excess parameters related to s and  provide valuable information regarding interaction between polar-polar liquid mixtures. It also gives

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www.ijpbs.com (or) www.ijpbsonline.com the information of formation of multimers in the mixture due to intermolecular interaction. From Graph 4 it is observed that the maximum

E

negative value of S when mole fraction of EE is 0.4428 for all observed temperatures. It represents that effective value of dipole moment is get reduced due to anti-parallel alignments of dipoles in the mixture. Maximum negative value

E

of S at 288K confirms the formation of strong H-bond hetero molecular interactions in the mixture and these interactions are relatively stronger compare to other observed E temperatures. Excess permittivity S is maximum positive value is observed when mole fraction of EE is 0.7607. It indicates the molecular interaction of DMF & EE increases the effective dipole moment. There is a tendency of dipole aligned in parallel direction also it is maximum at temperature 278K. As temperature increases alignment of dipoles in parallel direction decreases. E

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1   From Graph 5 it is observed that    is negative for entire range of concentration and for all observed temperatures. The negative value of inverse relaxation time indicates molecular interaction produced hindering field making effective dipole rotation slowly. The maximum negative value is observed when mole fraction of EE is 0.4428 for all observed temperatures. It is observed in Graph 6, that there is a non-linear relationship between fB and mole fraction. The Bruggeman factor shows a positive deviation throughout from the ideal value. Maximum deviation from ideal value is observed when mole fraction of EE is 0.7607. This indicates the existence of inter molecular interaction in the mixture. According to Graph 7 the values of geff are greater than unity for all temperatures shows parallel

orientation of electric dipoles. The maximum g eff value is observed for all temperatures when mole fraction of EE is 0.7607 shows maximum inter molecular interaction within the system. From graph 8 linear nature of Arrhenius plot shows that equivalent changes in values of molar enthalpy of activation H in the observed temperature range from 278 K to 308 K. The slope of Arrhenius plot changes with concentration, which shows the change in activation energy of the system. It is observed that molar enthalpy of activation H decreases till mole fraction of EE is 0.4428 and then it increases. It means less energy is needed for group of dipole reorientation up to mole fraction of EE 0.4428. Negative value of entropy S for all mole fractions of EE except zero value of EE confirms relatively high ordered arrangement of molecules in the system.

CONCLUSION The dielectric parameters, Kirkwood correlation factor, Bruggeman factor, thermo dynamic parameters have been reported for DMF-EE binary mixture for different concentrations at various temperatures. The relaxation time of the mixture is found to be higher for mole fraction of EE is 0.4428. The maximum negative value of E

1   excess relaxation time    indicates the slower rotation of effective dipoles of the system. The more deviation of Bruggeman factor from its ideal value for mole fraction of EE 0.7607 shows more energy is needed for group dipole reorientation.

REFERENCES 1. 2.

G.M. Dharne, A.P. Maharolkar, S.S.Patil, P.W.Khirade and S.C. Mehrotra. IJPBS V1 (2), (2010). R.J. Sengwa, Sonu Sankhela, Vinita Khatri. J. Mol. Liq., 151, 17-22, (2010).

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www.ijpbs.com (or) www.ijpbsonline.com 3.

R.H. Fattepur, M.T. Hosmani, D.K. Deshpande, R.L. Patil and S.C. Mehrotra. Pramana, Vol. 44 No.1, 33-44, (1995). 4. B.Gestblom and J. Sjoblom, J. Acta. Chem. Scan. A38, 575, (1984). 5. Prabhakar Undre, S.N.Helambe, S.B.Jagadale, P.W.Khirade & S.C.Mehrotra, Pramana J.Phys. Vol. 68, No 5, 851-861, (2007). 6. P.W. Khirade, A.S. Chaudhari, J.B. Shinde, S.N. Helambe and S.C. Mehrotra, J. Soln, Chem. 28 (8), 1031-1043, (1999). 7. V.P. Pawar and S.C. Mehrotra, J. Mol. Liq., 108/1-3, 95105, (2003). 8. K. Dharmalingam, K. Ramachandran, P. Shivgurunathan, B. Prabhakar Undre, P.W. Khirade, S.C. Mehrotra., J. Appl. Polym. Sci. Vol. 107 No.4, 2312-2316, (2008). 9. C.E. Shanon, Proc. IRE, 37, 10, (1949). 10. H.A. Samulan, Proc. IRE, 39, 175, (1951). 11. S. Havriliak and S.Negami, J. Polym, Sci. C14, 99, (1966).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|483-494 12. K.S. Cole, R.H. Cole, J. Chem Phys. 9, 341, (1941). 13. D.W. Davidson, R.H.Cole, J. Chem. Phys. 18, 1484, (1950). 14. P. Debye, Polar molecules, Chem. Catalog Co., New York, (1929). 15. D.A.G. Bruggeman, Ann. Phys. (Leipzig) 5, 636, (1935). 16. S.M. Puranik, A.C. Khumbharakhane and S.C. Mehrotra J. Mol. Liq. 59, 173, (1993). 17. M.I. Aralaguppi, T.M. Aminbhavi, R.H. Balundugi and S.S. Joshi, J. Phys. Chem. 95, 5299, (1991). 18. A.C. Kumbharakhane, S.M. Puranik and S.C. Mehrotra, J. Mol. Liq. 51, 261, (1992). 19. S.S. Dubal, S.B. Sayyad, S.S. Patil, P.W. Khirade, E-I. Sci. J. V (3) Issue-3, 2094-1749, (2012). 20. S. Glasstone, K.J. Laidler and H. Eyring, Theory of rate Process, Mc Graw-Hill, New York, 541, (1941). 21. R.H. Fattepur, S.B. Sayyad, N.H. Ayachit. P.W. Khirade and S.C. Mehrotra. Int.M.S. 9, (2010).

*Corresponding Author:

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Email:krishnafattepur@yahoo.com Telephone:+91 9480702543

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Research Article Biological Sciences IN-VITRO ASSAY OF ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY OF LEAF EXTRACT AND LEAF DERIVED CALLUS EXTRACT OF ACALYPHA INDICA L. ANIRBAN MULLICK1, SUNANDA MANDAL1, RITUPARNA BHATTACHARJEE1 AND ANINDITA BANERJEE* Department of Microbiology St. Xavier’s College, 30 Mother Teresa Sarani, Kolkata-700016. West Bengal, India. 1 The authors contributed equally to the work *Corresponding Author Email: anni79in@gmail.com

ABSTRACT Acalypha indica (Indian Acalypha), commonly known as Mukhtajhuri in West Bengal is an important medicinal plant of India. The leaves are known to possess antimicrobial and antifungal properties. Leaf decoction is said to have anti-inflammatory properties. The present study shows the presence of antioxidant and antibacterial properties of Acalypha indica leaf extract and leaf-derived callus extract. Antioxidant activity was studied through DPPH assay. Whereas, in vitro antibacterial study of the extracts were carried out by adopting agar well diffusion technique using the pathogens Escherichia coli (E. coli), Klebsiella sp, Salmonella sp, Bacillus sp. After 24 hours of incubation maximum zone of inhibition was found against Bacillus sp. for leaf extract and Salmonella sp. for callus extract. These activities may be due to presence of flavonoids, phenolic compounds and other secondary metabolites present in these extracts.

KEY WORDS Acalypha indica, Antibacterial, Callus extract, Leaf extract, Radical Scavenging Activity.

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INTRODUCTION Acalypha indica, a common weed in many parts of Asia including of India, Bangladesh, Pakistan, Sri Lanka as well as tropical Africa and South America [1] belongs to the family Euphorbiaceae. In Ayurveda, it is known as “Kuppi”, “Muktavarchaa” or “Haritamanjari”. Previous reports have also shown that Acalypha indica (A. indica) contains acalyphine which is used in the treatment of sore gums and to have a post-coital antifertility effect [2], anti-venom properties [3], and wound healing effects [4], antioxidant activity [5], anti-inflammatory effects [6], acaricidal effects [7], diuretic effects [8] and antimicrobial activity [9]. The roots of Acalypha indica is used as laxative and leaves for scabies and others

cutaneous diseases [10]. The leaf sap is used to treat wounds [11] as well as eye and skin infections. Historically these plants have provided a good source of anti-infective agents in treatment of asthma [12] and pneumonia and so have increasingly gained importance during recent years. Moreover, antioxidant compounds [13] like phenolic acids, polyphenols and flavonoids [14] present in this plant extract, scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and inhibit the oxidative mechanisms that lead to degenerative diseases [15]. This weed also has antimicrobial property. Previous reports have shown flavonoids [16] (kaempferol glycosides mauritianin, clitorin, nicotiflorin and biorobin) and secondary

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www.ijpbs.com (or) www.ijpbsonline.com metabolites present in leaves and flowers of this medicinal plant can be efficiently used in treatment of diseases caused by antibiotics resistant strains of bacteria. For these reasons, various parts of this plant are utilized in production of valuable drugs to cure nosocomial infections of Staphylococcus sp [17]. Moreover, in vitro production of callus from somatic plant tissues can be helpful in future drugs production for infectious diseases. So, this has prompted us to investigate antioxidant and antibacterial activity of leaf and callus derived from the leaves of this plant. MATERIALS AND METHOD Acalypha indica used for sample preparation

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Figure 1: Collection of whole plant of Acalypha indica from Barisha in South Kolkata, West Bengal, India. (i) Plant material: The whole plant of A.indica (Figure 1) was collected from the adjoining areas of Barisha (22.470N, 88.310E), South Kolkata locality and was used in preparation of leaf extract, callus induction and preparation callus extract. (ii) Callus Induction: The leaf explants were washed with sterile water. Then the leaves were dipped in 1% sodium hypochlorite solution for 30 minutes, followed by rinsing twice with sterile water. The leaves were washed with 0.1% mercuric chloride solution for 30 seconds followed by rinsing twice with sterile

water. Finally, the leaves were washed with fungicide Bavistin for 45 seconds followed by rinsing with sterile water. (iii) Callus Production: The surface sterilized leaves were trimmed and placed on Murashige and Skoog’s (MS) medium (HiMedia, Mumbai) supplemented with 5µM 2,4dichlorophenoxyaceticacid (2,4-D) and kept in culture rack at 22-250C temperature and alternative light for 16 hours and dark for 8 hours at Relative Humidity maintained at 60-70%. The complete formation of calli was obtained in 14 days and was allowed to propagate for 3 months (Figure 2) before taken for extraction. Completely formed Callus

Figure 2: Induction of leaf-derived callus in MS medium supplemented with 5µM 2,4-D. (iv) Preparation of leaf and callus extracts: The air dried leaves were crushed with mortar and pestle and were extracted with methanol for three consecutive days at room temperature. The methanolic extract was evaporated to dryness and the crude residue left behind was weighed down. The residue was named as methanolic extract (LME). The callus obtained was also air dried, crushed, extracted using methanol and filtered. The filtrate was evaporated and dissolved in methanol to obtain callus methanolic extract (CME).

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www.ijpbs.com (or) www.ijpbsonline.com (v) Determination of total phenolic content: The extracts were dissolved in methanol to obtain a concentration of 1mg/ml. 100µl of these solutions were taken in a test tube and to it 100µl of 50% Folin Ciocalteau (Merck Specialist Pvt. Ltd) reagent was added. The mixture was then incubated for 3 minutes at room temperature and 2ml of 2% sodium carbonate solution was added. The volume was made up to 3ml with double distilled water [18]. The mixture was kept for 1 minute in water bath at 1000C and allowed to cool in dark. The absorbance of the samples was noted at 720 nm using a UV-Vis spectrophotometer. The total phenolic content of LME and CME was calculated from standard curve of Gallic acid 1mg/ml.

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(iv) Determination of total flavonoid: To determine total flavonoid content, 150 μl of 5% NaNO2 was added to 20 μl of LME and CME (1 mg/ml) and was incubated at room temperature for 5 minutes, followed by the addition of 2.5ml of 10% AlCl3 solution and incubated further for 6 minutes at room temperature [19]. The absorbance of the samples were noted at 510 nm. The total flavonoids contents of LME and CME was calculated from standard curve of quercetin 1mg/ml. (vi) Determination of Radical Scavenging Activity: Antiradical activity was measured by a decrease in absorbance at 517 nm of DPPH (2,2-Diphenyl1-Picrylhydrazyl) solution [20] brought about by plant extracts. In this assay DPPH acts as an indicator for “Radical Scavenging Activity” and changes its deep violet colour to colourless or pale yellow in presence of antioxidant and help us to determine Radical Scavenging Activity (RSC) of the substances. Therefore, to determine RSC of the extracts a stock solution of DPPH (0.12 mg/ml)

was prepared in methanol and the samples mixture taken in the test tubes was of 2ml out of which LME and CME were present in varied amount (5µl, 10µl, 20µl) as per the concentration and rest was methanol (1995µl, 1990µl, 1980µl) .Then to these test tubes, 1ml of DPPH solution was added to achieve the final volume of 3ml and kept for 20 minutes incubation in dark. After 20 minutes of incubation in dark the absorbance was measured at 517 nm. Decrease in the absorbance of the DPPH solution indicates an increase of the DPPH antioxidant activity and percentage of Radical Scavenging Activity (% RSC) was calculated from the following equation: % RSC =

(Ao−As) Ao

x 100

(Ao = DPPH solution without the sample, As= DPPH solution with the sample.) (vii) Determination of Antibacterial Activity: Antibacterial property of A. indica was determined using Kirby Bauer method. The antibacterial activity was studied by spreading 100 µl of 24 hours old culture of (Bacillus sp., E. coli, Salmonella sp. and Klebsiella sp.) on Mueller Hinton Agar (HiMedia,Mumbai) and 100 µl of LME and CME were loaded in the wells of these plates and their zone of inhibitions were calculated in millimeter (mm) after 24 hours of incubation at 370C and the zone of inhibitions were compared with methanol as a negative control [21].

RESULTS 1. Calculation of total phenolic and flavonoid content of extracts: Total phenolic and flavonoid content of LME and CME are summarized in Table1. Total phenolic concentration was found out using Gallic acid (GAE) as standard. Total phenolic content for LME was found out to be 26.6 ± 2.5mg GAE/g of methanol extract. In the same way, total flavonid concentration was found out using Quercetin (QE) as standard. The total flavonoid content LME

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was found to be 21.5 mg Quercetin/g of extract. Whereas, the total flavonoid content of CME was found to be 19.0 ± 3 mg/g of extract. Table 1: Total phenolic and flavonoid content of extracts: Sample

Total phenolic content (mg GAE± 2.5/g of extract)

Total flavonoid content (mg QE± 3/g of extract)

LME

26.60 ± 2.5

21.50 ± 3

CME

26.00 ± 2.5

19.0± 3

2. Calculation of Radical scavenging activity: Percentage of RSC (Table2) was determined by DPPH assay at different concentrations for the

extracts of which 250µg of LME showed maximum 43.57 % of RSC. Whereas, same concentration of CME had only 8.65% RSC.

Table 2: Percentage of Radical scavenging activity Sample Concentration (µg) % RSC LME CME

250 250

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3. Antibiotic sensitivity test: Antibacterial test was done by measuring the diameter of the zone of inhibition (Table 3) by all the extracts on the test organisms and the antibacterial activity was classified [22] into the following types of test organism(s): >12 mm zone of inhibition is high sensitivity 9-12 mm zone of inhibition is moderate sensitivity 6-9 mm zone of inhibition is less sensitivity and < 6 mm zone of inhibition is resistant. Thus, from the classification it was seen that Bacillus sp. was maximum sensitive to LME on

43.57 8.65 with zone of inhibition 17±0.5 mm followed by Salmonella sp with diameter of zone of inhibition 15±0.5 mm. Whereas, E. coli has shown moderate sensitivity with diameter of zone of inhibition 10.06 ± 0.4mm in LME. The assay has also shown Klebsiella sp. was resistant to LME with diameter of zone of inhibition 3±0.2mm. This assay has also shown Salmonella sp. was less sensitive to CME with zone of inhibition of 8±0.2 mm. Whereas, Klebsiella sp., E. coli and Bacillus sp. was found to be resistant to CME with zone of inhibitions 2±0.3 mm,2±0.2 mm and 2.3±0.2 mm respectively.

Table 3: Antibiotic sensitivity of test organisms: Test Organism Zone of inhibition(mm) LME CME Klebsiella sp. 3.3± 0.2 2.0± 0.3 E. coli 10.06± 0.4 2.0± 0.2 Bacillus sp. 17.00± 0.5 2.3± 0.2 Salmonella sp. 15.00± 0.5 8.0± 0.2

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www.ijpbs.com (or) www.ijpbsonline.com In recent times due to beneficial effect of Acalypha indica researchers have gained interest in studying the antioxidant activity of this type of medicinal plant extracts to treat degenerative diseases [23] because antioxidant compounds of these medicinal plants may acts as radical scavengers when added to the food products and prevent the radical chain reaction of oxidation and increase shelf life by retarding the processes of lipid peroxidation [24]. This antioxidant activity may be due to presence of phytochemicals like phenolics and flavonoid compounds. In this investigation we have also found that all the extracts have significant phenolic and flavonoid compounds in them. The result has also shown LME has maximum phenolic and flavonoid contents, compared to CME. So, due to presence of maximum phytochemical compounds, 250 µg of LME has shown 43.57% Radical scavenging activity and may be due to presence of less phytochemical compounds, 250 µg of CME has shown 8.65% of Radical scavenging activity. Previous reports have also stated that Euphorbiaceae [25] showed antimicrobial activity due to presence of high concentration of different compounds like flavonoids, phenols and alkaloids in it. This may be the reason in our investigation we have found out both extracts have antibacterial property against many common pathogens. Maximum antibacterial property is shown by LME against Bacillus sp. (zone of inhibition 17±0.5 mm). This extract has also shown highly sensitivity to Salmonella sp. (zone of inhibition 15±0.5mm), moderate sensitive to E. coli (zone of inhibition 10.06±0.4mm) and resistant to Klebsiella sp. (zone of inhibition 3.3±0.2 mm).Whereas on the other hand, CME has shown only less sensitivity to Gram negative Salmonella sp. (zone of inhibition 8±0.2 mm) and resistant to all other test microorganisms. From the different zone of inhibitions, we can conclude that the maximum antibacterial activity of LME

may be due to the presence of maximum phenolic and flavonoid content in it. Our maximum antibacterial activity of LME towards Gram positive Bacillus sp. may be due to difference in cell wall compositions compared to Gram negative bacteria. This fact can be checked by employing more bacterial strains to find out the actual reason behind it. Moreover, various reports [26, 27] have already shown entrances of antibiotics through cell envelope (the outer and cytoplasmic membrane) are highly efficient for Gram positive bacteria depending on reaction with the protein layer (mucopolysaccharides or peptidoglycans). Moreover, the factors responsible for more sensitivity of the leaf extract towards bacteria are not exactly known but may be the presence of soluble secondary plant metabolites in LME. Hence, the present study showed both LME and CME have antioxidant and antibacterial property but LME has a potential to be a better antioxidant and antimicrobial agent in future.

CONCLUSION This is first time a comparison is made on antioxidant and antibacterial activity of leaf extract (LME) and callus extract (CME). However, CME did not show any significant antioxidant and antibacterial activity even at high doses. So, further studies are required to increase the antioxidant and antibacterial activity of CME and identify the exact composition of the phenols, flavonoids and other secondary metabolites of both LME and CME.

ACKNOWLEDGEMENT We express our sincere thanks to Rev. Fr. Dr. J. Felix.Raj S.J, Principal of St. Xavier’s College (Autonomous), Kolkata, India for his support to carry out these experiments in the college. We are also grateful Dr. Arup Kumar Mitra, Head Department of Microbiology of St. Xavier’s

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www.ijpbs.com (or) www.ijpbsonline.com College (Autonomous), Kolkata, India and other faculty members for their help and cooperation in this work. Lastly, we would like to extend our sincere thanks to our laboratory attendants for their time to time help in this work.

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zeylanicum and Acalypha indica in rats. J Ethnophharmacol, 79: 249-51. (2002). H. Panda Herbal Soaps & Detergents Hand Book National Institute of Industrial Research Delhi. 48 â&#x20AC;&#x201C; 49. (2003). Isolation of potential antibacterial and antioxidant compounds from Acalypha indica and Ocimum basilicum. K. Ramya Durga, S. Karthikumar and K. Jegatheesan. Journal of Medicinal Plants Research 3(10).703-706. (2009). V.T.Narwade, A. A.Waghmare, A. L. Vaidya. Detection of Flavonoids from Acalypha indica L. Journal of Ecobiotechnology.3 (11): 05-07(2011). Halliwell, B. Free radicals, antioxidants and human disease: curiosity, cause or consequence. Lancer, 344: 721-724.(1994). Nahrstedt A, Hungeling M, Petereit F. Flavonoids from Acalypha indica. Fitoterapia. 77(6):4846.(2006). Rubin, R.J., Harrignton, C.A., Poon, A., Dietrich, K., Grene, J.A. and Moiduddin, A.The economic impact of Staphylococcus infection in New York City hospitals. Emerging infectious diseases. 5: 9-17. (1999). Waterman, PG and Mole, S. Analysis of Phenolic Plant Metabolites, Blackwell Scientific Publication.83-85. (1994). Jia ZS, Tang M C, Zhu XR. Study on effect of scavenging superoxide free radical on mulberry flavonoids. J. Zhejiang Agric. Univ., 22(5): 519-523. (1996). R.Shanmugapriya,T.Ramanathan, .Thirunavukkarasu. Evaluation of Antioxidant Potential and Antibacterial Activity of Acalypha indica Linn. Using in vitro model. Asian Journal of Biomedical and Pharmaceutical Sciences 1 (1), 18-22. (2011). P. Saranraj, D. Stella, K. Sathiyaseelan and Sajani Samuel. Antibacterial Potentiality of Ethanol and Ethyl Acetate Extract of Acalypha indica against Human Pathogenic Bacteria. Journal of Ecobiotechnology 2(7): 23-27. (2010). Uma Devi, P., S. Murugan, S. Suja, S. Selvi, P. Chinnasamy, E. Vijayaanad.Antibacterial invitro lipid per-oxidation and phytochemical observation on Achyranthes bidentata blume. Pak. J. Nutrition, 6(5): 447-451. (2007). Willcox, J.K., S.L. Ash, G.L. Catignani. Antioxidant and prevention of chronic disease. Crit. Rev. Food Sci. Nutrition, 44: 275-295. (2004). Young, I.S. and J.V. Woodside. Antioxidants in health and disease. J. Clinical Pathol, 54: 176-186. (2001).

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www.ijpbs.com (or) www.ijpbsonline.com 25. Falodun A, Ali S, Mohammed Quadir I, Iqbal MI, Choudhary IMI. Phytochemical and biological investigation of chloroform and ethylacetate fractions of Euphorbia heterophylla leaf (Euphorbiaceae). J. Med. Plants Res., 2(12): 365-369. (2008).

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|504-510 26. Baron EJ, Peterson LR and Finegold SM. Baily & Scott's Diagnostic Microbiology.9th ed. Mosby-Year book, Inc., St. Louis. (1994). 27. Lennette, EH, Balows A, Hausler WJ and Shadomy HJ. Manual of Clinical Microbiology. 4 th ed. Amer. Soc. Microbiol., Washington. (1985).

*Corresponding Author: “Dr. Anindita Banerjee” Assistant Professor Department of Microbiology St. Xavier’s College 30 Mother Teresa Sarani Kolkata-700016 E-mail: anni79in@gmail.com Telephone: +91-(033)-22551276

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Research Article Biological Sciences CT SCAN – AUTOMATIC TUBE CURRENT MODULATION Nimma purna chander reddy1, Suresh sukumar2, Sushil Yadav3,Albin Babu M Wilson4 1

Clinical SME, Apollo Health street,. Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal 3 Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. 4 Lecturer. MSc. Medical Imaging Technology, Department of Medical Imaging Technology, KSHEMA,Nitte university, mangalore. 2

*Corresponding Author Email: suresh.medicalimaging@gmail.com

ABSTRACT AIM OF THE STUDY: To compare the dose difference and state the significance of Automatic Tube Current Modulation (ATCM) in abdominal MDCT examination while maintaining optimum image quality for diagnosis. Results: The varying in mAs values according to ACS for different body counter of the patients was observed. In group A resulted in estimations of dose saving in range of 10-27 %.In group B 8.1-36.6 %, in group C 8.3-33.4%, group D 5.7-19 % with application of ATCM technique. The overall radiation output results shown for forty patients as follows the least is 5.7% and highest is 36.6%.According my study statistics shows there is no correlation between BMI and the estimated dose savings. CONCLUSSION: The use of Automatic Tube Current Modulation (angular dose modulation and z-axis dose modulation) helps in  Radiation Dose reduction Up to 36.6% was achieved with acceptable diagnostic image quality.  Reduces over all irradiation time.  Helps in reduce stochastic and genetic effects.  Helps in Not only reduce the patient dose as well as occupational dose.  Scan duration is less compare to standard protocol so we can save the time at clinical side.  Effective in breath hold scans (Coronary Angio, Pulmonary Angio, Thorax and Abdomen).  Required dose is applied according to patient body counter.  Helps in decrease scan time in uncooperative and trauma patients.  I conclude that according to the observed results there is no requirement to

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INTRODUCTION The invention of computed tomography (CT) has revolutionized the practice of radiology and is so remarkable that in many cases it generates a dramatic increase in diagnostic information in a short duration comparing to other imaging modalities such as MRI and ultrasonography. CT imaging continues to be on the increase due to its varied advantage, despite the large radiation

dose imparted to patients. Due to wide spread use variety of geometries has been developed to acquire transmission data in a shortest time period &according to anatomical region such as heart. The advancement of the MDCT makes possible rapid volume acquisition and has opened new diagnostic fields such as CT cardiac angiography, virtual colonoscopy, and bronchoscopy. Fulfilling the demands for

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www.ijpbs.com (or) www.ijpbsonline.com effective diagnosis has led to a steady increase in the use of CT. With this trend of increasing use of CT department strive to scan with ALARAprinciple. According to literature the risk of radiation induced cancer in patients from CT examination is not negligibleÂš. Generally the radiation doses to patients are about 30-50% greater with the use of MDCT as due primarily to scanoverlap, positioning of the xray tube closer to the patient, over beaming, increased significance of over scanning and possibly increased scattered radiation with wider x-raybeams. A reduction in the radiation dose delivered from CT has become an important issue and various dose reduction and optimization techniques have been formulated.Modulation of the x-ray tube current during scanning is one effective method of reducing the dose and the adaption of Automatic Tube Current Modulation (ATCM) technique should permit overall reduction in radiation exposure. Automatic tube current modulation in CT is analogous to the automatic exposure control (AEC) or photo timing technique used for automatically terminating radiographic exposure. The techniques used are angular (x and y-axis) and z-axis tube current modulations. The x and y-axis modulation involves variation in tube current as the x-ray tube rotates about the patient, while the z-axis modulation involves variation in tube current along the z-axis of the patientÂ˛. The current study aims to compare radiation dose and image quality achieved with weight-based protocol, along with the dose modulation software available in the machine i.e. dynamic dose modulation (D-DOM) and z-axis dose modulation (Z-DOM) dose modulation techniques using a sixty-four slice, Philips Brilliance CT scanner. The D-DOM and Z-DOM are based on the angular and z-axis tube current modulation respectively.

AIM OF THE STUDY To compare the dose difference and state the significance of Automatic Tube Current Modulation (ATCM) in abdominal MDCT examination while maintaining optimum image quality for diagnosis.

REVIEW OF LITERATURE Livingstone RS, et al in year 2009 studied426 patients on a six-slice CT scanner for comparison of radiation dose and image quality using dose modulation techniques and weight- based protocol exposure parameters for biphasic abdominal CT. The use of dose modulation technique resulted in a reduction of 16 to 28% in radiation dose with acceptable diagnostic accuracy. A reduction of current-time product of approximately three to five percent using D-DOM and 37 to 55% using Z-DOM was achieved for arterial and portal venous phases compared to the weight based protocol settings. A reduction of approximately 30 to 50% of tube current-time product was noted within D-DOM and Z-DOM respectively for arterial and portal venous phases. Yoshinori Funama, et al in year 2007 investigated64 patients with known or suspected lung or abdominal disease about the possibility of obtaining adequate images at uniform image noise levels and reduced radiation exposure with automatic tube current modulation (ATCM) technique for 64-detector CT. The mean image noise ranged from 8.40 at the center of the left ventricle to 11.31 at the porta hepatis; the mean tube current ranged from 105.9 mAs at the center of the left ventricle to 169.6 mAs at the center of the spleen. The mean dose reduction rate per constant tube current at 175 mAs ranged from 3.1 to 39.5%. By use of the ATCM technique, it is possible to maintain a constant image noise level with a 64-detector CT.

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www.ijpbs.com (or) www.ijpbsonline.com Campbell J, et al in year 2005 studied one hundred and forty-eight consecutive chest CT examinations (70 men, 78 women; age range, 1590 years) with theobjective to determine additional radiation dose associated with scanning beyond the anatomic limits of the thorax in chest CT protocol and to assess the effect of zaxis modulation on the additional radiation dose associated with the scanning protocol. With z-axis modulation, the mean DLP for supraapical and infrapulmonary extra images was 39.98 mGy x cm and 132.59 mGy x cm, respectively. With fixed tube current, the mean DLP for supraapical and infrapulmonary extra images was 30.31 mGy x cm and 95.91 mGy x cm, respectively

MATERIALS AND METHODS MACHINERY FEATURES – SPECIFICATIONS Philips (Brilliance) 64 row MDCT Output Capacity-60KW KV-80, 120,140 MA-A.800mA -B. Dose modulated mA Anode storage capacity-8MHU Dose right ACS (Automatic current selection) Optimizes the dose for each patient based on the planned scan by suggesting the lowest possible setting mAs to maintain constant image quality at low dose throughout the exam.

Dose Right Z- DOM (Longitudinal Dose Modulation) Automatically controls the tube current along z axis adjusting the signal along the length of the scan, increasing the signal over regions of higher attenuation (shoulder, pelvis) and decreasing the signal over regions less attenuation(neck, legs). Study designis a randomized prospective, blinded study involving40 patients undergoing abdominal CT scan, performed using a sixty-four row CT scanner (Brilliance, Philips medical systems at Kasturba Hospital Manipal. The tube potential, tube current-time product, volumetric CT dose index (CTDI vol) and dose length product (DLP) values will be recorded. The tube potentials available in the machine are 80kV, 120 kV and 140 kV. Various other parameters such as the total time duration of the scan, field of view and pitch selection will be recorded. This protocol involves a complete examination of the region of interest along with a topogram, spiral or sequential ranges and reconstruction modes. The preprogrammed scan protocols used are based on recommended exposure factors specified by the manufacturers as a starting point for clinicalwork. During the course of the study, exposure parameters will be selected according to the patient's body weights and will be lower than the preset protocols. An appropriate tube current time product will be used for patients based on their body weight for arterial and venous phases. INCLUSION CRITERIA  Age20 to 80 years, both genders  Advised CT scan abdomenby respective clinician for clinical condition. EXCLUSION CRITERIA  Larger body matrix, children,  Critical patients.

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Dose Right D-DOM (Dynamic Dose Modulation) Automatically controls the tube current x and y axis rotationally increasing the signal over areas of higher attenuation (lateral) and decreasing signal over area of less attenuation (AP).

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 Clicked save as to rename the protocol before saving given name as ACS Abdomen Protocol.  Set dose right standards When dose right is set to yes, we have several options for setting and altering the protocol standards.  Selected the reference image The patient details were entered in the console including height and weight and ACS Abdomen Protocol was selected, entered the length of the scan and acquired the scano by using surview test later axial sections are planned on scano from diaphragm level to the Symphysis pubis level. No of images, scan time, CTDI & DLP entered in the data chart as DLP from standard technique and after the scan values are taken as DLP after applying ATCM technique and noted down estimation of dose savings according displayed on the console. PROTOCOL FOR CT ABDOMEN  Patient preparation: 4 hrs fasting.  Blood parameters: Urea: 8-38, Creatinine level: 0.6 - 1.6 mg/dl  Oral Contrast –neutral or positive depending on clinical indications

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Study of forty patients will be performed on sixtyfour slice CT scanner (Brilliance, Philips medical systems at Kasturba Medical College, Manipal) as per the method described above.  Following procedure to enable the ACS 1. In the processing window clicked the preference button. 2. Clicked scanner. 3. Selected dose right. 4. Selected desired dose right ACS setting: Yes or Auto. 5. Clicked ok to accept the settings and close the dialog box.  Creating specific protocol  We can also enable Dose Right ACS for specific protocol 1. Clicked home on the workflow bat. 2. Clicked generate protocols. 3. Selected the abdomen protocol. 4. Selected the helix scan step advanced tab. 5. Selected the Dose Right ACS. 6. Clicked ok. 7. Selected from generate main form age group as adult and weight as all. 8. Selected save option :

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www.ijpbs.com (or) www.ijpbsonline.com Scan Protocol Patient Position Scano Area Coverage Scan Direction Gantry Angle Breathing instructions Routine Plain + Contrast Scan

PROTOCOL Abdomen Helical Supine- Feet First PA-1800 Domes of diaphragm- Symphysis pubis Cranio caudal NO Yes

Start Location

Domes of diaphragm

End Location

Symphysis pubis

Slice thickness Increment

5 mm 5 mm

kV, mAs/slice

120,250

Resolution Filter

Standard Standard (C)

Collimation Rotation Time FOV Matrix Image Enhancement Contrast(Bolus Tracking)

64X0.625 0.75sec Varies 512X512 0.0 volume and flow rate depending on clinical index

Image Quality

AcceptableNot acceptablePlain scan with ATCM-

Radiation dose

515

OBSERVATION

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There were forty adult patients in our study including 27 males and 13female.Divided into four groups depends upon body mass index. That is Group-A underweight with BMI less then18.5, Group-B normal weight with BMI 18.5-24.9, Group-Overweight with BMI 25-29.9, Group-D obesity with BMI of 30 or greater. In each group we studied male female ratio, height and weight, differences between the dose from the standard techniques and dose from the ATCM technique and estimations of dose saving percentage varies individual to individual due to applied ATCM technique.

In group A There were total seven patients 4 males, 3 females with patientâ&#x20AC;&#x2122;s height in range of 153-183 centimeters and weight in range of 31-61 kilograms. A difference between the dose from the standard technique is in range of 196.8-645.1 mg y*cm and dose from the ATCM technique is in range of 155-492mgy* was observed which resulted in estimations of dose saving in range of 10-27 % with application of ATCM technique. In group-B, There were total twenty two patients 16 males, 6 females with patientâ&#x20AC;&#x2122;s height in range of 152-175 centimeters and weight in range of 42.5-71 kilograms. A difference between the dose

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www.ijpbs.com (or) www.ijpbsonline.com from the standard technique is in range of 259.61279.5 mg y*cm and dose from the ATCM technique is in range of 202-1067mgy* was observed which resulted in estimations of dose saving in range of 8.1-36.6 % with application of ATCM technique. In group-C, there were total eight patients 4 males, 4 females with patient’s height in range of 131-171 centimeters and weight in range of 51-86 kilograms. A difference between the dose from the standard technique is in range of 361.71196.8 mg y*cm and dose from the ATCM technique is in range of 331.6-1098mgy* was observed which resulted in estimations of dose saving in range of 8.3-33.4% with application of ATCM technique. In group-D, There were total three patients 3 males, no females. With patient’s height in range of 151-165 centimeters and weight in range of 7182 kilograms. A difference between the dose from the standard technique is in range of 623.5-992.5 mg y*cm and dose from the ATCM technique is in range of 524-935.5 mg y* was observed which resulted in estimations of dose saving in range of 5.7-19 % with application of ATCM technique. Total There were total forty patients 27 males, 13 females. With patient’s height in range of 131-183 centimeters and weight in range of 31-86 kilograms. A difference between the dose from the standard technique is in range of 199.61279.5mgy*cm and dose from the ATCM technique is in range of 155-1098 mg y* was observed which resulted in estimations of dose saving in range of 5.7-36.6 % with application of ATCM technique. Results The varying in m As values according to ACS for different body counter of the patients was observed. In group A resulted in estimations of dose saving in range of 10-27 %.

In group B 8.1-36.6 %, in group C 8.3-33.4%, group D 5.7-19 % with application of ATCM technique. The overall radiation output results shown for forty patients as follows the least is 5.7% and highest is 36.6%. According my study statistics shows there is no correlation between BMI and the estimated dose savings.

DISSCUSSION The standard protocol setting is giving same radiation dose output for every patient who is going for CT scan examination. There are various factors being used for CT image acquisition like mA, KVP, m As(1dr roshan Livingstone) and they are fixed in standard protocol. These fixed factors should not be used in every patient as they have different body habitus and body contours(synthia teaching manual).Fixed factors not only degrade image quality in the case of obese patients but are also giving excess radiation dose in the case of thin patients(chap bell). Image quality is mainly depends upon number of transmitted photons which are detected by the detector (teaching manual). Decrease in photon number lead to statistical noise that is due to quantum mottle. This can be overcome in two ways that is by increasing the mA or increasing the mAs. But according to justification of laws increase in exposure time results in increasing patient dose twice as well as increases scanning time for the particular region(synthia teaching manual). So the other option left is to change the mA to get the optimum image quality and decrease the radiation dose at the same time (denis tack). In modern modalitiesmanufacturers’ implemented Automatic Exposure Control system (AEC), which permit empirical adjustment of radiological technique factors according to size of the patient (from dr roshan 13). According to them we should enter weight and height of the

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www.ijpbs.com (or) www.ijpbsonline.com patient in control console but my study results shown there is no correlation when we compare BMI with the estimation of dose saving percentage. According to my study the output results shown dose reduction not depending upon weight and height of the patient its upon the tissue characteristics of the patient tissues attenuation. As I noticed without entering height and weight of the patient details in console can lead to dose reduction. According to previous referred study ATCM technique may fail to control the tube current if a pitch value larger than 1.0 is adopted.They found that the image noise in overweight and obese patients was significantly higher than that in normal weight and underweight patients, although with the ATCM technique it was possible to maintain an almost constant image noise level in these individuals. The use of Automatic Tube Current Modulation (angular dose modulation and z-axis dose modulation) helps in  Reduces over all irradiation time.  Helps in reduce stochastic and genetic effects.  Helps in Not only reduce the patient dose as well as occupational dose.  Scan duration is less compare to standard protocol so we can save the time at clinical side.  Effective in breath hold scans (Coronary Angio, Pulmonary Angio, Thorax and Abdomen).  Required dose is applied according to patient body counter.  Helps in decrease scan time in uncooperative and trauma patients.  I conclude that according to the observed results there is no requirement to enter weight and height of the patient in the console so that we can save the

time especially in the case of bedside patients very useful.

CONCLUSSION The use of Automatic Tube Current Modulation (angular dose modulation and z-axis dose modulation) helps in  Radiation Dose reduction Up to 36.6% was achieved with acceptable diagnostic image quality.  Reduces over all irradiation time.  Helps in reduce stochastic and genetic effects.  Helps in Not only reduce the patient dose as well as occupational dose.  Scan duration is less compare to standard protocol so we can save the time at clinical side.  Effective in breath hold scans (Coronary Angio, Pulmonary Angio, Thorax and Abdomen).  Required dose is applied according to patient body counter.  Helps in decrease scan time in uncooperative and trauma patients.  I conclude that according to the observed results there is no requirement to enter weight and height of the patient in the console so that we can save the time especially in the case of bedside patients very useful.

BIBILOGRAPHY 1.

A Comparison of radiation doses using weight-based protocol and dose modulation techniques for patients undergoing biphasic abdominal computed tomography examinations. Livingstone RS, Dinakaran PM, Cherian RS, Eapen A. Journal, The official journal of Association of Medical Physicists of India,Year-2009, Volume34,issue- 4,page- 217-222. Automatic tube current modulation technique for multidetector CT, it is effective with a 64-detector CT? Yoshinori Funama , Kazuo Awai, Masahiro Hatemura, Masamitchi Shimamura, Yumi Yanaga, Seitaro Oda and

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16. McCollough CH, Bruesewitz MR, James M, Kofler T Jr. Dose reduction and dose management tools: Overview of available options1. RadioGraphics 2006;26:503-12. 17. Tack D, Maertelaer VD and Gevenois PA. Dose reduction in multidetector CT using attenuation-based online tube current modulation. AJR Am J Roentgenol 2003;181:331-4. 18. Rizzo S, Kalra M, Schmidt B, Dalal T, Suess C, Flohr T, et al. Comparison of angular and combined automatic tube current modulation techniques with constant tube current CT of the abdomen and pelvis. AJR Am J Roentgenol 2006;186:673-9. 19. Huda W, Nickoloff EL, Boone JM. Overview of patient dosimetry in diagnostic radiology in the USA for the past 50 years. Med Phys 2008;35:5713-28. 20. European Commission. European guidelines on quality criteria for computed tomography. Brussels, Belgium: Report EUR 16262 EN 1999. 21. Kalra MK, Prasad S, Saini S, Blake MA, Varghese J, Halpern EF, et al. Clinical comparison of standard- dose and 50% reduced-dose abdominal CT : Effect on image quality. AJR Am J Roentgenol 2002;179:1101-6. 22. ACR. Computed Tomography Accreditation program: Phantom testing criteria. 1891, Preston White Drive, Reston VA20191. 23. Wade JP, Weyman JC, Goldstone KE. CT standard protocols are of limited value in assessing actual patient dose. Br J Radiol 1997;70:1146-51. 24. Tsapaki V, Aldrich JE, Sharma R, Staniszewska MA, Krisanachinda A, Rehani M, et al. Dose reduction in CT while maintaining diagnostic confidence: Diagnostic eference levels at routine head, chest, and abdominal CT-IAEA-coordinated research project. Radiology 2006;240:828-34. 25. Goddard CC, Al-Farsi A. Radiation doses from CT in the Sultanate of Oman. BJR 1999;72:1073-7. 26. Van der Molen AJ, Veldkamp WJ, Geleijns J. 16-slice CT: Achievable effective doses of common protocols in comparison with recent CT dose surveys. Br J Radiol 2007;80:248-55. 27. Brix G, Nagel HD, Stamm G, Veit R, Lechel U, Griebel J, et al. Radiation exposure in multi-slice versus singleslice spiral CT: Results of a nationwide survey. Eur Radiol 2003;13:1979-91. 28. Shrimpton PC, Hillier MC, Lewis MA, Dunn M. Doses from computed tomography examinations in the UK 2003 review. Report NRPB-W67. [Published on 2005 March].

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Yasuyuki Yamashita. Journal, Radiological physics and technology, publisher springer Japan.Year-january2008, volume-1, number-1, pages- 33-37. Scanning beyond anatomic limits of the thorax in chest CT: findings, radiation dose, and automatic tube current modulation. AJR Am J Roentgenoel Campbell J, Kalra Mk, Rizzo S, Maher MM, Sheppard JA.Year-Dec2005, volume-185, number-6, pages- 1525-30. Mulkens TH, Bellinck P, Baeyaert M, Ghysen D, Van Dijck X, Mussen E, et al. Use of an automatic exposure control mechanism for dose optimisation in multidetectorrow CT examinations: Clinical evaluation. Radiology 2005;237:21323. Nakayama Y, Awai K, Funama Y, Hatemura M, Imuta M, Nakaura T, et al. Abdominal CT with low tube voltage: Preliminary observations about radiation dose,contrast enhancement, image quality, and noise. Radiology 2005;237:945-51. Brenner DJ, Hall EJ. Computed tomography: An increasing source of radiation exposure. N Engl J Med 2007;357:2277-784. Rehani MM, Berry M. Radiation doses in computed tomography: The increasing doses of radiation need to be controlled. BMJ 2000;320:593-4. McCollough CH, Zink FE. Performance evaluation of a multi-slice CT system. Med Phys 1999; 26:2223-30. Hidajat N, Maurer J, Schroder RJ, Wolf M, Vogl T, Felix R. Radiation exposure in spiral computed tomography: Dose distribution and dose reduction. Invest Radiol 1999; 34:51-7. Brenner D, Elliston C, Hall E, Berdon W. Estimated risks of radiation-induced fatal cancer from pediatric CT. AJR Am J Roentgenol 2001;176:289-96. Brenner DJ, Elliston CD. Estimated radiation risks potentially associated with full body CT screening. Radiology 2004; 232:735-8. Brenner DJ. Radiation risks potentially associated with low-dose CT screening of adult smokers for lung cancer. Radiology 2004; 231:440-5. Yoshinaga S, Mabuchi K, Sigurdson AJ, Doody MM, Ron E. Cancer risks among radiologists and radiologic technologists: Review of epidemiologic studies. Radiology 2004;233:313-21. Wagner LK. Overconfidence, overexposure, and overprotection. Radiology 2004;233:307-8. Hart D, Hillier MC, Wall BF. Doses to patients from medical x-ray examinations in the UK - 2000 review. Chilton, NRPBW14 2002.

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Appendix A Communication of the decision of the institutional ethics committee Protocol title : Automatic Tube Current Modulation Date of review (DD/M/YYYY) : Please note*  Informed IEC immediately in case of any adverse events and serious adverse events  Informed IEC any amendments to the protocol, change of study procedure, site and investigator and premature termination of study with reasons along with summery.  Final & six months reports to be submitted to IEC.  Members of IEC have right to monitor the trial with prior information.  A copy of the consent document to be given study participant giving the concern.

Appendix B Informed consent form Serial Number : Hospital Number : Name : Age : Sex : I ……………………. hereby voluntarily give consent to Mr. PURNA CHANDER REDDY for taking Computed Tomographic Scan (CT Scan) of my Abdomen for studying the Automatic Tube Current Modulation, which will be used for the dissertational study. I have been explained about the procedure in the language that I understand and I am aware that I can refuse unconditionally.

Signature of the Individual Place:

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Date:

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Appendix C Proforma DATA SHEET Name: Serial. No

Date: Hosp No

Age

Sex Male

Weight

Height-

Female

Clinical Information Dose Measurements No of images Pre Scan time Measurements mA Post Measurements

kVp

120

CTDI DLP mAs

No of images

CTDI-

Scan time Est. Dose saving mA varies

DLP kVp

120

mAs

250

FOV

*Corresponding Author: Suresh Sukumar

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Assistant professor, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal.udupi â&#x20AC;&#x201C; 576104. Email suresh.medicalimaging@gmail.com. 9886118811

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ResearchArticle BiologicalSciences PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF METHANOL EXTRACT OF TRIDAX PROCUMBENS Razia Muthusamy, Karthiga Vasu, Lavanya Kanagaraj, Deboral Ponnampallam, Bernala Wilson Department of Biotechnology,Mother Teresa Womenâ&#x20AC;&#x2122;s University,Kodaikanal-624101, Tamilnadu *Corresponding AuthorEmail:razia581@gmail.com

ABSTRACT The leaves of Tridax procumbens were collected, grounded and subjected to methanol for extraction with soxhlet apparatus. The extract was screened phytochemically for its chemical components. The presence of alkaloids, reducing compounds (carbohydrates), cardiac glycosides, flavonoids, saponins, terpenes and steroids was recorded. Antibacterial activity bydisc diffusion method revealed that the methanol extract have a broad spectrum activity on gram positive, negative organisms respectively. The highest activity was shown in S. typhiS. flexneri and least activity on E.coli.

KEY WORDS Tridax procumbens,Escherichia coli, Salmonella typhi, Shigella flexneri

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INTRODUCTION Tridax procumbens Linn. (Asteraceae) is one of the medicinally important plants commonly found in subtropical countries growing primarily during raining season. It is a common weed in Tamilnadu present along with economically important crops (Suseela etal., 2002). It habitats waste places, road sides and hedges throughout India. The leaves of the plant are known to be used for the treatment of wound in traditional medicine (Collier 2001). The extracts of T. procumbens have been reported to have various pharmacological effects, antimicrobial activity, wound healing property and immunomodulatory activity on the experimental animals (Taddel and Rosas, 2000). Phytochemical constituents are the basic source for the establishment of several pharmaceutical industries (Savithramma et al., 2011). Pathogenic microorganisms are always trying to develop resistance to the various commercial antibiotic drugs for their control

(Beegum and Devi, 2003). High cost and adverse side effects are commonly associated with popular synthetic antibiotics (such as hypersensitivity, allergic reactions, immunosupression etc.) and are major burning global issues in treating infectious diseases (Schinor et al., 2007). Hence, recent attention has been paid to biologically active extracts and compounds from plant species used in herbal medicines (Essawi and Srour, 2000). The present study was designed to study the preliminary phytochemical analysis and antibacterial activity of Tridax procumbens.

MATERIALS AND METHODS Plant collection The Leaves of Tridax procumbens were collected from kodaikanal region of dindigul district of Tamilnadu.

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Preparation and Extraction of Plant material measuring the diameter of the zone of inhibition The plant materials (leaves of Tridax (mm) surrounding bacterial growth. procumbens) were air-dried at room temperature for 2 weeks and grinded to a RESULTS AND DISCUSSION uniform powder. The Methanol extract was The phytochemical screening of methanol prepared by soaking 10 g of powdered plant extract of T. procumbens was presented in Table materials in 100 ml of methanol at room 1.T. procumbens leaves contains alkaloids, temperature for 48 h. Extract was filtered after tannin, flavonoids, terpenoids, phenols, saponin, 48 h, with Whatmann filter paper No. 42 carbohydrate, glycosides, and cardiac glycosides. (125mm) and then through cotton wool. The Flavonoids are known to be synthesized by extract was concentrated using a rotary plants in response to microbial infection. Tannins evaporator with the water bath set at 40Â°C. (commonly referred to as tannic acid) are also Phytochemical screening known as antimicrobial agents (Sharma and The methanol extract was screened for Sharma 2010). Tannins have been reported to phytochemical constituents for the presence of prevent the development of microorganisms saponins, tannins, alkaloids, flavonoids, (Taddei, and Rosas-Romero 2000). The result of anthraquinones, glycosides and reducing sugars antibacterial activity of Tridax procumbens by (Sharma and Sharma, 2010). disc diffusion method showed in the (Table 2). Antibacterial assay The methanol extract showed significant zone of The agar disc diffusion method as described by inhibition against selected bacterial species. S. (Parekh and Chanda, 2006) was used in the flexneri, S. typhi, P. aeruginosa, and P. mirabilis antibacterial screening procedure. Muellershowed greater zone of inhibition than Klebsiella Hinton (MH) agar plates were prepared using pneumoniae and Escherichia coli which showed petridishes. For the agar disc diffusion method, lesser inhibition zone. Similar antibacterial the disc was saturated with 100 Îźl of the test activity has been reported (Janovska et al., compound, allowed to dry and then placed on 2003). the upper layer of the seeded agar plate. Antibacterial activity was determined by Table1: Phytochemical analysis of T. procumbens S.No Test T. procumbens(Leaf) 1. Alkaloids + 2. Flavonoids + 3. Terpenoids + 4. Phenols + 5. Tannins + 6. Anthraquinone _ 7. Free anthroquinine _ 8. Saponin + 9. Carbohydrate + 10. Carotenoid _ 11. Glycoside + 12. Cardiac glycoside + International Journal of Pharmacy and Biological Sciences (e-ISSN: 2230-7605)

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Table 2. Antibacterial activity of methanolic extract of Tridaxprocumbens S.No Organisms (Zone of Inhibition mm) Tridaxprocumbens 25µg 50µg 75µg 100µg 1. E. coli 4 6 12 14 2. 3. 4.

K. pneumoniae P. aeruginosa P. mirabilis

5 6 5

7 9 10

13 18 15

16 19 18

S. flexneri

S. typhi

CONCLUSION The study revealed that Tridax procumbens was rich in secondary metabolites particularly tannins and flavonoids which are responsible for antibacterial activity. Further, detailed investigation of the active compounds of the plant for the exact mechanism of action will contribute greatly to the development new pharmaceuticals.

REFERENCES o

o o

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Beegum B.N.R., and Devi T.G. Antibacterial activity of selected sea weeds from Kovalam South West coast of India.Asian Journal of Microbiol Biotech Env Sci, 5(3): 319-322,(2003) Collier M., An assessment plan for wound management. J comm. Nurs, 16 (6): 22-26, (2001) Essawi T., Srour M. Screening of some Palestinian medicinal plants for antibacterial activity. J Ethanopharmacol, 46: 343-349,( 2000) Janovska, D., Kubikova K., and Kokoska L. Screening for antimicrobial activity of some Medicinal plant species

of traditional Chinese medicine. Czech J. Food Sci., 21: 107-111,(2003) Parekh J, Chanda S. In vitro antimicrobial activities of extracts of Launaeaprocumbens Roxb. (Labiateae), VitisviniferaL. (Vitaceae) and CyperusrotundusL. (Cyperaceae). Afr. J. Biomed. Res., 9: 89-93, (2006). Savithramma N., Rao M. L., and Bhumi G. Phytochemical screening of Thespesiapopulnea (L.) Soland and Tridax procumbens L. J. Chem. Pharm. Res., 3(5):28-34, (2011) Sharma.M.K. and Sharma.S. Phytochemical and Pharmacological Screening of Combined Mimosa pudica Linnand Tridax procumbens for In vitro Antimicrobial Activity. Intl. J. Microbiol. Res., 1 (3): 171174, (2010) Schinor EC, Salvador MJ, Ito IY, Dias DA. Evaluation of the antimicrobial activity of crude extracts and isolated constituents from Chrestascapigera. Brazilian J. MIcrobiol,38: 145-149, (2007) Suseela L, Sarsvathy. A, Brindha. P. Pharmacognostic studies on Tridax procumbensL.(Asteraceae). Journal of Phytological Research. 15 (2): 141-147, (2002). Taddel A., Romero AJ.R. Bioactivity studies of extracts from Tridax procumbens. Phytomedicine. 7(3): 235238. (2000).

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Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal– 624 102 TamilNadu, India. Email*: razia581@gmail.com Mobile: +91 8012137535

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Case Report Biological Sciences ANEURYSMAL BONE CYST OF THE LATERAL END OF CLAVICLE IN A FOURTEEN YEAR OLD BOY â&#x20AC;&#x201C; CASE REPORT Nagakumar J S1, Nazeer BS2, Samarth Arya3 1

Assistant Professor, Department of Orthopaedics, SDUMC, Kolar, India 2 Professor, Department of Orthopaedics, SDUMC, Kolar, India 3 Jr.Resident, Department of Orthopaedics, SDUMC, Kolar, India

ABSTRACT Aneurysmal bone cysts are enigmatic lesions of unknown cause and presentation and are difficult to distinguish from other lesions. We present a case of aneurysmal bone cyst involving clavicle. It presented difficulties in diagnosis because of the uncommon Location. The boy was treated surgically with curettage and autologous bone grafting as discussed.

KEY WORDS Aneurysmal bone cyst (ABC), Clavicle.

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INTRODUCTION Primary bone tumours of flat bones like clavicle are rare. True benign tumours are much uncommon than metastatic or malignant lesions. Aneurysmal bone cyst (ABC) is a benign but locally aggressive lesion of the bone which accounts for 3% of all bone tumours. Its histology is characterized by multiloculated cystic tissue filled with blood. Etiology and pathogenesis of this lesion remains unclear and it affects 0.14 per lakh every year 1. It is a disease mainly of the young with a peak incidence in the second decade. However it may on occasion occur in the elderly and the very young 2. ABC may involve almost any bone but the most frequent sites are long tubular bones and vertebrae. Among flat bones, the pelvis and scapula are well known locations. Despite very characteristic radiological features, the unusual age coupled with the uncommon site led to diagnostic difficulties in present case. The clavicle is a rare site for these lesion and not many have been reported in literature. Smith in 1965

could find only 25 cases in the medical literature, textbooks and atlases 3. Because of these factors, this report is felt to be of interest.

CASE REPORT A 14 year old boy presented with swelling in his left clavicular region that had been increasing in size progressively since last six months to reach the size of a lemon. The swelling at the acromial end had distinct edges, was smooth surfaced (Fig. 1) and overlying skin temperature was normal. The mass was bony hard, non-tender and the skin over the swelling was pinchable. Swelling was immobile and Egg shell crackling sensation was noted. No functional impairment, skin changes or dilated veins or signs of neurological deficits and lymphadenopathy was noted. There was no history of trauma or history of recurrent fever and loss of weight. No other lumps or swellings were present anywhere else. Radiograph showed a cystic expansile lesion of the lateral end of the left clavicle bounded by a

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www.ijpbs.com (or) www.ijpbsonline.com thin layer of bone (Fig. 2). Based on the appearance various possibilities including simple bone cyst, ABC, eosinophilic granuloma, and enchondroma were considered. Basic hematological work up including complete blood count, ESR, CRP and alkaline phosphatase were within normal limits. The lesion was further studied with MRI left shoulder (Fig.3) and fine needle aspiration cytology. MRI pointed to the possibilities of ABC, Giant cell tumour and Chondroblastoma. FNAC report came out to be inconclusive. Therapeutic options which were considered at that point were resection of lesion and curettage with autologous bone grafting. The conservative approach was preferred as resection could have resulted in weakening of the shoulder. Intraoperatively, initially a 10cc disposable

syringe was used to aspirate the contents of the cavity. The content was found to be a blood-fluid (Fig. 4). The lesion was approached after incising the periosteum longitudinally. It was a multiloculated cyst containing streaks of thrombi (Fig. 5). The inner wall was curetted and electrocautery done to seal the bleeding walls of the cavity. Cavity was further irrigated with iodine containing alcohol solution and cavity was filled with corticocancellous strip of autologous iliac crest bone graft. Periosteal tube was repaired and limb was immobilized in cuff and collar sling. Postoperative period was uneventful. The preoperative diagnosis was confirmed with the histopathological examination of the curetted specimen.

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Figure 1: Photograph depicting swelling at the left clavicular region.

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Figure 2: Radiograph showing cystic expansile lesion bounded by a thin layer of bone at the lateral end of the left clavicle.

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Figure 3: MRI Left shoulder (Plain and contrast) Scan showing multilobulated T1 hypointense, T2 hyperintense arising from end of left clavicle with destruction of lateral end and peripheral enhancement, central non-enhancing areas.

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Figure 4: Intraoperative photograph showing blood-fluid in the cyst.

Figure 5: Photograph depicting multiloculated cyst containing streaks of thrombi.

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DISCUSSION Despite the long experience of orthopedicians, radiologists and pathologists, there is limited knowledge regarding the cause of the lesion, its natural history, and the results of treatment4-8.An interesting theory about the aetiology of primary

ABCs is that the lesions occur because of haemorrhage in the bone as a result of increased venous pressure. The haemorrhage is thought to lead to osteolysis. The osteolysis, in turn, causes further haemorrhage, leading to exponential growth of the tumour. This theory would perhaps

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www.ijpbs.com (or) www.ijpbsonline.com explain why ABCs are uncommon in the clavicle and bones of the facial skeleton, where thevenous pressure is low. On the other hand, ABCs are common in long bones, where the venous pressure is high and the marrow content is greater9. Conventional thinking; however, ascribes these lesions either to a reaction to physical injury, which may sometimes be remote, or to a vascular disturbance. The concept that the lesion represents a vascular degenerative process for some benign bone lesions is an attractive one, but the pathologic findings, with rare exception, do not really support this proposal. Few pathologic specimens contain tissues that are highly characteristic or diagnostic of giant cell tumor, chondroblastoma, hemangioma, osteoblastoma, non ossifying fibroma, 6, 7, 8, 10 chondromyxoid fibroma & others .Thus, it is often thought that ABC is more of a pathophysiological change in a pre-existing primary bone lesion rather than a single, unique entity11. Difficulty can occur in diagnosing these lesions. The imaging studies, even CTs and MRIs, sometimes do not provide clearly diagnostic criteria for the diagnosis of ABC, and ABC is sometimes added on to a list of diagnoses including eosinophilic granuloma, giant cell tumor, non ossifying fibroma, unicameral bone cyst, fibrous dysplasia, chondroblastoma, chondrosarcoma, chondromyxoid fibroma, 6, 7, 12 Ewing's tumour etc. . In the past, curettage alone was employed in the treatment later methods like saucerisation, resection, radiotherapy, cryotherapy and vascular occlusion are being employed. Nevertheless, there is no consensus among treating physicians regarding how these methods should be used. As a result, there are quiet contradictory reports regarding results and complications13. Resection of lesion offers low recurrence rate but this option cannot be exercised everywhere. A

combination of cryosurgery and curettage has been reported by few authors that reported local control after the first treatment in 82% patients14. Radiotherapy can result in radiation induced sarcomas and can cause radiation induced injury to physis13. Thus radiotherapy is reserved in cases that cannot be operated because of their location and to prevent damage to the function of important structure7. In some cases embolisation of a feeding vessel may help to decrease vascularity, making the surgical procedure less bloody, especially in difficult locations such as spine and pelvis but it is a highly demanding technique and may not be available at all centres. Recurrence rate in young children with ABC may be as high as 100%1. Autograft implantations or utilization of intercalary allografts are quite successful and for the most part, are used for patients with lesions that are large or seem to threaten the integrity of the bone. Our patient responded nicely to this form of treatment and we feel that this case enriches existing data regarding treatment option of an ABC in an unusually young patient and in unusual location.

REFERENCES 1.

3. 4.

Leithner A, Windhager R, Lang S.,Aneurysmal bone cyst: A population-based epidemiologic study and literature review. Clin Orthop Relat Res. 1999; 363:176-79. Dominok G. W., Knoch H. G., Manza B., SchulzeK. J., Die aneurysmatischeKnochencyste.LangenbecksArchievefu rChirurgie. 1971; 328: 153-168. Smith J, Aneurysmal bone cyst of clavicle.Br J Radiol. 1977; 50:706-9. Aho HJ, Aho AJ, Einola S: Aneurysmal bone cyst: A study of ultrastructure and malignant transformation. Virchows Arch A PatholAnatHistol. 1982; 395:169-79. Aho HJ, Aho AJ, Peliniemi LJ, Ekfors TO, Foidart JM: Endothelium in aneurysmal bone cyst.Histopathology. 1985; 9:381-87. Campanacci M: Aneurysmal bone cyst:In Campanacci M - Bone and Soft Tissue Tumors 2ndedn. New York, NY, Springer Verlag, 1999, 812-840. Dorfman HD, Czerniak B: Cystic lesions, In Dorfman HD, Czerniak B: Bone Tumors. St Louis, MO, Mosby, 1998, 855-912.

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www.ijpbs.com (or) www.ijpbsonline.com 8.

Szendroi M, Aratgto G, Ezzati A, Huttl K, Szavcsur P., Aneurysmal bone cyst: Its pathogenesis based on angiographic, immunohisto chemical and electron microscopic studies. PatholOncol Res. 1998; 4:277-81. 9. Boyd RC. Aneurysmal bone cysts of the jaws. Br J Oral Surg. 1979; 16: 248-53. 10. Bollini G, Jouve JL, Cottalorda J, Petit P, Panuel M et al., Aneurysmal bone cyst in children: Analysis of twentyseven patients. J PediatrOrthop B.1998; 7:274-285. 11. Kransdorf MJ, Sweet DE. Aneurysmal bone cyst: concept, controversy, clinical presentation, and imaging. Am J Roentgenol. 1995; 164: 573-80.

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|525-530 12. Mahnken AH, Nolte Ernsting CC, Wildberger JE, et al: Aneurysmal bone cyst: Value of MR imaging and conventional radiography. EurRadiol. 2003; 13: 118-24. 13. Tillman B., Dahlin DC, Lipscomb PR, Stewart JR., Aneurysmal bone cyst: an analysis of ninety-five cases. Mayo Clin Proc. 1968; 42:478-95. 14. Marcove R,Sheth DS, Takemoto S, Healey H., The treatment of aneurysmal bone cyst. ClinOrthop. 1995; 311: 157-63.

*Corresponding Author: Dr. Nagakumar J S

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Assistant Professor, Dept of Orthopaedics, SDUMC, Kolar, India, Ph: 9448543804

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Research Article Biological Sciences DIURETIC ACTIVITY OF JALAMANJARI CHENDOORAM IN RATS NITHYAMALA I *, KUMAR A, BANUMATHI V, VELPANDIAN V, PITCHIAHKUMAR M, AYYASAMY S Post Graduate Department of Gunapadam (Pharmacology), Government Siddha Medical College, Chennai, Tamilnadu, India. Pin Code: 600 106. *Corresponding Author Email: inithyamalabsms@gmail.com

ABSTRACT Aim: In the Siddha System of medicine, innumerable drugs are available, one such valuable diuretic drug Jalamanjari Chendooram (JC) from Siddha literature, has been identified, which till now not scientifically evaluated has been chosen and a detailed study has been done to evaluate diuretic activity of the drug. Therefore the present study was planned to evaluate the diuretic potential and effect on urinary electrolytes of JC in male Wistar rats. Methods: For the evaluation of diuretic activity the methods of Lipchitz et al., 1943 and Murugesan et al., 2000 were followed. Different concentrations of JC (25 mg and 50 mg/kg of body weight) and the standard drug Furosemide (20 mg/kg) were administrated orally to hydrated male Wistar rats and their urine output was measured at several intervals of time after a single dose administration. The parameters measured for diuretic activity were urine volume at different time intervals, sodium, potassium and chloride content. Results: JC 50mg/kg showed remarkable increase in volume of urine, sodium, potassium and chloride content. Conclusion: Conclusively, JC is an effective diuretic confirming the traditional use of the drug.

KEY WORDS Jalamanjari Chendooram, diuretic activity, electrolytes, urine volume, furosemide.

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INTRODUCTION The most common condition which causes a major financial and emotional burden on the community is renal diseases. It is also a medical condition with limited treatment options in the modern medicine. But since ancient times innumerable complementary and alternative medicines especially Siddha system of medicines possess with it a treasure of renal protective and effective treatments and are followed here and there successfully. One among the important renal protective drugs is the diuretic group of drugs. Diuretics increase the urine formation either by increasing the glomerular filtration rate (or) by decreasing the rate of reabsorption of fluid from the tubules. Such drugs which induce diuresis are known as

diuretics. Diuretic compounds are those which stimulate water excretion potentially from our body. So diuretics play a vital role in many oedematous diseased conditions such as congestive heart diseases, nephritis, and toxemia of pregnancy also in hypertensive conditions and pulmonary congestion. The mode of action of diuretics is that they decreases cardiac work load, oxygen demand, plasma volume and thereby decreases blood pressure [1]. Diuretics cure clinical conditions like acute and chronic renal failure, hypercalciuria, and cirrhosis of liver. Though these are the good effects of synthetic diuretics, many adverse effects have also been reported. These are hyperuricaemia, acidosis, gastric irritation & high blood sugar level [2] .

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www.ijpbs.com (or) www.ijpbsonline.com So there arises an emergent need to search for a safe renal protective diuretic drug and innumerable such drugs are available in Siddha literatures but yet to be standardized since it is deficit in scientific valuation. One such valuable diuretic drug Jalamanjari Chendooram from Siddha literature, has been identified, which till now not scientifically evaluated has been chosen and a detailed study has been done to evaluate diuretic activity in rats.

MATERIALS AND METHODS Preparation of the drug Jalamanjari [3] Chendooram The raw drugs required for the preparation of Jalamanjari Chendooram as mentioned in “Yoogi Karisal - 151” are Salt Petre, Borax, Conch shells, Alum, Ammonium Chloride, crystallized foliated Gypsum, Asbestos, Red ochre, Magnetic oxide of iron, Asphaltum, Sulphur , Iron Filings. The raw drugs were subjected to ‘Suddhi’ (purification process) as per Classical Siddha text [4, 5]. The purified drugs were powdered separately. The powders were mixed and grinded again into a very fine powder. A shallow container was heated and some amount of the powder was sprinkled in to it. The mixture first melted and then solidified. The solid was taken and allowed to cool. Similarly all the powders were used and the solidified products obtained were grinded into a very fine powder.

PHARMACOLOGICAL ACTIVITY

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Drugs and Chemicals Furosemide was procured from Himedia Laboratories, Mumbai, Other chemicals and reagents used in this study were analytical grade was purchased from SRL labs.

concentration at room temperature for oral administration by gastric intubation method. Animal selection: For the diuretic study, male Wistar rats weighing between 180-220 g were used. The animals were acclimatized to standard laboratory conditions (temperature: 25±2°C) and maintained on 12-h light: 12-h dark cycle. They were provided with regular rat chow and drinking water ad libitum (Approval number: XIII /VELS /PCOL /17 /2000 /CPCSEA /IAEC / 08.08.2012). Evaluation of Diuretic activity: The methods of Lipchitz et al., 1943 and Murugesan et al., 2000 were followed [6-8]. The screening was performed on healthy rats. Furosemide (20 mg/kg) was used as reference standard and Jalamanjari Chendooram were dissolved in saline solution for administration while normal saline (25 ml/kg) was used as vehicle. The rats were divided in 4 groups each containing 6 rats (n = 6). Rats were kept for fasting for 18 hrs before the study. The control group received normal saline and test groups received 25 and 50mg/kg of Jalamanjari Chendooram dissolved in normal saline. The doses of Jalamanjari Chendooram were decided on the basis of acute toxicity study. The doses were given by oral route and rats were kept in specially designed metabolic cages for the collection of urine for 6 hrs. The urine volume during 6 hrs is measured and urine electrolyte estimation was carried out for Na+, K+ using flame photometer and Cl – was estimated by titration. Na+, K+ estimation was carried out using flame photometry [9, 10]. The Cl – ion concentration was estimated by titration with 0.02 N AgNO3 using 5% potassium chromate solutions as indicator [11].

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www.ijpbs.com (or) www.ijpbsonline.com STATISTICAL ANALYSIS All results were expressed as mean ± standard error. The data was analyzed statistically using ANOVA followed by Dunnet’s Multiple Comparison Test.

RESULTS AND DISCUSSION Jalamanjari Chendooram has traditional use as a diuretic, the effect of this drug, standard drug Furosemide and control group on urination and

Group

Control Standard Test 1 Test 2

other parameters related to diuretic assay were investigated in Wistar rats and the results of the evaluation carried out were tabulated in Table 1 & 2. Table 1 shows the urine volumes collected at different time intervals control group, Furosemide and trial drug Jalamanjari Chendooram treated orally at dose levels of 25 and 50mg/kg. Table 2 shows the parameters related to electrolyte excretion (Na+, K+ and Cl– concentrations in mMol/L).

Table-1: Showing the urine volume at different intervals in rats Treatment Urine volume at different time intervals (in ml)

Normal Furosemide (20 mg/ kg) JC 25mg/kg JC 50mg/kg

15 min

30 min

45 min

60 min

120 min

0.270.04 0.340.05

0.510.02 1.460.1**

1.060.05 2.280.12**

1.020.08 3.380.18

1.540.22 4.870.24**

0.250.03 0.740.01* 1.240.05 1.880.11 2.150.30 0.290.04 0.620.01 1.360.07* 2.690.10 3.960.42** Values are mean ± SEM, * p< 0.01, ** p< 0.05 when compared to normal saline (control)

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Table 2: Effect of Jalamanjari Chendooram on electrolyte levels in urine Group Treatment Sodium Potassium Chloride (mMol/l) (mMol/l) (mMol/l) Control Normal saline (25 ml/ kg) 62.380.08 92.101.24 10.201.21 Standard Furosemide (20 mg/ kg) 104.110.51** 118.494.15** 14.341.74* Test 1 JC 25mg/kg 87.211.02** 92.400.18 14.010.04* Test 2 JC 50mg/kg 96.100.66** 92.562.88 13.650.80 Values are mean ± SEM, * p< 0.01, ** p< 0.05 when compared to normal saline (control) Effect on urine volume Two dose levels of Jalamanjari Chendooram (25 mg/kg and 50mg/kg) were selected for study and the urine volume after 15 minutes of administration of drug were found to be 0.250.03 and 0.290.04 respectively. Furosemide (20mg/kg) treated group was found to be 0.340.05. Urine volume after 120 minutes of administration of two dose levels of drug were

found to be 2.150.30 and 3.960.42 respectively. Furosemide (20mg/kg) treated group was found to be 4.870.24. Furosemide treated rats showed a significant increase in volume of urine as compared to control while Jalamanjari Chendooram 25mg treated rats did not show any significant increase in urine volume but Jalamanjari Chendooram 50mg/kg showed remarkable increase in volume of urine.

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Fig No.1. Effect of Jalamanjari Chendooram on electrolyte levels in urine

Concentration (mMol/l)

140

Effect of Jalamanjari Chendooram on electrolyte levels in urine

120 100 80 60 40 20 0 Normal

JMC 25mg/kg Sodium

Potassium

JMC50mg/kg

Frusemide (20 mg/ kg)

Chloride

Fig No.2. Effect of urinary volume of Jalamanjari Chendooram treated rat Diuretic activity of Jalamanjari Chendooram in rats 6 5 4 3 2 1 0

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15 min 30 min 45 min 60 min 120 min Normal JMC 25mg/kg JMC50mg/kg Frusemide (20 mg/ kg) -1 Urine volume at different time intervals (in ml)

Effect on urinary electrolyte excretion The effect of standard drug Furosemide and different doses of Jalamanjari Chendooram on electrolyte (Na+, K+ and Cl-) excretion in urine is tabulated in Table 2. The dose of 25mg/kg Jalamanjari Chendooram produced a moderate increase in Na+, K+ and Cl-

excretion, compared with the control group (Na+ =87.211.02, K+=92.400.18 and Cl-= 14.010.04). The dose of 50 mg/kg Jalamanjari Chendooram produced a significant increase in excretion of sodium, potassium and chloride ions in the urine to an extent similar to that of

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www.ijpbs.com (or) www.ijpbsonline.com Furosemide (Na+ = 96.100.66, K+ = 92.562.88 and Cl- =13.650.80).

CONCLUSION From this study it can be suggested that the drug Jalamanjari Chendooram is an effective and significant hyponatraemic, hypochloraemic and hypokalaemic diuretic with values close to the standard drug Furosemide, which supports the claim about the Jalamanjari Chendooram being used as a diuretic in Siddha system of medicine. On the basis of the results of present investigation, we can conclude that Jalamanjari Chendooram might be a good diuretic.

ACKNOWLEDGEMENT The authors are very much grateful to the Principal and HOD, Government Siddha Medical College, Chennai for their constant support to carry out this work successfully.

REFERENCES 1.

Finkel R, Clark MA, Cubeddu LX.Lippincott’s Illustrated Reviews: Pharmacology 4th Edn, Lippincott Williams &Wilkins, Florida, 2009, 190.

Deniels, T.C. and E.C. Jorgensen, Text Book of Organic Medicinal and Pharmaceutical Chemistry, 7th ed., Lippincott Co. Ltd., Toronto, 1977,575. 3. Ramachandran, Yoogi Karisal 151, Edn 2, Tamarai Press, Chennai, 2004,46 4. Thiyagarajan R. Gunapadam Thathu – Jeeva Vaguppu Part (2 & 3) Edn 2, Indian Medicine and Homeopathy Dept. Chennai. 2006, 305, 397, 408, 437, 442, 530,534, 546. 5. Aanaivaari Anandan, Sarakku Suddhi Sei Muraigal, Edn 1, Indian Medicine and Homeopathy Dept. Chennai106. 2008, 25, 28 & 100. 6. R. A. Turner, The Organization of Screening. In: Screening Methods in Pharmacology, Vol. I, New York and London, Academic Press; pp. 21(1965). 7. W.L. Lipchitz, Z.Haddian and A.Kerpscar. Bioassay of diuretics. J. Pharmacol. Exp.Ther. 79: 97-110 (1943). 8. T. Murugesan, L. Manikandan, K.B. Suresh, M. Pal and B.P. Saha. Evaluation of diuretic potential of Jussaea suffruticosa Linn extract in rats. Indian J.Pharm.Sci. 62(2): 150-151(2000). 9. Jeffery GH, Basset J, Mundan J, Denny R. 5th ed. England: Addison Westerly Longmann Ltd; 1989. Vogel's Textbook of Quantitative Chemical Analysis; p. 801. 10. Bhakuni DS, Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN. Screening of Indian plants for biological activity, II. Indian J Exp Biol. 1969; 7:250–62. 11. Beckett AH, Stanlake JB. 1st Edn. New Delhi: CBS Publishers and Distributor; 1997. Practical pharmaceutical chemistry; p. 197.

*Corresponding Author: Dr.I.Nithyamala

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Email: inithyamalabsms@gmail.com Phone No: 044-26222682 / 9444183646 Fax: 044 - 26222683

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Research Article Biological Sciences COMPARISON BETWEEN CT SCAN AND MANUAL METHOD OF AREA MEASUREMENT OF MASTOID PROCESS IN SEX DETERMINATION OF SOUTH INDIAN POPULATION Albin Babu M Wilson1, Nimma purna chander reddy2, Suresh sukumar3, Sushil Yadav4, Karuna paliwal5 1

Lecturer. MSc. Medical Imaging Technology, Department of Medical Imaging Technology, KSHEMA, Nitte University, mangalore. 2 Clinical SME, Apollo Health Street. 3 Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. 4 Assistant professor, MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. 5 MSc. Medical Imaging Technology, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. *Corresponding Author Email: suresh.medicalimaging@gmail.com

ABSTRACT Aim: The purpose of this study was to evaluate the significance for sex determination of the measurement of the area formed by projection of 3 craniometric points related to the mastoid process (the porion, asterion, and mastoidale points) of 3D reconstructed computed tomography (CT) of skull and the result is compare the result of manual measurement of the area formed by projection of 3 craniometric points related to the mastoid process. Method: 3D reconstructed CT of 40 males and 40 females were analysed. The three craniometric points were located and marked on both side of the 3D skull and the measurement was done by advanced post processing techniques. In manual measurement 40 males and 40 female skull were analyzed by digital caliper (0.01mm). The area of mastoid triangle was calculated by means of the Heron’s formula. The result of CT reading and Manual reading is analyzed for the sex determination and the both result is compared. Result: From this study the areas of the male CT measurement of Mastoid is 828.53±118.54 mm2which is greater than female Mastoid which is 578.24±71.53 mm2 (T test value 9.901and P Value 001). Area of the male Mastoid which is measured by manual 805.87±90.99 mm2 which is greater than female Mastoid which is 620.56±90.63 mm2 (T test value 10.202and P Value 001). Conculsion:The area of the mastoid triangle measured by manual and 3D reconstructed CT skull are used to determine the sex of the skull and there is no significant deference between CT scan and Manual method of area measurement of mastoid process in sex determination of south Indian population .

KEY WORDS 3D reconstructed computer tomography, Sex determination, Mastoid process

INTRODUCTION

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In the skull, the temporal bone is highly resistant to physical damage; thus it is commonly found as

remainder in skeletons that are very old; of this, the petrous portion has been described as important for sex determination (Kalmey &

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www.ijpbs.com (or) www.ijpbsonline.com Rathbun, 1996). Paiva & Segre (2003) introduced an easy technique for sex determination starting from the temporal bone, with a small observational error and with a high predictability degree. The technique is based on the triangular area calculation obtained between the point’s porion, mastoidale, and asterion, measured from xerographic copy of skulls. They found significant differences in the area between the right and left mastoid triangle when comparing male and female skulls, but owing to the asymmetries present in the skulls, it is recommended to observe the value of the total area (adding right and left sides), which was also significant, so that when it is higher than or equal to 1447.40 mm2, the skull is diagnosed as male skull, and a value near to 1260.36 mm2 or less is indicative of female skull (De Paiva & Segre). The present study is aimed at describing radiological methods identification sex of skull by 3D computer tomography image.

OBJECTIVE The purpose of this study was to evaluate the significance for sex determination of the measurement of the area formed by projection of 3 craniometric points related to the mastoid process (the porion, asterion, and mastoidale points) of 3D reconstructed computed tomography (CT) of skull and the result is compare the result of manual measurement of the area formed by projection of 3 craniometric points related to the mastoid process.

METHOD

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3D reconstructed CT of 40 males and 40 females were analysed. The three craniometric points were located and marked on both side of the 3D skull and the measurement was done by

advanced post processing techniques. In manual measurement 40 males and 40 females skull were analyzed by digital caliper (0.01mm). The area of mastoid triangle was calculated by means of the Heron’s formula. The result of CT reading and Manual reading is analyzed for the sex determination and the both result is compared.

RESULT From this study the areas of the male CT measurement of Mastoid is 828.53±118.54 mm2 which is greater than female Mastoid which is 578.24±71.53 mm2 (T test value 9.901and P Value 001). Areas of the male Mastoid which is measured by manual 805.87±90.99 mm2 which is greater than female Mastoid which is 620.56±90.63 mm2 (T test value 10.202and P Value 001) and there is no significant deference between CT scan and Manual method of area measurement of mastoid process in sex determination of south Indian population Table – 1:- The areas of male and female mastoid measured using manual and 3D computer Tomography imaging. (About here)

DISCUSSION The analysis of the mastoid process characteristics is important in the determination of sex for forensic purposes. The mastoid region used in this study, being a part of the temporal bone, is recognized as being the most protected and resistant to damage, due to its anatomical position at the base of the skull. This has been demonstrated by Kloiber (1953), Wells (1960), Schäefer (1961), Gejval (1963), and Spence (1967), as cited by Wahl and Henke10 (1980) According to Paiva & Segre (2003), When it is higher than or equal to 1447.40 mm2 singel side, the skull is recognized as male skull and When

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www.ijpbs.com (or) www.ijpbsonline.com the total area was lower than or equal to 1260.36 mm2 single side, the skull is recognized as female skull. In our present study The areas of the male right and left side From our study the areas of the male CT measurement of Mastoid is 828.53±118.54 mm2 which is greater than female Mastoid which is 578.24±71.53 mm2 for single side (T test value 9.901 and P Value .001). Areas of the male Mastoid which is measured by manual is 805.87±90.99 mm2 for single side which is greater than female Mastoid which is 620.56±90.63 mm2 for single side (T test value 10.202and P Value .001).

3. 4.

CONCULSION The area of the mastoid triangle measured by manual and 3D reconstructed CT skull are used to determine the sex of the skull and there is no significant deference between CT scan and Manual method of area measurement of mastoid process in sex determination of south Indian population .

De Paiva, L. A. & Segre, M. sexing the human skull through the mastoid process. Rev. Hosp. Clin. Fac. Med. São Paulo, 58:15-20, 2003. Standring S, ed. Gray’s Anatomy. 40th Ed., Philadelphia, Elsevier, Churchill Livingstone. 2005. Kemkes, A. & Gobel, T. Metric assessment of the "mastoid triangle" for sex determination: a validation study. J.Forensic Sci., 51:985-9, 2006 Walsh M, Reeves P, Scott S. When disaster strikes; the role of the forensic radiographer. Radiography 2004; 10:33-43. Al Ekrish AA, Ekram M. A comparative study of the accuracy and reliability of multidetector computed tomography and cone beam computed tomography in the assessment of dental implant site dimensions. Dental Maxillofacial Radiol 2011; 40:67-75. SUAZO, G. I. C.; ZAVANDO, M. D. A. & SMITH, R. L. Sex determination using mastoid process measurements in Brazilian skulls. Int. J. Morphol., 26(4):941-944, 2008

ACKNOWLEDGEMENT Author acknowledges the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors /publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. The author is highly thankful to the referees for their very constructive, valuable suggestions and useful technical comments, which led to a significant improvement of the paper.

REFERENCES

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Kalmey, J. K. & Rathbun, T. A. Sex determination by discriminant function analysis of the petrous portion of the temporal bone. J. Forensic Sci., 41:865-7, 1996

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Table â&#x20AC;&#x201C; 1:- The areas of male and female mastoid measured using manual and 3D computer Tomography imaging. CT scan Vs. Manual Measurement

gender

Mean

Std. Deviation

T test

P Value

CT scan Measurement

male

828.53

118.54

9.901

<0.001

female

578.24

71.53

male

805.87

90.99

10.220

<0.001

female

620.56

90.63

Manual Measurement

*Corresponding Author: Suresh Sukumar

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Assistant professor, Department of Medical Imaging Technology, Manipal College of Allied Health Sciences, Manipal University, Manipal. udupi â&#x20AC;&#x201C; 576104. Email: suresh.medicalimaging@gmail.com.

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Research Article Biological Sciences MANAGEMENT OF COMPOUND FRACTURES OF TIBIA BY EXTERNAL FIXATION: A PROSPECTIVE STUDY FROM A RURAL HOSPITAL OF SOUTH INDIA NAGAKUMAR J S1, S S GUBBI2, S B KAMAREDDY3. 1

Assistant professor in department of orthopaedics, Sri Devaraj Urs medical college, Kolar, Karnataka 2 Professor in department of orthopaedics, M R medical college, Gulbarga, Karnataka 3 Associate Professor in department of orthopaedics, M R medical college, Gulbarga, Karnataka

ABSTRACT Background: Introduction of external fixator is a revolution in the management of compound fractures tibia for it has saved many limbs from amputation. Objectives: to study the usage of external fixator in the treatment of compound tibial fracture and to assess the functional outcome of patient. Methods: During October 2005 to September 2007, 20 cases of open fracture tibia were selected based on Gustilo Andersonâ&#x20AC;&#x2122;s classification with exclusion of Type 1 and type 3C wounds. Fractures were managed by using bilateral frame with transfixing pins and biplanar fixators. Patients were followed at 4 weeks interval with clinical and radiological assessment. The results were classified as good, moderate and poor depending upon the degree of deformity, degree of shortening, range of motion at neighbouring joint. Results: All patients were male belonging to age group 20 - 40 years with road traffic accidents. Eighty percent of the fractures were of Type III with middle 1/3 of leg common site. Good outcome was noted in 14 cases (70%) while 15% each of moderate and poor outcome. Conclusions: External fixators could be the choice of fixation in open fracture tibia and was found to be simple, economical and effective.

KEY WORDS Open tibial fracture, external fixation, rural hospital

INTRODUCTION The management of compound fractures of tibia is a challenge to orthopedic surgeon. Number of methods have described and tested with varying results on the management of compound fractures1-3.Introduction of external fixator is a revolution in the management of compound fractures tibia, for it has saved many limbs from amputation4-7.

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OBJECTIVES 1. To study the usage of external fixator in the treatment of compound tibial fracture, 2. To assess the functional outcome of patient with reference to rate of fracture union and

range of movement at ankle joint and knee joint and to study the restoration of function of the limb.

METHODOLOGY The study was conducted in Basaveshwar Teaching & General Hospital attached to Mahadevappa Rampure Medical College, Gulbarga. During October 2005 to September 2007, 20 cases of open fracture tibia were selected based on Gustilo Andersonâ&#x20AC;&#x2122;s classification 8(table 1) as Type 1, 2, 3A, 3B and # based on the size of wound, degree of soft tissue injury, level of contamination, degree of bony injury and presence of neurovascular injury. Type

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www.ijpbs.com (or) www.ijpbsonline.com 1 and type 3C wounds were excluded as type I were treated with primary intra-medullary interlocking nailing and type 3C were referred to vascular surgeon due to associated vascular injury. Patients belonging to age groups 20 to 40 years were included. Patients were initially examined in casualty regarding head injury, respiratory, cardiovascular and abdomen status. Intravenous fluids, antibiotics and intramuscular tetanus toxin and tetanus immunoglobulin were given. After haemo-dynamically stabilized were shifted to major OT for wound debridement and external fixator application within 24 hours.

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OPERATIVE TECHNIQUES (Fig 1 & 2) The basic frame components are the adjustable clamps used for articulation of the schanz pin to the steel tube. The clamps allow screw insertion in any desired plane, Hollow tubes with outside diameter of 11mm and length from 100-600mm, Schanz pins of diameter 4.5mm and Steinmann pins are used. The triple trocar is a universal instrument for guiding insertion of the schanz pin with conus. It consists of 5mm and 3.5mm drill sleeve with a 3.5mm trocar. The pin is first predrilled with a 3.5mm drill bit, then over drilled in the near cortex with a long 4.5mm drill bit. The universal chuck with a T-handle holds the schanz pins during insertion, while the wrenches are used to tighten the clamp nuts. Other instruments include the open compressor and distractor9-10. The fixator components are generally assembled into one of two basic frame types of configurations namely unilateral uniplanar, unilateral biplanar. The one plane configurations are less obstructive and generally suffice for most injury situations. Two plane frames are more effective in neutralizing multi directional bending and torsional movements. However, they are only needed when dealing with severe comminuted

fractures or with bone loss. The safe corridor for schanz pin insertion in the tibia is at level proximal to the tibial tubercle, schanz pins can be safely inserted within the arc of 220 degree. At level B, just below the tibial tubercle, the safe arc decreases to 1400. At level C, in the distal third of the leg, the safe arc remains 1400 but anterior tibial vessels and deep peroneal nerve become vulnerable as they cross the lateral tibial cortex. At level D, above the ankle joint, the safe arc is 120 degree. At levels E and F, steinmann pins in the tarsal or metatarsal bones may be used to splint the ankle joint if neurological or soft tissue injuries prevent the application of an external support. External fixators are usually applied under general or regional anaesthesia with the limb draped free so as to leave all pertinent skeletal land marks visible11. The insertion of schanz pin should be done in the following manner. a. Assemble the triple trocar and penetrate soft tissue (through a stab incision) down to the bone surface. b. Remove the trocar and drill through both cortices using a long 3.5mm drill bit. c. Remove the drill sleeve, through the remaining 6 or 5mm sleeve over drill the near cortex using a long 4.5mm drill bit. The use of oscillating attachment combined with the three fluted drill bit is recommended. d. Insert the depth gauge probe through the drill sleeve hooking the far cortex. e. Loosen the locking pin, advance the knurled disk to the top of the drill sleeve and tighten the locking pin. f. Remove the probe, place the threaded tip of the schanz pin in to the schanz pin recess of the knurled disk g. Advance the universal chuck over the nonthreaded end of the schanz pin until the tip of the probe touches the end of the universal

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chuck. Tighten the universal chuck on to the schanz pin in this position. h. Insert the schanz pin until the universal chuck nearly touches the top of the drill sleeve, the schanz pin is now fully inserted into far cortex. i. Remove the drill sleeve and attach the adjustable clamp. Unilateral uniplanar frame: This is applied as follows Step I: Application of schanz pin close to the distal joint. The tube with the planned number of adjustable clamps is fixed to the schanz pin. Step II: Application of second schanz pin across the most proximal adjustable clamp. At this time, three dimensional reduction of fracture is easy, observing axis and rotation of the foot by comparing it with uninjured leg. Step III: The inner schanz pin is inserted about 2cm from the fracture area. The tubular fixator allows individualization of schanz pin placement according to fracture configuration. Prevention of drop foot by connecting metatarsal I and II to tube by means of schanz pin. Unilateral Biplanar Frame: This consists of 2 interconnects simple unilateral frames to allow optimal wound access. The plane for the second frame should lie at an angle of 60-100 degree with the plane of the first frame. First the ventral fixator is applied in a nearly sagittal plane aiming towards the medial posterior tibial cortex. Next the medial fixator is applied at an angle between 60-100 degree and fixed with either 2 or 4 schanz pins. The tubes are interconnected by smooth pins. Maneuvers for reduction of Fracture: In simple transverse fractures, stabilization at the fracture site is achieved by compressing main fragments against each other, taking care to avoid the tendency to angulate the fragments. The fixators are then used as a neutralization frame. In

diaphyseal fractures, however this maneuver should only be considered in treating simple two fragment fracture with relatively long contact areas. In comminuted fracture neutralization frame is applied. To diminish motion at fracture site and increase the stiffness of frame, the following was considered: a. Principal frame should be applied in saggital plane. b. Preloading of schanz pins c. Increasing the number of pin in each bony fragment d. Increasing the pins spread with in each fragment e. Reducing the distance between bone and the longitudinal tube. Frame application: It depends on site of wound that is opposite to the site of wound. If a soft tissue coverage procedure is required lateral on, then the side of frame application should be such, as to leave enough free area for the plastic surgery. The fixator was placed in neutralization mode in case of comminuted and butterfly fragment fractures, compression mode incase of transverse, oblique and segmental fractures so as to narrow fracture gap and improve stability. Relaxing skin incisions were placed around the pin tracts to avoid skin compression, bones were covered with overlying muscles, skin approximated with stay sutures. The foot and ankle were manipulated at the end of procedure to ensure absence of musculotendinous tethering by transverse pins. All these patients were followed at 4 weeks interval. Clinical and radiological assessment of the patients at follow up comprised of wound healing, tenderness at fracture site, degree of weight bearing, presence of callus, gap at fracture site, sclerosis at fracture ends and obliteration of medullary canal. Once the wound is clean and covered with healthy granulation tissue, plastic surgeon opinion sought

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www.ijpbs.com (or) www.ijpbsonline.com and treated accordingly. Static Quadriceps exercise was started immediate post-operatively. Knee and ankle motion was allowed 4 week from the operative day. Partial weight bearing was allowed in non-comminuted fractures 4-6 weeks later. In case of comminuted fractures 10-12 weeks from the day of surgery. Full weight bearing was allowed when there was radiological evidence of union. The results were classified as good, moderate or poor depending upon the degree of deformity, degree of shortening, range of motion at neighbouring joint. The degree of

deformity and limb length discrepancy was assessed using the modified Anderson and Huntchins Criteria (table 2). The ankle and knee movements were graded as Full range-Normal; Significant loss of movement -In the knee - loss of extension up to  100, and flexion up to 400, ankle - 250, but < 500 of flexion / extension; Insignificant loss - Anything less than but above the normal. Severe loss - both in knee and ankle, with loss of > 500 of flexion and extension. The results were classified as good, moderate and poor (Table 2).

Table 1: Gustilo – Anderson’s classification of type of fracture Types

No. of Patients

Type-I

Type -II

Type – III A

Type III B

Type – III C

Table 2: Modified Anderson &Huntchins Criteria to assess degree of deformity&limb length discrepancy. Shortening

Grade of deformity in Angulation (Malunion)

Good

<1 cm

Up to 50Varus / Valgus up to 100 Anterior / Posterior

Moderate

1-2cm

5-100 Varus / Valgus 10-200 Anterior / Posterior

Poor

> 2cm

> 100 Varus / Valgus > 200 anterior / Posterior

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Results

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Table 3: Management of Associated Injuries Associated Injury

Gustilo’s Type

Side/Type

Management

Closed fracture both bone Right Forearm

Type –II

Right

Closed reduction and internal fixation with square nail

Closed Fracture Femur M/3rd

right

Type III-B

Right

Intra-medullary interlocking nail for the fracture femur

Crush injury Right foot with fracture 1st and 2nd metatarsal bone

Type III A

Right

Wound debridement and fixed with k-wire

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Fig 1: Wound debridement

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Fig 2: Open wound

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Fig 3: Operative procedure in progress

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Fig 4: Operative procedure completed with fixators

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Fig 5a: Results showing good outcome

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Fig 5b: Results showing good outcome OBSERVATIONS All patients were male belonging to age group 20 - 40 years with road traffic accidents. Eighty percent of the fractures were of Type III with

middle 1/3 of leg common site. Good outcome was noted in 14 cases (70%) while 15% each of moderate and poor outcome (Fig 3a & 3b). Eight patients needed split skin grafting. One patient

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www.ijpbs.com (or) www.ijpbsonline.com had contra lateral closed fracture of both bone for which close reduction and internal fixation with square nailing was done. In two patients, one with associated ipsilateral closed fracture shaft middle third of right femur was treated by IM interlocking nail and another patient associated with ipsilateral crush injury of right foot with metatarsal I and II fractures was managed by Kwire fixation and split skin grafting (Table 3). In the 4 Gustilo – Anderson type 2 fractures which were 1 oblique, 1 segment, transverse and 1 butterfly external fixator was removed after 6 weeks and intramedullary interlocking nailing was done. In 8 Gustilo –Anderson Type 3 A fractures which were 2 butterfly and 6 comminuted, the butterfly fractures were treated with intramedulary nailing 6 weeks after being in external fixator. The remaining 6 which were comminuted, 5 were treated with external fixator for 4 months followed by POP cast (PTB cast) for another 6 weeks. One of them showed signs of sclerosis of 1 to 2 fragments which were excised after 4 months. In the 8 cases of Gustilo – Anderson type – 3B all were comminuted fractures, 6 were treated with external with external fixator for 5-6 months followed POP cast for another months 2 cases showed. Signs of nonunion and had cancellous bone grafting, after 6 months of external fixator application followed by POP cast above Knee next 2 months.

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DISCUSSION Seventy percent of patients in the present study had good results. All were male with mean age of 28years whereas in Thakur and Patanakar12 study, females and males represented 16.5% and 83.5% respectively with mean age of 38 years suggesting higher level of activities and mobility in the these age groups. The present study documents road traffic accident as the cause of injury in all cases whereas on an average 85.9% and 87.3% road traffic accidents were recorded by Pedro

AntichAdrover et al13 and Thakur and Patankar12 series respectively. An equal number of cases of Type III A and Type IIIB (40% each) and type II (20%) noted in the present study in comparison with Thakur and Patankar12 series where 12 spiral (or) long oblique, 27 Transverse (or) short oblique, 40 comminuted fractures were documented. The present study also records 50% of the fracture middle third, 30% fracture distal third and 20% fracture proximal third while it was 78% middle third, 10% distal third and 4% proximal third from a study by Henley MB, et al14. In our series, 8 patients underwent split skin grafting (40%), 1 patient muscle pedicle flap (10%) and one case of type III A and 2 cases of type IIIB bone grafting where as in the Thakur and Patankar12 series, skin grafting was required in 43 patients, 5 flap coverage and 44 cases (60.3%) of bone grafting. Superficial wound infection (20%) and pin tract infection (10%) were the common complications in the present study whereas superficial and deep wound infections of 42.2% and 16.1% respectively were noted in the series by Bhandariet al15suggesting infection rate was lower in the present study and was successfully managed with parenteral antibiotics.

CONCLUSION Open fractures of tibia are quite common, because of its subcutaneous location, high energy trauma, which is quite often encountered during high speed moving vehicles, especially on national highway. The study shows that reasonable outcome may be attained in open tibial fractures with the external fixation technique allowing early definitive treatment. Complications are minimal with good range of movements in knee and ankle. Competing interest: NIL

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Author’s contributions: NAGAKUMAR J S participated in acquisition of data, literature search and carried out analysis, interpretation, and drafting the manuscript. S S GUBBI, S B KAMAREDDY did acquisition of data and participated in drafting and revising the manuscript. All authors read and approved the article.

REFERENCES

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Dillin L, Slabaugh P: Delayed wound healing, infection, and non-union following open reduction and internal fixation of tibial plafond fractures. J Trauma 1986;26 (12):1116–1119. McFerran MA, Smith SW, Boulas HJ, Schwartz HS: Complications encountered in the treatment of pilon fractures. J Orthop Trauma 1992;6(2):195–200. Teeny SM, Wiss DA: Open reduction and internal fixation of tibial plafond fractures. Variables contributing to poor results and complications. ClinOrthopRelat Res 1993, 292:108–117. Sisk TD: General principles and technique of external fixation. ClinOrthopRelat Res 1983; 180: 96-100. Sisk TD. External fixation: historic review, advantages, disadvantages, complications and indications. Clin Orthop Relat Res1983; 180: 15-22. Edwards CC. Staged reconstruction of complex open tibial factures using Hoffman external fixation: Clinical decisions and dilemmas.ClinOthop 1983; 178: 180.

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Caudle, R.J., and Stern, P.J. Sever open fractures of the tibia. J Bone Joint SurgAm 1987; 69(6): 801-807. Gustilo RB, Mendoza RM, Williams DN. Problems in the management of type III (Severe) open fractures: New classification of type III open fractures.JTrauma 1984; 24: 742. Weber, B.S. and Mageri F. The external fixator. New York, spring – Verlag. 1985. Weber B.G and Magerl F. “A.O. Manual of external fixation”, Berlin, Hedelberg: Springer – Verlag. 1985, pp459. Behrens F, and Searls K. External fixation of the Tibia Basic concepts and prospective evaluation.J Bone Joint Surg 1986, 68B (2):246-254. Thakur AJ andPatnakar J. Open tibial fractures – Treatment by uniplanar external fixation and early bone grafting. J Bone Joint Surg (Br) 1991; 73-B: 448-51. Pedro Antich – Adrover, David Martin – Garin, Juan Murias-Alvarez, Carlos Puente-Alonso. External fixation and secondary intramedullary nailing of open tibial fractures. J Bone Joint Surg (Br) 1997; 79-B: 433-7. Henley MB, Chapman JR, Agel J, Harvey EJ, Whorton AM, Swiontkowski MF. Treatment of type II, III A, and III B open fractures of the tibial shaft: a prospective comparison of unreamed interlocking intramedullary nails and half-pin external fixators: JOrthop Trauma 1998; 12(1): 1-7. Bhandari M, Guyatt GH, Swionkowski MF, Schemitsch EH. Treatment of open fractures of the shaft of tibia. J bone Joint Surg Br2001; 83(1): 62-8.

*Corresponding Author: NAGAKUMAR J S

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Assistant professor in department of orthopaedics, Sri Devaraj Urs medical college,Kolar, Karnataka

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Research Article Biological Sciences COMPARATIVE EFFECTS OF CITRULLUS LANATUS AND CAJANUS CAJAN DIETS ON THE LIPID PROFILE AND BODY WEIGHT OF ALBINO RATS *Andrew Nwaka C.1, Christian Odunze S.1, John Ihedioha I.2 Ugwu Okechukwu P.C.3 Ossai Emmanuel C3., Ada Ikeyi4 and Bayim P.R.5 1

Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria. Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria. 3 Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria. 4 Department of Science Laboratory Technnology, Institute of Management and Technology, Enugu, Nigeria. 5 Department of Sciences, Cross-River State University of Technology, Calabar.

*Corresponding Author Email: andynwaka@yahoo.com

ABSTRACT This study evaluated the comparative effects of diets containing 50 and 70% Citrullus lanatus or Cajanus cajan on the serum lipid profile and body weight of albino rats. Thirty male albino rats randomly assigned into five groups (A – E) of six each were used for the study. The groupings and their diets were as follows: A – Standard rat feed (control), B – diet containing 50% C. lanatus, C – diet containing 70 % C. lanatus, D - diet containing 50% C. cajan, and E - diet containing 70% C. cajan. The rats were fed their group specific diets for 28 days during which they were weighed at weekly intervals. At the end of the 28 days of feeding, blood was collected from the rats and the serum lipid profile was assayed following standard procedures. Results showed that the rat groups fed diets containing 50 and 70 % C. lanatus had a significantly higher (p < 0.05) serum total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDL-C) but significantly lower (p < 0.05) serum triglyceride when compared to the control group fed on standard rat feed. The rat group diet fed containing 70% C. cajan however had a significantly lower serum TC, HDL-C, VLDL-C and triglyceride when compared to the control group fed standard rat feed. The rat group fed diet 70% C. cajan also had a significantly higher (p < 0.05) weight gain all through the study period when compared to all other groups. It was concluded that findings in this study suggest that diets containing 70% C. cajan can be recommended for its ability to lower serum cholesterol and triglycerides, and thus can possibly be used to prevent and manage atherosclerosis.

KEY WORDS Serum lipid profile, body weight, rats, Citrullus lanatus, Cajanus cajan

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INTRODUCTION Citrullus lanatus and Cajanus cajan are legume staples widely consumed in Nigeria and other parts of the world. Legumes are edible seeds of leguminous plants, belonging to the Leguminosae family. Legumes can be divided into two main broad classes, pulses and oil seeds. Pulses are the dried edible seeds of cultivated legumes and they

include peas, beans, pigeon pea (Cajanus cajan) etc. (Olusanya, 2008). The oil seeds are legume seeds that contain appreciable amount of oil and they include soya beans, melon (Citrullus lanatus) and groundnut. Legumes constitute the second largest family of seed plants and contain about 600 genera with 13 species. Apart from their nutritive value to human beings, they are also

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www.ijpbs.com (or) www.ijpbsonline.com important in fixing atmospheric nitrogen, thereby making the element available to other plants in the soil (Olusanya, 2008). Legumes are rich in protein, but their protein has a well-recognized deficiency of the sulphur amino acids, methionine and cystein but is comparatively rich in lysine (Ihekoronye and Ngoddy, 1987). Cereals on the other hand contain these amino acids which legumes lack. Hence a combination of legumes and cereals such as beans and maize would provide an ideal source of dietary protein for human beings. Cajanus cajan (pigeon pea) is a tropical legume grown mainly in India. The true origin of pigeon pea is still disputable. However, immigrants who moved to Africa to become railway workers and storekeepers (Hillocks et al., 2000) most likely introduced the crop into East Africa from India in the 19th century. The legume is increasingly becoming an important sustenance crop in the whole of Africa (Johansen et al., 1993). Some potential uses of pigeon pea in Africa include the production of noodles (Singh et al., 1989) and other fermented products (Onofiok et al., 1996). Pigeon pea leaves have been used to treat malaria (Aiyeoloja and Bello, 2006) in Nigeria, while in the Southern African; pigeon pea is currently one of the indigenous crops being promoted for potential medicinal uses (Mander et al., 1996). Also, clinical studies have reported the seed extracts to inhibit red blood cell sickling and as potential benefit for people with sickle cell anaemia (Akinsole and Solanke, 2011). Citrullus lanatus (melon) is a creeping annual legume which belongs to the Eucurbitaceae family (Olusanya, 2008). It is a water-loving crop and therefore is cultivated predominantly in the southern part of Nigeria (Olusanya, 2008). The flat seeds are embedded in white fleshy material in a circular fruit. Melon seed is rich in protein and oil (Fuller and Harvey, 2006; Olusanya, 2008) and low in carbohydrate but contains good amounts of

minerals especially phosphorus, magnesium and potassium and a fair amount of carotene and vitamin D (Olusanya, 2008). Melon is a high calorie source because of its high oil content. Melon seeds are an excellent source of tryptophan and arginine (Olusanya, 2008; Ojieh et al., 2008). Cholesterol and triglycerides are the major blood/serum lipids of clinical significance in humans and animals (Ononogbu, 1988; Oslon, 1998; Nelson and Cox, 2000). Cholesterol is an essential component of mammalian cell membranes which play major roles in membrane permeability and fluidity and also as precursor of bile acids, steroid hormones and fat soluble vitamins (Oslon, 1998; NCEP, 2002). Triglycerides play important role in metabolism as energy sources and transporters of dietary fat (Ononogbu, 1988; Nelson and Cox, 2000). Though cholesterol and triglycerides are physiologically important in the body, high levels of them in the blood have been found to be a major risk factor for the development of atherosclerosis (Brown and Goldstein, 1992; Oslon, 1998; Schoen, 2004; Brunzell et al., 2008). The possible pathological consequences of atherosclerosis include myocardial infarction (heart attack), cerebral infarction (stroke), aortic aneurysms, peripherial vascular disease, sudden cardiac death, chronic ischaemic heart disease etc (NCEP, 2002; Schoen, 2004; Brunzell et al., 2008). The consumption of food items that will significantly reduce the overall blood levels of cholesterol and triglyceride and / or those components of cholesterol that have been associated with increased risk of atherosclerosis is one of the major strategies at prevention and management of atherosclerosis (Law, 1999; NCEP, 2002; Brunzell et al., 2008). Hence the present study, which evaluated the comparative effects of feeding diets containing 50 or 70 %

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MATERIALS AND METHODS The rats used for the study were mature male Sprague-Dawley albino rats weighing 150 – 250 grams, procured from the Laboratory Animal House of Faculty of Veterinary Medicine University of Nigeria, Nsukka. The rats were randomly assigned to five groups (groups A – E) of six each and acclimatized for seven days before the commencement of the study. The rats were kept in clean cages in a fly-proof animal house and provided with feed and water ad libitum all through the study. Guidelines for the humane use and handling of laboratory animals for research (NAS, 2011) were followed all through the study. The standard rat feed used in this study was sourced from Grand Cereals and Oil Mills Ltd., Jos Nigeria. The C. lanatus and C. cajan seeds were procured and processed into a mash. The proximate composition of the standard rat feed, C. cajan and C. lanatus used for the study were determined following standard procedures. The protein content was determined by micro Khjelldahl method, while the ash, moisture and fat contents were determined by gravimetric method (AOAC, 2000; Changsam, 2003). The total carbohydrate was then estimated using the standard formula (Bemiller, 2003). After acclimatization, the rats were weighed and the different groups were fed their group-specific diets for 28 days. The group specific diets were as follows: Group A – Standard rat feed (control), Group B – diet containing 50% C. lanatus, Group C – diet containing 70 % C. lanatus, Group D - diet containing 50% C. cajan, and Group E - diet containing 70% C. cajan. The rats were further weighed at weekly intervals and weight gain was computed by comparing with the body weight before commencement of feeding of the experimental diets. After the 28 days of feeding

the experimental diets, 3 ml of blood was collected from the orbital sinus of the retrobulbar plexus of the rats following the orbital technique (Bolliger and Everds, 2010). The blood dispensed into clean plain glass test tubes and allowed to stand for 30 minutes to clot. After clotting, it was centrifuged at 3,000 revolutions per minute to separate the serum from clot. The clear serum was aspirated into clean labeled sample bottles and used immediately for the lipid profile assay following standard procedures. The serum lipid profile was assayed using Quimica Clinica Aplicada (QCA) test kits (QCA, Spain). The serum total cholesterol (TC) was determined by the enzymatic colorimetric method (Allain et al., 1974). The serum high density lipoprotein cholesterol (HDL-C) was determined by the dextran sulphate-magnesium (II) precipitation method (Albers et al., 1978). The glycerol phosphate oxidase enzymatic method was used to determine the serum triglyceride (Bucolo and David, 1973). The very low density lipoprotein cholesterol (VLDL-C) was calculated by dividing the serum triglyceride by 5, while the serum low density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formular (Friedewald et al., 1972; Warnick et al., 1990). Data generated from the study were subjected to one way analysis of variance and variant means were compared with the control post hoc using the least significant difference (LSD) method. Significance was accepted at p < 0.05.

RESULTS Results of the proximate analysis for the standard feed, C. cajan and C. lanatus showed that C. cajan had the highest percentage composition of crude protein (26.52%) when compared to that of the standard feed (19.53%) and that of the C. lanatus (23.4%) (Table 1). The proximate analysis also showed that C. lanatus had the highest percentage composition of fat (45.7%) when

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www.ijpbs.com (or) www.ijpbsonline.com compared to that of the standard feed (7.86%) and C. cajan (3.14%) (Table 1). The standard rat feed had the highest percentage composition of

carbohydrate (56.91%) while C. lanatus has the least (10.6%) (Table 1).

Table 1. The proximate composition of the rat feed, Cajanus cajan and Citrullus lanatus used for the study. Composition Means ± standard deviation. Standard rat feed Cajanus cajan Citrullus lanatus Crude protein 19.53 ± 0.44

26.52 ± 0.43

23.40 ± 0.20

Moisture

6.11 ± 0.26

7.46 ± 0.25

5.80 ± 0.18

Ash

5.76 ± 0.44

4.25 ± 0.31

4.60 ± 0.30

Crude fibre

3.83 ± 0.11

6.99 ± 0.34

12.00 ± 0.11

Fat

7.86 ± 0.27

3.14 ± 0.28

45.70 ± 0.17

51.64 ± 0.54

10.60 ± 0.20

Carbohydrate 56.91 ± 0.62

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Table 2. The serum lipid profile of albino rats fed for 28 days with diets containing varied percentages of Cajanus cajan or Citrullus lanatus. Means ± standard deviation. Groups Total HDL-C (mg/dl) LDL-C VLDL-C Triglyceride (Diets fed) cholesterol (mg/dl) (mg/dl) (mg/dl) (mg/dl) Group A 56.10 ± 11.63 27.15 ± 1.92 16.85 ± 7.83 12.10 ± 3.37 60.49 ± 18.66 (Standard rat feed) Group B 85.61 ± 8.64* 49.41 ± 14.60* 28.95 ± 7.78* 7.24 ± 3.94 36.20 ± 19.68* (50% C. lanatus) Group C 75.96 ± 11.04* 39.61 ± 9.20* 30.38 ± 6.31* 5.96 ± 1.84* 29.80 ± 9.20* (70% C. lanatus) Group D 50.82 ± 5.55 21.64 ± 0.63* 20.79 ± 8.05 8.30 ± 3.36 41.94 ± 16.80* (50% C. cajan) Group E 43.71± 7.52 20.83 ± 0.95* 16.62 ± 5.18 6.27 ± 1.48* 31.34 ± 7.41* (70% C. cajan) * Asterisk superscript on any mean indicates that it is significantly different from the control group (A) fed standard rat feed (p < 0.05).

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Table 3. The body weight gain of albino rats fed for 28 days with diets containing varied percentages of Cajanus cajan or Citrullus lanatus. Means ± standard deviation. Groups (Diets fed) Day 7 Day 14 Day 21 Day 28 Group A 10.06 ± 3.29 24.12 ± 9.27 35.05 ± 5.64 38.69 ± 3.89 (Standard rat feed) Group B 7.34 ± 2.86 18.68 ± 7.87 35.87 ± 6.30 39.94 ± 5.94 (50% C. lanatus) Group C 7.77 ± 3.62 19.54 ± 8.46 32.17 ± 4.18 36.77 ± 4.80 (70% C. lanatus) Group D 14.67 ± 5.91 33.34 ± 8.14 36.17 ± 4.18 36.77 ± 4.80 (50% C. cajan) Group E (70% C. cajan)

28.03 ± 8.87*

60.06 ± 7.94*

65.53 ± 9.83*

69.16 ± 6.41*

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* Asterisk superscript on any mean indicates that it is significantly different from the control group (A) fed standard rat feed (p < 0.05).

The rat groups fed diets containing 50% and 70% C. lanatus (Groups B and C) had a significantly higher (p < 0.05) serum TC, HDL-C and LDL-C when compared to the control group fed standard rat feed, while the group fed 70% C. cajan (Group E) had a significantly lower (p < 0.05) serum TC, and HDL-C when compared to the control group fed standard rat feed (Table 2). The serum VLDL-C of the rat groups fed 70% C. lanatus (Group C) and 70% C. cajan (Group E) were significantly lower (p < 0.05) than that of the rat group fed standard rat feed (Table 2). However, the serum triglyceride of groups B, C, D and E rats were significantly lower (p < 0.05) than that of the rat group fed the standard rat feed (Table 2). The body weight gain computations showed that the rat group fed with 70% C. cajan diet (Group E) had significantly higher (p < 0.05) body weight gain when compared to all other rat groups all through the study (Table 3).

DISCUSSION

The significantly higher total serum TC, HDL-C and LDL-C recorded for the rat groups fed diets containing 50% and 70% C. lanatus can be attributed to the high fat composition of C. lanatus as indicated by its proximate composition. This suggests that consumption of diets with up to 50 and 70 % C. lanatus could predispose the consumer to development of atherosclerosis and its associated pathological consequences (Brown and Goldstein, 1992; Oslon, 1998; Schoen, 2004; Brunzell et al., 2008). In contrast, the significantly lower serum TC, VLDL-C and triglyceride recorded for the rat group fed 70% C. cajan could be as a result of its low fat composition, and by implication, consumption of diets with up to 70% C. cajan could be used therapeutically to reduce blood TC, VLD-L and triglyceride and thus prevent/manage atherosclerosis. The findings in this study of the ability of diet containing 70% C. cajan to reduce blood cholesterol is in agreement with the reports

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www.ijpbs.com (or) www.ijpbsonline.com of Luo et al. (2008) that showed that administration of extracts of C. cajan was able to significantly reduce serum total cholesterol of hyperlipidemic mice. The findings in this study of significantly higher serum HDL-C in the rat groups fed 50 and 70 % C. lanatus is a positive finding as HDL-C, often referred to as “good cholesterol”, is known to facilitate the removal of “bad cholesterol” (LDL-C and VLDL-C) from the blood vessels and transferring them to the liver where they are metabolized and excreted (Libby et al., 1998; NCEP, 2002; Barter et al., 2007). The significantly lower serum triglyceride recorded for the rat groups fed diets containing 50 and 70% C. lanatus and C. cajan and also the significantly lower serum VLDL-C recorded for the rat groups fed 50 and 70% C. cajan are considered positive developments when viewed against the background of the role that LDL-C and VLDL-C play in the development of atherosclerosis and its pathological consequences (Libby et al., 1998; Barter et al., 2007). These findings in the C. cajan fed rats is in agreement with the reports of Luo et al. (2008) Results of the body weight gain computation which showed that the rats fed with 70% Cajanus cajan diet (Group E) had significantly higher body weight gain all through the study when compared to other groups is believed to be as a result of the high carbohydrate and protein content of C. cajan when compared to the standard feed and C. lanatu. It was worthy of note that the higher weight gain was not associated with a corresponding higher serum TC, rather the C. cajan fed rats had a lower serum TC. Based on the results of this study, it was concluded that rats fed diets containing 50 and 70 % C. lanatus had significantly higher serum TC, HDL-C and LDL-C but significantly lower serum triglyceride when compared to the control fed standard rat feed, while the rat group fed 70 % C.

cajan had significantly lower serum TC, HDL-C, VLDL-C and triglyceride. In addition, rats fed diets containing 70% C. cajan had a significantly higher body weight gain when compared to all other groups.

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Aiyeoloja, A.A., Bello, O.A. (2006). Ethnobotanical potentials of common herbs in Nigeria: A case study of Enugu State. Educational Research and Review, I: 16 – 22. Akinsole, O.A., Salonke, O.O. (2011). Clinical evaluation of extract of Cajanus cajan (ciklavit) in sickle cell anaemia. Nigerian Journal of Animal Production, 28: 243 – 250. Albers, J.J., Warnick, G.R. and Cheung, M.C. (1978) Quantification of high density lipoproteins. Lipids, 13: 926 – 932. Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W. and Fu, P.C. (1974), Enzymatic determination of total cholesterol. Clinical Chemistry, 20: 470 – 475. AOAC (2000). Official Method of Analysis of Association of Official Analytical Chemist. Washington D.C., 16th Edition, Pp. 12 – 18. Barter, P., Gotto, A.M., LaRosa, J.C., Maroni, J., Szarek, M., Grundy, S.M., Kastelein, J.J.P., Bittner, V., and Fruchart, J. (2007) HDL Cholesterol, Very Low Levels of LDL Cholesterol, and Cardiovascular Events. New England Journal of Medicine, 357: 1301 - 1310. Bemiller, N.J. (2003). Carbohydrate Analysis. Food Analysis Third Edition. Kluwer Academic publisher, New York. Pp. 143 – 146. Bolliger, A. P. and Everds, N. E. (2010) Hematology of laboratory rodents: mouse (Mus musculus) and rat (Rattus norvegicus). In: Weiss D. J. and Wardrop K. J. Eds., Schalm’s Veterinary Hematology, 6th edition, Wiley-Blackwell, Iowa, pp 852 – 862. Brown, M.S. and Goldstein, J.S. (1992) Koch’s postulates for cholesterol. Cell, 71: 187 – 188. Brunzell ,J.D., Davidson, M., Furberg, C.D., Goldberg, R.B., Howard, B.V., Stein, J.H. and Witztum ,J.L. (2008) Lipoprotein management in patients with cardiometabolic risk – Consensus statement from the American Diabetes Association and the Amarican College of Cardiology Foundation. Diabetes Care, 31: 811 – 822. Bucolo, G. and David, H. (1973) Quantitative determination of serum triglycerides by use of enzymes. Clinical Chemistry, 19: 476 – 482.

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Changsam, K.C (2003). Protein Analysis. Food Analysis. Third Edition. Kluwer Academic / Plenum Publisher, New York, pp. 134 – 135. Friedewald ,W.T., Levy R.I., Fredrickson ,D.S. (1972) Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clinical Chemistry, 18: 499 – 502 Hillocks, R.J., Minja, E., Nahdy, M.S,Subrahmanyam, P. (2000). Diseases and pests of pigeon pea in Eastern Africa. International Journal of Pest Management, 46: 7 – 18. Ihekoronye, A.I. and Ngoddy, P.O. (1987). Integrated food science technology for the trpics. Macmillian Publication, London, pp. 330 – 332. Johansen, C., Silim, S.N., Laxman, S. (1993) Towards a database for pigeon pea in Africa. International Pigeon Pea Newsletter, 18: 2 -5. Law, M.R. (1999) Lowering heart disease risk with cholesterol reduction: evidence from observational studies and clinical trials. European Heart Journal Supplement, 1:S3-S8. Libby, P., Schoenbeck ,U., Mach, F., Selwyn, A.P., Ganz, P. (1998) Current concepts in cardiovascular pathology: the role of LDL cholesterol in plaque rupture and stabilization. American Journal of Medicine, 104: 14S 18S. Luo ,Q.F., Sun, L.,Si. J.,Y., Chen ,D.H., DU, G.H. (2008). Hypocholesterolemic effect of stilbene extract from Cajanus cajan on serum and hepatic lipid in diet induced hyperlipidemic mice. Phytomedicine, 43: 145-149 Mander, M., Mander, J., Brean, C. (1996). Promoting the cultivation of indigenous plants for markets in domestication and commercialization of non-timber forest products in Agronomy system. Non-wood Forest Products, 9: 298– 104. NAS (National Academy of Sciences) (2011) Guide for the Care and Use of Laboratory Animals, 8th ed. The National Academies Press, Washington DC.

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NCEP (National Cholesterol Education Program) (2002) Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults – Adult Treatment Panel III, Final Report, NCEP, National Heart, Lung, and Blood Institute, National Institutes of Health, USA. Nelson, D.L. and Cox, M.M. (2000) Lehninger, Principles of Biochemistry, 3rd Ed. Worth Publishing, New York. Olusanya, J.O. (2008). Legume nuts. Essential of Food and Nutrition.1st ed. Apex Books Limited, Lagos, pp. 95 – 96. Onofiok, N., Nnanyelugo, D.O., Ukwondi, B.E. (1996).Usage patterns and contribution of fermented foods to the nutrient intakes of low income house holds in Emene, Nigeria. Plant Foods for Human Nutrition, 49: 199 – 211. Ononogbu, I.C. (1988) Lipid and Lipoproteins: chemistry, methodology, metabolism, biochemical and physiological importance. New Africa Publishing Co. Ltd. Owerri, Nigeria. Oslon, R.E. (1998) Discovery of the lipoproteins, their role in fat transport and their significance as risk factors. Journal of Nutrition, 128: 439S – 443S. Schoen, F.J. (2004) Atherosclerosis. In: Kumar V., Abbas A.K., Fausto N. (Eds.), Robbins and Cotran Pathologic Basis of Disease, 7th ed. Saunders, Philadelphia, pp. 515 – 525. Singh, U., Voraputhaporn, W., Roa, P.V., Jambunathan, R. (1989). Physicochemical characteristic of pigeon pea and mung bean starches and their noodle quality. Journal of Food Science, 54: 1293 – 1207. Warnick, G.R., Knopp R.H., Fitzpatrick V. and Branson L. (1990) Estimating low-density lipoprotein cholesterol by the Friedewald equation is adequate for classifying patients on the basis of nationally recommended cut points. Clinical Chemistry, 36: 15 – 19.

*Corresponding Author:

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Andrew C. Nwaka, Ph.D, Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria. E-mail: andynwaka@yahoo.com Phone: +234 (0) 8037834171.

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Research Article Biological Sciences IN VIVO RADIO PROTECTIVE PROPERTIES OF FUNGAL POLYSACCHARIDES Thulasi G.Pillaia and K.K.Janardhananb a

Division of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi, Thrissur- 680 653, Kerala, India. b Amala Cancer Research Centre, Amalanagar, Thrissur. *Corresponding Author Email: thulasigpilla@yahoo.co.in

ABSTRACT In vivo radio protective properties of polysaccharides isolated from Ganoderma lucidum, a macro fungi was examined. Swiss albino mice were exposed to 4 Gy gamma irradiation. Serum lipid peroxidation and tissue GSH were taken end points on 1st, 3rd, 5th, 7th and 9th day after irradiation. Polysaccharides were administered just after irradiation at 10 and 20 mg/kg body wt. Administration of polysaccharides reduced the Serum MDA levels compared to the irradiated group. Tissue GSH was maintained at normal levels after administration of polysaccharides. The polysaccharides possess radio protective property.

KEY WORDS Ganoderma lucidum, polysaccharides, radioprotection, radiotherapy.

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INTRODUCTION Ganoderma lucidum, commonly known as Reishi in Japan and Ling Zhi in China, is well known for its medicinal properties. G.lucidum contains a number of compounds among which the polysaccharides and triterpenoids have been identified as the major active components. Crude or partially purified polysaccharides of G.lucidum have been reported to inhibit tumor metastasis in mice1. The immunomodulating property of this mushroom provides a promising approach for cancer prevention and its administration is found useful alone or in combination with chemotherapy and radiotherapy [1]. Our earlier studies suggest that the aqueous extract of this mushroom has significant radioprotective activity ex vivo [2]. Polysaccharides are among the major source of pharmacologically active constituents of the aqueous extract. Polysaccharides from G.lucidum was reported to markedly restore the mitotic activity of bone marrow cells that has

been suppressed by anti-neoplastic drugs [3] The present study was undertaken to examine the protection offered by the polysaccharides from the macro fungi G.lucidum against radiation induced damage.

MATERIALS AND METHODS Chemicals All chemicals used in the study were of analytical grade obtained from reputed local manufactures. Animals Swiss albino mice, 6-8 weeks of age and weighing 28 Âą 2 g, were selected for the study. They were maintained in air-conditioned animal house and fed on standard mouse food and water ad libitum. Animal handling and experiments were done according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, and were approved by Institutional Animal Ethics Committee.

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www.ijpbs.com (or) www.ijpbsonline.com Isolation of polysaccharides The fruiting bodies of G.lucidum were collected from the outskirts of Thrissur district, Kerala, South India. The type specimen was deposited in the herbarium of Centre for Advanced Studies in Botany, University of Madras, Chennai, India (HERB. MUBL. 3175). Polysacchrides were isolated by the method of Mizuno [4] with slight modification [5] The confirmation of polysacchrides were done by Anthrone [6] and phenol sulphuric acid test [7] Structural confirmation was done by IR and NMR spectrum which were recorded at Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Mumbai, India. From the Gel filtration analysis the molecular weight of Polysaccharides were found to be 1.5 x 106 Daltons. The powder was dissolved in double distilled water and administered orally in the experiments. Irradiation The cobalt therapy unit with Gamma Cell 220 (AECL, Canada) facility of Amala Cancer Hospital, Thrissur was used for irradiation. Anaesthetized animals, were exposed to 4 Gy -irradiation at a dose rate of 1 Gy/min. Experimental Design Five groups with 15 animals each were used for the study. Group I – Normal Control (Double distilled water) Group II – Radiation alone (4 Gy) Group III – Amifostine (300 mg/Kg body wt) + Radiation 4 Gy Group IV - Radiation 4 Gy + Polysaccharides (10 mg/ Kg body wt) Group V - Radiation 4 Gy + Polysaccharides (20 mg/ Kg body wt) Animals were sacrificed in alternate days from 1 to 9 (1, 3, 5, 7, and 9) Polysaccharides were administered orally just after irradiation.

Tissue protein and serum protein Tissue and serum protein were determined by Bradford’s [8] method. The protein determination was done according to the procedure given in the kit purchased from Banglore Genei. BSA was used as standard and diluted to get 1mg/ml.Different concentrations of BSA and unknown samples were pipetted into different test tubes. 2 ml of Bradford’s reagent was added, mixed and kept at room temperature for 10 mins. OD was recorded at 595 nm using Varian DMS 200 UV-visible spectrophotometer. The value of unknown sample was recorded from the standard graph. Serum lipid peroxidation Serum lipid peroxidation was determined by Ohkawa et al [9] after precipitating the protein according to the method of Satoh [10] to 0.5 ml serum, 2.5 ml of 0.02% TCA was added and the tube is left to stand for 10 min at room temperature. After centrifugation at 3500 rpm for min, the precipitate was washed. A 4ml reaction mixture containing 0.4 ml of serum, 1.5 ml of 0.8% TBA, 1.5 ml of acetic acid (20% pH 3.5) and distilled water was kept for 1 hour in a boiling water bath at 950 C. After 1hour, the reaction mixture was removed from water bath, cooled and added 1 ml of distilled water. 5 ml of butanol: pyridine mixture (15:1) was added to the reaction tube, mixed thoroughly and centrifuged at 3000 rpm for 10 min. Absorbance of the clear supernatant was measured at 532 nm against butanol: pyridine mixture. The MDA was calculated with the help of a standard graph made by using different concentrations (1-10 nanomoles) of 1’1’3’3 – tetramethoxy propane in 1 ml distilled water and is expressed as nmol of MDA/mg protein. Determination of tissue GSH Reduced glutathione (GSH) in tissue was determined by the method of Moron et al,[9] 0.5ml of tissue homogenate was mixed with 0.1 ml of 25% TCA and kept on ice for few minutes

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www.ijpbs.com (or) www.ijpbsonline.com and then subjected to centrifugation at 3000g for few minutes to settle the precipitate. 0.3ml of the supernatant was mixed with 0.7ml of 0.2M sodium phosphate buffer (pH8). The yellow colour obtained was measured after 10 min at 412 nm against a blank which contained 0.1 ml of 5 % TCA in place of the supernatant. A standard graph was prepared using different concentrations of GSH in 0.3 ml of 5% TCA. The GSH content was calculated with the help of this standard graph and expressed as n mole/mg protein.

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RESULTS AND DISCUSSION Tissue GSH was found to be reduced from 3rd to 7th day in a radiation alone group. Administration of polysaccharides at 20 mg/kg body wt restored GSH on the 7th and 9th day (Figure1). Serum MDA was increased at 4 Gy gamma radiation (Figure.2). Administration of polysaccharides at 20 mg/kg body wt reduced significantly on 5th, 7th and 9th day. The presence of amifostine at 300mg/kg body wt also reduced MDA significantly on 5th day after radiation exposure. Antioxidant enzymes are among the endogenous system that are available for the removal or detoxification of these free radicals and their products formed by ionizing radiation. The GSH/GST detoxification system is an important part of cellular defense against a large array of endogenously or exogenously formed injurious agents. GSH offers protection against oxygenderived free radicals and cellular lethality following exposure to ionizing radiation. GST enzymes also possess peroxidase activity and can directly attack the peroxides that may be generated via oxidative reduction recycling, resulting in decreased cytotoxicity. The present study demonstrates that a significant reduction in GSH in radiation treated group. This could be due to the enhanced utilization of antioxidant defense system in an attempt to detoxify the radicals generated by radiation. In the intact and healthy

cells the enzymes are restored immediately after each interaction and GSH is also restored by synthesis [11]. But in the irradiated animals, the normal synthesis/repair will be disrupted due to damage to DNA and membranes. As a result, restoration will be delayed till the cells are recovered. This could explain the slow recovery in the levels of GSH and antioxidant enzymes after radiation treatment.The antioxidant property of the polysaccharides scavenge free radicals and neutralize it, thus reducing its capacity to damage. The balance between the production of free radicals and the antioxidant defences in the body has important health implications. This antioxidant property may be a contributing factor for the radioprotective properties offered by the polysaccharides. The interaction of ionizing radiation with biological system results in generations of free radicals, H and OH radicals, H2 and H2O2. Radiations induced free radicals in turn impair the antioxidant defense mechanism leading to increased membrane lipid peroxidation, which results in the damage of membrane bound enzymes [12-13] .The increased lipid peroxidation is due to the low concentration of GSH. The membrane damage due to lipid peroxidation is confirmed by the activity of hepatic enzyme GPT. Antioxidant enzymes are among the endogenous system that are available for the removal or detoxification of these free radicals and their products formed by ionizing radiation. Polysaccharides reduced chromosomal aberrations in mice exposed to 4 Gy gamma radiation [14] As a result, restoration will be delayed till the cells are recovered. This could explain the slow recovery in the levels of GSH and antioxidant enzymes after radiation treatment. The polysaccharides from Ganoderma administered to mice (5g/kg p.o for 30 days) produced no changes in body wt, organ wt or hematological parameters and produced no

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adverse effect [13]. Amifostine is an FDA approved radioprotector used clinically. Amifostine was used as a standard drug to compare the activity of Ganoderma polysaccharide. The protection offered by amifostine at 300mg/kg body wt, a dose which provided maximum protection with minimum toxicity and by the polysaccharides at 20mg/kg body wt was comparable. Thus the dose at which the polysaccharide renders protection is much lower than that of amifostine. Moreover, the polysaccharide is effective by oral administration,

which is the most convenient mode of administration in treatment of human diseases. In conventional radiotherapy, the use of a radioprotector, which can be administered orally is of significant advantage.

CONCLUSION The present finding that polysaccharides gives significant radioprotection when given after irradiation points to its advantage over the other pre-administered radioprotectors and potential for use both in medical non-medical exposures.

4.5

Serum MDA nmoles/ml

3.5

2.5

1.5

** **

0.5

0 1

No of days Normal RT 4 Gy + Amifostine (300mg/kg body wt) Polysaccharides PS 20mg/kg body wt + RT 4Gy

Radiation alone(RT) 4 Gy Polysaccharides PS 10mg/kg body wt + RT 4Gy

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Figure .1. Effect of polysaccharides on serum MDA after gamma Irradiation (4 Gy). *p < 0.01 compared to radiation alone. ** p< 0.05 compared to normal control.

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Tissue (GSH U/mg protein)

* 15

0 1

No of days Normal Rt 4 GY + Polysaccharides (10 mg/kg body wt)

Radiation alone (RT) 4 Gy RT 4 Gy + 20 mg/kg body wt)

RT 4 Gy + Amifostine (300 mg/kg body wt)

Figure .2. Effect of polysaccharides on tissue GSH after 4 Gy gamma irradiation. *p < 0.01 compared to radiation alone.

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REFERENCES [1] Gao Y., Zhou S., Cancer prevention and treatment by Ganoderma, a mushroom with medicinal properties, Fd. Rev. Intl.19: 275-325. (2003) [2] Pillai T.G., Salvi V.P., Maurya D.K., Nair C.K.K., Janardhanan K.K. Prevention of radiation induced damages by aqueous extract of Ganoderma lucidum occurring in Southern parts of India, Curr. Sci. 91:341344. (2006) [3] Wasser SP., Reishi or Ling zhi (G. lucidum). Encyclopedia of Dietary supplements. Marcel Dekker. 5: 603-622.2005. [4]Mizuno T., Development of an antitumor biological response modifier from Phellinus linteus Teng. (Review). Int.J. of. Med. Mushrooms. 2: 21-23. (2000) [5] Pillai T.G., Nair C.K.K., Janardhanan K.K. Enhancement of repair of radiation induced DNA strand breaks in human cells by Ganoderma mushroom polysaccharides, Fd. Chem. 119: 1040-1043. (2009) [6] Yemn E.W., Wills A.J. The estimation of carbohydrate in plant extract by anthrone. Biochem. J 57: 508-514. (1954) [7] Duboi S.M., Gilles G.A., Hamilton J.K., Colourimetric estimation of Carbohydrates by Phenol Sulphuric acid method, Anal.Chem. 28:350-356. (1956)

[8] Bradford, M M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochemistry. 72: 248-254. (1976) [9] Ohkawa H., Ohishi N., Yagi K., Assay for lipid peroxide in animal tissues by thiobarbituric acid reaction. Ann. Biochem. 95: 351-358. (1979) [10] Moron MA., Depierre JW., Mannervik B., Levels of glutathione reductase and glutathione S transferase activities in rat lung and liver. Biochem Biophys Acta. 582: 67-78, (1979) [11] Satoh K., Serum Lipid Peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chem.Acta, 90: 37-43, (1987) [12] Meister A., Anderson M.E., Transport and Direct Utilization of -glutamylcysteine for Glutathione Synthesis. PNAS. 3: 707-711 (1983) [13] Halliwell B., John H., Gutteridge NC., Production of hydroxyl radicals in living systems, In- Free Radicals Biology and Medicine, Clarendon Press, Oxford, 1989, 31. [14] Thulasi G Pillai and Uma Devi., Mushroom beta glucan: Potential candidate for post irradiation protection.Mutation Research.751 (2013) 109-115.

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*Corresponding Author: Thulasi G.Pillai

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Division of Forest Ecology and Biodiversity Conservation, Kerala Forest Research Institute, Peechi, Thrissur- 680 653, Kerala, India.

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Research Article Biological Sciences EFFECT OF SOYABEAN DIET ON THE SERUM IMMUNOGLOBULIN PROFILE AND CD 4+ COUNT OF MALE DIABETIC RATS CHALLENGED WITH K.PNEUMONIAE *ANDREW C. NWAKA1, PETER N. UZOEGWU2, ONYECHI OBIDOA3, UGWU OKECHUKWU P.C.4, OSSAI EMMANUEL C.4 and CHIGOR VINCENT N.5 1

Department Of Biochemistry, Anambra State University, Uli, Nigeria. Tropical Diseases Research Unit, Department of Biochemistry, University of Nigeria, Nsukka, Nigeria. 3 Department of Biochemistry, Kogi State University, Anyingba, Kogi State. 4 Department of Biochemistry, University of Nigeria, Nsukka. 5 Department of Microbiology, University of Nigeria, Nsukka. *Corresponding Author Email: andynwaka@yahoo.com

ABSTRACT In this study, the effect of soyabean supplemented diet on the immunoglobulin profile and CD 4+ count of male diabetic rats challenged with Klebsiella pneumoniae were evaluated in a total of sixty-four mature albino rats, weighing 150-250 grams. The rats were separated into eight groups of eight rats each. The groups were as follows: A- rats fed on rat feed, B-rats fed on rat feed and soyabean meal, C-diabetes-induced rats feed on rat feed, Ddiabetes-induced rats fed on rat feed and soyabean meal, E-rats fed on rat feed but challenged with Klebsiella pneumoniae, F- rats fed on rat feed and soyabean meal challenged with Klebsiella pneumoniae, G-diabetes induced rats fed on normal rat feed but challenged with Klebsiella pneumoniae and H-diabetes-induced rats fed on rat feed and soyabean meal but challenged with Klebsiella pneumoniae. The study lasted for 28days. At the end of 28 days, the rats were bled and blood collected, processed and the serum obtained used for assessment of immunoglobulin profile and CD4+ T-cell count. Immunoglobulin profile was carried out using immunoturbidimetric assay with Randox(R) kit, while CD4+ T-cell count was done using CY-flow machine. Results showed that there were significant increase (P< 0.05) in serum lgA, 1gM, 1gG levels and CD4+ T-cell of diabetic rats fed on normal feed and soyabean meal when compared with their counterparts fed with only normal feed. Results of the study suggest that soyabean diet could assist in boosting the serum immunoglobulin levels and CD4+ cell of diabetic rats and hence protecting the rats from K. pneumoniae infection.

KEY WORDS

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Diabetes mellitus, Immunoglobulins, CD4+-T-cell, K. pneumoniae and Soyabeans.

INTRODUCTION Diabetes mellitus, often referred simply as diabetes, is a syndrome characterized by disordered metabolism and abnormally high fasting blood sugar (hyperglycaemia) resulting from insufficient levels of the hormone, insulin or its insensitivity (Tierney et al., 2002). It is a chronic disease characterized by elevated blood glucose levels and disturbances in carbohydrate, fat and

protein metabolism (Sky, 2000, Rother, 2007). The World Health Organization, recognizes three main form of diabetes mellitus type I , type II and gestational diabetes (occurring during pregnancy) (WHO, 1999). Type I diabetes mellitus formerly called juvenile onset or insulin dependent diabetes mellitus (IDDM) is characterized by loss of the insulin

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producing beta cell of the islets of langerhans of the pancreas, leading to a deficiency of insulin. The main cause of this loss of Î˛- cell is a T-cell mediated autoimmune attack (Rother, 2007). There is no known preventive measure which can be taken against type I diabetes. While type 2 diabetes mellitus, also known as non-insulin dependent diabetes mellitus (NIDDM), is due to insulin resistance or reduced insulin sensitivities combined with reduced insulin secretion (Rosenbloom and Silverstain, 2003). Furthermore, gestational diabetes mellitus (GDM) resembles type 2 diabetes in several respects, involving a combination of relatively inadequate insulin secretion and responsiveness (WHO, 1999). In Nigeria, diabetes mellitus has emerged as one of the biggest health problems and its prevalence is increasing at an alarming rate (Unadike, 20120).

immunity have been shown to be affected by protein-energy malnutrition in young children, depending on the severity of the protein deficiency relative to energy. The effects include impaired antibody formation (Pretorius and De Villiers, 1962, Reddy and Srikantia, 1964 and Reddy et al., 1977), decreased serum immunoglobulin (Aref et al., 1977 and Watson et al., 1987), decreased thymic function (Watts, 1969, Purtilo and Connor, 1975 and Chandra et al., 1982) and splenic lymphocytes, delayed cutaneous hypersensitivity (Simythe et al., 1971 and Chandra, 1974), decreased complement formation (Sythe et al., 1971, Suskind et al., 1976), decreased interferon and effects on nonspecific mechanisms that include anatomic barriers and secretory substances such as lysomes and mucus (Scrimshaw and Sangiovanni, 1997).

Soyabean, is a plant of the family leguminoseae. Legumes are noted for their relatively high protein content when compared with cereals, tubers, fruits and vegetables. Soyabean contains about 40% protein, 18% fat and has a high concentration of minerals such as calcium, iron etc (Anosike et al., 2007).

In this study, the effect of soyabean diet on the serum immunoglobulin profile (i.e. lgM, lgG and lgA) and CD4+ count of male diabetic rats challenged with Klebsiella pneumoniae (a Gramnegative bacteria) was evaluated.

Infections, no matter how mild, have adverse effects on nutritional status. The significance of these effects depends on the nutritional status of the individual, the nature and duration of the infection and the diet during the recovery period (Scrimshaw and Sangiovanni, 1997). Furthermore, it is not surprising that protein deficiency is so consistently observed to interfere with resistance to infection because most immune mechanisms are dependent on cell replication or the production of active protein compounds. However, because protein cannot be synthesized without a balance of essential amino acids, experimental amino acid deficiencies have the same effect as protein. Essentially all forms of

MATERIALS Chemical used: All the chemical used in this research were of analytical grade. Animal used: Mature apparently healthy male albino rats weighing 150-250 grams were procured from the Animal House of Faculty of Veterinary Medicine, University of Nigeria, Nsukka campus. The animals were allowed to acclimatize for fourteen days before the commencement of the study. Soyabean meal: Rats on soyabean meal were given 9.6g/kg body weight of soyabean powder dispersed in water by intubation each morning before the animals were fed for the day.

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www.ijpbs.com (or) www.ijpbsonline.com Rat feed: The normal rat feed used in this study is pelleted growers mash of Vital (R) feed. It was produced by Grand Cereals and Oil Mills, Jos, Nigeria. Pathogenic Organism: Klebisella pneumoniae was used as the pathogenic organism in this study. Its stock was procured from the Department of Veterinary Microbiology, Faculty

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Groups A B C D E F G H

of Veterinary Medicine, University of Nigeria, Nsukka.

METHODS Experimental Design Sixty-four mature male albino rats, weighing 150250grams were used in the study. The rats were separated into eight groups of eight rats each and kept in eight different standard cages as follows:

Treatment Rats fed on rat feed Rats fed on rat feed and soyabean meal Diabetes-induced rats fed on rat feed Diabetes-induced rats fed on rat feed and soyabean meal. Rats fed on rat feed but challenged with Klebsiella pneumoniae Rats fed on rat feed and soyabean meal challenged with Klebsiella pneumoniae Diabetes-induced rats fed on normal rat feed but challenged with Klebsiella pneumoniae Diabetes-induced rats fed on rat feed and soyabean meal but challenged with Klebsiella pneumoniae

All the protocols as approved by Institutional Animal Ethics Committee (IAEC) were observed in the study. At the end of the 28days, the rats were bled from the retro-bulbar plexus of the medical canthus of the eye. The blood samples were collected into EDTA tubes and plain sample bottles. The EDTA acted as anticoagulant. The blood samples in the EDTA bottles were used for CD4+ T-cell counts, while the blood sample collected in the plain tubes without EDTA were allowed to stand for 30 minutes and centrifuged at the speed of 3,000 revolutions per minutes for 10minutes. The serum was extracted and used for lgA, lgG and lgM assays using standard biochemical methods.

the alloxan administration, the rats were fasted for 12hours and their blood glucose level was measured. Rats having blood glucose level of 400mg/dl and above were selected and used as diabetic rats in this study. Rats that were described as fasted were deprived of food for at least 12hours but were allowed free access (ad libitum) to drinking water.

Induction of Diabetes Diabetes was induced in the rats by injecting 180mg/kg body weight of alloxan monohydrate intraperitoneally in 0.9% w/v NaCl (normal saline) after fasting the rats for 12hours. Seven days after

Proximate Analysis The proximate composition of soyabean and the normal animal feed used in the study were evaluated. Their protein content were determined by micro khfelldal method as was described by

Pathogen Challenge The rats were challenged with Klebsiella pneumoniae by intraperitioneal administration of 0.4ml of the bacterial suspension containing 108 c.f.u/ml in 0.9% (w/v) normal saline on the 22 nd day of the study.

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www.ijpbs.com (or) www.ijpbsonline.com AOAC (2000) and Changsam (2003), while their ash, moisture and fats contents were determined by gravimetric method as described by AOAC (2000). Crude fiber were determined by gravimetric method as described by James (1995). The total carbohydrate were then estimated using the formula as was given by Bemiller (2003). Immunoglobulin Profile and CD4+ T-cell Determination The serum lgm, lgA and lgG were determined using immunoturbidimetric assay (Whicher et al., 1983) with Randox(R) kit, while CD4+ count was carried out using CY Flow SL Green Machine.

RESULTS The result of proximate analysis for the soyabean powder used in this study (Fig.1) indicated that its moisture content was 7.40%, protein 48.22%,

fibre 3.80%, fat 15.90%, ash 4.60% and carbohydrate content 20.08%. Also, the result of proximate analysis of normal feed (pelleted growers mash of vital(R) feed) used in this study (Fig. 2.) showed that the feedâ&#x20AC;&#x2122;s moisture content was 16.75%, fat 5.80%, ash 7.25%, fibre 2.60% and protein 17.95% and crbohydrate content was 49.65%. Furthermore, when the different groups of rats were challenged with Klebsiella pneumoniae, it was observed that only the group G with diabetic rats fed with normal feed came down with the infection. This was indicated by the sickly appearance of the rats in the group and mortality of half of the rats in the group G before the end of the study.

Moisture 6% Carbohydrate 21%

Ash 5%

Protein 48% Fat 16%

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Fibre 4%

Fig. 1: Soyabean proximate composition

Fig.3.1: Result of soyabean proximate analysis

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Moisture 17% Ash 7%

Carbohydrate 49%

Fat 6% Fibre 3%

Protein 18% Fig. 2: Normal feed proximate composition Fig.3.2:Result of normal feed proximate analysis

250 208.41

IgM conc.(mg/dl)

200

182.64 163.14

150 120.55

133.75

100

123.39 79.21 62.49

Series1

0 A

Groups of albino rat used

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Fig. 3: Mean serum IgM concentration for different groups of rats used

Figure 3.4.1:Results of mean serum IgM concentration for different rat groups used

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250 224.43

200 169.61 IgA conc.(mg/dl)

193.67

188.71

181.63 167.04

150 132.72 124.12 Series1 100

0 A

Groups of rat used

Fig. 4: Mean serum IgAofconcentration differentfor groups of rat rats usedused Fig.3.4.2:Results mean serum IgAfor concentration different groups

1200

IgG conc.(mg/dl)

1000

899.31 757.55

800 597.96

627.26

612.92

600

502.94 413.11

400

Series1

311.57

200 0 A

Groups of rats used

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5: Mean serum IgG concentration for different groups of rats used FigureFig. 3.4.3:Results of mean serum IgG concentration for different rat groups used

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400 350

320

331.25

CD4 (cells/microlitre)

300 250

226.5

211.75

219.75

200 161.75 150

125

Series1

99.75 100 50 0 A

Groups of albino rats used

Fig. 6: Mean serum CD4+ concentration for different groups of rats used

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Figure 3.5:Results of mean CD4 count for different groups of albino rats used

The result of the mean serum immunoglobulin M (IgM) for different groups of rats used in the study (Fig 3) indicated that the mean serum IgM of groups E, F and H were significantly higher (P<0.05) than that of the control (group A). The result (Fig. 3) also showed that there were significant reductions (P<0.05) in the serum lgM concentrations of diabetic rats fed on normal feed only, group C (79.21+ 20.73 mg/dl) and diabetic rats fed on normal feed but challenged with Klebsiella pneumoniae, group G (62.49+22.96mg/dl) when compared to control, group A (120.55 + 9.29mg/dl). Fig.3 indicated that the mean serum lgM of diabetic rats fed on soyabean diet (group D) was significantly higher (P<0.05) than that of diabetic rats fed on normal feed. Result (Fig.3) also showed that the mean serum lgM of diabetic rats fed on soyabean diet challenged with klebsiella pneumoniae (group H) was significantly higher (P< 0.05) than that of diabetic rats fed on normal feed but challenged with Klebsiella pneumoniae (group G). Fig.3 also

indicated that the mean serum IgM of normal rats fed on soyabean diet (group B) and normal rats fed on soyabean diet but challenged with Klebsiella pneumoniae (group F) were higher than their counterparts fed on normal feed (groups A and E respectively). The increases were statistically not significant (P >0.05). The result of the mean serum lgA concentrations for the different groups of rats used in this study (Fig. 4) revealed that the lgA levels of normal rats fed on normal rat feed and soyabean meal but challenged with klebsiella pneumoniae, group E (188.71 + 15.09mg/dl) and diabetic rats fed on normal rat feed and soyabean meal but challenged with Klebsiella pneumoniae, group H (193.67 + 19.43 mg/dl) were significantly higher (P<0.05) than that of the control, group A (169.61 + 17.53mg/dl). Fig.4 further indicated that the mean serum lgA concentration of diabetic rats fed on normal feed, group C (124.12+ 25.21 mg/dl) was significantly lower (P<0.05) than those of the

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control, group A (169.61 + 17.53mg/dl) and those of the diabetic rats fed on normal feed and soyabean meal, group D (167.04 + 18.61mg/dl). Fig.4 also indicated that the mean serum lgA of rats fed on soyabean but challenged with Klebsiella pneumoniae (group F) and diabetic rats fed on soyabean but challenged with Klebsiella pneumoniae ( group H) were significantly higher (P<0.05) than their counterparts fed on normal feed (group E and G respectively). The result of the mean serum lgG concentrations for the different groups of rats used in this study (Fig.5) showed that the mean serum lgG concentrations of normal rats fed on normal feed but challenged with Klebsiella pneumoniae, group E (757.55 + 63.22mg/dl) and normal rats but also challenged with Klebsiella pneumoniae group F (899.31 + 56.23mg/dl) were significantly higher (P< 0.05) than that of the control, group A (599.96 +45.06mg/dl). Fig.5 also revealed that the serum mean lgG concentration of diabetic rats fed on normal feed, group C (311.57+ 53.36mg/dl) was significantly reduced (P<0.05) when compared to the control, group A (597.96 + 45.06mg/dl) and to that of the diabetic rats fed on normal rat feed and soyabean meal, group D (502.94+ 41.54mg/dl). Figure 5 further indicated that the mean serum lgG of rats fed on soyabean diet challenged with Klebsiella pneumoniae (group F) and diabetic rats fed on soyabean diet challenged with Klebsiella pneumoniae (group H) were significantly higher (P< 0.05) than their counterparts fed on normal feed only (groups E and G respectively). The result also revealed that there was no significant difference (P< 0.05) between the mean serum lgG of rats fed on soyabean diet (group B) and that of rats fed on normal feed only (group A). The result of CD4+ cell count for the different groups of albino rats used in this study as shown in Fig.6 indicated that the mean CD4+ cell count of

normal rats fed on normal feed and soyabean meal, group B (331-25 + 22.50 cells/µl) and normal rats fed on soyabean diet but challenged with Klesibiella pneumoniae (group F) were significantly not different (P>0.05) from their counterparts fed on normal feed only (groups A and E respectively). However, with the exception of group B, the mean CD4+ count of all the other groups were siginificantly lower (P< 0.05) than that of the control, group A. Moreover, the results shown in Fig.6 indicated that there were significant reduction (P<0.05) in CD4+ count of all the groups challenged with Klebsiella pneumoniae when compared with their counterparts not challenged with the pathogen. The results also showed that the mean CD4+ counts of diabetic rats fed on normal rat feed and soyabean meal, group D (226.50+ 21.41 cells/µl) was significantly higher than that of the diabetic rats fed on only normal feed, group C (161.75+ 28.74 cell/µl).

DISCUSSION Over the years, most investigations on diabetes have concentrated on the effect of various diets or herbal extracts on glucose levels without adequate consideration of their effect on the immune response. However, the effect of soyabean diet on some immune markers (lgG, lgA, lgM and CD4+cells) of diabetic rats were evaluated in this study. The results of the immunoglobulin profile indicated significant increase (P< 0.050 in the lgM concentrations (Fig.3) of normal rats fed on normal rat feed but challenged with Klebsiella pneumoniae (group E), normal rats fed on normal feed and soyabean meal challenged with Klebsiella pneumoniae (group F) and diabetic rats fed on normal feed and soyabean meal but challenged with Klebsiella pneumoniae when compared to the control group (group A). This

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suggested that the presence of Klebsiella pneumoniae (antigen), the state of health of the rats and the nutritive value of the soyabean diet used in the study could have likely been the reason for the increased lgM antibodies. This observation corroborated Edelman (1973) observation that in active immunity, the effective contact with antigens causes the host to produce antibodies in response to the antigens. When the lgM concentration of diabetic rats fed on normal rat feed (group C) was compared to the control, a significant reduction (P<0.05) was observed in group C, suggesting that diabetes also causes depletion of the lgM level, thereby reducing immune competence in the rats. However, when the lgM concentration of diabetic rats fed on normal feed (group C) was compared with those of their counterparts fed on normal feed and soyabean meal (Group D), it was observed that the reduction in lgM concentration in group C was restored to normalcy with the incorporation of soyabean diet in group D, an observation that could be attributed to soyabeanâ&#x20AC;&#x2122;s high protein composition with its high content of essential amino acids. These amino acids could have helped in boosting the plasma proteins which could have led to increase in the IgM concentration, since immunoglobulins are synthesized by plasma cells. This observation is in accord with McGee and McMurray (1977) report that soyabean (protein) could help in boosting immunoglobulin concentration in the intestine of mice. Immunoglobulins are proteins. So the increase in IgM concentration observed in the diabetic rats fed on soyabean diet (group D) when compared to that of the diabetic rats fed on normal feed (group C) could be as a result of high essential amino acids present in the soyabean used. Also, the results of IgA and IgG concentrations for normal rats fed on normal feed challenged with Klebsiella pneumoniae (group E), normal rats fed

on normal feed and soyabean meal challenged with Klebsiella pneumoniae (group F) and diabetic rats fed on normal feed and soyabean meal but challenged with Klebsiella pneumoniae (group H) indicated significant increases (P<0.05) in IgA and IgG concentrations when compared with control group A (Fig. 4 and 5). This also suggested that the production of more antibodies (IgA and IgG) in both diabetic and non-diabetic rats (except in group G) as soon as they were challenged with Klebsiella pneumoniae in order to protect the rats from invading antigen could have been affected by the presence of Klebsiella pneumoniae, the state of health of the rats (i.e. whether diabetic or non-diabetic) and the nutritive value of the diet (soyabean) used. This observation is also in consonance with the report that the presence of antigen could trigger off the production of antibodies as protective measure of the host against invading antigen (Edelman, 1973). It was also observed that more IgM, IgA, and IgG antibodies were synthesized in the groups in which soyabean meal was incorporated into their diet when compared with their counterparts fed on only normal feed. This suggests that soyabean diets could have helped in boosting their immunoglobulin levels, due to the presence of essential amino acids in the soyabean. These essential amino acids could have helped in boosting the immunoglobulin level. This observation also corroborates McMurray (1977) which stated that protein (essential amino acids) helps in boosting immunoglobulin concentration in the intestine of mice. Furthermore, the IgM, IgA and IgG concentrations of diabetic rats fed on normal feed (group C) were significantly reduced (P<0.05) when compared to the control, an indication that diabetes causes depletion of immunoglobulin levels. This could be due to the diabeticsâ&#x20AC;&#x2122; inability to utilize blood glucose for energy generation. Muscle proteins are therefore

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metabolized for energy generation, leading to loss of muscle amino acids which could result to the shrinking of the muscles. The depletion of the muscleâ&#x20AC;&#x2122;s amino acids could have caused the decrease in the immunoglobulin levels, since immunoglobulins are synthesized from amino acids. However, it was observed that the reduced immunoglobulin concentrations in group C was restored almost to normal level in diabetic rats fed on Soyabean diet (group D). The suspicion is that the essential amino acids in soyabean could have been replenished the lost amino acids for energy generation. The replenished essential amino acids from Soyabean Diet could have assisted in boosting the immunoglobulin concentration in the rats fed with Soyabean supplemented diet, since immunoglobulins are proteins. Moreover, Fig.6 indicated that with the exception of normal rats fed on normal rat feed and soyabean meal (group B), that the mean CD4+ count of all other groups (groups C, D, E, F, G and H) were significantly lower (P< 0.05) than the control (group A). This suggests that diabetes and the presence of pathogen (Klebsiella pneumoniae) could have caused reduction in CD4+ cells. The utilization of muscle protein for energy generation could have brought about the depletion of essential amino acids in the diabetic rats. This in turn could have caused the decreased CD4+ cell count in diabetic rats as observed, since CD4+ cells are produced from essential amino acids. Also results in Fig.6 showed that both diabetic and non-diabetic rats challenged with Klebsiella pneumoniea but fed on soyabean incorporated diet had higher CD4+ count than their counterparts fed on only normal rat feed. This is because soyabean is rich in essential amino acids. These essential amino acids could have helped in replenishing the lost muscles and tissues amino acids during energy generation in diabetic rats fed in soyabean diet. The replenished essential amino

acids in diabetic rats fed on Soyabean supplemented diet could have boosted their CD4+ cell count when compared to that of the diabetic rats fed on normal feed. This should also be expected considering the high nutritive value of Soaybean (Fig.1) with high percentage of protein, minerals and vitamins including antioxidants (Regel et al., 2000; Anosike et al., 2007; and Villegas and Gao, 2008). The above observation also corroborates the report that decreased dietary intake of antioxidants could lead to a concomitant decrease in CD4+ T-cells (Oguntibeju et al., 2005 and 2006). Since the CD4+ T-cells, perform a central and coordinating role in the immune response (Hughes et al., 1997; Vajpayee et al., 2009), and Soyabean diet helped in raising the CD4+ cells in diabetic rats as observed in this study, Soyabean diet therefore could help in the general boosting of the immune system of diabetics.

CONCLUSION This study revealed significant increases (P<0.05) in serum IgA, IgM and IgG levels and CD4+ T-cells concentration of diabetic rats fed with normal feed supplemented with soyabean meal (group D) and the rats infected with K. pnemoniae and fed with normal feed and supplemented with soyabean meal (group H) when compared with their counterparts fed with only normal feed (groups C and F). Moreover, it was observed that when various groups of rats were challenged intraperitoneally with 0.4ml of Klebsiella pneumonia suspension containing 108 c.f.u/ml, establishment of infection and high mortality rate of the infected rats were observed in diabetic rats fed with normal feed only (group G), whereas no mortality was observed in diabetic rats fed with normal rats feed supplemented with soyabean meal (group H). These results suggest that soyabean diet could be useful in the general

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SUGGESTIONS FOR FURTHER STUDIES It is suggested that further studies be carried out as follows:  Inducing diabetes that could last for more than 3 months in order to appreciate the effect of soya protein more, in chronic complications of diabetes.  Feeding the animals with soyabean diet for more than 3 months (i.e. longer period), since the longer the animal is exposed to the diet, the more the effect of the diet could be discovered.  Using more than two different pathogenic organisms that are infectious to albino rats in subsequent study, to confirm these results.

We are grateful to the following for assistance rendered towards the success of this research work: Prof. L.S.U. Ezeanyika and Prof. I.N.E. Onwurah, both from Department of Biochemistry, University of Nigeria, Nsukka. Also to Prof. J. I. Ihedioha, Prof. S.V.O Shoyinka and Prof. K. F. Chah, all from the Faculty of Veterinary Medicine, University of Nigeria, Nsukka. We will also not forget the Head of Hematological Section of Nnewi Teaching Hospital, Mr. A. Ozoabia, who assisted us in carrying out CD4+ count with their machine.

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ACKNOWLEDGEMENT

Anosike, C.A., Ezeanyika, L.U.S. and Obidoa, O. (2007). Effect of roasted soyabean (glycine max) diet on the histology of selected rat tissues. Bio-Research, 5 (2): 237-240. AOAC (2000). Official method of analysis of Association of Official Analytical Chemists. Washington D.C., 16th Edition. Pp. 12-18. Aref, G.H., el-Din, M.k. and Hassan, A.J., (1970). Immunoglobulins in Kwashiorkor. Journal of Tropical Medicine and Hygiene, 73:186-191.

Bemiller, N.J. (2003) Carbohydrate Analysis. Food Analysis. Third edition. Kluwer Academic publisher, New York Pp. 143-146. Chandra, R.K., Gupta, S. and Singh, H. (1982). Inducer and suppressor T-cell subsets in protein-energy malnuitrition: analysis by monoclonal antibodies. Nutrition Research, 2:21-26. Changsam, K.C. (2003). Protein Analysis. Food Analysis. Third edition kluwer Academic/Plenum publisher, New York. Pp. 134-135. Hughes, M. D., Johnson, V. A. and Hirsch, M. S. (1997). Monitoring plasma HIV–I RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. Annals of Internal Medicine, 126:929-938. James, O.K. (1995). Gravimetric method of analysis employed in twenty different cereals and legumes, Wayton, U.K. Oguntibeju, O. O., Van den Heever, W. M. J. and Van Schakwyk, F. E. (2005). An analysis of based line dietary intake of HIV-positive/AIDS patients. South African Journal of Medical Technology, 19 (2): 3-9. Oguntibeju, O. O., Van den Heever, W. M. J. and Van Schakwyk, F. E. (2006). The effect of a liquid nutritional supplement on viral load and haematological parameters of HIV-positive/AIDS patients. British Journal of Biomedical Science, 63 (3): 134-139. Pretorius, P.J. and Villiers, L.S. (1962) Antibody response in children with Protein Malnutrition. American Journal of Clinical Nutrition, 10: 379-383. Purtilo, D.T. and Connor, D.H. (1975). Fatal infection in protein-calorie malnourished children with thymolymphatic atrophy. Archieves of Disease in Childood 50: 149-152 Reddy, V. and Srikantia, S.G. (1964). Antibody response in Kwashiorkor. Indian Journal of Medical Research, 52: 1154-1158. Reddy, V., Bhaskaram, C. and Raghuramulu, N . (1977). Immunological responses in malnourished children. Indian Journal of Padiatrics, 14: 255- 258 Regel, F. J., Fraser, G. D., Weeks, E. C. and Greenberg, A. N. (2000). Dietary phytoestrogen have antiinflammatory activity in guinea pig. Proceedings of the Society for Experiment Biology and Medicine. 223 (4): 372-378 Rosenbloom, A. and Silverstain, J.H., (2003) Type 2 Diabetes in children and Adolescents: A Clinicians Guide to Diagnosis, Epidemiology, Pathogenesis, Prevention and Treatment. American Diabetes Association, U.S.I., Rother, K.I. (2007). “Diabetes Treatment Bridging the Divide”. The New England Journal of Medicine, 356 (150: 1499-1501.

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Scrimshaw, N.S. and san Giovanni, J.P. (1997). Synergism of Nutrition, Infection and Immunity. American Journal of clinical Nutrition, 66:464s-477s. Sky, G.M. (2000). Kinetics of insulin secretion underlying notabolism events in diabetes mellitus. Diabetes mellitus: A fundamental and clinical text. Lippicott willians and Wilkins, Plucadelphia Pp. 2-11. Smythe, P.M., Brereton-stiles, G.G. and Grace, H.J. (1971). Thymolymphatic deficiency and depression of cell mediated immunity in protein-calorie malnutrition. Lancet, 2: 939-943. Suskind, R., Edelman, R., KulapongsP., Pariyanonda, A. and Sirisinha, S. 91976). Complement activity in Children with Protein Calorie Malnutrition. American Journal of Clinical Nutrition, 29: 1089-1092. Tierney, I.M., Mcphee, S.J. and Papadakis, M.A. (2002). Current Medical Diagnosis and Treatment of Diabetes. Large Medical books. McGraw-Hill, New York.Pp. 12031215. Unadike, B.c. (2010). Awareness and knowledge about diabetes mellitus among nursing students in the Niger Delta region of Nigeria. Middle East Journal of Nursing, 4(1): 1-8.

o o o o

o o

Vajpayee, M., Kaushik, S., Sreenivas, V., Mojumdar, K., Mendiratta, N. and Chauhan, K. (2009). Role of immune activation in CD4+ T-Cell depletion in HIV â&#x20AC;&#x201C;I infected Indian patients. European Journal of Clinical Microbiology and Infectious Diseases, 28: 69-73. Villegas, R. and Gao, Y. T. (2008). Legume and soy food intake and incidence of type 2 diabetes in the Shanghai womens health study. American Journal of Clinical Nutrition. 87 (1): 162-167. Watson, B.B, Reyes, M.A. and Mc Murray, D.N., (1978) Influence of malnutrition on the concentration of lgA, lysozymes, amylase and aminopeptidase in childrenâ&#x20AC;&#x2122;s tears. Proceedings of the Society for Experiment Biology and Medicine, 157: 215-219. Watts, T. (1969). Thymus weights in malnourished children. Journal of Tropical Pediatrics, 15:155-158. Whicher, J.J., Price, C.P. and Spencer, K. (1983). Immunoturbidimetric assay. Critical Reviews in Clinical Laboratory Science, 18: 213-216. WHO (World Health Organization) (1999). Department of Non Communicable Disease Surveillance (1999). Definition, Diagnosis and Classification of Diabetes Mellitus and its complications.

*Corresponding Author:

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Andrew C. Nwaka, Ph.D, Department of Biochemistry, Anambra State University, Uli, Anambra State, Nigeria. E-mail: andynwaka@yahoo.com; Phone: +234 (0) 8037834171.

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ReviewArticle PharmaceuticalSciences HERBAL MEDICINES USEFUL FOR THE TREATMENT OF DIABETES IN NORTH-EAST INDIA: A REVIEW Prodyut Mondal*, Niroj Bhuyan, Sonjit Das, Mritunjay Kumar, Sudarshana Borah, Kabita Mahato. Department of Pharmaceutical Sciences, Dibrugarh University. Assam 786004.India. *Corresponding AuthorEmail:mondalprodyut0@gmail.com

ABSTRACT Aim: Diabetes mellitus (DM) is a group of metabolic disorder characterized by hyperglycemia, which is associated with abnormalities in carbohydrate, fat and protein metabolism result in chronic complications. The main objective of the study to presenting the medicinal plants used in North-East India for anti-diabetic purposes. This study emphasizes potential sources for the development of new anti-diabetic drugs from indigenous medicinal plants found in North-East India. Methods: The North-east India is the indigenous for its biodiversity aspect. The herbal drugs which have the anti-diabetic properties are documented here on the basic of the information of articles published on the popular journal in different times and based on the information of extensive field survey. Results: There are 141nos. of medicinal plant have documented. Traditionally most of the plants show promising anti-diabetic activity. Whole plant and parts of the plant such as bark, leaf, fruit etc. are being used by the ethnic community to cure diabetes. In compares to the other parts of the plant the leaves are the mostly used by the traditional people as anti-diabetic purposes of the different plant. Conclusion: The traditionally used medicinal plants have possessing potent anti-diabetic properties that has not still explored scientifically. The article summarize the significance of medicinal plants that are used in north-east India as anti-diabetic purposes and the requisite level of activity as well as toxicity would be considered for further scrutiny to develop the potential drug molecules.

KEY WORDS Medicinal Plants, anti-diabetic drugs, diabetes mellitus, North East India.

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INTRODUCTION The prevalence of diabetes is rising all over the world due to population growth, aging, urbanization and an increase of obesity and physical inactivity. Unlike in the West, where older persons are most affected, diabetes in Asian countries is disproportionately high in young to middle-aged adults. This could have long-lasting adverse effects on a nationâ&#x20AC;&#x2122;s health and economy, especially for developing countries. The International Diabetes Federation (IDF) estimates the total number of people in

India with diabetes to be around 87.0 million by 2030 [1]. Diabetes mellitus is a common and very prevalent disease affecting the citizens of both developed and developing countries. It is estimated that 25% of the world population is affected by this disease [2]. Diabetes is the condition where the fasting and post prandial (after food) blood glucose level become high (normal 120mg/dl after 2 hours of meal, adult male) due to mainly improper metabolism of carbohydrates, fats and proteins. Diabetes mellitus is a group of syndromes characterized

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www.ijpbs.com (or) www.ijpbsonline.com by hyperglycemia altered metabolism of lipids, carbohydrates, and proteins and an increased risk of complicates form vascular disease. Most patients can be classified clinically as having either type 1-diabetes mellitus (type IDM, formerly known as insulin–dependent diabetes or IDDM) or type 2- diabetes mellitus (type 2DM, formerly known as non –insulin-dependent diabetes or NIDDM) [3]. Generally the four types of cells (α, β, γ and Δ) are in the islets of langerhans gland of pancreas in a human body. Within these the alpha (α) and the beta (β) cells take important role to secrete insulin and glucagon hormone respectively for the balancing of blood glucose level. In case of Type I diabetes mellitus (IDDM) there is the destruction of pancreatic beta cells majority of cases due to autoimmuno antibodies. This type of diabetes mellitus is less common to the population. The International Diabetes Federation and the World Health Organization estimate that, worldwide over 100 million people suffer from type II diabetes and 50% of those cases are undiagnosed. Over 90% cases of diabetes mellitus is in Type II (NIDDM) where generally no loss or moderately loss of β-cell mass occur; insulin in circulation is low and no β cell antibody is demonstrable [4]. Plants have been the basis of many traditional medicine systems throughout the world for thousands of years and continue to serve mankind with new remedies. At present, there is a worldwide movement or assessing the plant resources which are of medicinal and economical value and importance. Researchers are focusing mainly on ethnobotanical & ethnomedicinal investigations to fulfill the increasing demand of herbal products. In the last few decades there has been an exponential growth in the field of herbal medicine and these drugs are gaining popularity both in developing and developed countries because of their natural origin and less

side effects [5]. WHO estimate that, about 80% of the population in the developing countries depends directly on plants for its medicine [6, 7]. WHO listed 20,000 medicinal plants globally, [8] within these India’s contribution is 15-20% [9]. In India 2,000 medicinal preparations used are of plant origin. Indian has an ancient heritage of traditional medicine. Indian Materia Medica includes about 2000 drugs of natural origin almost all of which are derived from different traditional system and folklore practices. Out of these drugs derived from traditional system, 400 are of mineral and animal origin while the rest are of vegetable origin. The plant based indigenous knowledge was passed down from generation to generation in various parts of the world throughout its history and has significantly contributed to the development of different traditional systems of medicine [10]. India has a rich heritage of traditional medicine and the traditional health care system have been flourishing for many centuries. It mainly consists of three major systems namely the Ayurveda, the Siddha and the Unani. Lot of efforts has been taken by the government and private sectors for the development of the traditional system based on these three methods. The North-East region of India (220-290 N; 890970 E) comprises the Sikkim and the seven sister states namely Assam, Arunachal Pradesh, Nagaland, Meghalaya, Mizoram, Manipur and Tripura. This region of India has several hill ranges interspersed with valleys and is by large sparsely populated. Nearly 40% of the total geographical area of this region is covered by evergreen forest. Each state of the North–East inhabited by number of ethnic tribes characterized by their native languages, rituals, costumes and housing pattern. There are more than 180 major tribal communities of the total 427 tribal communities found in India [11, 12].

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www.ijpbs.com (or) www.ijpbsonline.com The north-east region is also rich in medicinal plant wealth. Many medicinal plant species having industrial potential are growing wild in this region [13]. A large no of people belonging to various groups of the north-eastern region of India still practicing their own traditional heath care systems. The traditional treatment systems adopted by these ethnic communities are being used generation wise without any scientific validation. They have the vast knowledge about various plants that are used for food and as medicine. Since the discovery of insulin several synthetic oral hypoglycemic drugs (OHDs) are available in market. But these synthetic drugs are not sufficient to treat this deadly disease, particularly type II. Most of these synthetic drugs have some serious side effects. In this respect our traditional herbal therapeutic system can act as an alternative to synthetic drugs. Generally these phytochemicals have fewer side effects. Many of them are helpful in preventing the secondary complications of diabetes [14]. The herbal

medicines are generally cost effective and readily accessible in compares to synthetic drugs. The adverse side effects and higher cost of the existing anti-diabetic drugs necessitate the search for novel, well tolerated, efficient and easily available herbal remedies to tackle the deadly diabetes.

MATERIALS AND METHODOS Medicinal plants provide a promising bargain since they are easily accessible and affordable. The North-eastern communities of India use traditional herbal medicines for controlling the disease some of which are reported to be highly potent and promising. Herbal preparation of different plant parts such as leaf, root, fruit, bark and also the whole plant are used by numerous communities [15,16].The data were collected based on the available literature published in the form of journal [14, 17, 18, 19, 20], popular articles and active field survey. Some medicinal plants that are used as anti-diabetic purposes are listed in Table No 1.

Table Noâ&#x20AC;&#x201C; 1. Medicinal Plants used as anti-diabetic purposes in North-East India. Sl. No

Botanical Name & Family

Local Name

Part Used

Preparation to used

Acacia Concina DC (Mimosaceae)

Khangthur

Leaves

Aconiotum hetrophylloides Ranuculaceae Adhatoda vasica Nees Acanthaceae Aechmea fasciata Bromeliaceae

Bish, Seli, Chandu

Dried tuberous roots

Bogabahok

Roots

Aechmea

Whole plant

Aegle marmelos correa] Rutaceae Ajuga bracteosa

Bel

Leaves

Neel-kantha

Leaves

The leaves infusion is taken orally. Decoction of root is used Infusion of roots is used Crushed and mixed with water Leaves boiled with water Leaves boiled

3. 4.

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5. 6.

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Koroi Siris Mankachu

Roots, leaves, flowers

Ghrita kumari

Leaves

Satiana

Leaves, Bark

Anana scomosus (L) merr Bromeliaceae Andrographis paniculata Nees Acanthaceae Annonareticulata Linn Annonaceae

Matikathal

Fruits, Whole plant

Kalmegh

Whole plant

Aatlas

Barks

14.

Annona squamosa Linn Annonaceae

Manga

Leaves

15.

Annona reticulata L. Annonaceae Antidesmaacidum Retz Euphorbiaceae Antocarpus heterophyllus Linn Moraceae Areca catecheu Linn Arecaceae

Atlas

Leaves, Fruits

Nikhutenga

Leaves

Kothal

Leaves

Tamul

Nut

Argyreia speciosa Linn. F. Convulaceae Artocarpus lokoocha Roxb Moraceae Artemisia maritma Asteraceae Averrhoacarambola Linn Oxalidaceae Beta vulgaris L. Chenopodiaceae

Takoria alu

Stem, Leaves

Diwatenga

Barks

Chinglaibaknag

Leaves

Kardoi

Barks

Beet

Roots

8. 9. 10.

11. 12.

13.

16. 17.

18.

19. 20. 21.

578

22.

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Lamiaceae Albizzia procera Benth Mimosaceae Alocacia indica schott Araceae Aloevera tournex.Linn Liliaceae Alstonia scolaris R.Br. Apocynaceae

23.

Rhizomes

with water Leaves juice is taken orally Dried rhizome is used Leaves paste is used Leaves paste and bark extract is taken. Fruit juice is used. Crushed and boiled with water Dried barks decoction is used Leaves boiled with water Leaves, Fruits juice is used Leaves juice is used orally Leaf juice is used orally Dried and soaked with water. Stem, Leaves paste is used. Barks infusion is used Boiled leaves extract Bark infusion is used. Root juice taken orally

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Roots

Simolu

Roots

Bouganvillea Willd. Nyctaginaceae

27.

Brassica juncea(L)Czern. Brassicaceae

Sorioh

Leaves, Seeds

28.

Caesalpinia crista Linn. Caesalpiniaceae Caesalpinia pulcharrima Caesalpiniaceae Cajanus cajan Mill Papilionaceae

Lataguti

Seeds

Guletura

Flowers

Rahar

Leaves

Calotropis gigantea (L) W. Akon Aiton. Asclepiadaceae Canna indica Linn Parijat Cannaceae Cannabis sativa Linn Bhang Cannabaceae

Leaves

34.

Carrica papaya Linn Carricaceae

Amita

Seeds

35.

Casia alata Linn. Caesalpiniaceae Cassia angastifolia Linn Caesalpiniaceae Cassia fistula L. Caesalpinaceae Cassia occidentalis Linn. Caesalpinaceae Cassia sophera Linn Caesalpinaceae Catharanthus roseus (L.) G. Don.

Khorpat

Tender leaf

Channa

Leaves

Sunaru

Barks

Bonoriadadol

Seeds, stem

Bonmadelua

Seeds, stem, bark

Nayantara

Whole plant

30.

31.

32. 33.

36. 37. 38. 39.

579

Yomri, Nukmam

26.

29.

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Boenninghausenia albiflora Rutaceae Bombox malabaricum DC Bombacaceae

40.

spectabilis Kagaz phul

Root juice is taken orally Mixed with water or root juice is used. Leaves paste is anti-diabetic but not used by human. Seed powder with milk taken orally Crushed powder is used. Flower juice is used Powder leaves mixed with water. Leaves, Flower paste is used

Leaves

Leaves, Stem Leaves, stem, flowers

Dried leaves are used. Leaves and stem soaked with water Seed powder mixed with water Leaves mixed with water Infusion of leaves is used Bark powder is used Seeds powder is used Seeds and bark powder is used Boiled extract of leaves,

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Apocynaceae 41.

Centella asiatica(L.)Urban. Apiaceae

Manimuni

Whole plant

42.

Ciceracida Linn Euphorbiaceae Cichorium intybus Asteraceae Cinnamomum tamala fr. Nus Lauraceae Citrus aurantifolia Linn Rutaceae Clerodendrum infortunatum L. Lamiaceae Coccinia indica cogn Cucurbitaceae Cocos nucifera L. Arecaceae Coix lacrymajobi Poaceae Colocasiaesculenta(L) Scholl Araceae Coptis teeta Ranuculaceae

Holfoli

Leaves

Kasni

Seeds

Tezput

Bark. roots

Nemu

Fruits

Vetetita

Leaves

Balipoka

Fruits, root

Narikol

Oil

43. 44. 45. 46.

47. 48. 49. 50. 51.

52. 53.

54.

Page

580

55. 56. 57.

Chaning angouba Roots (Manipur) Kolakachu Roots Rings, Mishimetectga

Dried root rhizomes.

Coriandrumsativum Linn Dhania Apiaceae Costus speciosus Jamlakhati (Koeing).Smith Zingiberaceae Curcuma domestica val Halodhi Zingiberaceae

Leaves

Curcuma aromatic Salisb Zingiberaceae Cucumis melo Roxb Cucurbitaceae CynodondactylonPers

Bonoria Halodhi

Rhizomes

Sal kumura

Seeds

Dubori

Whole plant

Rhizomes

Leaves, rhizomes

flowers taken orally Whole plant juice is taken in empty stomach Infusion of leaves is used Seeds powder is used Bark powder made infusion Fruit juice is used Leaf paste is taken orally. Fruits juice is used. Oil is taken out and consumed. Crushed extract of roots is used. Roots boiled with water and Roots and rhizomes juice is orally taken in empty stomach. Leaves infusion is used Rhizome paste taken oral Powdered rhizomes are used. Powder rhizome is used Seed powder is used. Crushed and

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Poaceae 58.

Dalbergia sisco Roxb. Papilionaceae Daucas carota Linn Apiaceae Debregeasia longiafoliaWedd. Urticaceae Dilleniaindica Linn. Dilleniaceae Dioscorea alata Linn. Diosconeaceae

Sishu

Stem barks

Gajor

Roots

U-khajing Manipur

Tubers

Aautenga

Fleshy perianth

Juice is used.

Kath alu

Rhizome

63.

Eichhornia cussipes F- NF

Kachuripana

Whole plant.

64.

Enhydra fluctuanas Lour Asteraceae

Komperek Manipur

Nodes of the plant

65.

Equisetum debile Roxb. Equisteraceae

Lai-utang Manipur

Whole plant

66.

Erythairina indica Linn Papilionaceae Eugenia jambolana Linn Myrtaceae

Moder Gadela (Assam) Kala Jamu

Roots

Boiled with water and taken. Crushed powder infusion is used. Boiling the nodes of plant with water Boiled with root of male Carica papaya. Root powder is used Fruit juice is used orally.

Euphorbia hirta Linn. Euphorbiaceae Ficus benghalensis Linn. Moraceae

Gakhirotibon

Whole plant

Bot

Stem, barks

Ficus religiosa L Moraceae Flacourita jangomas Lour Flacourtiaceae Garcinia padunculata Roxb. Clusiaceae

Dhup

Leaves, Fruits

Heitroi

Fruits

Borthekera

Fruits

Gloriosa superba Linn.

Gloriosa

Whole plant

59. 60.

61. 62.

67.

68. 69.

70. 71.

581

72.

Page

boiled with water Bark powder is used. Paste or juice is used. Thirst of diabetic patient

73.

Fruits, Barks, Seeds.

Crushed and made infusion Bark infusion or decoction is used Leaf, fruit taken orally Raw fruits are used. Fruit juice or raw fruit is used. Made infusion

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Seeds

Gomari

Leaves, fruits

Petuk

Fruits

Hatisur

Aerial part

Holoptelia integrifolia Planch Ulmaceae Holorrhena antidysenterica wall Apocyanaceae Hydrocotyle rolundifolia Roxb Apiaceae Ichnocarpus frutescent R. Bn. Apocyanaceae

Holoptelia

Barks

Kutaz

Barks, fruits

Soru manimuni

Whole plant

Crushed to make paste.

Syamalota

Roots

Imperata cylindrical Beauv Poaceae Ipomoea aquatica Forssk. Convulaceae

Ulukher

Roots

Kalmou

Leaves

84.

Ipomoea batata (L) Lam. Convulaceae

Mitha alu

Leaves

85.

Jatropha curcus Linn. Euphorbiaceae Jussieua repens Onagraceae

Bongaliara

Leaves, twigs

Ishing-kundo

Leaves, whole plant

87.

Justicia adhatoda Acanthaceae

Nongmangkhaangouba

Leaves

88.

Kyllinga triceps Rottb

Chumthang

Whole plants

Roots decoction is used Root infusion is used. Dried leaf powder mixed with Piper nigrum and taken orally. Leaf boiled and juice is taken orally. Leaves juice is used Boil extract of the plant is used. Boil the leaves with the leaf of Clerodendrun spihonanthus. Boiled extract

76. 77.

78. 79.

80.

81.

82. 83.

86.

582

and used. Seed soaked with water. Leaves juice is used. Fruit juice is used. Dried and infusion is filtered before used. Bark powder is used Fruit juice is used

Soyabeen

75.

Page

Liliaceae Glycine max Merr Papilionaceae Gmelinaarborea Roxb Verbenaceae Grewia abutelifolia Juss Tiliaceae Heliotropium indicum Linn. Heliotropiaceae

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namthibi Jetuka

90.

Leucaena glauca Mimosaceae

Cialag Manipur

Leaves

91.

Leucas aspera Spreng. Lamiaceae

Doron

Whole plant

92.

Lindernia bracheata Linn Kachidoria Scruphulariaceae Litseaglutinosa(lour) CB Haluca Robins Lauraceae Ludwigia octovalvis Jacq. Kabo Kaji

Whole plant

95.

Madhuka indica Gmel Sapotaceae

Mohua

Barks, Seeds

96.

Mangifera indica Linn Anacardiaceae

Aam

Fruits

97.

Meliaazadirachta Linn Meliaceae Mentha arvensis Lamiaceae Meyna spinosa Roxb. Rubiaceae Mimosa pudica Linn. Mimosaceae

Mohaneem

Leaves

Nungshi-hidak

Plant part

Lam-heibi

Fruits

Nilazibon

Whole plant

Mirabilis jalapa L. Nyctaginaceae Moringaoleifera Linn Moringaceae Mormordica charantia Linn Cucarbitaceae

Gopal godhuli

Roots

Sogina

Barks, flowers, leaves

Tita-Karela

Leaves, fruits

89.

93.

94.

98. 99. 100.

101.

583

102.

Page

103.

of the plant. Leaves juice mixed with cowâ&#x20AC;&#x2122;s milk taken once in a week. Leaves decoction is used. Stomach along with Monopterus cuchia fish. Juice used.

Leaves

Leaves juice is used.

Whole plant

Boiled extract is used. Bark and seed crushed and infusion is used. Fruit juice or raw fruit is used. Leaves juice is used. Mixed with honey. Boiled extract of fruits Whole plant boiled with water. Root juice is taken. Barks infusion is used. Leaves and fruit juice is used

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Morus indica Linn Moraceae Mucunapruriens DC. Papillionaceae

Nuni

Tender leaves

Bandarkakura

Roots

Murraya koeningii Sprang Rutaceae Musa paradisiacal Linn Musaceae Nelumbo nucifera Gaertn Nelambaceae

Narasingha

Leaves

Kashkol

Flowers, fruist

Podumful

Tender leaves

109.

Nictanthus arbor tristis Linn Oleaceae

Sewali

Leaves, flowers

110.

Nigella sativa Ranuculaceae Ocium sanctum L. Lamiaceae

Keman

Seeds

Tulsi

Leaves

Osbeckiane palensis Hook Melastomaceae Oxalis corniculata L. Oxalidaceae

Photkola (Boga) Tengesi

Tender shoot

Parkia timoriana (A.DC) Mimosaceae

Yongchak

Barks

105.

106. 107. 108.

111.

112. 113.

Page

584

114.

Aerial part

orally. Eat with any roasted fish Roots powder soak with water Leaves juice is used Fruit juice is used. Crushed and pinch of powder taken with water. Young Leaf juice, flower paste is taken orally. Seeds powder is used. Leaf powder taken with honey to treat Diabetes. Crushed powder is used 100 gm aerial part are wrapped with banana leaf and after roasted in firewood is given with salt once daily 3 days interval. Decoction of bark with Centella asiatica and Ficus glomerata

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www.ijpbs.com (or) www.ijpbsonline.com 115.

Leaves plants Barks

117.

Phyllanthuse emblica Linn Euphorbiaceae

Amlakhi

Fruits

118.

Phyllanthus niruri Linn Euphorbiaceae Phyllanthuse urinaria Linn

Bonamlokhi

Whole plant

Heikruman

Leaves or whole plant

120.

Plumeria acuminata Apocyanaceae

Sun-champa

Barks

121.

Portulaca oleraceae Portulaceae Pouzolzia zeylanica (L) Benn Urticaceae PremnalatifoliaRoxb Verbenaceae Psidium guyava Linn Myrtaceae Swertia chirata L Gentianaceae

Kulfa

Whole plant

Borali bakua

Whole plant

Agnimantha

Leaves, Barks

Madhuri

Fruits, leaves

Chirata

Whole plant

Saraca indica Linn Caesalpiniaceae Scleria terristris (Linn) Cyperaceae

Ashok

Fruits

Thangjou

Plant part

Sesamum orientale Pedaliaceae Sesbaniaseban (Linn) Merr. Fabaceae Smilax lanceifolia Roxb. Smilaceae Solanum indicum Linn. Solanaceae

Senum

seeds

Chuchurangkei

Leaf or tender shoot

Kwa manbi

Roots

Bhakuritita

Fruits

119.

122. 123. 124. 125.

126. 127.

128. 129.

585

130. 131.

fruits is used. whole Extract of the plant is used Decoction of bars with decoction of Zingiber officinale Fruit juice or raw frit is taken orally.

Peristrophe fera C.B. Clarke Ishing langthrei Acanthaceae Phologocanthus tubiflorus Sang-chi Nees. Acanthaceae

116.

Page

Boiled extract is used orally. Powder bark decoction is used. Crushed powder is used. Decoction is used Leaves juice is used. Leaves infusion is used. Whole plant extract is consumed. Fruit juice is used. Boiled extract of the plant part is used. Seeds. Boiled extract of leaves. Boiled extract of root. Fruit juice is used.

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www.ijpbs.com (or) www.ijpbsonline.com 132. 133. 134. 135.

136. 137.

138.

139.

Spondias mangifera wild Anacardiaceae Sterculia villosa Roxb Starculiaceae Syzigium cumini (Linn.) Myrtaceae Tabernaemontanadivericota (L) R. Br. Apocynaceae Terminalia chebuta Roxb. Combretaceae Thevetia peruviana (pers) Merill Apocyanaceae Inospora cordifolia Miers Menispermaceae

Amara

Fruist

Udal

Roots

Jamhei

Seeds

Kothal pool

Leaves and flowers

Selekha

Fruits

Halodhia-korobi Utonglei

Barks

Sidhilota

Leaves, bark

Trigonella foenumgraceum Mithi Linn Papilionaceae

586

Seeds

140.

Vinca rosea Linn Apocyanaceae

Nayantora

Leaves

141.

Zanthoxylum armatum DC. Rutaceae

Muthrubi

Leaves and roost

RESULT AND DISCUSSION

Page

Herbs have been used for healing purposes and to promote wellness since from the ancient times and are not categorized as medicines but treated as food since they are natural products. Nowadays, herbal medicines, health and dietary supplements are flooding the markets. The use in the right way provides effective and safe treatment for many ailments and the effectiveness is mostly subjective to the Patient [21]. One of the major advantages of the herbal

Raw fruit is used. Root infusion is used. Boiled extract of seeds. Fresh flower soaked with water. Fruit juice is used. Powder bark is used. Powder bark infusion is used. Seeds soaked with water and taken in the morning. Leaves chewed at morning or juice is used. Roots and leaves decoction is used.

drug is that it is cost effective and easily affordable. In compares to the synthetic drugs they became less toxic or least side effects. From the ancient times the people of the northeastern part of India have been using the herbal medicines for curing the diabetes. Most of the plant has possessing prominent activity in this literature there are 141 nos. of plants have mentioned from the North-east India that are used for anti-diabetic purposes. The figure 1 shows the percentage use of plant part of the

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Rhizomes and tubers 13%, Bark: 13%, and Flowers: 4%.

Figure 1: It shows the percentage of plant parts is used for anti-diabetic purposes. PERCENTAGE OF PLANT PART

35 30 25 20 15 10

PERCENTAGE OF PLANT PART

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587

The ethnomedicinal information from the above table and figure (Tab-1 & Fig-1) shows that the peoples of this region are too much habituated for the use of herbal drugs in diabetes. The ethnomedicinal knowledge emphasizes the significance of the crude drugs. The northeastern communities have the ethnic knowledge to use the anti-diabetic plant. They use these plants generaously without any scientific modification. So it is utmost necessary to screen these traditional medicines scientifically for proper investigation into their use. It is necessary to keep in mind that most of the medicinal plants have number of allied species that are morphologically similar and are difficult to distinguish. Plant parts used in preparations are usually collected by common people without any attention to botanical authentication. So, there is the possibility of incorporation of wrong plant or parts of the plant with less activity or without any activity. Therefore, it is very essential to evaluate the botanical identity and quality of the

medicinal plants used in the traditional preparation. Pharmacognostic studies are usually adopted for standarization and quality evaluation of botanical drugs.

CONCLUSION The North-East India is stands for it is glory of eminent bio-diversity aspect and the ethnic communities of this region provide the footstep for the traditional knowledge. By the advancement of the concept of living peoples of the ethnic communities practicing less dependency to their traditional knowledge. So the knowledge which is generously rolled, facing a critical barrier for advancement, gradually. So it will be the aim to documented of the ethnic knowledge and preserved thereof. Differentnatural products with anti-diabetes activity have been described in the literature. There is a need for a multidisciplinary approach to develop potentially effective drugs. Within the list many of the medicinal plants have possessing potent anti-diabetic properties that has not still

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ACKNOWLEDGEMENTS The authors gratefully thanks to Dr. KamaruzZaman (Asst. Professor) Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam-786004, India, for his kind advice to the present work.

REFERENCES

[9]Singh, H.B., Alternative source for some conventional drug plants of India, Ethnobotany and ethnomedicinal plants of Indian subcontinent (edsMaheshwari J.K.,), Science Publishers, Jodhpur, India, 2000; 63-78. [10] ZamanKamaruz, Pharmacognostical and phytochemical investigations on some anti-malarial herbal drugs of north-east India: Department Of Pharmaceutical Sciences, Dibrugarh University, Assam; 2010; 2-3. [11]Sajem A.L., Rout. J and Nath. M. Traditional tribal knowledge and status of some rare and endemic medicinal plant of North Cacher Hills district of Assam, North-east India. Ethnobotanical Leaflet, 2008; 12: 261275. [12] Kala C.P., Ethnomedicinal botany of the Apatani in the Eastern Himalayan Region of India. Journal of Ethnobiology&Ethnomedicine, 2005; 1:11. [13] Bhagabati AK, Kalita MC and Barua S. (eds). In: Biodiversity of Assam: Status stretagy and action plan for conservation, EBH Publishers, Guwahati (India) on behalf of Assam Science Society, 2006; 60-62. [14]Sarmah PC. Ethno Antidiabetic Plants Of Assam; International Journal of Applied Biology and Pharmaceutical Technology: Volume: 2: Issue-4: Oct Dec -2011. [15] Bora U., Sahu A., Saikia A.P., Ryakala V. K.,andGoswami P., Medicinal plants used by the people of north-east India for curing malaria. Phytotherapy Research, 2007; 21:800-804. [16] Kumar. Sudhir., The medicinal Plants of North-East India. Scientific Publishers (India), Jodhpur 2002; 22,59,63,82. [17]Barukial J.,Sarmah J.N., Ethnomedicinal Plant used by the people of Golaghat district, Assam India. Int. J. Med. arom.Plants. Vol. 1, No. 3: December 203-211. [18] Chakravarty S., Kalita CJ. An Investigation On Anti Diabetic Medicinal Plants Used By Villagers In Nalbari District, Assam, India. IJPSR (2012), Vol. 3, Issue 06. [19]Khan Md. H., Yadav PS., Anti-diabetic plants used in Thoubal district of Manipur, Northeast India. Indian Journal of traditional knowledge; Vol.9 (3), July 2010; 510-514. [20] Das T, Mishra SB., Saha D., Agarwal S., Ethnobotanical Survey of Medicinal Plants Used by Ethnic and Rural People in Eastern Sikkim Himalayan Region. African Journal of Basic & Applied Sciences 4 (1): 16-20, 2012. [21]G.B. Kavishankar, N. Lakshmidevi, S. Mahadeva Murthy, H.S. Prakash, S.R. Niranjana. Diabetes and medicinal plants-A review. Int J Pharm Biomed Sci 2011, 2(3), 6580

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[1] A Ramachandran, AK Das, SR Joshi, CS Yajnik, S Shah, KM Prasanna Kumar, Current Status of Diabetes in India and Need for Novel Therapeutic Agents: Special Issue On Human GLP1 Analougues Volume 58. [2]Maiti R, Jana D, Das UK, Ghosh D.Antidiabelic effect of aqueous extract of seed of Tamarindusindicain streptozotocin induced diabetic rats. J Ethnopharmacol 2004, 92, 85-91. [3]TiinamaijaTuomi., Type I and Type II diabetes, International Journal of Pharmacy and Pharmaceutical Sciences, 2005; 34: 3214-3218. [4]Tripathi KD. Essentials of medical pharmacology, 6thediton, Jaypee Brothers Medical Publishers (P) Ltd. New Delhi: 254-255. [5]Grover J K., Yadav S. & Vats V., Medicinal plants of India with anti-diabetic potential, Journal of Ethnopharmacology, 2002; 81: 81. [6]Pareek, S.K., Medicinal plants in India: Present status and future prospect. In prospect of medicinal plants (edsGautam P. L. et al), Indian society for plant genetic Resources, New Delhi, 1998; 5-14. [7] Mukhopadhaya, S., Conservation, protection and biodiversity of medicinal plants. In Prospects of medicinal plants (edsGautam P. L. et al), Indian society for plant genetic Resources, New Delhi, 1998; 15-28. [8] Gupta R. and Chadha, K. L., Medicinal and aromatic plant in India. In advance in Horticulture, Medicinal and aromatic plants (eds. Chadha K.L. and Gupta R.), Malhotra Publishing House, New-Delhi, 1995; 1-44.

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*Corresponding Author:

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Prodyut Mondal* Department Of Pharmaceutical Sciences Dibrugarh University, Assam 786004. India.

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ResearchArticle BiologicalSciences MULTIPLE Β LACTAMASE ENZYMES PRODUCING CLINICAL ISOLATES OF GRAM NEGATIVE BACTERIA IN A TEACHING HOSPITAL VidyaPai*, Sunil Rao P, Bhaskaran Nair Department of Microbiology, Yenepoya Medical College, Nithyanandanagar, Mangalore-575018, India. *Corresponding AuthorEmail:

ABSTRACT Context: Gram negative bacilli producing Beta lactamases have been increasingly reported worldwide and infections with such bacteria are difficult to treat. It is also not unusual to find a single isolate that expresses multiple beta lactamase enzymes further complicating the treatment options. Aims: The present study was designed to investigate the coexistence of different beta lactamase enzymes in clinical isolates of gram negative bacilli. Material and Methods: A total of 321 isolates of gram negative bacilli obtained from various clinical specimens were included in the study. Antimicrobial susceptibility testing was performed for all the isolates in accordance with CLSI guidelines. All bacterial strains were tested for ESBL, Amp. C & MBL production. Statistical analysis used: Descriptive statistics was used and the percentage of ESBL, Amp C and MBL carrying gram negative bacilli isolates were calculated. Results: ESBL production was seen in 100 (31.1%) isolates with maximal incidence in Citrobacter species (52.1%), followed by P. aeruginosa (30.4%). Amp C production was detected in 67 (27.8%) isolates with highest percentage (25.4%) among non-fermenters. Conclusions: Early detection of these multiple β lactamase producing isolates in a routine laboratory could help to avoid treatment failure, as often such isolates show a susceptible phenotype in routine sensitivity testing. Unless strict measures to limit the indiscriminate use of cephalosporins and Carbapenems in the hospitals are undertaken, the multiple β lactamase producing pathogens would spread with no treatment options left to treat nosocomial infections with such pathogens.

KEY WORDS ESBL, Amp C, MBL.

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INTRODUCTION Gram negative bacilli account for the majority of bacterial pathogens isolated from clinical specimens.1The incidence of infections due to Gram negative bacilli resistant to β lactam agents has increased in recent years. Till 2006, ESBL production by GNB was considered as the most important threat to clinical therapeutics. 2, 3.This led to a parallel increase in the usage of β Lactam/ β lactamase inhibitor combinations, monobactams and carbapenems. Eventually, in the last few years, reports from worldwide show resistance to these drugs as well. 4, 5

The resistance to monobactams and carbapenems is due to the production of Amp.C and Metallo beta lactamases respectively. The genes coding for these β lactamases are carried on plasmids, facilitating rapid spread between micro-organisms and often are co-expressed in the same isolate.6 The treatment options for such infection are limited and hence of great concern. Hence the present study was designed to investigate the presence of different classes of β lactamase enzymes in the clinical isolates of gram negative bacilli.

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inhibition adjacent to the Fox disc were taken as Amp C producers. MBL detection method: The isolates were screened for the presence of MBLs by the combined disc test (CDT). Two Imipenem 10 µg discs were placed on the surface of an agar plate with bacterial inoculum and 5 µl EDTA was added to one of them to obtain a concentration of 750 µg. The zones of inhibition of IPM alone and IPM-EDTA were compared after 16-18 hours incubation in air at 35oC. An increase in zone size of >7mm was taken as positive. Descriptive statistics was used and the percentage of ESBL, Amp C and MBL carrying gram negative bacilli isolates were calculated.

RESULTS A total of 321 bacterial isolates were included in the study. Table 1 depicts the different bacterial species tested and their resistance pattern. ESBL production was noticed in 100 (31.1%) isolates with maximal incidence in Citrobacter species (52.1%, n=12), followed by P. aeruginosa (30.4%, n=32). Amp C production was detected in 67 (27.8%) isolates. Majority of P.aeruginosa strains (28.4%, n=30) produced Amp C β lactamases. imipenem resistance was seen in a mere 11(30.9 %) strains, whereas resistance to ciprofloxacin was seen in 108 (95.5 %) strains (Table 2). Co-production of Amp C β lactamase and ESBL was seen in 17.1% (n=55) strains and MBL production was detected in 11 isolates. Coproduction of Amp C and Metallo β lactamases was found in 1.2% (n=4) isolates with maximal occurrence among Acinetobacter species (2.3 %) isolates. (Table 3)

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The study was conducted in the department of microbiology of our medical college hospital. A total of 321 consecutive, non-duplicate isolates of gram negative bacilli obtained from various clinical specimens were included in the study. The isolates were characterized by using standard microbiological techniques.7 Antimicrobial susceptibility testing was performed for all the isolates by using the commercially available discs [Himedia, Mumbai, India] in accordance with CLSI guidelines.8 The antibiotics which were tested include, Piperacillin 100µg ([PIP), Ceftazidime 30 µg (CAZ), Imipenem 10 µg (IPM), Ciprofloxacin 5 µg (CIP), Gentamycin 10 µg (GEN), Amikacin 30 µg (AK) and Aztreonam 30 µg (ATM). Quality control was achieved using standard strains of E.coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. All bacterial strains were tested for ESBL, Amp. C & MBL production by the following methods. ESBL detection method: ESBL status of the isolates was detected by combined disc diffusion using Cefotaxime 30 µg [CTX] &Ceftazidime 30 µg [CAZ] disc alone and in combination with Clavulanic acid [CEC & CAC] as per CLSI recommendations. Amp C detection method: The isolates were tested for Amp C production by the disc antagonism test. A test isolate [with a turbidity equipment to that of 0.5 McFarland standard) was spread over a Mueller Hinton agar plate. Cefotaxime 30µg (CTX) and Cefoxitin 30 µg (Fox) discs were placed 20mm. apart from centre to centre. Isolates showing blunting of CTX zone of

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Table 1: Resistance pattern of bacterial isolates: Isolate No. PIP CIP GEN AK ATM E.coli 60 40 39 60 14 9 Klebsiella species 72 72 30 24 16 12 Citrobacter species 23 23 23 23 23 2 Proteus species 18 2 6 4 --Pseudomonas species 105 92 84 75 53 34 Acinetobacter 43 41 35 40 31 10 Total 321 270 217 226 137 67

CAZ 19 23 14 1 32 11 100

IPM 1 4 1 -3 2 11

Table 2: ESBL & Amp C production in bacterial isolates. Clinical isolate E.coli Klebsiella species Citrobacter species Proteus species Pseudomonas species Acinetobacter Total

No. of isolates 60 72 23 18 105 43 321

ESBL producers (%) 16 (26.6%) 20(27.7%) 12 (52.1%) 1(5.5%) 32 (30.4%) 11 (25.5%) 100 (31.1%)

Amp C producers 9(15%) 11 (15.2%) 2(8.6%) - (-) 30 (28.4%) 9 (20.9 %) 67 (20.8%)

Both ESBL & Amp c 5 (8.3%) 8(11.1%) 6 (26.0%) - (-) 21 (20.0%) 9(20.9%) 55(17.1%)

Table 3: MBL & Amp C production Amp C 9(15%) 11 (15.2%) 2(8.6%) - (-) 30 (28.4%) 9 (20.9 %) 67 (20.8%)

Both MBL + Amp C -- ( - ) 2 (1.9) 2 (2.3) 4(1.2)

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E.coli Klebsiella species Citrobacter species Proteus species Pseudomonas species Acinetobacter Total

MBL producers 1 (1.6) 4 (5.5) 1(4.3) -- (-) 3(2.8) 2(4.6) 11(3.4)

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DISCUSSION Gram negative bacterial isolates show a multiplicity of resistance mechanisms. ESBL producing strains of GNB have emerged as a major problem in hospitalized on well as community based patients.9 The incidence of ESBL in major hospitals of India has been reported as high as 60%-80%.10,11 Their prevalence worldwide has been non-uniform. US hospitals have reported 40% of Klebsiellapneumoniae isolates as ESBL producers whereas reports from Taiwan show 94% of Klebsiella species as ESBL producers. 12,13 In our study 31.1% of total GNB included, showed ESBL production with the highest incidence in Citrobacter species (52.1%) followed by P.aeruginosa (30.1%). Higher percentages of ESBL producing GNB were shown by other studies 14,15whereas reports from Chennai 16 and Hyderabad 17 show lower percentages of ESBL producers. Shortly after ESBLs, Amp C β lactamase emerged which were resistant to 3rd generation Cephalosporin including β-lactam/ β lactamase inhibitor (in contrast to ESBL) but sensitive to 4th generation cephalosporins. In 2003, 20.7% Amp C producers were reported from Delhi, 18 37% from Chennai.19 The numbers of Amp C producers has been increasing over the years. In our study 27.8% of GNB isolates showed Amp C production with highest percentage (25.4%) among non-fermenters. Some hospitals have reported high percentage (up to 80%) of Amp C producers.20 The only β lactam active against Co-Amp C and ESBL producers are Carbapenems, however, recently resistance to Carbapenems has been increasing, which is mostly due to production of MBL.21 Our findings showed 3.4% of the bacterial isolates produced MBLs and 1.2% strains produced both Amp C and MBLs. Carbapenemases have been reported in E.coli,

Klebsiella species. Pseudomonas species and Acinetobacter species from different parts of the globe. The percentage various widely with some centres reporting low figures (48% in Acinetobacter) where as others showing upto 80% (Acinetobacter species). 22 Lower resistance to imipenem in our centre may probably due to the reserved use of thus drug. An interesting finding was that 2 isolates were sensitive to imipenem by routine disc diffusion method but showed MBL production by CDT (IPM-EDTA). These carbapenem susceptible isolates carrying hidden MBL genes, may spread unnoticed and may lead to untoward infection control problems. As there is no single method proven as ideal method for MBL detection in all the isolates, we used the CDT, which is recommended by CLSI and proven by many other studies.

CONCLUSION The present study emphasizes the prevalence of gram negative bacilli producing β lactamase enzymes of diverse mechanisms. Early detection of these multiple β lactamase producing isolates in a routine laboratory could help to avoid treatment failure, as often such isolates show a susceptible phenotype in routine sensitivity testing. Unless strict measures to limit the indiscriminate use of cephalosporins and Carbapenems in the hospitals are undertaken, the multiple β lactamase producing pathogens would spread with no treatment options left to treat nosocomial infection with such pathogens.

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Eisenstein BI, Zaleznik DF. Enterobacteriaceae. In: MandellGL, Bennett JE, Dolin R, editors. Principles and practiceof infectious diseases, 5th ed. Philadelphia, Pa: ChurchillLivingstone; 2000 p. 2294-310.

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and Plasmid mediated AmpC enzymes among clinical isolates of E. coli and Klebsiellapneumoniae from seven medical centres in Taiwan. Antimicrob Agents Chemother, 2006; 50 (5):1861-1864. Rajini E, Sherwal BL, Anuradha . Detection of ExtendedSpectrum β-lactamases in AmpC β-lactamaseProducing Nosocomial Gram-negative Clinical Isolates from a Tertiary Care Hospital in Delhi Vol. 4, No. 6 (2008-01 - 2008-02) Mathur P, Kapil A, Das B, Dhawan B: Prevalence of Extended spectrum beta lactamase producers in a tertiary care hospital. Ind J Med Res.2002; 115:153-7. Kumar MS, Lakshmi V, Rajagopalan R: Occurrence of Extended spectrum beta lactamases among Enterobacteriaceaespp isolated at a tertiary care institute. Ind J Med Microbiol. 2006; 24 (3):208-211. Menon T, Bindu D, Kumar CPG, Nalini S, ThirunarayanMA:Comparision of double disc and three dimensional methods to screen for ESBL producers in a tertiary care hospital. Ind J Med Microbiol. 2006; 24 (2):117-120. Manchanda V, Singh NP: Occurrence and detection of AmpC β lactamases among gram negative clinical isolates using a modified three dimensional test at Guru TeghBahadur Hospital, Delhi, India. J AntimicrobChemother, 2003; 51: 415-418. Subha A, Devi VR, Ananthan: AmpC β lactamase producing multidrug resistant strains of Klebsiellaspp and E.coli isolated from children under five in Chennai. Ind J Med Res.2003; 117:13-18. Woodford N, Reddy S, Fagan EJ: Wide geographic spread of diverse acquired AmpC β lactamases among E. coli and Klebsiellaspp in the UK and Ireland. J AntimicrobChemother, 2007; 59(1):102-5. Livermore DM, Woodford N: Carbapenemase (2000) A problem in waiting? CurrOpinMicrobiol 3:489-95 Shobha KL, Lenka PR, Sharma M K, Ramachandra L , Bairy I .Metallobetalactamase production among Pseudomonas species and Acinetobacter species in coastal Karnataka. Journal of Clinical and Diagnostic Research [serial online] 2009 October [cited: 2009 October 5]; 3:1747-1753.

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Livermore DM. b-lactamase mediated resistance and opportunities for its control. J AntimicrobChemother1998; 41 (Suppl D): 25-41. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum beta lactamase producing gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002; 115: 153-7. Navaneeth BV, Sridaran D, Sahay D, Belwadi MRS. A preliminary study on metallo b-lactamase producing Pseudomonas aeruginosain hospitalized patients. Indian JMed Res 2002; 116: 264-7. Chitnis SV, Chitnis V, Sharma N, Chitnis DS. Current status of drug resistance among gram-negative bacilli isolated from admitted cases in a tertiary care center. J AssocPhysiciansIndia2003; 51: 28-32. Chatterjee SS, Karmacharya R, Madhup SK, Gautam V, Das A, Ray P. High prevalence of co-expression of newer β-lactamases (ESBLs, Amp-C-β-lactamases, and metallo-β-lactamases) in gram-negative bacilli. Indian J Med Microbiol 2010; 28:267-8 Crichton P.B. Enterobacteriaceae: In: Mackie and McCartney Practical MedicalMicrobiology, 14th ed. Collee J.G, Fraser A.G, Marmion B.P, Siminous A, editors.(Churchill Livingstone: New York) 1996. 361-4. Performance standards for Antimicrobial Disc Susceptibility Tests, Clinical and Laboratory Standards Institute. Vol.29 No.3, Jan 2009. An update on newer [beta]-lactamases Ind J Med Res Nov, 2007. Hansotia JB, Agarwal V, Pathak AA, Saoji AM. Extended spectrum beta-lactamase mediated resistance to third generation cephalosporins in Klebsiellapneumoniae in Nagpur, central India. Indian J Med Res 1997; 105 : 158-61. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum beta lactamase producing Gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002; 115 : 153-7. Jacoby GA, Munoz-Price LS: The new beta lactamases. N Eng J Med 2005; 352: 380-91. Yan JJ, Hsueh PR, Chang FY, Shyr JM, wan JH, Liu YC, Chuang YC, Tsao SM, Wu HH, Wang LS, Lin TP, Wu HM, Chen HM, Wu JJ: Extended spectrum beta lactamases

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*Corresponding Author:

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Dr.VidyaPai, Professor, Dept. Of Microbiology, Yenepoya Medical College, Nithyanandanagar, Mangalore-575018.

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Review Article Pharmaceutical Sciences A REVIEW ON PARENTERAL PRODUCTION TECHNOLOGY B. Venkateswara Reddy1*, B.Rasmitha Reddy1, K.Navaneetha1, V.Sampath Kumar2 1

St.Pauls College of Pharmacy, Turkayamjal, Ranga Reddy (Dist), A.P-501510 Sree Datta Institute of Pharmacy, Ibrahimpatnam, Ranga Reddy (Dist), A.P.

ABSTRACT The main objective of this paper is to facilitate the area planning, utilities, environmental control for production of parenteral. Compare to other dosage forms parenterals are efficient. This gives quick onset of action and provides a direct route for achieving the drug effect within the body. So by producing these under necessary requirements we can yield better economic and therapeutical performance.

KEY WORDS Area Planning, change rooms, environmental control, personnel flow.

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INTRODUCTION: Parenteral preparations are sterile, pyrogen-free liquids (solutions, emulsions, or suspensions) or solid dosage forms containing one or more active ingredients, packaged in either single-dose or multidose containers. They are intended for administration by injection, infusion, or implantation into the body. The dosage form for conveying a drug by means of injection through the skin or mucous membranes. Parenteral drugs are administered directly into the veins, muscles or under the skin or more specialized tissues such as the spinal cord. Circumvented the highly efficient first line body defense that is skin and mucus membrane. Thus they should be free from microbial contamination and should have high purity Preparations such as vaccines, human blood and products derived from human blood, peritoneal dialysis solutions, and radioactive pharmaceuticals require special formulation, methods of manufacture, or presentation appropriate to their particular use and may not comply with certain parts of this monograph.

TYPES: There are four main forms of parenteral preparations:  Injections,  Intravenous infusions (large volume parenterals),  Powders for injections, and  Implants. Certain injections and intravenous infusions may be presented in the form of sterile concentrated solutions, which must be suitably diluted before use. FACILITIES REQUIRED FOR PARENTERAL PRODUCTION: PRODUCTION: Parenteral preparations may contain excipients such as solvents, suspending agents, buffering agents, substances to make the preparation isotonic with blood, stabilizers, or antimicrobial preservatives. The addition of excipients should be kept to a minimum. When excipients are used, they should not adversely affect the stability, bioavailability, safety, or efficacy of the active

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www.ijpbs.com (or) www.ijpbsonline.com ingredient(s), or cause toxicity or undue local irritation. There must be no incompatibility between any of the components of the dosage form. Water for injections is used as the vehicle for aqueous injections. It should be freshly distilled by the process described under "Aqua pro Injection", be free from carbon dioxide, and comply with Test for bacterial endotoxins. Sterilization at this stage may be omitted,

provided that the solution or preparation is immediately sterilized upon finalization. For nonaqueous injections, fixed oils of vegetable origin are used as vehicles. Unless otherwise specified in the individual monograph, sodium chloride or other suitable substance(s), may be added to an aqueous solution for injection in order to render the preparation isotonic.

Planning & scheduling

Figure: 1 Overview of manufacturing process

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Figure: 2 Flow of materials Types of sterile products processing: 1 Terminally sterilized â&#x2020;&#x2019; prepared, filled and sterilized 2 Sterilized by filtration 3 Aseptic preparations

Manufacture of sterile preparations:1. Terminally sterilized: - usually involves filling and sealing product containers under high-quality environmental conditions. Products are filled and sealed in this type of environment to minimize the microbial and particulate content of the in-

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process product and to help ensure that the outside or recycled air must be first filtered to subsequent sterilization process is successful. In remove particulate matter and then HEPA filters most cases, the product, container, and closure are used to get CLASS-100 air systems. have low bio-burden, but they are not sterile. The product in its final container is then subjected to GMP Requirements for Sterile Products a sterilization process such as heat or irradiation. ▶ Specific points relating to minimizing risks of 2. Sterilization by Filtration:contamination.  Previously sterilized container are taken. – Microbiological – Particulate matter  Filters having nominal pore size 0.22 μm – Pyrogen or less are used for filtration General Requirements  Remove bacteria and moulds but Not ▶ Production in clean areas viruses & Mycoplasmas  Double filter layer or second filtration ▶ Airlocks for entry  No fiber shedding or asbestos filters – Personnel entry. – Material entry  Filter integrity testing ▶ Separate areas for operations 3. Aseptic Preparation: - In an aseptic process, the drug product, container, and closure are first – Component preparation subjected to sterilization methods separately, as – Product preparation appropriate, and then brought together. Because – Filling there is no process to sterilize the product in its – Sealing etc… final container, it is critical that containers be ▶ Level of cleanliness filled and sealed in an extremely high-quality ▶ Filtered air environment Before aseptic assembly into a final ▶ Air classification: Grade A, B, C and D. product, the individual parts of the final product ▶ Laminar air flow: are generally subjected to various sterilization – Air speed (horizontal versus vertical flow) processes. Any manual or mechanical – Number of air changes manipulation of the sterilized drug, components, – Air samples containers, or closures prior to or during aseptic ▶ Conformity to standards assembly poses the risk of contamination and ▶ Work station and environment thus necessitates careful control. ▶ Barrier technology and automated systems. Note: - In area occupied by personal, the air must be exchanged with the frequent intervals. Fresh Space requirements:[QUANTITATIVE LAYOUT OF PARENTERAL MANUFACTURING] Area Function Square meter Percentage Production Warehouse Utility Quality control Administration

11,094 7,606 1,716 1,716 1,018

45.1 30.9 4.1 7.0 4.1

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1,014 1,014

39 0.9 24,607 100.0 Table: 1 space requirements

599

AREA PLANING AND ENVIRONMENTAL CONTROL:Area planning may be addressed by functional groups ground this critical area with particular attention given to maintaining cleanliness. The goal of the designer is to group manufacturing operations so that the flow to people, product, and components proceeds in the direction of successive steps of increasing cleanliness likewise, the flow of waste materials and products must be thoroughly separated from the flow of clean personnel and product in order to prevent contamination.

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Functional groupings:Warehousing:o Basic warehousing functions include receiving, shipping, and in-process storage. o Receiving areas include unpacking, sampling and incoming quarantine. o Shipping includes quarantine prior to shipment. o The storage of spare parts, air filters, change parts, water treatment chemicals, office supplier, laboratory supplies, janitorial supplies, uniforms, an so on may be handled as central storage or individually by department. o Finished product and certain raw materials need special environmental storage conditions, such as, temperature and humidity control. o The first and most basic warehouse function is received and holds incoming materials.

Warehouse space is usually of greater height than production areas, is less rigidly controlled from an environmental and sanitation stand point, and usually has a relatively high density of flammable materials. Thus a separate but adjoining area separated by a firewall is usually the best arrangement. Administrative areas:Administrative area planning requires careful analysis of the direct and indirect administrative requirements of a particular plant. Successively higher levels of supervision are usually provided successively larger office areas. Some offices are individual, while some are grouped in an “open area concept”. The relative location of administrative areas demands particular attention. For the necessary to maintain production, a close proximity is desirable. Any other support offices should be separated from the production area because, production area contaminations can be related to people. The reduction of numbers of people will reduce the challenge to the plant cleanliness. Many of the “fringes” normally associated with administrative areas-plants, flowers, closets, outside windows are potential contamination sources. Finally the traffic of visitors, vendors, employment application, and so on, who are not particularly acquainted with pharmaceutical discipline can be reduced. Requirements are related more to the relationship between the plant and the company or corporate headquarters. These indirect administrative requirements will usually include functions not directly related to plant operation,

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such as company or corporate management and staff functions. Environmental control zone grouping:1st. Zones as per the c GMP: Zone 7:- Filling line  Zone 6:- Filling area  Zone 5:- Weighing, mixing & transfer area.  Zone 4:- Clean area  Zone3:-General production  Zone 2:- Warehouse  Zone 1:- Exterior

1st. Zones as per Gazette of India

WHITE GRAY

BLACK

GRAY Figure: 3 environmental control zone groups

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ZONES AS PER GAZZETE OF INDIA:  White zone:-Final step (filling of parenteral)  Grey zone:-weighing, Dissolution & filtration.  Black zone:-Storage, Worst area from contamination view point a) Have a per-cubic-particle count of not more than 100 in a size range of 0.5 micron and larger through the entire work area upstream of the work piece. b) Be supplied at the point of use as specified in section 212.77.

The layout of the plant must be carefully developed in coordination with the needs of the HVAC system. Zone-7:-filling line:The walls of the filling area are the last physical barrier to the ingress of contamination, but within the filling area a technique of contamination control known as laminar flow may be considered as the barrier to contamination. Zone-6:-filling area:Zone 6 is a distinct zone of the controlled environment area for an aseptic filling process but may not be distinct zone for non-aseptic filling processes.

Figure: 4 Aseptic filling

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Non asepting filling, followed by terminal sterilization, normally requires less rigid environmentalcontrol.

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Figure: 5 Non aseptic filling Zone-5:-weighing, mixing, and transfer area:Zone5 encompasses those activities of â&#x20AC;&#x153;weighing, mixing, filling or transfer operationsâ&#x20AC;? addressed by c GMP section 212.81 which are not handled as zone 6 but which require a controlled environment. Zone-6:-clean area:Activities in these may include washing and preparations of equipment or accumulation and sampling of filled product. Zone-3:-general production and administration area:The third zone of environmental control is formed by the periphery of the general production area. Openings into the area are usually well sealed and large enough for only essential material-handling equipment and personnel. Zone-2:-plant exterior:The environmental with in which a plant located is first environmental control zone. It is a base point from which to work in determining the requirements for the various control barriers. Management actions to control zone 1 might include the maintenance of sterile areas around the facility where weeds, insects and rodents are controlled or eliminated.

The design of filling areas or more generally, controlled environment areas involves attention to many seemingly minor details. The basic cleanlability requirement includes smooth, cleanable walls, floors, ceilings, fixtures, and partition exposed columns, wall studs, bracing, pipes, and so on are unacceptable. The need for cleanability also eliminates the open floor system commonly used in the microelectronics industry for laminar airflow rooms. The goal of the designer, when creating the details for the architectural finishes and joining methods, is to eliminate all edges or surfaces with in the room where dirt may accumulate. All inside walls must be finished; common methods of finish are block, plaster, or gypsum board. Concrete block walls are sturdy and easily constructed. The porosity of concrete block walls can be reduced by coating with block filler prior to painting. But even filled concrete block walls have a surface texture that is not conductive to cleaning. Painted concrete block walls are particularly susceptible to peeling if they are subjected to moisture as from leakage or rain on the backside. Use of ceramic-faced block can overcome the surface finish problems of concrete block. Epoxy

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www.ijpbs.com (or) www.ijpbsonline.com paint is normally used to increase the durability and impermeability of the surface. When gypsum board is used, an epoxy point system is normally employee to create a surface that is resistant to cleaning compounds. Gypsum board is not an acceptable surface for use in powder-filling operations without incorporating an additional surface coating or vapor barrier. By itself, gypsum is susceptible to vapor barrier. By itself, gypsum is susceptible to vapor migration which presents problems in a low humidity controlled area. To overcome the surface weaknesses of most walls, various heavy coverings are available. A few spray on and brush on coatings have provide a much harder and more durable surface than gypsum, but are still relatively economical to install and do not present the installation difficulties of vinyl sheeting. The use of modular systems has increased substantially in the last few years that provide a much harder and more durable surface than gypsum, but are still relatively economical to install and do not present the installation difficulties of vinyl sheeting. The use of modular wall systems has increased substantially in the last few years because they arrive at the construction site prefinished and are much faster. Selection of floor materials poses a particularly difficult problem since they must be durable, and easily cleaned and sanitized. To achieve good floor results, the application must be matched to the particular characteristics of the floor system. Hardeners may be added to concrete to increase to surface hardness by as much as a factor of 3, greatly improving the floor’s resistance to scratching and dusting and are available in colors to improve the appearance of the floor. A sealed concrete floor is therefore not acceptable for use in controlled areas with in a parental filling plant because of the potential for

cracking of the soil beneath the concrete when laid as a coating over a cured concrete surface. The plants in many parenteral plants are constructed of epoxy terrazzo. Finally, the floor is sealed with several coats of urethane to protect the surface finish. The result is a very attractive floor that is extremely impact and abrasion-resistant. A third general type of floor is composed of large sheets vinyl or polyvinylchloride laid on a concrete base floor and “welded” together with heat or sealed at the seams with cement. Selection of compatible material-handled equipment whets and for floors will reduce floor damage. All floors in areas where water can accumulate should toward one or more drain points. 3. LIGHTNING FIXTURES: Lighting fixtures should be reduced flush with the ceiling. Since most lighting fixtures are not tightly sealed, the diffuser should be sealed integrally with the ceiling, and the lamps changed from outside the room. Either recessed or surface mounted fixtures can be used. Special “washdown” fixtures are well sealed, but protrude obtrusively into the room and have clips and sealing lips which are difficult to sanitize. Areas having a full HEPA ceiling obviously cannot accommodate recessed lighting fixtures. In these areas, fixtures are of a special “teardrop” shape which minimizes disruption to the laminar airflow pattern. 4. CHANGE ROOMS: Personnel access to all controlled areas should be through change rooms. Change rooms concepts and layouts vary from single closet size rooms to expensive multi-room complexes. Entrance to a change area is normally through vestibules whose doors are electrically interlocked so that both cannot be opened simultaneously, thus maintaining the necessary

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www.ijpbs.com (or) www.ijpbsonline.com air pressure differential to prevent the entry of airborne contamination. Upon entry into the change room wash sinks are provided for scrubbing hands and forearms. Although commercial hands are often used, they may create undesired airflow patterns and may circulate particular laden air. Special filtered driers are available to minimize the creation of particulate contamination. Further control may be achieved by using filtered and heated compressed air for drying to reduce further particular potential. In some facilities, a foamed type of alcohol is dispensed on the hands, which then evaporates. This is used to eliminate need

for tap water and sinks in the gowning rooms, since these can be a potential source of contamination. After hands are dry, garments are taken from dispensers and donned while moving across a dressing bench. As a final gowning step, aseptic gloves are put on and sanitized. Exit from the change room to the controlled area is, like entrance, through an interlocked vestibule. Depending on the degree of disrobing required, separate gowning facilities facilities may be provided for men and women. Separate â&#x20AC;&#x153;degowningâ&#x20AC;? rooms are provided where the clean room garments can be discarded prior to leaving the controlled zone.

Figure: 6 Change room personnel traffic in or near work areas where controlled substances are handled. The flow of material and personnel through corridors are inefficient and unsafe paths for moving materials, particularly if heavy forklifts are required. Parenteral plants, like any other plant have visitors and the degree of access to be granted must be determined. A glassed mezzanine or balcony provides absolute solution yet may give an excellent view of the processes, but may not be adaptable for single-floor layouts.

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5. PERSONNEL FLOW:The movement of personnel should be planned during the design of individual plant areas. Each individual production area may have a smooth and efficient personnel flow pattern, a discontinuous or crowded pattern may develop when several individual production area plants are combined. The separation of people and products is greatly facilitated by the use of the third dimension. Security concerns about personnel flow may include minimizing access to controlled substances and minimizing the

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× Design

√

Design

Figure: 7 personnel flow

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Discontinuous and crowded flow patterns can decrease production efficiency, increase security problems, and increase the problems of maintaining a clean environment. Personnel flow path from zone to zone must be such that access to higher level of cleanliness is only through change rooms, gowning rooms, locker rooms, or other areas as may be required to prepare the personnel for the cleaner area. 6. UTILITIES AND UTILITY EQUIPMENT LOCATION:Utilities:Piping system in particular, must be initially and often periodically cleaned and serviced. Exposed overhead piping is not acceptable from a cleanliness or contamination standpoint since it collects dirt, is difficult to clean and may leak. Buried or concealed pipe may require unacceptable demolition for cleaning or repair. Whenever possible, major utility distribution services should located outside of clean areas. The actual utility connections are distributed with in the plant, building codes usually require that their distribution systems be exposed and not buried with in walls or ceilings. Utilities equipment location:Public utilities require space for metering. In addition to meeting, electrical power system require for switchgear and transformers. Water systems usually require treatment to ensure

consistent quality. Plant generated utilities typically require steam boilers, air compressors, and distillation, the typical “boiler room” approach. Although a central location minimizes distribution problems and minimizes service distribution distances. Proper equipment maintenance is difficult in foul weather, especially winter. Heavy equipment may damage the roofstructure, particularly if the equipment location requires numerous penetrations through the roof which, coupled with equipment vibration, will invariable lead to leakage. A mezzanine equipment platform eliminates the problems of operation in a harsh environment and roof loading.

MATERIALS: The selection of materials for a piping system depends on the product tube handled, the product purity desired, material cost, and installation cost. Carbon steel: Carbon steel pipe, manufactured according to ASTM standard A53 of A106 is commonly available in various schedules or wall thicknesses. The standard schedule is number 40. Common uses include water, compressed air, oil, nitrogen, steam and steam condensate. Copper: Copper is commonly used for water and compressed air piping because of easy

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www.ijpbs.com (or) www.ijpbsonline.com installation. Either type of K or type L, tubing is available in annealed form, making it more flexible. Copper has a smooth surface finish compared to that of carbon steel and is relatively resistant to corrosion. Copper loses strength rapidly at higher temperatures and is not recommended for steam use. Type 304 Stainless Steel: It contains approximately 18% chromium and 8% nickel, being nonmagnetic and non-harden able. Type 304 is a good general purpose alloy for pharmaceutical applications where pitting corrosion is not a problem. Type 316 Stainless Steel: It is similar to type304 except that type 316 has 24% higher nickel content. 2% less chromium and has 2-3% molybdenum. The molybdenum gives type 316 improved resistances to pitting corrosion as compared to type 304 and slightly improved general corrosion resistance. Both type 304 and 316 stainless steel are susceptible to intergranular corrosion adjacent to welded areas Type 316L piping is typically used for distribution of water for injection, clean steam, deionized water, compressed air to be used in controlled environmental areas and or product transfer piping. Plastics: Plastic piping has been used in drain lines and chemical treatment systems. Additionally, some companies have used poly vinyldene fluoride (PVDF) piping for dematerialized water. This poly fluroplastic has an advantage in that a system is constructed by thermal fusion of the joints rather than welding. Surface finish: Surface finish specifications after refer to 3-A sanitary standards. According to these standards a product contact surface should be polished to a number 4 finishes, a finish obtained by polishing

with a 150-grit sanding belt. In addition to mechanical polishing, electro polishing has been used to improve further the surface finish of stainless steel. The electro polished surface exhibits somewhat better corrosion resistance than mechanically polished surfaces. Joining techniques: Piping system can be joined by threading, welding or clamping. Threaded connections are common for non-electrical applications where iron pipe may be used.Sanitary tubing is welded by using an automatic fusion welding machine that fuses the two sections of tubing together, using an electric current and a purge of inert gas on the inside of the tubing to yield a high quality weld. The quality of the weld is checked internally by the use of a video boroscope. Following the welding, the piping is passivated with nitric acid to form an oxide layer on the inside of the pipe, thereby providing increased corrosion resistance. Valuing: A typical ball valve as ported ball that is rotated 90째 to regulate flow. A diaphragm valve, control flow by compressing a diaphragm against a wire placed across his direction flow. A number of new valves came into the market recently to deal with the limitations of existing valves. One of the best is pinch valve. The pinch valve is a cylindrical valve that is modulated by pinching the inner tubing wall of the valve. Utility services connection arrangements: Utilities must be carefully connected to avoid stagnant areas and to avoid difficult to clean areas just as would be done for the utility distribution system. To minimize contamination potential, typical utility arrangements and typical service connections should be defined during planning. Utilities can be arranged so that the service connections enter a room vertically upward,

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horizontally, and vertically downward, with various advantages and disadvantages. Vertical upward service connections, with connections under machinery, create a very neat appearance, a low full unobstructed machine access, and require only short connection lengths. Horizontal service connections are often used in single level facilities to avoid floor excavation during equipment relocation or utility maintenance. Horizontal service do limit machine access, create some congestion, and may necessarily be longer than vertical service connections. Vertical downward services create a visually cluttered appearance and may restrict access to the working surface of equipment. This type of connections may also be undesirable if laminar flow coverage of the equipment is necessary. 7. Engineering and maintenance:From an engineering stand point, even a location outside the plant can serve well if access to the production area by engineers for field wok is not too difficult often particularly in small or less complex plants, maintenance or other plant service functions such as utilities or combined with engineering, making an in-plant location desirable. Although often associated with engineering, maintenance is a unique and distinct function. Maintenance responsibilities cover all areas of the plant and can generally be grouped into two categories: Plant maintenance and production maintenance. Production maintenance is a direct production support function and includes all the routine and recurring operating maintenance work. Production maintenance facilities are usually minimal, often only a place to store a tool box, and seldom have more than a small workbench. Plant maintenance operations, in contrast, are more diverse. They vary from heavy maintenance on production equipment to cosmetic work on

the building exterior and often include plant service functions such as sanitation, ground sweeping, or waste disposal. Facilities required are extensive and mostly include provisions for equipment cleaning. Disassembly major rebuilding of equipment and painting. These operations can present a contamination risk to pharmaceutical operations and must be isolated. Although maintenance requires access to all parts of a plant, it must be located to be able to receive and handle cumbersome and bulky groups. An absolute must is that the plant maintenance shop be located so that its personnel have easy access to major plant utilities and service equipment. Types of containers: 1. Ampoules: They are intended for single use only; ampoules are opened by breaking the glass at a score line on the neck. Because glass particles may become dislodged during ampoule opening, the product must be filtered before it administered. Because of their unsuitability for multiple-dose use, the needs to filter solutions before use and other safety considerations have markedly reduced ampoule use. 2. Vials: are glass or plastic containers are closed with a rubber stopper and sealed with an aluminum crimp. Advantages over ampoules: ď&#x201A;ˇ They can be designed to hold multiple doses (if prepared with a bacteriostatic agent). ď&#x201A;ˇ It is easier to remove the product. ď&#x201A;ˇ They eliminate the risk of glass particle contamination during opening. 3. Prefilled syringes -These designed for quickest administration and maximum convenience. Drugs administered in an emergency (e.g., atropine,

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www.ijpbs.com (or) www.ijpbsonline.com epinephrine) may be available for immediate injection when packaged in prefilled syringes. 4. Infusion solutions are divided into two categories: small volume parenteral (SVP), those having a volume of 100 ml; and large volume parenteral (LVP), and those having a volume of 100 ml or greater. Infusion solutions are used for the intermittent or continuous infusion of fluids or drugs. LIST OF EQUIPMENTS (as per schedule-M): The following equipment's is recommended: a) Manufacturing area: 1. Storage equipment for ampoules, vials bottles and closures. 2. Washing and drying equipment. 3. Dust proof storage cabinet 4. Water still. 5. Mixing and preparation tanks or other containers. 6. Mixing equipment where necessary. 7. Filtering equipment. 8. Hot air sterilizer. b) Aseptic filling and sealing rooms 9. Benches for filling and sealing.

10. Bacteriological filters. 11. Filling and sealing unit under laminar flow work station. c) General Room. 12. Inspection table. 13. Leak testing table. 14. Labeling and packing benches. 15. Storage of equipment including cold storage and refrigerators if necessary. An area of minimum sixty square meters partitioned into suitable sized cubicles with air lock arrangement, is recommended for the basic installation.

EQUIPMENTS: Sterile Garment Cabinet:  Made up of Stainless steel.  Ensure a clean storage space by making use of UV disinfectant and heating through IR lamps.  These cabinets may be designed in horizontal air flow system and clean air through HEPA filters

Syringe Filling Machine:

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Figure: 8 Syringe Filling Machine Characteristics: o Barrier isolators o In-process check weighing o Filling: rotary piston pumps.

o o o

Volume: 0.2 to 29 ml All types of syringe including glass, plastic can be filled. Filling Rate: 300 to 600 syringes in a minute.

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Ampoule Washing Machine:-

Figure: 9 Ampoule washing machine Process:  Water is sprayed onto the ampoules.  Turned to an angle of 180 degree with their mouth downward to remove water.  Finally the ampoules are filled with compressed air to remove residual water.  Certain machines have a high temperature zone meant for killing any bacteria. Vial Filling Machine:-



Recycled Water (WFI) Compressed Air DM Water Compressed Air Water for Injection (WFI) Compressed Air

Global solution: preparation and sterilization of components, handling, sterile filling, process control and vial laser etching. More than 15 years of proven reliability in sterile filling.

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Figure: 10 Vial filling machine  Fill vials and bottles  Liquids, viscous material and suspensions and powders.  Unique patented system for filling liquid products in sterile conditions.

Washing cycle:1st wash 2nd wash 3rd wash 4th wash 5th wash 6th wash -

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www.ijpbs.com (or) www.ijpbsonline.com PROCESS: The machine comprises of an intake section which loads the vials.  Transferred through an intermittent transport section.  Liquid filling section which fill the vials with predetermined quantity.  Finally the filled and rubber stoppered vials are released and discharged.

CONCLUSION The parenteral route of administration is the most effective route for the delivery of the active pharmaceutical substances with narrow therapeutic index, poor bioavailability especially for those drugs, prescribed to unconscious patients. The present article describes that area planning, facilities, design, construction and manufacturing of sterile products. It is more impartment to produce good quality of parenteral. Parenterals are the pyrogen free liquids these are manufactured and stored according to cGMP guidelines. Proper area, environmental control, personnel observation will gives excellent parenteral products and attain their described therapeutic effect.

REFERENCES 1.

Industrial pharmacy (sterile products) by Leon Lachman, 657-659 2. Good manufacturing practices for pharmaceuticals 6th edition, Joseph D.Nally, page no-37-113 3. Pharmaceutical science by Remington, 21th edition, vol.1, page.no-814-828 4. American Journal of Hospital Pharmacy, Vol. 38, Issue 8, 1144-114710. Dispensing for pharmaceutical students; 5. www.fda.gov. 6. Drugs & Cosmetics Act 1940. 7. www.GMP.online.coms 8. www.ispc.org 9. www.whqlibdoc.who.org 10. www.dwscientific.co.uk 11. www.pharmamachines 12. www.pharmamachines.com

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Main Advantages: Vial is closed and protected throughout the process.  Vial is opened in the final filling stage in a controlled environment with horizontal laminar flow.  No need for dry heat tunnel sterilization as it is carried out in an autoclave. Sterilization and depyrogenation combined with a HWFI washing cycle and an autoclave cycle. No need for a dry heat tunnel. SIP System:  For in-line sterilization of various processing equipments.  Handling various biological solutions and mixtures requires cleaning and sterilizing these equipments from time to time as they are susceptible to contamination.  Proper SIP integration with pharmaceutical equipment is very important for the overall success of the operation.

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*Corresponding Author:

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B. Venkateswara Reddy St.Pauls College of Pharmacy, Turkayamjal, Ranga Reddy (Dist), A.P-501510

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Review Article BiologicalSciences MARVELOUS MEDICINAL MUSHROOMS G.Hepzibah Beulah1, A. Anita Margret2*and Jeyakumar Nelson3 1

Assistant Professor, Department of Zoology, Rani Anna Government College, Tirunelveli- 627 012, Tamilnadu, India Phone: 91-96-77-565578 2 Assistant Professor, Department of Biotechnology, Bishop Heber College, Tiruchirappalli-620 017, Tamilnadu, India. 3 Unit of Microbiology, Faculty of Medicine, AIMST University, Semeling-08100, Bedong, Kedah DarulAman, Malaysia. *Corresponding AuthorEmail:anitamargret@gmail.com

ABSTRACT The significance of high-quality nutrition has long been known in relation to preventing numerous human health problems and the palatability of healthy foods are increasingly appreciated. Mushrooms are the heterotrophic macro fungi and their nutritional properties made them a better-quality dietary food. Its products bestow improvement of the human immune system and thereby known to increase disease resistance. Identification of new compounds from mushrooms which can aid to develop nutritional supplements needs to be encouraged. Medicinal mushrooms have plenty of novel bioactive compounds that are significant for enhancement of human life. In current scenario, global utilization of these mushrooms gained importance in glimpse and bloom of its vital role in modern medicine. Though several mushrooms have been studied for their usage in medicinal purpose, this review provides insight into prospective implications of medicinal mushroom applications.

KEY WORDS Macro fungi, Nutritional property, Medicinal mushrooms, Mushroom research, Health benefits, Therapeutic usage.

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INTRODUCTION Humans are constantly searching worldwide for the development of new therapeutic agents especially from natural sources. Nature has blessed mankind with diverse biota that creates an opportunity for potential findings. The same strategy can be more easily achieved using microbial origin. Beneficial microorganisms have valuable sources of compounds which influences human health either by direct or indirect ways. Mushrooms are a special group of macro fungi and are premier recyclers on the planet. According to recent estimates, mushrooms constitute at least 12,000 species in the ecosystem and 2,000 species are reported as

edible among the known. About 35 edible mushroom species are commercially cultivated and nearly 200 species were collected from wild and used for medicinal purposes. After the discovery of the first wonder drug, Penicillin from filamentous fungi much more attention has been carried out in therapeutic usage of fungus especially from medicinal mushrooms. The fruiting body of mushroom contains excellent source of nutrients, high proteins, low in calories, rich in minerals, fibers, essential amino acids and vitamins are considered as a vegetable meat. The modern cultivation technology has been implicated for economic growth of edible mushrooms. With an

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intensive advanced research in mushroom biology there is growing awareness about the utilization of high quality nutritional and medicinal mushrooms. The bioactive ingredients found in medicinal mushrooms are natural, simpler and make them easily ingestible in body system. At present, there is an enormous covenant of public interest in the use of medicinal mushrooms for health and healing.The future challenge is to correlate the significance of these mushrooms in healing the dreadful diseases of mankind. Mushroom research and development: Present scenario Edible mushrooms are widely used for industrial purposes like other microorganisms for the production of valuable substance. Mushroom biotechnology deals with the principles of mushroom biology and bioprocess technology to develop safe mushroom medicinal products. Though, the progress of mushroom technology has achieved tremendous improvement that allowed the scientist to address various issues raised for the new developmental approach. In order to increase the utility of mushroom, it is necessary to develop the following areas of concern. 1. Production strain selection 2. Techniques in strain improvement 3. Optimization of growth parameter 4. Post harvesting technology 5. Marketing surveillance Significant increases in the medicinal mushroom research and development were initiated especially during last four decades. The current scenario of mushroom production is fully exploited using standard cultivation methodology. About 5 million tons of mushrooms are now commercially cultivated in more than 100 countries. China is the major

producer of edible mushroom and accounts for over 64% of global production. It is generally recognized that in order to maintain high-yielding strains, the techniques employed in mushroom breeding should be frequently modified and improved by new findings, particularly in the field of Microbial biotechnology and Genetics. Development of economic cultivation technology, selection of disease resistance strains and formulation of cheap substrates has been studied in detail and recommended to mushroom growers to achieve the high yield target. As a result of this current research, 25% of the yield is increased in mushroom production. Mushrooms are not only considered as a nutritious protein-rich food but also serves as a potential source for producing pharmaceutical and nutraceutical compounds. Assuming that the proportion of useful mushrooms among the unknown shall be only 5 % and may be thousands of mushroom species yet to be discovered having possible benefits to human kind. However, valuable health benefits could be obtained from many edible and even non-edible mushrooms. Hence, there is a need to evaluate novel bioactive compounds which has drawn more attraction of researchers now days. The research area in mushroom science especially on medicinal mushrooms is now focused on characterization of such pharmaceutically important novel compounds. From a medical point of view, we have now realized the importance and benefits of mushrooms usage. In order to meet our future challenges, more innovative approaches in successful development of medicinal mushroom is essential. If research is geared up in right direction, certainly mushroom technology will bloom revealing the magic of health and healing.

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Health benefits of Medicinal mushrooms Traditionally, mushroom has been used as a delicacy for many centuries throughout the world. It has been shown that constant intake of either mushrooms or mushroom nutraceuticals (dietary supplements) can make people healthy and fit. It is conventionally used in many countries particularly in Asian countries like China, Japan and India to indulgence common diseases such as atherosclerosis, hepatitis, hyperlipidemia, diabetes, dermatitis and cancer. Due to the presence of its high protein, fiber with low fat contents which aid the dietician choice as a food for health related problems. Mushrooms such as Lingzhi (Ganodermalucidum), Shiitake (Lentinulaedodes) and Yiner (Tremellafuciformis) have been used by traditional healers. The common bioactive compound present in the mushroom includes polysaccharides, triterpeniods, glycoprotein and antibiotics. In particular, the presences of polysaccharides have been proved as potential antitumor and immune modulating properties. There have been a number of studies suggesting the possible role of these mushrooms with immune modulating, anti-diabetic, anti-tumor, anti-viral, and anti-inflammatory activities. Nutraceutical attributes of Mushrooms Edible mushrooms are good source of protein rich food, enriched minerals,B-complex vitamins, riboflavin, niacin, thiamin, folic acid, pantothenic acid, vitamins C and D. Mushrooms can be served as potential prebiotics which contains carbohydrates like chitin, hemicellulose, Betaglucans, mannans, xylans and galactans which enhance the immune function as well as improve digestion.The nutritional benefits of edible Oyster mushroom have high protein, low fat and also a natural source of bioactive compound lovastatin, a chemical used in pharmaceutical drugs to reduce the total cholesterol level in

human and as an important food supplement for patients suffering from hypercholesterolemia. Grifolafrondosahas more protein content, a high proportion of unsaturated fatty acids, vitamin including B1, B2,C, D, Niacin and minerals. Shiitake has rich in several anti-oxidants, antitumor substances and cardiovascular benefits. Dried and whole mushroom have been utilized in traditional medicine since long time. The mushroom, G.lucidumhas 400 different bioactive compounds which were extracted from the fruiting body, mycelia and spores. The bio active compounds especially polysaccharides, organic germanium, triterpenoids and ganoderic essence shows a significant health benefit. It reduces the incidence of tumor and also recommended as a chemo preventive agent against cancer. Numerous reports suggested its anti-viral, antiinflammatory and immunomodulating activities.The extract of this mushroom protect DNA damage related with free radicals and radiation.Novel bioactive molecules isolated from Shiitake known as lentinan (â&#x20AC;&#x153;Elixir of Lifeâ&#x20AC;?) has been licensed as an anti-cancer drug by Japanese FDA which has proved to heal on bowel cancer, liver cancer, stomach cancer, ovarian cancer and lung cancer. Its immune modulating activity shows increased host resistance to bacterial and viral infections. Lenthionine, a cyclic organosulfur compound of Shiitake mushrooms has antimicrobial activity. The mushroom, Flammulinavelutipeshas a major polysaccharide named flammulin which is proved to have an effective anti-tumor property. Frequent consumption of Enoki mushroom reduces cancer rate in the community of the Nagano city in Japan was recorded. The experiment of this mushroom extract also proved that it can be used for the treatment of liver diseases and gastric ulcer. Grifolan, a polysaccharide from Maitake mushroom (Grifolafrondosa) has been commercialized as a

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www.ijpbs.com (or) www.ijpbsonline.com chemo preventive agent against cancer. Moreover, it improves the body immune system by activates helper T cells, cytotoxic T cells, natural killer cells and macrophages in the treatment of breast, liver and lung cancer. It has anti-diabetic properties and reduces some side effects of anti-cancer drugs such as hair loss, pain, nausea etc. Anti-cancerous properties and chemo preventive activity in Agaricus mushroom was confirmed by The Japanese Cancer Association and the Japanese Pharmacological Society. Hericiumerinaceusis used for the treatment of gastric and esophageal carcinoma and ingestion of this mushroom extending the life of cancerridden patients. Tremellafusiformis extracts are used to protect liver cells from radiation damage. Anti-inflammatory, anti-viral and antioxidant activities had also been reported from Pleurotus mushroom. Bioactive compounds which aid fibrinolysis were isolated from variety of medicinal mushrooms which substantiate their significant role in treating cardiovascular diseases. Chitosan have been extracted from several mushrooms such as Pleurotusostreatus, Agaricushortensis and Lycoperdonperlatum which exhibit attractive role in multiple industrial applications, pharmacological, biomedicine and cosmetic fields. All these research has strongly recommended that medicinal mushrooms can be used as a potential source for the development of new therapeutic agents.

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CONCLUSION The increasing global population in the twentyfirst century demands high quality food with health care. Mushroom has been recognized as an alternative potential source of food and medicine to overcome the needs. More research in this area, may lead to the development of new cultivation technology using mushroom biotechnology hence, larger quantities of novel

mushrooms can be massively harvested. There is an urgent need to study the medicinal importance of wild mushrooms and it creates an opportunity for scientists to elucidate the active principle behind these mushrooms. Besides research, extension activities for exploration and establishment of medicinal mushroom farms need to be initiated. Furthermore, a healthy diet formulation can be generated which provides bioactive ingredients that promote human health and healing potential. According to the father of Medicine, Hippocrates, “Let food is your medicine and medicine is your food”, it may be concluded that mushrooms are the most potent, natural immune force ever discovered and hence it can be considered as a priceless asset for human welfare.

REFERENCES [1] Aida F.M.N.A., Shuhaimi M., Yazid M. and Maaruf A., Mushroom as potential sources of prebiotics: a review. Trends in Food science and Technology. (20):567-575, (2009). [2] Douglas L. C. and Sanders. M. E., Probiotics and prebiotics in dietetics practice. Journal of the American Dietetic Association, (108): 510-521, (2008). [3]Gao Y., Zhou. S.H., Chen G., Dai X. and Ye J., A phase I-II study of a Ganodermalucidum (Curt.:Fr.) P. Karst. Extract (ganopoly) in patients with advanced cancer. International. Journal of Medicinal Mushrooms. 4(3): 207–214, (2002). [4]Hobbs C., Medicinal Mushrooms: an Exploration of Tradition, Healing and Cultures; 1995 Kidd P. The use of mushroom glucan and proteoglucans in cancer treatment; Alternative Medicine Review .5(1):427(2000) [5]Khor E. and Lim L.Y., Implantable applications of chitin and chitosan, Biomaterials. 24(13): 2339–2349 (2003). [6]Nanba H. and Kubo K., Effect of Maitake D-fraction on cancer prevention. Annals of the New York Academy of Sciences, (833): 204-207(1997). [7]Sanchez C., Modern aspects of mushroom culture technology. Applied. Microbiology Biotechnology. (64):756-762(2004). [8]SeonJoo Yoon, MyeongAe Yu, Yu RyangPyun, Jae Kawn Hwang, Djong Chi Chu, Lekh Raj Juneja and Paulo A.S. Mourao., The nontoxic mushroom Auriculariaauricula

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www.ijpbs.com (or) www.ijpbsonline.com contains a polysaccharide with anticoagulant activity mediated by anti-thrombin. Thrombosis Research. (112):151-158(2003). [9]Stamets P., MycoMedicinals, an informative booklet on medicinal mushrooms. Mycomedia, (3): Olympia, Washington (1999). [10] Synytsya A., Mickova k., Jablonsky I., Spevacek and Erban V., Glucans from fruit bodies of cultivated mushrooms Pleurotusostreatus and Pleurotuseryngii:

IJPBS |Volume 3| Issue 1 |JAN-MAR |2013|611-615 structure and potential prebiotic activity. Carbohydrate Polymers. (10):1016. 2008. [11]Wasser S.P., Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Applied Microbiology and Biotechnology, (60):258274(2002). [12]Zang M., Cui S.W, Cheung P. C. K. and Wnag Q., Antitumor polysaccharides from mushrooms:a review on their isolation. Trends in Food Science Technology, (18):4-19(2007).

*Corresponding Author: A. Anita Margret*

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Assistant professor, Department of Biotechnology, Bishop Heber College, Tiruchirappalli- 620 017. Tamil Nadu, South India E-mail: anitamargret@gmail.com Phone: 91-97-87-395808 / 00-91-431-2770136/2770158/2772345 Fax: 00-91-431-2770293

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ResearchArticle BiologicalSciences EFFECT OF LONG TERM ADMINISTRATION OF ALUMINIUM CHLORIDE ON OXIDATIVE STRESS AND ACETYLCHOLINESTERASE ACTIVITY IN RAT BRAINS 1

*Nirmala Rajesh Naidu, 2ShankarBhat, 3Urban Dsouza

PhD Scholar, Physiology Department, Yenepoya Medical College, University Road, Deralakatte, Mangalore 575018 2 Professor & HOD, Physiology Department, Yenepoya Medical College, University Road, Deralakatte, Mangalore 575018. 3 Professor, physiology department,KVG medical college,Sullia *Corresponding AuthorEmail:Nimmiraj.naidu@gmail.com

ABSTRACT Oxidative modifications are the hallmark of oxidative imbalance in the brain of individuals with Alzheimers, Parkinsons and Prion diseases and their respective animal models. The aim of the research was to study the impact of aluminum chloride (AlCl3) administration in drinking water (7mg/kg body weight) and Dgalactose(i.p).The results revealed that the levels of lipidperoxidation were significantly increased, while the activities of superoxide dismutase (SOD) as well as reduced glutathione (GSH) content were significantly decreased in the brains of rats. Additionally, brain acetylcholinesterase (AChE) activities were significantly increased. It can be concluded that Al-induced neuronal oxidative stress and inhibition of the antioxidant system and enzyme activities could be the mechanisms of AlCl3 neurotoxicity. The stained samples were examined by means of light microscope for histological changes. Histological examinations showed clumpy of cell neurons, or reduced pyramidal cells and scanty neurofibrillary tangle which was an indication of neurodegeneration in the treated groups when compared to the control. It was however, concluded that the oral administration of aluminium chloride could induce brain damage which may impair memory and learning as seen in Alzheimer disease.These results suggest that AlCl3, enhances oxidative stress in the brain, thereby disturbing the antioxidant defense of rats. Increased oxidative stress could be one of the mediating factors in the pathogenesis of AlCl 3, toxicity in the brain.

KEY WORDS Aluminium chloride, Acetylcholinesterase, Hippocampus, Oxidative stress.

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INTRODUCTION Aluminum (Al) has the potential to be neurotoxic in humans and animals. It is present in many manufactured foods and medicines and is also added to drinking water for purification purposes (1). Al is widely used in antacid drugs, as well as in food additives and tooth paste (2). Environmental pollution with different aluminum containing compounds, especially those in industrial waste expose people to higher than

normal levels of Al (3). Particulate matters distributed by cement â&#x20AC;&#x201C; producing factories contain, high amount of Al, and animals and populations residing in the vicinity are exposed to the pollution (4). Although aluminum has been implicated in Alzheimer's disease, Parkinsonism, Dementia complex and causes extensive damage to the nervous system, to date the mechanism of Al neurotoxicity has not been fully elucidated (5). In recent researches,

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www.ijpbs.com (or) www.ijpbsonline.com aluminum has been reported to accelerate oxidative damage to biomolecules like lipid, protein and nucleic acids (6.). Therefore, the present study was carried out to investigate the alteration in biochemical parameters including free radicals, enzymes activities and histopathological alterations induced by AlCl3 in brain tissue of rats.

METHOD AND MATERIAL

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Animals Adult wistar rats weighing between 180-200g of either sex were procured from the central animal house, Yenepoya University, Mangalore. The protocol was approved by the Institutional Animal Ethics Committee (CPCSEA- registration no 347/ CPCSEA) and was carried out in accordance with the Indian National Science Academy Guidelines for the use and care of animals. Animals were acclimatized to laboratory conditions at room temperature prior to the experiments. The rats were acclimatized for one week in the animal house facility. They were housed in polypropylene cages at an ambient temperature of 25±1°C with a natural dark-light cycle. They had free access to standard pellet diet and water given ad libitum. All experiments were conducted in the forenoon (9:30 AM to 1:00 PM). Treatment group Rats were divided into two group of six animals each: a) Control group was orally administered distilled water for 90 days b) Treatment group was orally administered Aluminium chloride (7mg/kg body weight) dissolved in distilled water for 90 days and intra peritoneal injection of D-galactose (84mg/kg body) (7).

EXPERIMENTAL PROCEDURE: Preparation of the Tissue Homogenate: Brain tissues were washed with cold saline and dried. Each of these tissues was separately transferred to a glass homogenizer containing 10ml of 10mM cold phosphate buffer saline (PBS - pH 7.4). The tissues were homogenized using an electrical homogenizer (Remi 8000 RPM). The unbroken cells and cell debris were removed by centrifugation at 3000 RPM for 10 minutes by using Remi C 24 refrigerated centrifuge (-4°C). The obtained supernatant was used for the biochemical estimations. Estimation of Lipid Peroxidation Lipid peroxidation was estimated according to the method of Kartha and Krishnamurthy. (8). This assay is based upon the reaction of TBA with malondialdehyde (MDA) which is one of the aldehyde products of lipid peroxidation. Estimation of GSH GSH was estimated by Beaulter et al (9) and glutathione content was expressed as (g/gm protein). Estimation of SOD SOD was estimated by the technique explained by Fridovich(10). The activity was expressed as unit/ mgprotein Protein Estimation Protein content of the tissue samples was determined by Lowry et al method (11) Histological Study Brain Specimen used for histological study was fixed in neutral formalin for a week at room temperature dehydrated and embedded in paraffin wax. The paraffin section were cut at 20m thickness and stained with hematoxylin and eosin. Statistical Analysis The Biochemical data were subjected to one way ANOVA followed by Turkey-Kramer multiple comparison post hoc test, using Graph Pad Instat

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RESULTS Effect of Aluminium chloride on selected biochemical parameters in rat brain is present in Table 1. Administration of Aluminium chloride and D-galactose to rats for 3 months resulted in statistically significant increase in lipid peroxidation and elevation of AchE activity in comparison with control. Aluminium group

exhibited significant reduction in SOD activity as well as GSH content compared to control. The staining shows that there were typical neuropathological changes in the hippocampus of AD model rat. In the control group the neurons were full and arranged tightly, the nuclei were light-stained. By comparison in the model group rat, the cytoplasm of neurons were shrunken, the nuclei were side-moved and darkstained, neurofibrillary degeneration and neuron loss were observed in hippocampus.

Table 1: Brain antioxidants/oxidants system in rats treated with oral AlCl3 and D-galactose (i.p) Acetylcholinesterase GSH SOD LP nmol/mg activity Treatment nmol/mg nmol/mg tissue protein (μmole enzyme/mg tissue protein tissue protein protein/min)

Control group Aluminum receiving group Statistical significance

0.4960±0.10

0.4030±0.04

0.65±0.03

4.84±0.06

1.2594±0.07

0.3375±0.016

0.28±0.02

8.46±0.89

***P‹0.001

**P‹0.01

***P‹0.001

Statistical significance test done by ANOVA followed by Turkey-Kramer multiple comparisons test Values: Mean ± SD. *Significance of the results: p < 0.001.

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Figure 1:Microscopic study of hippocampus in mouse brain. Grossly (x40). Histological sections of brain were stained with hematoxylin& eosin (H&E). Control (A). Exposed rat to 7mg/kg/day AlCl (3) during 3 months (B)

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Figure 2:Microscopic study of cerebral cortex in mouse brain. Grossly (x40). Histological sections of brain were stained with hematoxylin & eosin (H&E). Control (A). Exposed rats to 7 mg/kg/day AlCl(3) during 3 months (B).

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DISCUSSION In this study, during three months observation of rats receiving aluminium chloride, decreases in water and food intake and transient diarrhoea occurred, which resulted inlowering of final body mass of animals in comparison to the controls (differences statistically significant). The rats’ body mass after three months of the study was 322±21g in the control group and 269±23g in the investigated group. In the course of the experiment no changes were observed in the behavior of animals. In humans, chronic exposure to aluminium ions may result in mood changes, dysmnesia, convulsions, muscular weakness, and pathological fractures of bones. Aluminium accumulates mainly in bones, spleen, liver and lungs [3, 4, 7, 8]. In our study the content of aluminium was not investigated in the mentioned organs. Assessment of harmful effect of aluminium ions was based on the analysis of selected biochemical parameters. Statistically significant increase of brain lipid peroxidation and decrease of reduced glutathione, SOD and AchE in animals receiving aluminium chloride is of interest. While aluminum is not a transition metal and cannot initiate peroxidation, many studies have looked for a correlation between aluminium accumulation and oxidative damage in the brain.

It has been pointed out in in-vitro studies that aluminium significantly accelerates ironmediated lipid peroxidation under acidic and neutral conditions (12). The elevation of brain lipid peroxidation in this study suggests the participation of free-radical- induced oxidative cell injury in mediating the toxicity of AlCl 3 as proposed by other studies (13); it is reported that the neurotoxicity of AlCl3 was due to the increase in brain lipid peroxidation as a result of being AlCl3 able to cross the blood–brain barrier as an L-glutamate complex and it deposits in a rat’s brain (14). As oxidative damage is mediated by free radicals, it was necessary to investigate the status of endogenous antioxidant enzymes like superoxide dismutase and glutathione, which are the first line of defense against free radical damage under oxidative stress conditions (15). In our study, chronic administration of aluminium chloride resulted in marked oxidative stress as indicated by increases in lipid peroxidation, as well as decreases in reduced glutathione, superoxide dismutase, compared to the control groups is in accordance with what has been previously reported by (16). The decreased activity might have resulted from the oxidative modification of genes that control these enzymes. Under the oxidative stress

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www.ijpbs.com (or) www.ijpbsonline.com conditions, SOD presents the first line of defense against superoxide as it converts the superoxide anion to H2O2 and O2 (17). It also has an important role in detoxifying superoxide radical to H2O2, which is then converted to H2O by CAT and GSH at the expenditure of GSH. Therefore the increased lipid peroxidation may be interpreted here by an inhibition of SOD and GSH activities and other antioxidants in the brain tissue leading to membrane damage and neuron death (17). In the present study, aluminium may have altered the cellular redox state by inhibiting the enzymes involved in antioxidant defense which functions as blockers of free radical processes as postulated (18). The results are in accordance with Nehru B, Anand P (18) who observed significant decrease in the activities of SOD in cerebrum, cerebral hemisphere and brain stem after Al exposure. Glutathione in its reduced form is the most abundant intracellular antioxidant and is involved in direct scavenging of free radicals or serving as a substrate for the glutathione peroxidase enzyme that catalyzes the detoxification of H2O2. It is also known that SOD and catalase are protective enzymes and both function in very close association for the detoxification of highly reactive free radicals. Cholinergic neurons are positive markers for the evolution of memory and related disorders affecting acetylcholine and resulting in decreased activity of acetylcholinesterase and choline acetyl transferase [19]. Recent findings suggested that administration of aluminium was found to increase acetylcholinesterase in mouse brain [20]. We also demonstrated that chronic administration of aluminium to rats significantly increased acetylcholinesterase The hippocampus and the cerebral cortex are the key structures of memory formation. Because the hippocampus is especially

indispensable in the integration of spatial information, a decline in learning ability may be induced by the deterioration of hippocampal function [20]. In this study the brains of experimental animals, studied by optical microscopy, displayed a massive cellular depletion in the hippocampal formation with neurofibrillary degeneration. We observed numerous ghost-like neurons with cytoplasmic, nuclear vacuolations and necrosis of the cerebral cortex which are form of neurodegeneration which can be due to the accumulation of Aluminium in these regions [21]. Other experimental protocols have provided evidence that Al can accumulate in hippocampus and cortex [22]. Evidence for stronger glia activation was observed in Al-exposed animals, indicative of an acceleration of pathological and inflammatory events by Al [23,]. Inflammatory responses are known to play an important role in neurodegenerative disease such as AD [24]. Recently, it has also been suggested that there may be an important link between Al, oxidative stress, inflammation and AD [25]. This is supported by our data and by other studies indicating that Al facilitates iron-induced oxidative stress in vitro [26], this may be the cause for Al-induced learning and memory deficits observed before severe neurodegeneration can be identified. This action may also be the basis for Al as a putatively contributing factor in AD.

CONCLUSION The result summarized here indicates that chronic ingestion of aluminium chloride leads to oxidative stress which is a hallmark of oxidative imbalance in the brain of individuals with Alzheimerâ&#x20AC;&#x2122;s. All our observation in the present study provides conclusive evidence that the aspects of Aluminium toxicity to human beings

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14.

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10.

11.

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Newairy A.S., Salama A.F., Hussien, H.M. and Yousef, M.I. (2009): Propolis alleviates aluminium-induced lipid peroxidation and biochemical parameters in male rats. Food ChemToxicol, 47(6):1093-8, (2009) Abbasali K.M., Zhila T. and Farshad N.: Developmental Toxicity of aluminum from High Doses of AlCl3 in Mice. The Journal of Applied Research, 5: 575-579, (2005) Kloppel H., Fliedner A. and Kordel W. :Behaviour and endotoxicology of aluminium in soil and water. Review of the scientific literature. Chemosphere. 35: 353-363, (1997) Shehla K.F., Prabhavathi P.A., Padmavathi P. and Reddy P.P.: Analysis of chromosomal aberrations in men occupationally exposed to cement dust. Mutat Res., 490: 179-186. (2001) Niu Q. Yang Y., Zhang Q., Niu P., He S., Di Gioacchino M. AndBoscolo P.: "The relationship between Bcl-2 gene expression and learning & memory impairment in chronic aluminum-exposed rats. Volume 12, Number 3, 163-169, (2007) Jyoti A., Sethi P. and Sharma D.: Bacopamonniera prevents from aluminium neurotoxicity in the cerebral cortex of rat brain. Journal of Ethnopharmacology ,111, 56–62, (2007) Pan Rui, Qiu sheng, Lu Da-Xiang and Dong Jun. Curcumin improves learning and memory ability and its neuroprotective mechanism in mice. Chin Med J., 121(9):832-839,(2008) Med Of Aryreiaspeciosa in Mice.journal of Health Science,53(4) 382-388..1963, 61; 882-890,(2007) kartha R, Krishnamurthy S. factors affecting in vivo lipid peroxidation in rat brain homogenates. Ind. J. Physiol. Pharmacol. 22(1); 44-52, (1978) Charles Beauchamp and Irwin Fridovich. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gel. Analytical Biochemistry., 276-287, (1971) Oliver H. Lowery, Nira J., Rosebrough, A.Lewis Farr, and Rose J. Randall.Protein measurement with the folin phenol reagent .265-274, (1971) Ohyashiki T, Karino T, Suzuki S, Matsui K. Effect of aluminum ion on Fe2_-induced lipid peroxidation in phospholipid liposomes under acidic conditions. J Biochem . 120:895–900, (1996) Anane R, Creppy EE Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase_

15.

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17. 18.

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21.

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25.

26.

catalase and vitamins E and C. Hum ExpToxicol 20:477– 481, (2001) Deloncle R, Huguet F, Babin P, Fernandez B, Quellard N, Guillard O . Administration of aluminum L-glutamate in young mature rats: effects on iron levels and lipid peroxidation in selected brain areas. ToxicolLett 104:65–73, (1999) Benzi G, Marzatico F, Pastoris O, Villa RF. Relationship between aging, drug treatment and the cerebral enzymatic antioxidant system. ExpGerontol 24:137– 148, (1989) Flora SJS, Mehta A, Satsangi K, Kannan GM, Gupta M. Aluminum- induced oxidative stress in rat brain: response to combined administration of citric acid and HEDTA. Comp BiochemPhysiol C ToxicolPharmacol 134:319–328, (2003) Fridovick I Superoxide radical: an endogenous toxicant. Annu Rev PharmacolToxicol 23:239–253, (1975) Nehru B, AnandP. Oxidative damage following chronic aluminiumexposure in adult and pup rat brains. J Trace Elem Med Biol .19:203–208. (2005) Dua R, Gill KD. Aluminium phosphide exposure: implications on rat brain lipid peroxidation and antioxidant defence system. PharmacolToxicol 89:315– 319, (2001) Platt, B., G. Fiddler, G., Riedel and Z. Henderson. Aluminium toxicity in the rat brain histochemical and immunocytochemical evidence Brain Res. Bull., 55: 257-267, (2001) Buraimoh, S.A. Ojo, Hambolu and S.S. Adebisi. Effects of Oral Administration of Aluminium Chloride on the Histology of the Hippocampus of Wistar Rats. Current Research Journal of Biological Sciences 3(5): 509-515,( 2011) Adebayo AdekunleBuraimoh. Effects of Aluminium Chloride Exposure on the Histology of the Cerebral Cortex of Adult Wistar Rats. Journal of Biology and Life Science. Vol. 3, No. 1, 87-113, (2012) Yokel, R.A. and J.P. O'Callaghan. An aluminum induced increase in GFAP is attenuated by some chelators. Neurotoxicol. Teratol., 20: 55-60, (1998) Rogers, J.P., S. Webster, L.F. Lue, L. Brachova W.H. Civin, M. Emmerling, B. Shivers, D. Walker and P. Mcgeer,. Inflammation and Alzheimer’s disease pathogenesis. Neurobiol. Aging, 17: 681-686, (1996) Campbell, A. and S.C. Bondy . Aluminum induced oxidative events and its relation to inflammation: a role for the metal in Alzheimer's disease. Cell. Mol. Biol., 46: 721-730,( 2000) Xie, X.X., M.P. Mattson, M.A. Lovell and R.A. Yokel. Intraneuronalaluminium potentiates iron- induced

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Yenepoya Medical College,University Road, Deralakatte, Mangalore 575018 Phone: +91 824 2204668/69/70, Fax: +91 824 2204667 E-Mail: Nimmiraj.naidu@gmail.com

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Research Article Pharmaceutical Sciences A NEW UV-METHOD FOR DETERMINATION OF BORTEZOMIB IN BULK AND PHARMACEUTICAL DOSAGE FORM CHANDRA K SEKHAR*1, P.SUDHAKAR1, G.RAMESH REDDY2, P.VIJAYA BABU3, N.LINGA SWAMY4 *Quality Control, Bio-Leo Analytical Labs India Pvt Ltd, IDA, Prasanthi Nagar, Kukatpally, Hyderabad, India *Corresponding Author Email: kudupudichandra@gmail.com

ABSTRACT A new simple easy UV-spectrophotometric method was developed for the estimation of Bortezomib in bulk and dosage form. The maximum absorption was found to be at 270 nm. Methanol was used as a diluent. The Calibration curve was linear over the concentration range of 3.5-21.0 µg/ml. The propose method was validated as per the ICH guidelines parameters like Linearity, precision, accuracy, robustness and ruggedness. The method was accurate, precise, specific and rapid found to be suitable for the quantitative analysis of the drug and dosage form.

KEY WORDS Method development and validation, Bortezomib, UV, Spectrophotometric.

1. INTORDUCTION [(1R)3methyl1({(2S)3phenyl2[(pyrazin2ylcarbonyl )amino]propanoyl}amino)butyl]boronic acid. There is no official UV-method for the Bortezomib. As per literature survey a few methods have been reported the estimation of Bortezomib individually 1-5. With this present proposed method Bortezomib estimates easy, simple and economical by UV-method in bulk and pharmaceutical formulation.

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2. MATERIAL AND METHODS 2.1 Spectrophotometric Conditions Shimadzu UV-Vis double beam spectrophotometer provided with matched 10mm quartz cuvettes equipped with UV-probe software from shimadzu corporation, Japan was employed in the study. AR grade methanol and Class-A glassware purchased from E.Merck Co; Mumbai, India were used in the study.

2.2 Drug Samples The reference samples were obtained from M/s. Bio-Leo Analytical Labs India Pvt Ltd, Hyderabad, India, the formulation samples were purchased from local market. 2.3 Preparation of stock and working standard solution of Bortezomib About 3.5mg of Bortezomib was weighed accurately on Sartorius semi micro balance model-CPA225D and transfers in to 25ml volumetric flask the solution was sonicated and the resulting solution was diluted with the methanol to get a working standard solution of 140 µg/ml. 2.4 Sample Preparation Weighed accurately equivalent to 3.5 mg of sample transferred to 25ml volumetric flask make up to the mark with methanol sonicated and filtered through 0.45µ membrane filter paper. Further dilute 10ml to100 ml with methanol.

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RESULTS AND DISCUSSION

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The present study was aimed at developing a simple economical precise and accurate UV method for the analysis of Bortezomib in bulk drug and in pharmaceutical dosage form. Methanol was used as a diluent. A good linear relationship (r2 = 0.993) was observed for Bortezomib. The regression concentration and absorbances are given in Table 1 & 2. When test solutions were analyzed by the proposed method for finding out intra and inter-day variation, low co-efficient of variation was observed. High recovery values obtained from the dosage form by the proposed method indicates the

method is accurate. The drug content in tablets was quantified using the proposed analytical method are given in Table 3. The deliberate changes in the method have not much affected the results. This indicated the robustness of the method. The lowest value of LOD and LOQ as obtained by the proposed method by calculated using 3.3xstdev/slope for LOD and 10xstdev/slope for LOQ. The standard solution of the drug was stable up to 24 hrs as the difference in percent assay during the above period is within limit system suitability parameters were studied with six replicates standard solution of the drug and the calculated parameters are within the acceptance criteria. The system precision was established by six replicate of the standard solution containing analytes of interest. The values of relative standard deviation were found within the limit, indicating the repeatability of the method. The relative standard deviation was found within the limit, indicating the injection repeatability of the method. The results were presented in Table 4. The diluted preparations of marketed tablets were injected in duplicate and the results were calculated and presented in Table 5. Hence it can be concluded that the proposed UV method is simple economical sensitive and reproducible for the analysis of Bortezomib in bulk and in pharmaceutical dosage form.

Table 1: Optical characteristics of the proposed method Parameter Value Absorption Maxima(nm) 270 Beer’s law 0.993 Regression equation (Y=mX+c) Y=0.025x+0.023 Slope(m) 0.025 Intercept(c) 0.023 LOD(µg/ml) 0.09939 LOQ(µg/ml) 0.3012

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Table 2: Calibration data of the proposed method Bortezomib Conc Mean Area (mcg/ml) 3.500 0.123 7.000 0.201 10.500 0.307 14.000 0.381 17.500 0.456 21.000 0.535 Table 3 : Accuracy data (Triplicate values at 50,100 &150 percent levels) Amount taken Amount found Percent Recovery Percentage of mean (µg) (µg) recovery 7.0 7.10 101.42 101.42 Bortezomib 10.5 10.45 99.52 99.52 17.5 17.42 99.54 99.54 *Each value is a mean of three readings Table 4: Precision Study S.No. Abs 1 0.307 2 0.306 3 0.306 4 0.307 5 0.305 6 0.306 avg 0.306167 stdev 0.000753 %RSD 0.246

% of Assay 100.57

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Drug Bortezomib

Table 5: Assay Results Amount present/ml 3.52 mg

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Figure 1: UV-spectrum of Bortezomib (10.5mcg/ml).

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Figure 2: Linearity of Bortezomib

ACKNOWLEDGEMENT The authors are thankful to M/s BIO-LEO ANALYTICAL LABS INDIA PVT.LTD, HYDERABAD for

providing a gift samples and laboratory facilities, India for encouragement.

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REFERENCES 1.

S.Venkat Rao, M.Srinivasa Rao, G.Ramu and C.Rambabu, UV Visible spectrophotometric determination of Bortezomib in its bulk and formulation dosage forms, DER PHARMACIA LETTRE, 2012, 4(3): 720-727. C.Rambabu, S.Venkat Rao, G.Rambabu and M.Ganesh, Estimation of Bortezomib in bulk and its pharmaceutical dosage forms by using a novel validated accurate RPHPLC, International journal of pharmacy and pharmaceutical sciences, 2011, 3(3): 303-305. Scott E Walker, Debbie Milliken and Shirley law, Stability of Bortezomib reconstituted with 0.9% sodium chloride

at 4 and 23 degrees, Can J Hosp Pharm, 2008, 1(1):1420. Kasa Srinivasulu, Mopidevi narasimha naidu, kadaboina rajasekhar, murki veerender, and mulukutla venkata suryanarayana, Development and validation of stability indicating LC method for the assay and related substances determination of a proteasome inhibitor Bortezomib, Chromatography Research International, 2012, doi:10.1155/2012/801720. Venkataramana M, K Sudhakar babu, and SKC Anwar, A validated stability-indicating UFLC method for Bortezomib in the presence of degradation products and its process related impurities, J chromatograph Separat techniq, 2012, doi.org/10.4172/21577064.100017

*Corresponding Author:

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CHANDRA K SEKHAR, Bio â&#x20AC;&#x201C;Leo Analytical Labs India Pvt Ltd, IDA, Prasanthi Nagar, Kukatpally, Hyderabad, India. Email: kudupudichandra@gmail.com

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Research Article Pharmaceutical Sciences SYNTHESIS AND SCREENING OF BIOLOGICALLY SIGNIFICANT INDOLE DERIVATIVES FOR ANTICONVULSANT ACTIVITY K.Swathi*and M. Sarangapani Medicinal Chemistry Laboratory, U.C.P.Sc., Kakatiya University Wararagal-506009, Andhra Pradesh, India *Corresponding Author Email: kswathi84@yahoo.co.in

ABSTRACT In the present work, some new 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-diones and 5-Hydroxyindole 3semicarbazone 2-ones were prepared from 5-hydroxy isatin. The structures of the products were characterized by IR, NMR, MASS Spectral studies. All the compounds were examined for anticonvulsant activity by maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) induced convulsion method. These compounds were also evaluated for their neurotoxicity study by rotorod method. Some of these compounds showed good anticonvulsant activity when compared with standard drug Phenytoin and all the compounds showed less neurotoxicity when compared with standard drug Diazepam.

KEY WORDS Synthesis, 5-[2(3)-dialkyl amino alkoxy] Indole 2, 3-diones, 5-Hydroxyindole 3-semicarbazone 2-ones, Anticonvulsant activity.

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1. INTRODUCTION Epilepsy, one of the common neurological disorders, is a major public health problem, affecting around 4% of individuals over their lifespan. About 20-30% of the epilepsy patients are resistant to the available medical therapies. This fact warrants the investigation for new antiepileptic drugs. Isatin is an endogenous compound isolated in 1988 and reported [1] to possess a wide range of central nervous system activities. Surendranath pandya [2] et al. reported the synthesis and anticonvulsant activity of some novel nmethyl/acetyl, 5-(un)-substituted isatin-3semicarbazones. In the last few years, Isatin derivatives have been discovered which show potential hypnotic [3], antibacterial [4-6] and MAO inhibitory [7] activity.

It is evident from the literature survey that Isatin derivatives, isatin semicarbazone derivatives and dialkylamino alkyl derivatives showing more promising central nervous system and anticonvulsant activities. Keeping in view of these two molecular moieties viz., 5-hydroxy isatin (Resembles serotonin) and dialkylamino alkyl (Resembles NT), it is our endeavor to bring such important moieties into a single molecular frame as a model for molecular conjunction by appropriate synthetic routes and to screen them for anticonvulsant activity and neurotoxicity.

2. MATERIALS AND METHODS The compounds were mostly synthesized by conventional methods and described in experimental selection and also by the methods established in our laboratory.

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2.1. Chemicals Leptazole, Diazepam, Dialkylaminoalkylhalides, semicarbazidehydrochloride purchased from Sigma- Aldrich Chemicals Private Limited, Hyderabad, India. p-amino phenol, hydroxylamine hydrochloride, sodium sulfate were purchased from Merck Chemicals Private Limited, Hyderabad, India. 2.2. Chemistry Solvents were dried or distilled before use. Melting points were obtained on a Thoshniwall melting point apparatus in open capillary tubes and are uncorrected. The purity of the compounds were ascertained by TLC on silica gel â&#x20AC;&#x201C;G plates(Merck).Infrared spectra(IR) were recorded with KBR pellet on a Perkin-Elmer BX series, Infrared spectrophotometer. Mass spectra were recorded by the direct inlet method on Thadmam-mass-quantam API 400H mass spectrophotometer.1H NMR spectra were recorded on Brucker spectrospin 400 MHz spectrophotometer in DMSO-d6. 5-hydroxy Isatin was synthesized from p- amino phenol by using Sandmayer[8] method It consists in the reaction of aniline with chloral hydrate and hydroxylamine hydrochloride in aqueous sodium sulfate to form an isonitrosoacetanilide, which after isolation, when treated with concentrated sulfuric acid, furnishes isatin in >75% overall yield. 2.3. Preparation of 5-Hydroxyindole 3semicarbazone 2-one 5-Hydroxyisatin was heated under reflux in methanol containing two or three drops of acetic acid with semicarbazide hydrochloride for half an hour. The product thus separated was filtered and purified by recrystalization from suitable solvent. (Yield 89%, m.p.270oc)

2.4 Preparation of 5-[2(3)-dialkyl amino alkoxy] Indole 2,3 diones and 5-Hydroxyindole 3semicarbazone 2-ones A mixture of 5-hydroxyisatin/5-Hydroxyisatin-3semicarbazone (0.01 Moles) and dialkylamino alkylhalide (0.01 Moles) placed in 10% alcoholic potassium hydroxide and this mixture was stirred at room temperature for 6 hours .The alcohol was reduced to half of its volume and cooled. The product separated was filtered, washed with small portions of cold alcohol repeatedly and dried .It was purified by recrystalisation from hydro alcoholic mixtures to get a crystalline solid. Similarly other 5-Hydroxy Isatin derivatives as shown in Scheme 1 were prepared and their melting points were determined in Open capillary tubes using Toshniwall melting point apparatus and are uncorrected. Purity of the compounds was checked by TLC. The physical data of the title compounds were presented in Table 1. The compounds were characterized by spectral data. 2.5. Spectral data The compounds have been characterized by the spectral data IR, PMR and Mass. IR spectrum (KBr) of compound (III) exhibited absorption bands (cm-1) 3421.47 (OH), 1630.08 (C = O), 1548 (Ar,C=C), 1282(C-O-C), 883.85579.8 (Ar). 1H NMR (300 MHz, DMSO-d6): 13.3 (s, 1H, OH), 10.36(s, 1H,-CONH), 6.65-7.29(m, 3 H, Ar-H). Mass spectrum of compound III showed molecular ion (M+) base peak at m/z (164.1). Compound (IIIa) showed characteristic IR peaks at 3276(NH), 1651.96 (C=O), 1569.82 (Ar, C=C), 1276(C-O-C), 807.93(Ar). 1H NMR (300 MHz, DMSO-d6):10.36(s, 1H,-CONH ), 7.01-7.29(m,3 H,Ar-H),3.2 (T,2H,O-CH2 s) ,2.9 (T,2H,N-CH2), 1.36 (S,6H,N-(CH3)2).Mass spectrum of compound IIIa showed molecular ion (M+) base peak at m/z 231 (100%).It also shows peak at m/z (71) may be due to the fragmentation of the alkyl chain from the molecule ion.

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Scheme 1: Synthetic protocol of the title compounds.

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Table I: Physical data of 5-[2(3)-dialkyl amino alkoxy] Indole 2,3 diones and 5-Hydroxyindole semicarbazone 2- ones

C12H14N2O3 C14H18N2O3 C13H16N2O3 C13H16N2O3

% YEILD 91% 86% 93% 85%

<320 <320 <320 <320

234 262 248 248

C16H24N2O3

81.8%

<320

292

1 1 2 1

NNHCONH2 NNHCONH2 NNHCONH2 NNHCONH2

C13H17N5O3 C15H21N5O3 C14H19N5O3 C14H19N5O3

92% 83% 92% 86%

<320 <320 <320 <320

291 319 365 365

NNHCONH2 C17H27N5O3

82%

<320

349

S.No

Compound

M.F

1 2 3 4

IIIa IIIb IIIc IIId

CH3 C 2H 5 CH3 CH3

H H H CH3

1 1 2 1

O O O O

H H H CH3 H

M.P

M.Wt

CH3

IIIe

CH2 H3C

6 7 8 9

IVa IVb IVc IVd

CH3 C 2H 5 CH3 CH3 CH3

IVe

CH2

Page

631

H3C

Compound (IIIb) showed characteristic IR peaks at 3274(NH), 1681.53 (C=O), 1570.21 (Ar ,C=C), 1243(C-O-C), 845.51(Ar). 1H NMR (300 MHz, DMSO-d6): 10.25(s, 1H,-CONH ), 7.03-7.45(m,3 H,Ar-H),2.99 (T,2H,O-CH2 s) ,2.72 (T,2H,N-CH2) ,1.24 (S,10H,N-(C2H5)2). Mass spectrum of compound IIIb showed molecular ion (M+) base peak at m/z 263 (100%).It also shows peak at m/z (71) may be due to the fragmentation of the alkyl chain from the molecule ion. Compound (IIIc) showed characteristic IR peaks at 3274(NH), 1651.96 (C=O), 1579.72 (Ar, C=C), 1266(C-O-C), 805.91(Ar). 1H NMR (300 MHz, DMSO-d6):10.46(s, 1H,-CONH ), 7.21-7.49(m,3 H,Ar-H),2.84 (T,2H,O-CH2) , 2.51 (M,2H, CH2),2.48 (T,2H,N-CH2), 1.25 (S,6H,N-(CH3)2).Mass spectrum of compound IIIc showed molecular ion (M+) base peak at m/z 247 (100%). Compound (IIId) showed characteristic IR peaks at 3257(NH), 1679.64 (C=O), 1546.86 (Ar, C=C), 1245(C-O-C), 812.71(Ar). 1H NMR (300 MHz, DMSO-d6):10.51(s, 1H,-CONH ), 7.12-7.42(M,3 H,Ar-H),2.76 (M,2H,O-CH2) , 2.45 (T,3H, R1=CH3),2.31 (M,1H,N-CH), 1.44 (S,6H,N-

(CH3)2).Mass spectrum of compound IIId showed molecular ion (M+) base peak at m/z 247 (100%). Compound (IIIe) showed characteristic IR peaks at 3257(NH), 1689.46 (C=O), 1576.34 (Ar, C=C), 1228(C-O-C), 814.53(Ar). 1H NMR (300 MHz, DMSO-d6):10.26(s, 1H,-CONH ), 7.34-7.51(m,3 H,Ar-H),2.96 (T,2H,O-CH2 s) ,2.82 (T,2H,N-CH2), 1.35 (S, 2H,N-CH) ,1.21 (D,12H,Csss -(CH3)2).Mass spectrum of compound IIIe showed molecular ion (M+) base peak at m/z 291 (100%).It also shows peak at m/z (71) may be due to the fragmentation of the alkyl chain from the molecule ion. Compound (IVa) showed characteristic IR peaks at 3276(NH), 1651.96 (C=O), 1569.82 (Ar, C=C), 1276(C-O-C), 807.93(Ar). 1H NMR (300 MHz, DMSO-d6): 10.36(s, 1H,-CONH ), 7.01-7.29(m,3 H,Ar-H),3.2 (T,2H,O-CH2 s) ,2.9 (T,2H,N-CH2), 1.36 (S,6H,N-(CH3)2).Mass spectrum of compound IVa showed molecular ion (M+) base peak at m/z 291 (100%). The mass spectrum shows its base peak at m/z 77 (100%) may be due to the fragmentation of the semicarbazone from the molecule ion.

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DMSO-d6): 10.26(s, 1H,-CONH ), 7.34-7.51(m,3 H,Ar-H),2.96 (T,2H,O-CH2 s) , 7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH),2.82 (T,2H,N-CH2), 1.35 (S, 2H,N-CH) ,1.21 (D,12H,Csss -(CH3)2).Mass spectrum of compound IVe showed molecular ion (M+) base peak at m/z 347 (100%). The mass spectrum shows its base peak at m/z 77 (100%) may be due to the fragmentation of the semicarbazone from the molecule ion. 3. Pharmacology 3.1. Anticonvulsant Activity Materials: Normal saline, test compounds, Leptazole, stop watch, phenytoin. Animals: Swiss mice. 3.1.1. Maximal electroshock seizure (MES) method [9] Method: The anticonvulsant activity was studied by Maximal Electroshock Induced Convulsion method by using electro-convulsometer. Healthy male mice weighing between 20-25g were fasted for overnight and divided into groups of six animals each. The test compounds suspended in normal saline were administered at a dose of 100 mg/kg body weight i.p. The control group animals received only vehicle (Normal saline). The test started 30 min after i.p. injection. Maximal seizures were induced by the application of electrical current to the brain via corneal electrodes. The stimulus parameter for mice was 50 mA in a pulse of 60 Hz for 200 ms. Abolition of the hind limb tonic extensor spasm was recorded as a measure of anticonvulsant activity; results are presented in Table 2.

Page

632

Compound (IVb) showed characteristic IR peaks at 3274(NH), 1681.53 (C=O), 1570.21 (Ar ,C=C), 1243(C-O-C), 845.51(Ar). 1H NMR (300 MHz, DMSO-d6):10.25(s, 1H,-CONH ), 7.03-7.45(m,3 H,Ar-H),2.99 (T,2H,O-CH2 s) ,2.72 (T,2H,N-CH2) ,1.24 (S,10H,N-(C2H5)2), 7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH).Mass spectrum of compound IVb showed molecular ion (M+) base peak at m/z 317 (100%). The mass spectrum shows its base peak at m/z 77 (100%) may be due to the fragmentation of the semicarbazone from the molecule ion. Compound (IVc) showed characteristic IR peaks at 3274(NH), 1651.96 (C=O), 1579.72 (Ar, C=C), 1266(C-O-C), 805.91(Ar). 1H NMR (300 MHz, DMSO-d6):10.46(s, 1H,-CONH ), 7.21-7.49(m,3 H,Ar-H),2.84 (T,2H,O-CH2) , 2.51 (M,2H, CH2), 7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH),2.48 (T,2H,N-CH2), 1.25 (S,6H,N-(CH3)2). Mass spectrum of compound IVcshowed molecular ion (M+) base peak at m/z 363 (100%). Compound (IVd) showed characteristic IR peaks at 3257(NH), 1679.64 (C=O), 1546.86(Ar ,C=C), 7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH) 1245(C-OC), 812.71(Ar). 1H NMR (300 MHz, DMSO-d6): 10.51(s, 1H,-CONH ), 7.12-7.42(M,3 H,Ar-H),2.76 (M,2H,O-CH2) , 7.41-7.46(d,2H, NH2) ,11.36(S,1H, NH), 2.45 (T,3H, R1=CH3),2.31 (M,1H,N-CH), 1.44 (S,6H,N-(CH3)2).Mass spectrum of compound IVd showed molecular ion (M+) base peak at m/z 363 (100%). Compound (IVe) showed characteristic IR peaks at 3257(NH), 1689.46 (C=O), 1576.34 (Ar, C=C), 1228(C-O-C), 814.53(Ar). 1H NMR (300 MHz,

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S.No 1

Table 2: Anticonvulsant activity and neurotoxicity study of 5-[2(3)-dialkylamino alkoxy] Indole 2,3-diones Animals protected in % Neurotoxicity Compound MES induced Chemically induced (%) Convulsions convulsions IIIa 65.54 60.9 7

IIIb

58.61

64.6

IIIc

48.42

56.6

6.7

IIId

41.5

49.8

3.4

IIIe

37.46

44.83

4.6

IVa

65.18

71.2

IVb

66.68

65.77

IVc

54.76

44.3

6.2

IVd

38.18

40.3

7.8

IVe

37.44

33.9

Phenytoin

100

89.36

Control

Diazepam

Page

633

Number of animals n=6, The compounds were tested at adose of 100mg/kg (b.w)

3.1.2. Pentylenetetrazole (PTZ) method [10] Materials: Normal saline, test compounds, Leptazole, stop watch, phenytoin. Animals: Swiss mice. Method: The anticonvulsant activity was studied by using Leptazole (Pentylene tetrazole) as a chemical convulsion inducer. Healthy male mice weighing between 20-25g were fasted for overnight and divided into groups of six animals each. The animals were injected with Leptazole (80mg/kg) given intraperitonially. Those animals which show convulsions were selected for the experiment. The test compounds suspended in normal saline were administered at a dose of 100 mg/kg body weight i.p. The control group animals received only vehicle (Normal saline).

The Leptazole is again given in the same dose and the time taken for convulsions to start was noted, results are presented in Table 2 and Figure 1. 3.1.3. Neurotoxicity study [11] Healthy male mice weighing between 20-25gm were fasted for overnight and divided into groups of six animals each. Turn of the rotating rod, select an appropriate speed (25 rpm), and place the animal one by one on the rotating rod. A normal mouse (untreated) generally falls off within 3-5 min. Test compound dissolved in saline were administered, intraperitonially in a dose of 100 mg/kg. The control group received saline only. One group of animals was administered diazepam as a standard (i.p 4

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diazepam treatment respectively, results are presented in Table 2 & Figure 2.

Page

634

Figure 1: Anticonvulsant activity of 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-dions and 5-Hydroxyindole 3-semicarbazone 2-ones.

Figure 2: Neurotoxicity study of the 5-[2(3)-dialkylamino alkoxy] Indole 2, 3-diones and 5-Hydroxyindole 3- semicarbazone 2-ones

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Physical data TLC, IR, H NMR and mass spectra confirmed the structures and purity of the synthesized compounds. All the title compounds decomposed before melting. All the synthesized compounds were evaluated for their in vivo anticonvulsant and skeletal muscle relaxant activity. It was observed that compounds IVa, IIIa, IVb, IIIb, exhibited more promising anticonvulsant activity. Among the test compounds IIIc, IVc, IIId, IVd, IIIe, IVe were found to be next in the order of reducing the duration of convulsions. Compounds with dimethyl amino and diethyl amino ethyloxy group at c5 of Isatin showed more protection against Maximal Electroshock Seizure (MES) induced convulsions where as compounds IIIa, IIIb and IVa, IVb exhibited more protection activity against chemically (Leptazole)induced convulsions. 5-[2(3)-dialkylamino alkoxy] isatin 3-semicarbazones showing more activity compared to 5-[2(3)-dialkylamino alkoxy] isatins .All the test compounds showed less (<20%) neurotoxicity (Skeletal muscle relaxant activity) when compared with Diazepam.

amino ethyl chain derivatives was found to be relatively superior in anticonvulsant activity and other compounds(IIIc, IVc, IIId, IVd, IIIe, IVe ) are next in the order of activity.All the compounds showed less neurotoxicity compared to Diazepam.

ACKNOWLEDGEMENTS The First author would like to thank the CSIR, New Delhi for providing financial support. Authors are thankful to Principal University College of Pharmaceutical Sciences, Kakatiya University for providing facilities.

REFERENCES 1.

K.Bhattacharya Salil, K.Mitra Shankarand B.AcharyaSatya, J.Psychopharmacol,5, 202(1991). 2. S.N. Pandeya and A. Senthil Raja, J. Pharm. Sci., 5(3), 275 (2002). 3. Pandeya SN, Yogeeswari P and Stables jp.Eur J Med Chem.35, 879-86 (2000). 4. A.K. Padhy, S.K. Sahu, P.K. Panda, D.M. Kar and P.K. Misro, Indian J. Chem., 43B, 971 (2004). 5. A. Raviraj, Kusanur, Manjunath Ghate and Manohar,V. Kulkarni, J. Chem. Sci., 116(5), 265 (2004). 6. S. Gupta, Raman, S.N.Vikas, Srivastava, Asian J. chem., 16(2), 779-783 (2004). 7. B. Gringberg, L. Imazylis and M. Benhena, Chemija, 2, 87 (1990). 8. C.S.Marvel and G.S.heirs,Organic synthesis Collect.,1,327(1941). 9. R.L. Krall, J.K. Penry, B.G. White, H.J. Kupferberg & E.A. Swinyard, Epilepsia., 19, 409(1978). 10. H.Gerhard Vogel(Ed), Drug Discovery and Evaluation of Pharmacological assays, IInd Edition 487 (2002). 11. H.Gerhard Vogel(Ed), Drug Discovery and Evaluation of Pharmacological assays, IInd Edition 398s (2002).

Page

635

5. CONCLUSION A new series of five 5-[2(3)-dialkyl amino alkoxy] Indole 2,3 dione derivatives were synthesized by reacting 5-hydroxyindole 2,3 dione with 2-N,N di alkylamino alkyl halides.Evaluation of these compounds as anticonvulsant and skeletal muscle relaxant activity revealed that the compounds IVa(R=CH3), IVb(R=C2H5), IIIa(R=CH3) and IIIb(R=C2H5) with a dimethyl and diethyl

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Medicinal Chemistry Laboratory, U.C.P.Sc., Kakatiya University, Wararagal-506009, A.P,India. Email: kswathi84@yahoo.co.in

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International Journal of Pharmacy and Biological Sciences

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