IJPBS JOURNAL VOLUME 5 ISSUE 4 2015 by jaypalreddy g

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INTERNATIONAL JOURNAL OF PHARMACY AND BIOLOGICAL SCIENCES A Peer Reviewed (Refereed/ Juried) International Journal www.ijpbs.com or www.ijpbsonline.com

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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Volume 5 Issue 4 Oct-Dec, 2015 CONTENTS EVALUATION OF IN VIVO CENTRAL ANALGESIC ACTIVITY AND PRELIMNARY 01-05 PHYTOCHEMICAL SCREENING OF METHANOLIC EXTRACT OF TERMINALIA BROWNII LEAVES Gomathi Periasamy*, Yewbnesh Alemayehu, Workagegnehu Tarekegn, Biruk Sintayehu, Mebrahtom Gebrelibanos, Gereziher Gebremedhin, Aman Karim

SYNTHESIS AND PHARMACOLOGICAL SCREENING OF NEW ISATIN-3-[N2-(BENZIMIDAZOL-1- 06-12 ACETYL)] HYDRAZONE J. Venkateshwar Rao*2, M. Sarangapani1, N.Jeevan Kumar2, A.Jyothsna2

ASSOCIATION OF BMI WITH INSULIN RESISTANCE IN TYPE 2 DIABETES MELLITUS-A STUDY 13-19 IN LOCAL TELANGANA POPULATION 1Madhavi

Kandregula, 2Noorjahan Mohammed and 3Priscilla Abraham Chandran

SYNERGISTIC ANTINEOPLASTIC ACTIVITY OF BERBERINE AND DOXORUBICIN ON A CHEMICALLY INDUCED HEPATOCELLULAR CARCINOMA IN RATS

20-31

Bakheet E. M. Elsadek1,* Gamal M. K. Atwa1, Hisham H. Taha1, Tahia H. Saleem2 1Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Assuit Branch, P.O. Box 71524 Assiut, Egypt 2Department of Biochemistry, Faculty of Medicine, Assiut Uniersity, P.O. Box No. 71526, Assiut, Egypt

MICROALGAE AS SOURCE FOR POTENTIAL ANTI-ALZHEIMER´S DISEASE DIRECTED 32-38 COMPOUNDS - SCREENING FOR GLUTAMINYL CYCLASE (QC) INHIBITING METABOLITES S. Krause-Hielscher1, H.-U. Demuth2, L. Wessjohann3, N. Arnold3, C. Griehl1* 1 Anhalt University of Applied Sciences, Department of Applied Biosciences and Process Technology, Group Algae Biotechnology, 06366 Köthen, Germany EFFECT OF ACUTE EXPOSURE OF FORMALDEHYDE ON PULMONARY FUNCTION TESTS OF FIRST YEAR M.B.B.S. STUDENTS Shital Rameshrao Mankar and Amita Rajesh Ranade

39-43

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III

EVALUATION OF IN VIVO CENTRAL ANALGESIC ACTIVITY AND PRELIMNARY PHYTOCHEMICAL SCREENING OF METHANOLIC EXTRACT OF Terminalia brownii LEAVES Gomathi Periasamy*, Yewbnesh Alemayehu, Workagegnehu Tarekegn, Biruk Sintayehu, Mebrahtom Gebrelibanos, Gereziher Gebremedhin, Aman Karim Pharmacognosy Course and Research Team, Department of Pharmacy, College of Health Sciences, Mekelle University, Mekelle, Tigray- 231, Ethiopia.

*Corresponding Author Email: pgoms@yahoo.com

ABSTRACT Background: Terminalia brownii is a herbal plant which has been used in traditional medicine in western, eastern and southern Africa to treat some disease conditions. Terminalia brownii Fresen (Combretaceae) locally known as “Abalo” in Amharic and “Weiba” in Tigrigna is a medicinal plant which is traditionally used in western, eastern and southern Africa to treat some disease conditions like malaria, hepatitis and yellow fever. Aim: The aim of this study is to evaluate the central analgesic activities of 80% methanolic extract of T.brownii leaves in experimental animals. The study was carried out in Pharmacognosy and Phytochemistry laboratory, Department of Pharmacy, College of Health Sciences, Mekelle University, Mekelle. Method: Thirty mice (28– 35g) of both sex were used for the study. Tail flick response method was used to assess the central analgesic activity of the plant extract. The methanolic extract of the leaves of T. brownii at the doses of 200 mg/kg 300 mg/kg and 400 mg/kg was used in the present study. Acute toxicity was carried out in mice to determine safe doses for use. Standard phytochemical screening methods were used to test the presence of phytoactive compounds in the plant. Results: The preliminary phytochemical screening of the extract showed the presence of flavonoids, phytosterols, polyphenols, tannins, saponin and coumarins. In oral acute toxicity study, no mortality was observed at a dose as high as 2000 mg/kg. The methanolic leaf extract of Terminalia brownii produced significant analgesic effect in a dose dependent manner and an appreciable analgesic effect was noticed at 400 mg/kg dose. Conclusion: The present study reveals that the methanolic leaf extract of T. brownii have potential analgesic activity against heat stimuli in the tested animals characterized by a profound increment in the reaction time of tail flick response when compared to the control group.

KEY WORDS Terminalia brownii, Analgesic activity, Tail immersion method

INTRODUTION Natural products continue to play an important role in the discovery and development of new pharmaceuticals, as clinically useful drugs, as starting materials to produce synthetic drugs, or as lead compounds from which a totally synthetic drug is designed1,2. The genus Terminalia is the second largest genus in the Combretaceae, the family is distributed throughout the tropical and subtropical regions of the world and approximately fifty species of Terminalia are naturally distributed throughout western, eastern and southern Africa3,4.

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Terminalia species provide economical, medicinal, spiritual and social benefit. Fruits and barks; are important sources of tannin, as well as gum and resins for glazing pottery5,6. Leaves, fruits, bark and roots of species such as T. mollis Lawson, T. ivorensis, T. laxiflora Engl. and Diels, T. catappa L. and T. superba; are sources of dyes of different colors (black, red, orange, yellow, brown) and used for decorating the walls of houses and buildings with murals, for dyeing clothes, mattings, rattan, spoons and walking sticks7. The phloem fibres are chewed and the solution swallowed in the treatment of yellow fever, particularly in children. An extract from Gomathi Periasamy* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. the leaves is used to treat pink-eye in livestock and a medicine from the bark is used in the local treatment 8,9 of hepatitis . It is believed that plants which are rich in a wide variety of secondary metabolites, belonging to chemical classes such as tannins, terpenoids, alkaloids, polyphenols are generally superior in their anti-microbial activities. Terminalia brownii Fresen (Combretaceae) is a well known plant in Ethiopia and widely used in Ethiopian traditional medicine to treat bacterial, fungal and viral infections10. The present study was carried out to evaluate the analgesic activity of T. brownii leaves in animal models.

MATERIALS AND METHODS Plant Collection and Extract Preparation Fresh leaves of Terminalia brownii were collected from “Ende-gebremariam”, Northwest Tigray and dried. The dried leaves were then powdered with a mechanical grinder and stored in airtight container. The powdered plant material (500gm) was macerated with 2.5 liter of 80% methanol for 3 days with occasional shaking and filtered. The filtrate was remacerated using 1.5 liters of the same solvent each for the next two consecutive three days and filtered. The macerates were then concentrated in a soxhlet rotary evaporator at 60ºC. The solvent was completely removed under reduced pressure and labelled TBL and used for the present study. Experimental animals Thirty swiss albino mice (24– 41g) of both sex equal in number were used for the present study. The animals were grouped and housed in cages with not more than six animals per cages and maintained under standard laboratory condition. They were allowed free access to water and standard livestock pellets. The animals were acclimatized to laboratory condition for ten days and examined to be free of wounds, swellings and infections before the commencement of the experiment. All experimental protocols were conducted in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animals. Phytochemical screening The phytochemical analysis was performed on the ground (powered) leaf of T. brownii for identification of the constituents. The constituents tested for were International Journal of Pharmacy and Biological Sciences

alkaloid, tannins, saponins, free anthraquinones, oanthraquinone glycosides, cardiac glycosides, flavonoids, polyphenols, coumarins and 11 phytosterols . Acute toxicity test The acute toxicity of methanol extract of T.brownii leaf was determined in mice according to the method of Hilaly et al., 2004 with slight modifications12. Mice fasted for 4h were randomly divided into 6 groups of five mice per group. Graded doses of the plant’s extract (100, 200, 400, 800, 1600, 2000 mg/kg p.o) and control (3ml/kg distilled water) were separately administered to the mice in each of the groups by means of oral route. All the mice in the groups were then allowed free access to food and water and observed over a period of 48 h for signs of acute toxicity. The number of deaths within this period of time was recorded. Analgesic Activity Tail immersion method Mice were divided into five groups of six animals each. Group 1, 2 and 3 received 200, 300 and 400 mg/kg of TBL respectively through oral route. Group 4 recived 2ml/kg of 0.5% methylcellulose (control). Group 5 received the standard drug pethidine (5mg/kg i.p). The animals were held in position with the whole tail extending out. The tail area (2-3 cm) was immersed in a hot water which was thermostatically maintained at 50 ± 2°C, The withdrawal time of the tail from hot water (inseconds) was noted as the reaction time or tail-flick latency. The maximum cut off time for immersion was 180 seconds in order to avoid injury of the tail tissues. The initial reading was taken immediately before administration of test and standard drugs and then 30, 60, 90, 120, 150 and 180 minutes following administration. The criterion for analgesia was postdrug latency which was greater than two times the predrug average latency. Tail-flick latency difference (TFLD) or mean increase in latency after drug administration was used to indicate the analgesia produced by test and standard drugs. Analgesia TFLD was calculated as follows: Analgesia TFLD = postdrug tail flick latency minus predrug tail flick latency13-16.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Statistical analysis Statistical analysis values for analgesic activity were expressed as "mean increase in latency after drug administration ± SEM" in terms of seconds. The significance of difference between means was determined by Student's t-test. Values of P<0.05 were considered significant and P<O.01 as highly significant.

RESULTS Phytochemical screening Preliminary phytochemicals analysis of TBL in this study revealed the presence of tannins, saponins, flavonoids, polyphenols, coumarins and phytosterols. Oral acute toxicity study In the present study, a preliminary toxicity study was designed to demonstrate the appropriate safe dose range that could be used for subsequent experiments rather than to provide complete toxicity data of the

Treatment

Control (methyl cellulose) Standard pethidine TBL TBL TBL

extract. The 80% methanolic extract of TBL did not show significant changes in behaviors such as alertness, motor activity, breathing, restlessness, diarrhea, convulsions and coma when compared with the normal control groups and none of the animals died up to a dose of 2000 mg/kg indicating that the LD50 is greater than 2000 mg/kg and hence three doses, 200 mg/kg, 300 mg/kg and 400 mg/kg were selected for the analgesic activity study. Analgesic activity Tail immersion test method In tail immersion method the extract considerably increased the animals reaction time to the heat stimuli. Values were found to be significant (p<0.05) and even strongly significant (p<0.01) at reaction time 60, 90, 120 and 180. Pretreatment with TBL at the doses of 200, 300 and 400mg/kg increased the reaction time at a dose dependent level.

Table 1 Effect of TBL on tail immersion reaction time in mice Reaction time (sec)

Dose (mg/kg) 2

0min 2.93±0.55

30min 8.6±4.12

60min 7.44±1.13

3.562±0.303 37.30±14.35 45.70±15.96**

200 300 400

5.67±4.32 27.72±24.37 13.30±8.11** 12.23±8.42 10.66±6.3131 7.76±2.68 7.78±2.49 15.27±14.17 10.03±7.77** 12.64±9.01 15.10±9.24 13.64±8.06 6.32±2.33 24.48±18.09 25.88±18.03 27.44±12.61** 23.54±13.77** 18.83±14.38 Each data represents the statistical mean latency of analgesic responses (sec) ± S.E.M. All data were found to be significant at 5% level of significance where P<0.05. ∗∗ Treatment groups were compared with control (P < 0.01).

DISCUSSION Plant products are in use for long time in folklore medicine for the cure of different diseases with minimal side effects and comparable efficacy. The plant kingdom has been explored for drugs relieving 17 pain . Accordingly this study was undertaken to evaluate the central analgesic activity of methanol extract from T.brownii leaf in experimental animal model by using tail immersion method. Here the painful reactions in animals were produced by thermal stimulus that is by dipping the tip of the tail in hot water. Analgesic effect against thermal noxious International Journal of Pharmacy and Biological Sciences

90min 7.2±1.64

120min 5.66±1.26

150min 5.30±1.2

180min 5.50±1.38

26.60±8.33**

17.60±6.38

17.70±7.85

21.10±10.74** 5.38±1.26** 9.98±5.14 13.83±10.51

stimuli may be elicited through opioid receptors or through modulation of several neurotransmitters 18 involved in relevant phenomena . In this study the extent of activity shown by the crude extracts are less than that of the standard drug pethidine but many fold more than that of the control group, which justifies its activity. Centrally acting analgesic drugs elevate pain threshold of animals towards heat and pressure19. The effect of the extract on this pain characteristic model indicates that it might be centrally acting. Pharmacological studies have shown that the Gomathi Periasamy* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. naturally -occurring opioid peptide, endorphin, interacts preferentially with µ receptors, the enkephalins with d receptors and dynorphin with k receptors. Morphine has considerably higher affinity for µ receptors than for other opioid receptors. The opioid antagonist, naloxone, inhibits all opioid receptors, but has highest affinity for µ receptors. All 3 receptors produce analgesia when an opioid binds 20 to them . Hence the mechanism of action of TBL may be due to activation of one of the three opioid receptors specially the µ receptors. Several secondary metabolites isolated from different medicinal plants have been discovered to posses’ inhibition of pro-inflammatory mediators such that reducing pain. The metabolites responsible for this effect are flavonoids, tritepeinoids, sterols (phytosterols), tannins, alkaloids, anthraquinones, coumarins, polyphenolic compounds etc. and in this study the preliminary phytochemical screening revealed the presence of flavonoids, saponins, 21 tannins, coumarins, polyphenols and phytosterols . Therefore, the analgesic activity of the extract is thought to be mediated by the presence of flavonoids, phytosterols, polyphenols, tannins, saponins and coumarins.

5. 6.

7. 8.

10.

11.

12.

13.

CONCLUSION In conclusion, this study has shown that the methanolic extract from the leaf of T.brownii does possess significant analgesic activity in laboratory animals at the doses tested and the results were comparable with the standard drug morphine. It is also suggested that the mechanism of analgesic action of TBL might be associated with activation of one of the three opioid receptors specially µ receptor.

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Soejarto DD and Farnsworth NR. Tropical Rain Forests: Potential Source of New Drugs? Perspectives in Biology and Medicine 1989, 32, 244. Farnsworth NR, Akerle O, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bulletin of World Health Organization, 1985; 63(6):965-81. Graham JG, Quinn ML, Fabricant DS, Farnsworth NR. Medicinal Plants. Journal of Ethnopharmacology 2000, 73: 347–350.

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Adebanjo AO, Adewumi CO, Essein EE. Anti-infective agents of higher plants. In: International Symposium of Medicinal Plants. Fifth ed., University of Ife, Nigeria, 1983, 152–158. Abelson PH. Medicine from plants. Science 1990, 247, 513. Batawila k, Koumaglo K, Gbe,assor M, de Foucault B, Bouchet PH, Akpagana K (2005). Antifungal activities of five combretaceae used in Togolese traditional medicine. Fitoterapia, 2005; 76: 264-268. Errington L, Chisumpa SM. Natural dyes of zambia. Mission Press, Ndola, 1987. Sokmen A, Jones BM, Erturk M. The antibacterial activity of Turkish medicinal plants. Journal of Ethnopharmacology 1999, 67: 79–86. Martin-Bettolo GB. Present aspects of the use of medicinal plants in traditional medicine. Journal of Ethnopharmacology 1980, 2: 5–7. Kokwaro JO. Medicinal Plants of East Africa. University of Nairobi Press, Nairobi, Kenya, 2009, 276. Debella A. Manual for phytochemical screening of medicinal plants. Ethiopian Nutrition and Research Institute, 2002, 45-71. Hilaly JE, Israili ZH, Lyoussi B. Acute and chronic toxicological studies of Ajuga iva in experimental animals. Journal of Ethnopharmacology 2004; 91:4330. Adeolu A, Adedapo and Sunday O, Ofuegbe. AntiInflammatory and Analgesic Activities of Soft Drink Leaf Extract of Phyllanthus amarus in Some Laboratory Animals. British Biotechnology Journal 2013, 3(2): 191-204, Moody JO, Robert VA, Connolly JD, Houghton PJ. Anti-inflammatory activities of the methanol extracts and an isolated furanoditerpene constituent of Sphenocentrum jollyanum Pierre (Menispermaceae). Journal of Ethnopharmacology 2006; 104:87-91. Sawadogo WR, Boly, Lompo M, Some N. Antiinflammatory, analgesic and antipyretic activities of Dicliptera verticillata. International Journal of Pharmacology 2006; 2:435-8. Gupta M, Mazumdar UK and Gomathi P. Antiinflamatory and Antinociceptive effects of Galega Purpurea Root. International Journal of Pharmacology 2007, 3(3): 210-218. Sini KR, Karpakavalli M and Sangeetha PT. Analgesic and Antipyretic Activity of Cassia occidentalis Linn. World Applied Sciences Journal 2010, 11 (10): 12161219 Uddin ME, Taufiqual AM, Chowdhury AU, Rahman K, Rafikul S. Sedative and analgesic activities of

Gomathi Periasamy* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Ludwigia repens. Phytopharmacology 2012, 2(2) 202211 19. Mutalik S, Utalik, Paridhavik K, Malikarjunarao C, Udupa N. Antipyretic and analgesic effect of leaves of Solanum melongena linn. In rodents. Indian Journal of Pharmacology 2003, 35: 312-315.

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Benyhe S. Morphine: new aspects in the study of an ancient compound. Life Sciences 1994, 55, 969â&#x20AC;&#x201C;79. 21. Jain PK and Himanshu Joshi. Coumarin: Chemical and Pharmacological Profile. Journal of Applied Pharmaceutical Science 2012, 02 (06): 236-240.

*Corresponding Author: Gomathi Periasamy* Email: pgoms@yahoo.com

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Gomathi Periasamy* et al

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SYNTHESIS AND PHARMACOLOGICAL SCREENING OF NEW ISATIN-3-[N2-(BENZIMIDAZOL-1ACETYL)] HYDRAZONE J. Venkateshwar Rao*2, M. Sarangapani1, N.Jeevan Kumar2, A.Jyothsna2 1

Medicinal Chemistry Laboratory, University college of Pharmaceutical Sciences, KakatiyaUniversity, Warangal-506 009, India 2 Talla Padmavathi College of Pharmacy, Urus, Kareemabad, Warangal-506 009, India

*Corresponding Author Email: pharmacyvenkat@gmail.com

ABSTRACT

Twenty new isatin-3-[N2-(benzimidazol-1-acetyl)] hydrazones were synthesized from ten different isatin-3-[N2â&#x20AC;&#x201C; (chloroacetyl)] hydrazones by reacting with benzimidazole and 2-methyl benzimidazole. The intermediates were obtained from isatin hydrazones on condensation with chloroacetyl chloride. These compounds were characterized by IR, 1H NMR and Mass spectra. All the compounds were screened for antimicrobial, antioxidant, cytotoxic activity. Some of the compounds showed promising antibacterial and antifungal activity.

KEY WORDS Benzimidazole, isatin, antibacterial, antifungal, antioxidant, cytotoxic.

INTRODUCTION In recent years several benzimidazole derivatives have been synthesized and reported to possess varied biological and pharmacological properties. They are found to be useful as antibacterial[1], antiviral[2], antitubercular[3], anthelmenthic[4], antiprotozoal[5], cytotoxic[6], ulcer inhibitor[7], anticonvulsant[8], antihistaminic[9], anti inflammatory[10]. A good number of them have been also marked as drugs like Benzitramide (analgesic), Cytostasan (anti-cancer), Clemizole (antihistaminic), Droperidol and Pimozide (psychopharmacological agent), etc. An interesting observation one could make from a careful survey of the literature is absence of any report on isatin derivatives containing benzimidazole system in the side chain at 3rd position. So, it has been felt worthwhile to undertake the present work of synthesizing such compounds for the first time by appropriate synthetic routes, with a view to evaluate for antibacterial, antioxidant and anticancer activities. The required isatins (I) and their hydrazones (II) were prepared by the standard methods available in literature. Isatin hydrazones on treatment with chloroacetylchloride afforded their respective isatin3-[N2-(chloro acetyl)] hydrazones (III). Each of these compounds was reacted with benzimidazole and 2methyl benzimidazole in presence of dry acetone with an anhydrous potassium carbonate to get the International Journal of Pharmacy and Biological Sciences

respective isatin-3-[N2-(benzimidazol / 2-methyl benzimidazol-1-acetyl)]hydrazones(IV). The intermediates and the title compounds were purified by recrystallization and characterized by analytical and spectral (IR, PMR & Mass) data.

MATERIALS AND METHODS Chemistry: All the chemicals were of synthetic grade and commercially procured from Sigma Aldrich, Mumbai, India. Melting points were determined by open capillary method and were uncorrected IR spectra were recorded on FTIR (Bruker Alpha-E) by KBr disc method.1H NMR spectra were recorded at 400MHz in CDCl3 as solvent and TMS as an internal standard using BRUKER ADVANCE 400 instrument. Mass spectra were recorded on PEP-SCIUX-APIQ pulsar mass spectrophotometer. Elemental analyses were performed on Perkin-Elmer EAL240 elemental analyzer. I) Synthesis of isatin hydrazones (II): An appropriate isatin (indole-2,3-dione) (I, 0.01 mol) was dissolved in alcohol (20 ml) and added hydrazine hydrate (99%, 0.015 mol) while shaking. The reaction mixture was stirred well, warmed on a water-bath for 10 min and left in the refrigerator for 3 hours. The resultant yellow crystalline solid was filtered, washed repeatedly with small portions of cold water and J. Venkateshwar Rao* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. finally with a small quantity of cold alcohol. The product was dried and purified by recrystallization from chloroform. The compounds thus obtained were characterized by comparison with their physical constants reported in the literature [11][12]. 2 II) Synthesis of isatin-3-[N -(chloroacetyl)] hydrazones [13] (III): An appropriate isatin hydrazone (II, 0.01 mol) was heated under reflux with chloroacetyl chloride (0.01 mol) in dry benzene under anhydrous conditions using calcium chloride guard-tube for 2 hours. The product thus formed was filtered and washed with small portions of benzene to remove the unreacted chloroacetyl chloride. It was purified by recrystallization from suitable solvent(s). III) Synthesis of benzimidazole[14]: Placed 27 g (0.25 mol) of O-phenylenediamine in a 250 ml round bottomed flask and added 17.5 gm (16 ml, 0.34 mol) of formic acid (90%). The reaction mixture was refluxed on a water bath for 2 hours, cooled, then added 10 per cent sodium hydroxide solution slowly, with constant rotation of the flask, until the mixture was just alkaline to litmus. Filter off the crude benzimidazole at the pump, washed with ice-cold water thoroughly. It was completely dried and purified by recrystallization from boiling water. The yield of pure benzimidazole, m.p. 171oC (lit. 172oC), is 25 gm (85%). Adopting this procedure 2-methylbenzimidazole was also prepared by the reaction of O-phenylenediamine and aceticacid. It was recrystallized from 10% aqueous ethanol, the yield is 2.2g (56%), m.p. 176oC (lit. 176oC). IV) Synthesis of isatin-3-[N2-(benzimidazol/2methylbenzimidazol-1-acetyl)] hydrazone(IV): 2 A mixture of an appropriate isatin-3-[N (chloroacetyl)]hydrazone (III, 0.01 mol) and a

benzimidazole (0.01 mol) or 2-methyl benzimidazole (0.01 mol) in dry acetone (20 ml) and anhydrous potassium carbonate, heated under reflux on a water bath for 5 hrs. The solvent was evaporated and poured in crushed ice. The product thus formed was filtered, washed with cold water and dried. The compound was purified by recrystallization from suitable solvent(s). Pharmacological activity: All the compounds were screened for antibacterial, antifungal, antioxidant and cytotoxic activity by following the standard protocols as available in the literature. Antibacterial activity [15]: The antibacterial activity of synthesized compounds was conducted against two gram positive bacteia viz., Bacillus subtilis and Staphylococcus aureusand two gram negative bacteia viz., Escherichia coli and Proteus vulgaris by using cup plate method. Ampicillin sodium was employed as standard to compare the results. Antifungal activity [16]: All those compounds screened for antibacterial activity were also tested for their antifungal activity by using potato-dextrose-agar medium against clotrimazole as standard. The fungi employed for screening were: Aspergillus niger, Cunninghamella verticulata. Antioxidant Activity: The method is based on the principle described by Blois et al[17] method. The model of scavenging the stable DPPH (1,1-diphenyl-2picryl-hydrazil) radical is a widely used method to evaluate antioxidant activities in a relatively shorter time compared with other methods. The effect of antioxidants on DPPH radical scavenging was thought to be their hydrogen donating ability. The reduction in absorbance is calculated as percentage inhibition as follows:

Absorbance of Blank â&#x20AC;&#x201C; Absorbance of Test % inhibition = ---------------------------------------------------------- x 100 Absorbance of Blank Cytotoxic Activity [18]: Cytotoxic activity was performed on against HBL-100 cell lines and HeLa cell lines using Microculture tetrazolium assay (MTT) method. It is based on the metabolic reduction of 3(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium bromide (MTT) to water insoluble formazan crystals with mitochondrial dehydrogenase enzyme, which gives direct correlation of viable cells.

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RESULTS AND DISCUSSION Chemistry: The reaction sequence use in the synthesis of Isatin3-[N2-(benzimidazol/2-methylbenzimidazol-1acetyl)] hydrazone (IV) is depicted in Scheme. The required isatin-3-[N2-(chloroacetyl)] bhydrazones (III) have been prepared by a reaction of respective isatin hydrazones with chloroacetyl chloride. The isatin hydrazones (II) on the other hand, have been obtained by the reaction of respective isatins (I) J. Venkateshwar Rao* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. with hydrazine hydrate (99%). These compounds (XVI) have been purified by recrystallization from suitable solvent(s) and characterized by their analytical and spectral data. The IR spectrum (in 2 KBr) of the isatin-3-[N -(chloroacetyl)]hydrazone (III, R1 = H) exhibited the absorption frequencies (in cm 1 ) at : 3238 (NH), 1700 (C=O, lactm), 1666 (C=O, acid hydrazide), 1624 (C=C, aromatic), 1533 (C=N), 951 (C=C, aromatic). 2 Each of the isatin-3-[N -(chloroacetyl)]hydrazones (XVI) has been subjected to a nucleophilic substitution reaction with benzimidazole and 2methyl benzimidazole in dry acetone and anhydrous potassium carbonate to get their respective Isatin-3[N2-(benzimidazol/2-methylbenzimidazol-1acetyl)]hydrazone (IV); similarly Twenty compounds

were prepared. The resultant products have been purified by recrystallization from suitable solvents and characterized by their physical data (Table 1) and spectral (IR, PMR and Mass) data. IVb, Yield -1 80%, mp 262ᵒC; IR (KBr) cm : 3422 (-NH str), 1696 (C=Ostr, lactam), 1625 (C=O, acid hydrazine), 1502 (C=Nstr); 1HNMR (δppm):12.71 (s, 1H, lactam), 11.35 (s, 1H, NHCO), 8.33 (s, 1H, -N-CH-N), 6.97-8.27 (m, 8H, Ar-H), 5.82 (s, 2H, COCH2); EI-MS: 319 (M+).

R1= H, 5-CH3, 5-Cl, 5-Br, 5-NO2, 5-SO2NO2, 5-COOH, 5-COOCH3, 6-Br, 7-CH3; R2= H, CH3 Reagents and Conditions: 1) isatin (I, 0.01 mol), ethanol, hydrazine hydrate (99%, 0.015 mol), Δ10min 2) chloroacetyl chloride (0.01 mol), dry benzene, reflux 2h 3) Benzimdazole or 2-Methyl benzimidazole (0.01mol), dry acetone, anhydrous potassium carbonate reflux 5h. Scheme: Synthesis of Isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1- acetyl)]hydrazone (IV), IVa-t

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Pharmacological activity: Antibacterial activity: 2 The antibacterial activity data of isatin-3-[N (benzimidazol / 2-methyl benzimidazol-1-acetyl)] hydrazones (IV, Table-2) indicates that the compounds have a noticeable degree of inhibition, specifically against gram-positive strain i.e., B. subtilis. Most significant of them has been found to be the compound IVa showed greater inhibitory effect against the organisms employed, particularly against B. subtilis and S. aureus with the zones of inhibition of 22 and 20 mm respectively, which has been comparable to that of the standard employed at the concentration of 10 ď g/ml. This has been closely followed by compound IVd with a 5-bromo substituent in indolinone showed significant inhibitory effect specifically against B. subtilis and E. coli with zones of inhibition of 18 mm, each. Some of the compounds showed moderate antibacterial activity against both the gram-negative organisms E.

coli and P. vulgaris. The compounds with 5carboxylic acid and 5-carbomethoxy substituents on indolinone have not exhibited any activity against gram-negative organisms. Antifungal activity: 2 The antifungal activity data of isatin-3-[N (benzimidazol/2-methyl benzimidazol-1acetyl)]hydrazones (IV) as depicted in Table-2 shows that the compounds of this series have shown antifungal action against A. niger and C. verticulata except compounds IVh, IVq and IVr, of course, with a degree of variation in their action. Compounds IVc (R1 = 5-Cl, R2 = H) and IVm (R1 = 5-Cl, R2 = CH3) have more activity against C. verticulata with the zones of inhibition of 17 mm and 16 mm respectively among all the test compounds. The data shows that this series of compounds have been found to be comparatively more effective among all the series tested for antifungal activity.

Table 1: Physical data of Isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1- acetyl)]hydrazone (IV) S.No. Compd. R1 R2 Mol. Formula m.p.(â&#x2014;ŚC) Yield (%) 1 IV a H H C17H13N5O2 262 85 2 IV b 5-CH3 H C18H15N5O2 241 80 3 IV c 5-Cl H C17H12N5O2Cl 212 78 4 IV d 5-Br H C17H12N5O2Br 279 64 5 IV e 5-NO2 H C17H12N6O4 210 88 6 IV f 5-SO2NH2 H C17H14N6O4S 272 70 7 IV g 5-COOH H C18H13N5O4 222 60 8 IV h 5-COOCH3 H C19H15N5O4 229 65 9 IV i 6-Br H C17H12N5O2Br 248 84 10 IV j 7-CH3 H C18H15N5O2 238 82 11 IV k H CH3 C18H15N5O2 259 80 12 IV l 5-CH3 CH3 C19H17N5O2 245 82 13 IV m 5-Cl CH3 C18H14N5O2Cl 240 75 14 IV n 5-Br CH3 C18H14N5O2Br 265 65 15 IV o 5-NO2 CH3 C18H14N6O4 205 75 16 IV p 5-SO2NH2 CH3 C18H16N0O4S 276 72 17 IV q 5-COOH CH3 C19H15N5O4 210 65 18 IV r 5-COOCH3 CH3 C20H17N5O4 232 68 19 IV s 6-Br CH3 C18H14N5O2Br 228 75 20 IV t 7-CH3 CH3 C19H17N5O2 252 78

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Table 2: Antibacterial activity and antifungal of Isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1acetyl)]hydrazone (IV)

S.No. Compd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

IV a IV b IV c IV d IV e IV f IV g IV h IV i IV j IV k IV l IV m IV n IV o IV p IV q IV r IV s IV t

R1 H 5-CH3 5-Cl 5-Br 5-NO2 5-SO2NH2 5-COOH 5-COOCH3 6-Br 7-CH3 H 5-CH3 5-Cl 5-Br 5-NO2 5-SO2NH2 5-COOH 5-COOCH3 6-Br 7-CH3

R2 H H H H H H H H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

Zone of inhibition (in mm) Bacterial species B. subtilis S. aureus E. coli 22 20 18 15 13 12 16 15 14 18 17 18 13 11 -13 12 11 11 --13 --17 16 15 16 15 -18 17 16 16 15 -16 15 14 18 16 17 13 12 14 15 16 -12 --14 12 -17 16 16 18 15 16 22 20 18 ----

Antioxidant activity: The IC50 values of antioxidant activity of isatin-3-[N2(benzimidazol/2-methylbenzimidazol-1-acetyl)] hydrazone derivatives (IV) are in the range of 10.45 to 17.56 M as shown in Table-3. The compound IVa (R1 = R2 = H) has shown highest percentage of free radical scavenging activity at a concentration of 10.45 M among these compounds. This has been followed by the compounds IVc, IVd and IVn with an IC50 of 10.52, 10.61 and 10.98 M respectively. Cytotoxic activity:

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P. vulgaris 17 11 12 16 ----13 -15 -13 15 13 ---14 15 17 --

Fungal species A. niger C. verticulata 15 14 12 13 13 17 10 14 8 10 10 12 8 6 --9 12 10 8 14 12 11 12 14 16 12 13 10 7 11 7 ----10 11 11 7 --19 20

IC50 values of cytotoxic activity of isatin-3-[N2(benzimidazol / 2-methyl benzimidazol-1-acetyl)] hydrazone (IV) are given in the Table-3. They are ranging from 345.59 to 473.9 M. The compound IVm (R1 = 5-Br, R2 = CH3) has shown highest cytotoxic activity among the series at a concentration of 345.59 M against HeLa cell lines. This has been followed by the compound IVd (R1 = 5-Br, R2= H) with IC50 of 347.69 M against HBL-100 cell lines. The compounds IVe, IVg, IVh, IVp, IVq, IVr and IVt have not shown any activity against HBL-100 cell lines and HeLa cell lines.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Table 3: Antioxidant activity and Cytotoxic activity of Isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1- acetyl)] hydrazone (IV). Antioxidant activity S.No.

Compd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

IV a H IV b 5-CH3 IV c 5-Cl IV d 5-Br IV e 5-NO2 IV f 5-SO2NH2 IV g 5-COOH IV h 5-COOCH3 IV i 6-Br IV j 7-CH3 IV k H IV l 5-CH3 IV m 5-Cl IV n 5-Br IV o 5-NO2 IV p 5-SO2NH2 IV q 5-COOH IV r 5-COOCH3 IV s 6-Br IV t 7-CH3 Ascorbic acid Cisplatin

R2 H H H H H H H H H H CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

IC50Value (M) 10.45 15.07 10.52 10.61 13.39 15.6 16.61 17.56 11.43 14.91 11.63 14.12 11.39 10.98 14.37 15.13 16.92 17.43 13.67 15.05 5.87 --

CONCLUSION It has been felt necessary, from the results of preliminary investigations that there is a need for further advanced studies, atleast on a few of the test compounds found to be superior.

ACKNOWLEDGEMENTS The authors are thankful to Department of Science and Technology, New Delhi for financial assistance. The authors are also grateful to Torrent Pharmaceutical, Ahmedabad for providing PMR & Mass spectra and to Prof. D.R. Krishna for helping in cytotoxic activity.

REFERENCES 1. 2. 3.

Habib, Nargues, Samuel, Abdel Hamid, Soad, EI-Hawas and M. Farmaco, 44(12) (1989) 1225. Neeru Srivastava and V.S. Misra, Indian J. Chem., 27B (1988) 298. Preeti Kagthara, Tejas Upadhyam, Rajeev Doshi and H.H. Parek, Indian J. Het. Chem., 10 (2000) 09.

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4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16.

Cytotoxic activity HBL-100 cell lines IC50 values (M) 433.13 441.86 365.28 347.69 NA NA NA NA 367.15 473.9 454.48 NA 375.59 357.79 401.58 NA NA NA 391.56 NA -25.00

HeLa cell lines IC50 values (M) 411.84 NA 349.84 325.46 NA NA NA NA 373.99 NA 463.36 NA 345.59 355.17 NA NA NA NA 365.49 NA -25.00

Polasi, Endre, Gonczi, Csaba, Korbontis, Dezso, Heja, Hing, Teljes Hu, 52, 067, 1990. E. Alcalde, I. Dinares, J. Elguero, C. Frigola, A. Osuna, S. Castanys, J. Med. Chem., 25(4) (1990) 309. Arch. Pharm., 332(4) (1999) 115. Masahiro Kise, Fusao Ueda, Shinchi Tada, Maso Murass, Ger. Offen, DE 3, 622, 036 (1987), Chem. Abstr., (10691987) 156473Z. S.P. Singh, S. Parmar and B.R. Pandey, J. Het. Chem., 14 (1977) 1093. Orjales, Aurelio, Rubi O Roya, Victor, E.S. Span, 2, 013, 41, May 1990. Kulkarni, Manohar V. Patil and D. Vemana, Arch. Pharm., 314(6) (1981), 440. N.P. Buu-Hoi and Guettier, Bull. Soc. Chim. Fr. (1946) 586. F. Knotz, Sci. Pharm., 38 (1970) 463. M. Sarangapani and V.M. Reddy, Indian Drugs, 36(6) (1999) 357. Vogel’s, Text Book of Practical Organic Chemistry, ELBS with Longman, 5 (1998) 1162. Indian Pharmacopoeia, Microbiological assay and test, ed. Vol. II (1996) A-100-107. British Pharmacopoeia (Pharmaceutical Press, London) (1953) 796.

J. Venkateshwar Rao* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. 17. M.S. Blois, Nature, 26 (1958) 1199. 18. B. Gringberg, L. Imazylis and M. Benhena, Chemija, 2 (1990) 87.

*Corresponding Author: J. Venkateshwar Rao* Email: pharmacyvenkat@gmail.com

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ASSOCIATION OF BMI WITH INSULIN RESISTANCE IN TYPE 2 DIABETES MELLITUS -A STUDY IN LOCAL TELANGANA POPULATION 1

Madhavi Kandregula, 2Noorjahan Mohammed and 3Priscilla Abraham Chandran Department of Biochemistry, Nizam’s Institute of Medical Sciences, Hyderabad

*Corresponding Author Email: m_noorjahan@yahoo.co.in

ABSTRACT Context: Type 2 diabetes (DM), a heterogeneous disorder characterized by impaired insulin secretion and insulin resistance, is closely related to obesity. But lack of the evaluation of insulin resistance syndrome in such patients could delay the initiation of therapeutic measures. Aims: we investigated the associations among BMI, insulin resistance and beta-cell function in type 2 DM. Settings and Design: A prospective study (carried out in local Telangana people) in a tertiary care hospital. Methods and Material: Total 81 subjects with DM were investigated. Fasting samples were collected for the estimation of FBS, HbA1c, Insulin and C-Peptide. Statistical analysis used: Statistical analysis was performed using Medcalc version 15.2.1 software. Data is expressed as the mean ± SD or median and 25th& 75th percentiles. A two-tailed p value of < 0.05 was considered statistically significant. Results: Study subjects were divided into 2 groups. BMI < 24.9 kg/m2 was taken as Group 1 and BMI > 25 kg/m2 was taken as Group 2. Statistically significant difference was found when BMI [22±2 vs 29±4.3 kg/m 2; p=<0.0001] HOMA IR [5.7(3.4-12.4) vs 9.74(6-22.6) p=0.008], HOMA-ß [100.1(42-153) vs 147.8(70.4-415.2)p=0.006] were compared between Group 1 &2. A higher BMI was associated with increased HOMA-IR and HOMA-β. Conclusions: A higher BMI in Diabetics is associated with insulin resistance. BMI could thus be a sensitive anthropometric marker of evaluation in obesity as well as insulin resistance in diabetics. The desirable approach would be to advocate an initiation of increasing the physical activity especially for weight reduction and if required therapeutic measures.

KEY WORDS BMI- Body Mass Index, HOMA - Homeostasis Model Assessment, IR Insulin Resistance, DM-Diabetes Mellitus

INTRODUCTION: Type2 diabetes, a heterogeneous disorder characterized by impaired insulin secretion and 1 resistance , is closely related to obesity. Insulin resistance (IR) is typically defined as decreased sensitivity or responsiveness to metabolic actions of 2 insulin . Several factors including BMI has complicated impact on insulin resistance and β-cell 3 function . Few studies analysed changes in insulin secretion depending on BMI using various tools used for quantifying insulin sensitivity and resistance directly and indirectly. HOMEOSTASIS MODEL ASSESSMENT (HOMA) uses fasting blood glucose and insulin concentrations to calculate IR and beta-cell function. HOMA-IR value of 2.5 is taken as an indicator of IR in adults4. The frequency of IR also varies among ethnic groups.

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Subjects and Methods: This study was conducted from June 2014 to July 2015 in a tertiary care hospital. Study was approved by institutional ethics committee and all participants gave informed consent. In total, 81 patients with type 2 diabetes mellitus were investigated. Diabetes was diagnosed based on the ADA criteria. Patient’s complete history including duration of diabetes, height, weight were recorded. BMI was calculated and expressed as kg/m2. Sample collection: Fasting plasma and serum samples were collected for the estimation of FBS, HbA1c, Insulin and C-Peptide. Biochemical estimations: All the parameters were estimated on automated analyzers. FBS was estimated using GOD-POD method, HbA1c by HPLC method, Insulin and CPeptide were estimated by chemiluminescence immunoassay method.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Determination of Homeostasis Model Assessment (HOMA) — IR HOMA IR and HOMA β are calculated by using the following formulas. HOMA IR = (FBS mg/dl) X (Fasting Insulin mU/L) / 450 HOMA β % = 360 X Fasting Insulin (mU/L) / FBS (mg/dl) – 63. Statistical analysis Statistical analysis was performed using Medcalc version 15.2.1 software. Data is expressed as the mean ± SD unless otherwise stated. Non-normally distributed variables were expressed as median and 25th& 75th percentiles and log-transformed for other analysis. Independent t test was used to compare the

variables between two groups. Pearson’s correlation analysis was done to observe correlation between variables. A two-tailed p value of < 0.05 was considered statistically significant.

RESULTS: Our study included 81 known cases of type 2 diabetes mellitus, out of which 36 were males and 45 were females. Study subjects were divided into two groups based on BMI. 2 Group 1 – BMI < 24.9 kg/m 2 Group 2 – BMI > 25 kg/m

Gender distribution of subjects in both the groups was shown in Table 1. Table 1: Gender distribution of study subjects Males Females Group 1 (n=28) 20 (71.4%) 8(28.5%) Group 2 ( n=53) 25 (47.1%) 28 (52.8%) Baseline characteristics of study subjects of both the groups were expressed as Mean ± SD. unpaired student t test is used to compare the difference between two groups and the results are shown in Table 2. Table 2: Baseline characteristics of study subjects Variable Group 1 (n= 28) Group 2 (n= 53) Age (yrs) 51.5 ± 9.2 52.7 ± 7.3 BMI(kg/m2) 22 ± 2 29 ± 4.3 Duration of DM (yrs) 6.9 ± 7.1 8.7 ± 7.9 As shown in table 2, Mean BMI between the two groups is different and is statistically significant (22 ± 2 kg/m2vs 29 ± 4.3 kg/m2; p <0.0001).Mean age among the two groups is almost similar.Mean duration of DM in group 2(8.7 ± 7.9yrs) is higher when compared to group 1(6.9 ± 7.1yrs), but not statistically significant (p= 0.3).

P value 0.4 <0.0001 0.3

Biochemical parameters of two groups are normally distributed and expressed as Mean ± SD and to know the significance of difference between two groups, unpaired student t test is used and the results are shown in Table 3.

Table 3: Biochemical parameters of study subjects Parameter Group 1 ( n= 28) Group 2 ( n= 53) P value FBS (mg/dl ) 156 ± 61.6 155 ± 73.1 0.9 HbA1c( % ) 8.3 ± 2.3 8.2 ± 1.8 0.8 C-Peptide ( ng/ml) 2.3 ± 1.5 3.2 ± 1.7 0.02 As shown in Table 3, mean C-Peptide levels (2.3 ± 1.5 vs 3.2 ± 1.7ng/ml; p= 0.02; Fig: 2) are significantly different between two groups.

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250 P=0.003

INSULIN (mU/L

200 150 100 50 0 Group 1

Group 2

Figure 1: Comparison of Insulin between two groups

8 7

p=0.02

6 5 C-peptide (ng/ml) 4 3 2 1 0 Group 1

Group 2

Figure 2: comparison of C-peptide between two groups FBS and HbA1c did not show any significant difference among two groups. Insulin, HOMA IR and HOMA β values are nonnormally distributed and expressed as median and

25th& 75th percentiles. The three parameters are log transformed and then compared between the two groups by using unpaired students- t test and the results are showed in Table 4.

Table 4: comparison of HOMA IR and HOMA β between two groups Group 1 Group 2 th th th th Median 25 and 75 Median 25 and 75 percentiles percentiles Insulin (mU/L) 15.4 9.2 & 36 29.3 16.7 & 62.4 HOMA IR 5.7 3.4 & 12.4 9.74 6 & 22.6 HOMA β 100.1 42 & 153 147.8 70.4 & 415.2 Parameter

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P value

0.003 0.0082 0.006

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1000

HOMA IR

p=0.008 100

HOMA IR 10

1 Group 1

Group 2

Figure 3: comparison of HOMA IR 10000 p=0.006 1000

HOMA

HOMA β (%) 100 10 1 Group 1

Group 2

Figure 4: comparison of HOMA β between two groups HOMA IR was compared between males and females in both the groups, but was not found to be significantly different.

Correlation of BMI with other variables: Pearsons’ correlation analysis was done to know the correlation of BMI with other variables and the results were shown in Table 5.

Table 5: Correlation of BMI with other variables Variable Age Duration of DM FBS HbA1c Insulin C-Peptide HOMA IR HOMA β As shown in table 5, significant positive correlation was observed between BMI with insulin (r=0.23,

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r value 0.04 0.05 -0.09 -0.10 0.23 0.3 0.3 0.22

p value 0.6 0.6 0.4 0.3 0.03 0.01 0.02 0.04

p=0.03), C-Peptide (r= 0.3; p=0.01), HOMA IR (r=0.3; p= 0.02) (fig: 5), HOMA β (r= 0.22;p=0.04),(fig : 6).

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45 40 35 BM 30 I 25 20 15 0

100

120

HOMA IR

Figure 5: correlation of BMI with HOMA IR 45 40 35 BMI

30 25 20 15 0

500

1000

1500

2000

HOMA β

Figure 6: correlation of BMI with HOMA β

DISCUSSION: With changes to modern lifestyles in recent years, the 5,6 prevalence of type 2diabetes has increased in India . An increasing BMI is known to be a contributing factor for the development of type 2 diabetes mellitus in India as well as in other countries7,8.In the present study, we investigated the associations among BMI, duration of diabetes, insulin resistance, and beta-cell function in patients with type 2 diabetes. This study has shown high prevalence of insulin resistance in women than in men. Gender difference was not significant in India9 though non-significant higher prevalence of DM was found among women in another investigation in India10. We found that a higher BMI was associated with higher values of HOMA-IR in patients with type 2 diabetes. Sung et al.11 reported that obesity is a risk International Journal of Pharmacy and Biological Sciences

factor for type 2 DM and that the relative risks for DM in subjects with a BMI of > 27 kg/m2 were significantly higher than those with a BMI of < 23 kg/m2. Chang et al.12 also reported that BMI was the most important determinant of insulin resistance even in non-obese patients with type 2 diabetes mellitus. In the present study, a higher BMI was associated with decreased insulin sensitivity, which supports the positive relationship between BMI and insulin resistance in type 2 diabetes mellitus. As insulin resistance increases, β-cells compensate by increasing insulin secretion, resulting in compensatory hyperinsulinemia and the maintenance 1 of normal glucose tolerance . In an autopsy-based study of individuals with normal glucose tolerance, a greater β -cell volume was found in obese 13 individuals . BMI was also positively correlated with Noorjahan Mohammed* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. relative β –cell volume in Korean patients with type 2 diabetes. These results suggest that increased BMI may be related to increased β -cell mass, but the impact of BMI on β -cell function is not fully understood in patients with type 2diabetes. Several previous studies have suggested that the contribution of insulin resistance and insulin secretory dysfunction might differ in nonobese and obese subjects. Arner et al.14 found the insulin response to an IV glucose infusion was impaired in both lean and obese diabetic patients, but during insulin infusion, glucose utilization was impaired only in obese volunteers. Park et al.15 reported that non-obese Korean patients with type 2 diabetes had lower levels of fasting serum C-peptide compared to obese subjects similar to this study. IR greatly reduces the sensitivity of cell walls to insulin. So the vital process whereby glucose passes through the cell wall via insulin to be converted into energy gets greatly impaired. As a result, excess glucose remains in the blood stream, causing elevated levels of blood sugar, which are sent to the liver16. Once it reaches there, the sugar gets converted into fat and carried via the blood stream throughout the body. This process can lead to weight gain and obesity. Evidences have revealed that normal function of Adipose tissue is disturbed during obesity and adipose tissue dysfunction plays a prominent role in the development and/or progression of insulin resistance17. EFFECT OF BMI ON HOMA BETA: Chang et al. 12 also reported that insulin-sensitive patients with diabetes were associated with low HOMA β. In the present study, patients with type 2 diabetes who had higher BMI also had increased values of HOMA β. In addition, BMI had a positive association for HOMA β, 18,19,20,21 similar to previous studies . Thus, our findings may suggest that increasing BMI possibly contributes to further deterioration of β -cell function with associated increasing insulin resistance.

impending Insulin Resistance (IR) and complications which are associated with IR.

CONCLUSION:

11.

A higher BMI in Diabetics is associated with insulin resistance. BMI could thus be a sensitive anthropometric marker of evaluation in obesity as well as insulin resistance in diabetics. The desirable approach would be to advocate an initiation of increasing the physical activity especially for weight reduction and if required therapeutic measures. Insulin resistance as per HOMA-IR could thus be included as a mandatory biochemical parameter, as it would forewarn the obese individuals about the International Journal of Pharmacy and Biological Sciences

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Ferrannini E. Insulin resistance versus insulin deficiency in non-insulin-dependent diabetes mellitus: problems and pros- pects. Endocr Rev 1998; 19:477-490. Singh Y, Garg M, Tandon N, Marwaha RK. A Study of Insulin Resistance by HOMA-IR and its Cut-off Value to Identify Metabolic Syndrome in Urban Indian Adolescents. Journal of Clinical Research in Pediatric Endocrinology. 2013; 5(4):245-251. doi:10.4274/Jcrpe.1127. Chung JO, Cho DH, Chung DJ, Chung MY. Associations among Body Mass Index, Insulin Resistance, and Pancreatic β-Cell Function in Korean Patients with New-Onset Type 2 Diabetes. The Korean Journal of Internal Medicine. 2012; 27(1):66-71. doi:10.3904/kjim.2012.27.1.66. Muniyappa R, Lee S, Chen H, Quon MJ. Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab. 2008; 294:15–26. Kim SM, Lee JS, Lee J, et al. Prevalence of diabetes and impaired fasting glucose in Korea: Korean National Health and Nutrition Survey 2001. Diabetes Care. 2006; 29:226–231. Qiao Q, Nyamdorj R. Is the association of type II diabetes with waist circumference or waist-to-hip ratio stronger than that with body mass index? Eur J Clin Nutr. 2010; 64:30–34. Kim SM, Lee JS, Lee J, et al. Prevalence of diabetes and impaired fasting glucose in Korea: Korean National Health and Nutrition Survey 2001. Diabetes Care. 2006; 29:226–231. Qiao Q, Nyamdorj R. Is the association of type II diabetes with waist circumference or waist-to-hip ratio stronger than that with body mass index? Eur J Clin Nutr. 2010; 64:30–34. Rising prevalence of NIDDM in an urban population in India. Ramachandran A, Snehalatha C, Latha E, Vijay V, Viswanathan M Diabetologia. 1997 Feb; 40(2):232-7. Impacts of urbanisation on the lifestyle and on the prevalence of diabetes in native Asian Indian population. Ramachandran A, Snehalatha C, Latha E, Manoharan M, Vijay V Diabetes Res ClinPract. 1999 Jun; 44(3):207-13. Sung et al. [19] reported that obesity is a risk factor for type 2 DM and that the relative risks for DM in subjects with a BMI of > 27 kg/m2 were significantly 2 higher than those with a BMI of < 23 kg/m Body mass index is the most important determining factor for the degree of insulin resistance in nonobese type 2 diabetic patients in Korea. Chang SA,

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Kim HS, Yoon KH, Ko SH, Kwon HS, Kim SR, Lee WC, Yoo SJ, Son HS, Cha BY, Lee KW, Son HY, Kang SK Metabolism. 2004 Feb; 53(2):142-6. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC Diabetes. 2003 Jan; 52(1):102-10. Different aetiologies of type 2 (non-insulindependent) diabetes mellitus in obese and non-obese subjects. Arner P, Pollare T, LithellH Diabetologia. 1991 Jul; 34(7):483-7. Past and current obesity in Koreans with non-insulindependent diabetes mellitus. Park JY, Lee KU, Kim CH, Kim HK, Hong SK, Park KS, Lee HK, Min HK Diabetes Res Clin Pract. 1997 Feb; 35(1):49-56. Niraj, A, Pradahan, J, Fakhry, H, Veeranna, V, & Afonso, L. Severity of coronary artery disease in obese patients undergoing coronary angiography: â&#x20AC;&#x153;obesity paradoxâ&#x20AC;&#x153;revisited. Clin Cardiol (2007). , 30, 391-6. Goossens, G. H. The role of adipose tissue dysfunction in the pathogenesis of obesity-related insulin resistance. Physiol Behav. (2008)

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Analysis of factors influencing pancreatic beta-cell function in Japanese patients with type 2 diabetes: association with body mass index and duration of diabetic exposure. Funakoshi S, Fujimoto S, Hamasaki A, Fujiwara H, Fujita Y, Ikeda K, Hamamoto Y, Hosokawa M, Seino Y, Inagaki N Diabetes Res Clin Pract. 2008 Dec; 82(3):353-8. C-peptide response to glucagon in patients with noninsulin-dependent diabetes mellitus. Juang JH, Huang HS, Huang MJ J Formos Med Assoc. 1992 May; 91(5):491-6. Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. Yoon KH, Ko SH, Cho JH, Lee JM, Ahn YB, Song KH, Yoo SJ, Kang MI, Cha BY, Lee KW, Son HY, Kang SK, Kim HS, Lee IK, Bonner-Weir S J Clin Endocrinol Metab. 2003 May; 88(5):2300-8. Determinants of insulin secretion and sensitivity in bangladeshi type 2 diabetic subjects. Roy MN, Biswas KB, Siddiqua N, Arslan MI, Ali L Metab Syndr Relat Disord. 2007 Sep; 5(3):275-81.

*Corresponding Author: *

Noorjahan Mohammed Email: m_noorjahan@yahoo.co.in

International Journal of Pharmacy and Biological Sciences

Noorjahan Mohammed* et al

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SYNERGISTIC ANTINEOPLASTIC ACTIVITY OF BERBERINE AND DOXORUBICIN ON A CHEMICALLY INDUCED HEPATOCELLULAR CARCINOMA IN RATS Bakheet E. M. Elsadek1,* Gamal M. K. Atwa1, Hisham H. Taha1, Tahia H. Saleem2 1

Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Assuit Branch, P.O. Box 71524 Assiut, Egypt 2 Department of Biochemistry, Faculty of Medicine, Assiut Uniersity, P.O. Box No. 71526, Assiut, Egypt

*Corresponding Author Email: b_elkot@yahoo.com

ABSTRACT Background: Hepatocellular carcinoma (HCC) is the most common type of liver malignancies. Yet, the outcome of the traditional chemotherapeutic agents in the management of HCC still unsatisfactory, most probably due to their limited therapeutic efficacies. Thus, there is an urgent medical need for alternative therapeutic approaches for fighting HCC. Aim: our study aimed at evaluating the possible synergistic antitumor activity of the herbal alkaloid berberine (BER) with the conventional doxorubicin (DOX) in a DENA-induced HCC rat model. Methods: HCC was induced in male Wistar rats by oral DENA administration in their drinking water (100 mg/L) for 8 weeks. In addition to a positive and negative control groups, a group of animals (10 rats) was given DOX (4Ă&#x2014; 2.5 mg/kg, i.v., weekly). Another group (10 rats) was given BER (100 mg/kg) by oral gavages daily for one month. A third group (10 rats) was given a combination of both BER and DOX in the above described dose and schedule. Results: Combination between DOX and BER was superior over their corresponding individual administrations as indicated by significant improvement in the overall estimated indices of liver function. Also, in comparison to individual therapy, this combination was obviously more potent to reduce the levels of novel HCC related tumor markers including serum AFP-L3 and tissue levels of Golgi protein 73, and glypican 3. Furthermore, histological investigations and assessments of hepatic tissue levels of some oxidative stress markers strongly confirmed the advantageous effects of combined BER and DOX in fighting DENA-induced HCC. Conclusion: our study conclusively revealed that combining BER with DOX exhibited a promising preclinical anticancer efficacy and could be considered as a novel strategy to synergistically combat HCC in clinical practices.

KEY WORDS Alphafetoprotein-L3; Apoptosis; Berberine; Doxorubicin; Hepatocellular carcinoma.

INTRODUCTION Hepatocellular carcinoma (HCC) is the main primary malignant tumor of the liver. Worldwide, it represents the third leading cause of cancer-related deaths in males and the fourth in females with a steadily increasing incidence of about 625000 new cases per year emerging around 600000 worldwide annual deaths [1]. Although the majority of the cases occur in Asia and Africa, the incidence has also been rising in the developed world. In Egypt, the burden of HCC has been increasing with a doubling in the incidence rate in the past 10 years [2]. The geographical variation in the incidence of HCC is explained by disparity in the prevalence of the major risk factors including chronic hepatitis B (HBV) or C virus (HCV) infection, alcoholinduced liver disease (ALD), non alcoholic fatty liver disease (NAFLD), primary biliary cirrhosis, exposure to International Journal of Pharmacy and Biological Sciences

environmental carcinogens (particularly aflatoxin), and then type 2 diabetes and obesity [3, 4]. Unfortunately, the prognosis of HCC is usually very poor due to the lack of effective early diagnostic tools and the actual diagnosis is usually made at an advanced stage that being too late for any effective treatment [5]. Similarly, the outcome of HCC is not satisfactory yet because only 10 - 20 % of HCC can be completely surgically removed, and in case of inefficient surgical removal of the whole malignant tissues, the overall survival without treatment is only in the range of 3 to 8 months. Moreover, treatment with conventional chemotherapeutic agent is usually of limited benefit either due to the development of drug resistance or due to their unlimited chronic and acute systemic toxicities [6, 7]. During the past 30 years, no consistent survival benefits for these Bakheet E. M. Elsadek* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. approved anticancer agents in HCC have been recorded in around 100 randomized studies including systemic and intra-arterial chemotherapy (predominantly doxorubicin-based or platinumbased), various hormonal therapies (tamoxifen and antiandrogens), and immunotherapy (usually interferon alpha) [8, 9]. Therefore, there is a pressing medical need for the identification and discovery of alternative therapeutic strategies with lower drawbacks, to wage a more humane war against HCC and hence to combat the current morbidity and mortality associated with such malignancies. Phytochemical remedies show promise in this area as their potential chemopreventive or chemotherapeutic actions in several types of cancer have been extensively indicated by epidemiologic and experimental studies [10]. For instance, herbal medicine has a long history of use in the treatment of cancer and it is significant that over 60% of the currently used anti-cancer agents are come from natural sources [11]. Naturally occurring drugs that are part of the war against cancer include vinca alkaloids (vincristine, vinblastine. vindesine, vinorelbine), taxanes (paclitaxel, docetaxel), podophyllotoxin and its derivative (etoposide, teniposide), camptothecin and its derivatives (topothecan, idarubicin) and others [12]. In the same respect, Berberine (BER), an isoquinoline derivative alkaloid that belongs to the camptothecin family of drugs, represents a new insight as a promising candidate of these natural compounds. It was initially isolated from the herbs Rhizoma coptidis (HuangLian) and was subsequently extracted from the roots, rhizome, and stem bark of a number of important medicinal plants such as Berberis vulgaris (barberry), Berberis aquifolium (oregon grape), Berberis aristata (tree tumeric), and Tinospora cordifolia [10]. The potential effectiveness of BER is indicated by its extensive use in traditional Indian and Chinese medicine for the treatment of several diseases such as diarrhea, rheumatic diseases, diabetes and some autoimmune diseases [13, 14]. Some years ago, cumulative evidences, in both in vitro and in vivo studies, have demonstrated that BER exhibits a promising potential to suppress growth, invasion, and metastasis of various lines of cancers such as glioma, lung cancer, nasopharyngeal carcinoma, and melanoma cell mostly via induction of apoptosis and cell cycle arrest with little resistance and low toxicity to normal cells [15-18]. More recently, other studies explored the chemosensitizing

International Journal of Pharmacy and Biological Sciences

influence of BER in combination with approved chemotherapeutic agents as an alternative approach for cancer control and treatment [19]. In this regard, emerging evidences confirmed that BER exhibited a synergistic anticancer activity with DOX, most likely through its own capability to enhance apoptosis and cell cycle arrest in cancer cells. Accordingly, this combinatorial approach can strongly inhibit mitogenic and survival signaling to inhibit cell proliferation and eventually induce cell death [19, 20]. In light of the above knowledge, we hypothesized that combining DOX with BER could be considered as a novel strategy to increase the in vivo antitumor efficacy of DOX against certain malignancies. The current study was therefore performed to test this hypothesis in a chemically induced HCC rat model.

MATERIALS AND METHODS Chemicals Berberine (BER) and diethylnitrosamine (DENA) were purchased from Sigma-Aldrich GmbH (Munich, Germany). Commercially available doxorubicin (DOX) vials (10 mg adriamycin hydrochloride) were purchased from Pharmacia Italia S.P.A. Gruppo Pfizer Inc. (Nerviano Italy). Alpha-Fetoprotein Lens Culinaris Agglutinin 3 (AFP-L3) rat specific ELISA assay kit was purchased from Glory Science. Co., Ltd (Hangzhou, China). Rabbit polyclonal anti-GP73 antibody and alkaline phosphatase-conjugated goat anti-rabbit secondary antibody were purchased from Novus Biologicals, LLC, Littleton (CO, USA). Mouse monoclonal anti Î˛-actin antibody was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). Total RNA purification kit was purchased from Jena Bioscience GmbH (Jena, Germany). RevertAid M-MuL V Reverse Transcriptase kit was purchased from Thermo Fisher Scientific inc. (MA, USA). Protease inhibitor cocktail was purchased from Cell Signaling Technology, Inc. (MA, USA). BCIP/NBT substrate detection kit was purchased from Genemed Biotechnologies, Inc. (CA, USA). Molecular screening agarose gel was purchased from Roche Diagnostics, GmbH (Mannheim, Germany). PCR primers were custom-made by Vivantis Technologies Sdn. Bhd. (Selangor Darul Ehsan, Malaysia) to amplify the rat GPC3 and Î˛-actin cDNA (Table 1). All other chemicals, reagents, and solvents were of analytical grade that obtained from standard suppliers and were used without further purification.

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Primer

GPC3

β-actin

Table-1: The primers used for amplification of GPC3 and β-actin in RT-PCR detection Amplified Sequence Gen Bank Accession Annealing Temp. Product Number °C (bp) 603 FORWARD: NM_012774.1 53 °C 5’-GGCAAGCTGACCACCACTAT-3’ REVERSE: 5’-CTTGTTCCCTGGAGCATCGT -3’ 243 FORWARD: NM_031144.3 54 °C 5’-CCACCATGTACCCAGGCATT-3’ REVERSE: 5’- ACGCAGCTCAGTAACAGTCC-3’

Animals and experimental design: Animal experiments were performed after approval by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Assiut University, Assiut, Egypt. All experiments were performed using 15-16 week old healthy male Wistar rats (weighting 130-140 g) that were purchased from the laboratory animal colony, Assiut University, Assiut, Egypt. Rats were housed (5 per cage) in wire-floored cages at a regulated environment (temperature, 22±2oC; humidity, 50±5%; night/day cycle, 12 hours) with free access to standard pellet diet and tap water add libitum. Animals' weights were taken every 3 days and animal behaviors were monitored daily. After two weeks acclimatization period, rats were randomly divided into two groups of 10 and approximately 40 rats, respectively. Animals in the first group (control group) were received only a suspending vehicle that consist of a mixture of 1% sodium Carboxymethyl cellulose and 1% Tween-80 (daily by oral gavages) throughout the experimental period. The animals of the second large group received DENA, given in their drinking water (100 mg/L) for 8 weeks [21]. The DENA solution was administered in dark bottles (additionally covered with aluminum foil) since the solution is light-sensitive and was prepared as a fresh solution every other day. One month after the end of DENA administration, the 40 rats of the second group were allocated to one control group (DENA group) which received the above mentioned suspending vehicle and 3 other treated subgroups of 10 animals each. Rats in the first subgroup (DENA+DOX group) were given doxorubicin 4× 2.5 mg/kg (i.v., weekly) via tail vein [19]. Rats in the second subgroup (DENA+BER group) were given daily BER by oral gavages (100 mg/kg) suspended in the above mentioned suspending vehicle for four weeks [19]. Rats in the third subgroup (DENA+DOX+BER group) were given a combination of both DOX and BER in the same above International Journal of Pharmacy and Biological Sciences

described doses and schedule. One week after the last treatment, animals were sacrificed by cervical decapitation under isoflurane anesthesia after blood collection from all animals via retro-orbital vein plexus for serum preparation. Subsequent to autopsy, the livers were excised, purified from adhering fat and connective tissues, washed in ice-cold isotonic saline and then divided into three parts; the first part was stored in formalin (10%) solution and subjected for histopathological examination and the remaining two parts were instantly flash frozen in liquid nitrogen and stored separately at –70 oC for subsequent western blotting and RT-PCR assays. Biochemical estimations Serum AFP-L3 was assayed by rat specific ELISA assay kit. Serum Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), and Alkaline Phosphatase (ALP) activities were assayed by kinetic procedures using corresponding kits according to the manufacturer's instructions. Serum total bilirubin was determined using colorimetric kit according to the manufacturer's instructions. Lipid peroxidation was determined spectrophotometrically in liver tissues as thiobarbituric acid reacting substance (TBARS) and is expressed as equivalents of malondialdehyde (MDA) [22]. Reduced glutathione (GSH) was assayed spectrophotometrically in liver tissues using Ellman assay method [23]. Superoxide dismutase (SOD) activity in liver tissues was estimated using the xanthine oxidase method [24]. Nitric oxide (NO) was assayed spectrophotometrically in liver tissues by measuring its stable metabolites, in particular, nitrite and nitrate [25]. Investigation of golgi protein 73 (GP73) protein expression by western blotting technique Liver tissue homogenates were prepared in ice-cold Tris-HCl lysis buffer, pH 7.4 containing 1% protease inhibitor cocktail using Potter-Elvehjem rotor-stator homogenizer (glass/teflon homogenizer), fitted with a Bakheet E. M. Elsadek* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Teflon pestle. Proteins in each corresponding homogenate were denatured at 95 oC for 5 minutes in 2× Laemmli buffer. SDS-PAGE electrophoresis was achieved by loading 50 µg protein per lane at 75 volts through resolving gel 12% followed by 125 volts during approximately 2 hours and transferred to a PVDF membrane using T-77 ECL semidry transfer unit (Amersham BioSciences UK Ltd) for 2 hours. Immunoblotting was performed by incubating the PVDF membrane in TBS buffer containing 0.1% Tween and 5% defatted milk for one hour at 4oC, followed by overnight incubation at 4oC with rabbit polyclonal anti-GP73 antibody at 1:2000 dilution. After being washed three times with TBST buffer, each membrane was incubated for one hour at room temperature with an alkaline phosphataseconjugated goat anti-rabbit secondary antibody at 1:5000 dilution. After being washed four times in TBST, the membrane bound antibody was detected with a commercially available BCIP/NBT substrate detection kit. Equivalent protein loading for each lane was confirmed by stripping and re-blotting each membrane at 4oC against mouse monoclonal anti βactin antibody at 1:5000 dilution. The analysis was repeated to assure reproducibility of results. Detection of glypican 3 (GPC3) mRNA using reverse transcriptase PCR (RT-PCR) In order to obtain a maximum intact RNA yield, a part of liver was harvested in a specific lysis buffer supplied in total RNA purification kit using a PotterElvehjem rotor-stator homogenizer according to the manufacturer's instructions. To avoid RNA destruction during or after procedure by active RNAses, outer stationary glass tube and inner turning teflon shaft of the homogenizer were washed with 0.1% diethylpyrocarbonate-treated water (DEPC-treated o water), incubated overnight at 37 C, and then autoclaved for 15 minutes to eliminate residual DEPC. The purity (A260/A280 ratio) and the concentration of the isolated RNA were determined using a GeneQuant 1300 spectrophotometer (Uppsala, Sweden). RNA quality was subsequently confirmed by gel electrophoresis. Then the first-strand cDNA was synthesized from 4 µg of total RNA using an Oligo (dT) 18 primer and RevertAid M-MuL V Reverse Transcriptase kit. This mixture was incubated at 42 oC for one hour, followed by incubation at 70 oC for 5 minutes to terminate the reaction. The resulting cDNA was amplified by PCR. Numbers of cycles and reaction temperature conditions were estimated to be optimal to provide a linear relationship between the amount of input template and the amount of PCR International Journal of Pharmacy and Biological Sciences

product generated over a wide concentration range: from 1 to 20 µg of total RNA. In brief, cDNA was first o denatured for 3 minutes at 95 C then amplified for 33 cycles consisting of: denaturing for 30 second at 95 oC; o o annealing for 30 second at 53 C for GPC3; and 54 C o for β-actin; primer extension for 30 second at 72 C followed by one cycle of primer extension for 5 o minutes at 72 C. RT-PCR of β-actin was performed in parallel as an internal control. The RT-PCR products were analyzed by electrophoresis using 2% molecular screening agarose gel, stained with ethidium bromide and visualized by UV light. Histopathological examinations For histopathological examination, liver tissue samples of all lobes were fixed in 10% formalin, embedded in paraffin and routinely stained with hematoxylin and eosin (H&E). Washing was carried out in sterile tap water, then in serial dilutions of alcohols (methyl, ethyl and absolute ethyl) which was used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 oC in a hot air oven for 24 hours. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns thickness with a sledge microtome and the obtained tissue sections were placed on glass slides, deparaffinized and stained by H&E stain and then examined under an electric light microscope for assessment of histopathological changes [26]. Statistical analysis Statistical analyses of the data were carried out using Graphpad prism version 5.0 (Graph pad software San Diego, USA). Data comparisons were performed using analysis of variance (ANOVA) followed by Tukey’s ttest. The levels of significance were accepted with p < 0.05 and all relevant results were graphically displayed as mean SEM.

RESULTS AND DISCUSSION Hepatocellular carcinoma (HCC) is a major worldwide public health concern that represents globally the fifth most common type of cancer [27]. Despite recent advances, there has been a little success in improving the survival of HCC patients, partly due to lack of effective therapy, tumor recurrence, and because the diagnosis is generally made at an advanced stage of the disease [28]. Therefore, the prevalence of HCC is expected to worsen in the coming years, plateauing between 2015 and 2020 [29]. In the last few decades, treatment with conventional chemotherapeutic agents such as doxorubicin (DOX) enjoyed considerable popularity as one of the most Bakheet E. M. Elsadek* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. practiced strategies in the management of unresectable HCC [30]. Nowadays, this approach is no longer widely used in clinical applications as a result of the limited efficacy of DOX, most likely due to the elevation of drug resistance, in addition to its undoubted implication in the appearance of numerous dose related side effects that leading eventually to unsatisfactory outcomes [31]. Accordingly, improving both the therapeutic efficacy and index of DOX is considered as an unmet golden goal to achieve a more efficient fighting potential against tumors. In this regard, a lot of evidences indicated that combined therapy with multiple drugs or modalities is a common practice in cancer treatment, which can achieve better therapeutic effects than a single drug or modality and can reduce the side effects and resistance to drugs [32]. As an option, chemosensitizing phytochemicals in combination with approved chemotherapeutic agents are being explored as an alternative more efficient approach for cancer control and treatment [33]. In the light of this knowledge, we aimed here in the current study to evaluate the potential of berberine (BER), a botanical alkaloid of the protoberberine type, to improve the in vivo anticancer efficacy of the

Control

DENA

conventional DOX against HCC in a chemically induced rat model. In the current study, we have observed that, combinatorial treatment with both DOX and BER was superior over their corresponding individual use in respect to signs of toxicity as was manifested for example by decrease in body weight gain. Our results have shown that the increase in the animal body weight was obviously attenuated in all DENA drinking animals during the course of the experiment before starting treatment, suggesting primary tumor burden or the metastatic spread of the tumor as a possible cause. A similar observation was also reported in previous works using this model [34]. As illustrated in Figure-1, following treatments, the induced decreases in body weight gain were hampered to large extent in the combination group (DENA+DOX+BER) and to less extent in the BER-treated group (DENA+BER) demonstrating decreased side effects which, on the contrary, was more pronounced in the DOX-treated group (DENA+DOX), most likely due to the direct toxic effects of DOX on the animal intestinal mucosa as well as its indirect actions arising from reduced food intake causing a decrease in secretion of internal hormones and resulting in decreased trophic effects to the mucosa [35].

DENA+BER

DENA+DOX

DENA+BER+DOX

400 Acclimatization

Animal body weight (g)

Autopasy

Waiting 1 week

350 DENA drinking

Waiting 4 weeks

Therapy

300 250 200 150 100 50 0 0

Week

Figure-1: illustration of the changes in the animalâ&#x20AC;&#x2122;s body weights during the experiment. We have also observed that representative macroscopic views of livers from the DENA group showed abnormal morphological features with several macroscopic tumor nodules scattered throughout the liver with an irregular rough surface, as appear in Figure-2. Similar morphological observations have also previously been reported in International Journal of Pharmacy and Biological Sciences

several experiments in which DENA was used as a carcinogen for animal models of HCC [34, 36]. In a comparatively similar style, livers of rats treated with individual DOX and BER macroscopically showed altered morphological features with irregular rough pale surface incorporating numerous scattered macro and micronodules of different sizes throughout the Bakheet E. M. Elsadek* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. liver â&#x20AC;&#x201C; see Figure-2. On the contrary, livers in the combination group (DENA+BER+DOX) showed relatively better morphological appearance with very

few micro-nodules. Ideally livers in the control group showed normal morphological aspects as seen in Figure-2.

Figure-2: Effect of tested compounds on the morphological aspect of the liver. Representative macroscopic views of livers from the DENA, DENA+BER and DENA+DOX groups showed abnormal morphological features with several macroscopic tumor nodules of different sizes (arrows) scattered throughout the livers with an irregular rough surface. Conversely, livers in the combination group (DENA+BER+DOX) showed relatively better morphological appearance with very few micro-nodules. Livers in the control group showed normal morphological aspects. Regarding hepatic performance, it was clearly observed in our study that in comparison to the healthy control group, animals in DENA group suffered from marked indications of impaired liver functions including significant hypoalbuminemia (p < 0.001), in addition to significant elevations in the serum activities of ALT (p < 0.001), AST (p < 0.001), GGT (p < 0.001) and ALP (p < 0.001) as well as total serum bilirubin level (p < 0.001) â&#x20AC;&#x201C; see table-2. These observed DENA-forced alterations in serum indices of International Journal of Pharmacy and Biological Sciences

liver functions could be secondary events following lipid peroxidation of hepatocyte membranes with the consequent increase in the leakage of ALT, AST, GGT, ALP and total bilirubin from damaged liver tissues as have been previously reported in many models of DENA-induced hepatocellular degeneration [37, 38]. Of note that neither BER nor DOX individual administration was able to significantly restore any of these impaired indices to its corresponding normal levels. Fortunately, the overall estimated liver Bakheet E. M. Elsadek* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. function indices were obviously improved in the group treated with BER and DOX combination (DENA+BER+DOX) as illustrated in table-2. In comparison to the DENA group, animals in the combination group showed significant increase in the serum albumin level (p < 0.05), in addition to significant decreases in the serum activities of ALT (p < 0.001), AST (p < 0.001), GGT (p < 0.01), and ALP (p < 0.01) as well as total level of serum bilirubin (p < 0.01). Notably, in comparison to individual

Albumin (g/dL) sALT (IU/L) sAST (IU/L)

Control 4.06 ± 0.20 63 ± 1.41 115 ± 7.07

sGGT (IU/L)

29.4 ± 4.49

therapeutic agent in DENA+BER, and DENA+DOX groups, combination therapy revealed much more superiority in the form of significant increase in the serum albumin level (p<0.05 and p<0.01 respectively), besides significant decreases in the serum activities of ALT (p < 0.001 and p < 0.001 respectively), AST (p < 0.001 and p < 0.001 respectively), GGT(p < 0.05 and p < 0.05 respectively) and ALP (p < 0.05 and p < 0.05 respectively) as well as total level of serum bilirubin (p < 0.05 and p < 0.05 respectively).

Table-2: Liver function tests: DENA DENA+BER ***

***

DENA+DOX

DENA+BER+DOX

***

*,†,§,‡‡

2.23 ± 0.24 *** 165 ± 3.53

2.26 ± 0.14 *** 160 ± 2.82

2.02 ± 0.20 ***,† 155 ± 1.41

3.15 ± 0.20 *,†††,§§§,‡‡‡ 75 ± 2.12

320 ± 14.14

300 ± 10.61

280 ± 7.07

170±1.21

***

118.0 ±15.34

101.9± 11.91

***

100.4 ± 12.21

***

*,†††,§§§,‡‡‡ ††,§,‡

54.9± 8.44

*** *** *** *,††,§,‡ ALP (IU/L) 75 ± 3.53 160 ± 14.14 155 ± 7.07 150 ± 3.53 115 ± 7.07 *** *** *** ††,§,‡ Total Bilirubin 0.38 ± 0.01 1.60± 0.21 1.50 ± 0.07 1.46 ± 0.13 0.90 ± 0.14 (mg/dL) Data are presented as mean ± SEM (n = 10). *,†,§,and ‡ indicate significant changes from control, DENA, DENA+BER, and DENA+DOX groups respectively. *,†, § and ‡ indicate significant change at P < 0.05; **,††,§§, and ‡‡ indicate significant change at p < 0.01; ***,†††,§§§ and ‡‡‡ indicate significant change at p < 0.001.

Additional evidences that clearly support this superiority of BER and DOX combination over either BER or DOX individual administration were presented by the outcome of assessments of a panel of novel HCC tumor markers including AFP-L3, GP73 and GPC3. These contemporary markers have been recently used by several investigators for the early detection and monitoring treatment response as well as poor differentiation, recurrence, and/or malignant invasion of HCC and also as surrogate markers of its clinicopathological variability with relatively satisfactory sensitivity and specificity [39-41]. As Control

appear in our results, serum AFP-L3 level was significantly increased in DENA group in comparison to normal healthy control group (p < 0.001). As obvious in Figure-3, while both BER and DOX solitary administration failed to significantly decrease the elevated AFP-L3 circulating levels, their combinatorial administration was more efficiently able to significantly reduce the elevated AFP-L3 levels earning approximately 56% (p < 0.001), 52% (p <0.01), and 46% (p <0.05) corresponding decreases in comparison to DENA, DENA+BER and DENA+DOX groups respectively.

Table-3: Hepatic tissue levels of oxidative stress markers DENA DENA+BER DENA+DOX

MDA (nmol/g wet tissue)

3.42 ± 0.51

11.51 ±1.00

NO (µmol/g wet tissue)

2.66 ± 0.43

11.85 ±1.95

GSH (µmol/g wet tissue)

10.61 ± 1.44

5.35 ± 0.83

SOD (U/mg wet tissue)

10.81 ± 1.40

4.99 ± 0.92

***

13.33 ± 2.33

13.21 ± 2.30

4.89 ± 0.64

4.24 ± 0.92

DENA+BER+DOX ***

13.35 ± 1.34

***

15.61 ± 2.10 *

5.62 ± 1.22

4.10 ± 1.00

***

14.09 ± 1.74

***

18.95 ± 1.88

4.92 ± 0.73

4.35 ± 1.50

Data are presented as mean ± SEM (n = 10). * indicates significant changes from control group. * indicate significant change at P < 0.05; ** indicate significant change at p < 0.01; *** indicate significant change at p < 0.001.

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400

***

AFP-L3 (ng/mL)

300

***

‡ §§

200

†††

* 100

A +B

A +D

E A +B N E D

A E N D

on tr

Figure-3: Serum levels of AFP-L3 as a circulating tumor markers of HCC in different groups. Data are presented as mean ± SEM (n = 10). *, †, §, and ‡ indicate significant changes from control, DENA, DENA+BER, and DENA+DOX groups respectively. *, †, §, and ‡ indicate significant change at P < 0.05; **, ††, §§, and ‡‡ indicate significant change at p < 0.01; ***, †††, §§§, and ‡‡‡ indicate significant change at p < 0.001. Additionally, western blotting assessments of GP73 as well as RT-PCR detection of GPC3 mRNA as HCCrelated molecular markers augmented the previously observed beneficial role of combined BER and DOX therapy, but not their individual use, in fighting HCC. As shown in figure-4, DENA administration induced strong expression of both GP73 and GPC3 genes, proving the occurrence of premalignant liver changes in this group. These results are in agreement with some previous researches which reported that GP73 and GPC3 are upregulated in HCC tumor tissues

compared with normal and benign liver diseases and contributed to promoting the growth of HCC by stimulating Wnt signaling [42, 43]. Auspiciously, combined BER and DOX therapy was able markedly to attenuate this elevated expression to be asymptotic to its expression pattern in the healthy control group, meanwhile singular BER or DOX administration were unable to hinder the elevated expressions of any of these molecular markers – See Figure 4.

Figure-4: illustration of the western blotting assessments of GP73 (A) and RT-PCR detection of GPC3 mRNA fragments (B) as HCC-related molecular markers in liver tissue homogenates. β-Actin was used in parallel as an internal control. The right panels represent corresponding quantification of each analysis measured by Image J software and expressed as a β-actin ratio. International Journal of Pharmacy and Biological Sciences

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As well, histological examinations of liver tissues presented another strong evidence for the combined BER and DOX advantageous effects to fight DENAinduced HCC. The representative photomicrographs of liver sections obtained from animals in DENA group showed hepatocellular carcinoma with apparent hepatic tissue alterations that reflect severe dilatation of central vein and degeneration in the surrounding adjustment hepatocytes incorporating several micronodular lesions characterized by focal necrosis in the hepatic parenchyma associated with fibroblastic cells proliferation and inflammatory cells infiltration beside hyperplasia and marked atypia as seen in figure 5. Similar histological observations have been previously reported in published DENA-induced rat models [44, 45]. Combination of BER and DOX, on

the other hand, showed relatively normal hepatic histological structures that were approximately comparable to those observed in the hepatic tissues of the healthy control group with only mild dilatation in the portal vein â&#x20AC;&#x201C; see figure 5. These observations firmly suggest the tumor suppressing potential of the blend incorporating both BER and DOX against DENAinduced HCC. Meantime, individual BER or DOX administration failed to completely buffer the DENA induced histological alterations in the hepatic tissues and notable dilatation and congestion of the portal vein with focal hepatic hemorrhage and periductal inflammatory cells infiltration surrounding the bile ducts as well as fibroblastic cells proliferation and malignant foci are still observed in these tissues â&#x20AC;&#x201C; see figure 5.

Figure-5: Representative photomicrographs of liver sections from different groups. [A] Liver tissues of the control group showing normal histological structure of hepatic lobule; [B] Liver tissues of the DENA group showing hepatocellular carcinoma distorting the normal trabecular structure of the liver with sever dilatation in the portal vein and focal necrosis, fibroblastic cells proliferation and inflammatory cells infiltrations in the hepatic parenchyma; [C] Liver tissues of the DENA+BER group showing dilatation in the portal vein with periductal inflammatory cells infiltration and fibroblastic cells proliferation as well as dilatation and congestion of hepatic sinusoids; [D] Liver tissues of the DENA+DOX group showing hepatocellular carcinoma with marked dilatation in the portal vein and focal hepatic hemorrhage as well as inflammatory cells infiltration and fibroplasia around bile duct; [E] Liver tissues of the DENA+BER+DOX group showing normal intact hepatic histological structure.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Additionally, signs of intense perturbations in the oxidant/antioxidant balance were reported clearly in our current investigation which revealed serious increases in oxidative and nitrosative damages in liver tissues of DENA group in the form of significantly increased lipid peroxidation products MDA (p <0.001) and NO levels (p < 0.001) associated concomitantly with significant depletion in the GSH content (p < 0.001) and SOD activity (p <0.001) in comparison to the healthy control. Increased generation of reactive oxygen species (ROS) and decreased antioxidant enzymes in liver tissues have been reported in many models of DENA induced HCC [46]. These studies concluded that oxidative stress and free radicals play a major role in tumor promotion through interaction with critical macromolecules including lipids, DNA, DNA repair systems, and other enzymes leading eventually to induction of DNA damage and lipid peroxidation [47]. As expected, individual DOX administration in DENA+DOX group clearly potentiates the DENA-induced oxidant/antioxidant disruption, most likely due to the attendant recognized prooxidant activities of DOX itself [48]. Unexpectedly, in comparison to the DENA group, administration of BER, either in individual or combination therapeutic protocols, failed to calmdown the abovementioned DENA-induced oxidative stress, rather, it aroused more disruptions in the levels of the estimated biomarkers of oxidative stress than their corresponding values in DENA group in the form of insignificant increases in hepatic tissue levels of MDA and NO with concomitant insignificant decreases in hepatic tissue GSH content and SOD activity. These data contradicted with results of several studies which established inhibitory effects of BER on chemically induced lipid peroxidation and oxidative stress in CCl4-induced toxic liver damage [49]. A possible accommodation for this contradiction is presented by the results of a recent report which attributed the BER-induced cytotoxicity in malignant HepG2 cells to the BER-forced ROS overproduction that may be the upstream event to procaspases activation and could be the critical initiator of apoptotic cell death through a mitochondrial/caspases pathway [15]. It is worth noting that, in the contrary, such BER-forced cell death is inapplicable in normal hepatocytes mainly due to its no influence on ROS production in these cells. Additional support for this hypothesis was proposed by another study which concluded that enhancement of ROS production represents the key element in the selective BER-induced apoptosis in International Journal of Pharmacy and Biological Sciences

human prostate cancer cells, but not in the normal prostate epithelial cells [17]. This inconsistent cytotoxicity of BER in prostate cancer cells and prostate epithelial cells strongly support the hypothesis that the resistance to BER-induced oxidative stress of normal cells but not their corresponding malignant cells appears non-specific to certain tissues. Finally, all the aforementioned results in our study strongly suggest that neither BER nor DOX singular administration exhibited a reproducible anticancer activity against chemically induced HCC in rat model. Their combinatorial use, on the other hand, revealed indubitable in vivo antineoplastic activity against this kind of cancer as confirmed herein through assessment of some biochemical, molecular and histological proofs. These results are in agreement with other research which reported that BER alone and DOX alone did not show any considerable effects on murine melanoma tumor growth; however their combination strongly inhibited the in vitro cell growth and induced cell death, and caused G2/M arrest in cell cycle together with a decrease in Kip1/p27. In a corresponding in vivo experiment, this combination significantly decreased murine B16F10 xenograft tumor volume (85%, p< 0.005) and tumor weight (78%, p <0.05) as compared to control indicating inhibition of proliferation and an increase in apoptosis [19]. Also, these results were in agreement with the study which reported that BER was able significantly to enhance the anticancer effect of DOX in A549 and HeLa cells in vitro, possibly mediated by inducing apoptosis [18]. Moreover, another recent study presented an additional testimony for the synergistic anticancer potential of BER and DOX combination in the treatment of lung cancer. The authors concluded that BER was able to sensitize lung cancer cells to the cytotoxic effect of DOX by suppressing the DOXmediated activation of the signal transducer and activator of transcription 3 (STAT3) which plays critical roles in malignant transformation and progression and was found to be constitutively activated in a variety of human cancers [50].

CONCLUSION In conclusion, our study revealed that combining BER with DOX could resemble a novel strategy to synergistically generate in vivo anticancer effects against HCC supposedly due to their dual apoptotic endeavor. Nevertheless, more investigations are definitely required to translate this promising Bakheet E. M. Elsadek* et al

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ACKNOWLEDGEMENTS The authors are grateful to Prof. Dr. Adel Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, Cairo University, for carrying out the histopathological examinations. Conflict of interest: The authors declare that there is no conflict of interests.

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12. Kinghorn, A.D., et al., Discovery of anticancer agents of diverse natural origin. Pure Appl Chem, 81 (6): 10511063, (2009) 13. Li, H., et al., Berberine ameliorates experimental autoimmune neuritis by suppressing both cellular and humoral immunity. Scand J Immunol, 79 (1): 12-9, (2014) 14. Tian, X., et al., Interaction of anticancer herbal drug berberine with DNA immobilized on the glassy carbon electrode. Bioelectrochemistry, 73 (1): 18-22, (2008) 15. Bing , L., et al., Berberine Inhibits Human Hepatoma Cell Invasion without Cytotoxicity in Healthy Hepatocytes. PLoS ONE, 6 214-216, (2011) 16. Katiyar, S.K., et al., p53 Cooperates berberine-induced growth inhibition and apoptosis of non-small cell human lung cancer cells in vitro and tumor xenograft growth in vivo. Mol Carcinog, 48 (1): 24-37, (2009) 17. Meeran, S.M., Katiyar, S., and Katiyar, S.K., Berberineinduced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation. Toxicol Appl Pharmacol, 229 (1): 33-43, (2008) 18. Tong, N., et al., Berberine sensitizes mutliple human cancer cells to the anticancer effects of doxorubicin in vitro. Oncol Lett, 3 (6): 1263-1267, (2012) 19. Anil Mittal, Saba Tabasum, and Singh, R.P., Berberine in combination with doxorubicin suppresses growth ofmurine melanoma B16F10 cells in culture and xenograft. Phytomedicine, 15 218-225, (2013) 20. Ma, X., et al., Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes. Biomaterials, 34 (18): 4452-65, (2013) 21. Di Stefano, G., et al., Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging. Dig. Liver Dis., 40 (4): 278-284, (2008) 22. Mihara, M. and Uchiyama, M., Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem, 86 (1): 271-8, (1978) 23. Ellman, G.L., Tissue sulfhydryl groups. Archives of biochemistry and biophysics, 82 (1): 70-77, (1959) 24. Sun, L., et al., Improved superoxide dismutase assay for clinical use. Clin Chem, 35 (6): 1265-6, (1989) 25. Sun, C., et al., Determination of nitrite in serum and tissue by oscillopolarography. Journal of West China University of Medical Sciences, 30 (2): 222-224, (1999) 26. Banchroft, J.D., Stevens, A., and Turner, D.R., Theory and practice of histological techniques. 4th ed. New York:Churchil Livingstone. 1996. 27. Yim, S.H. and Chung, Y.J., An Overview of Biomarkers and Molecular Signatures in HCC. Cancers (Basel), 2 (2): 809-23, (2010) 28. Abul Seoud, R.A. and Mabrouk, M.S., TMT-HCC: a tool for text mining the biomedical literature for hepatocellular carcinoma (HCC) biomarkers identification. Comput Methods Programs Biomed, 112 (3): 640-8, (2013)

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. 29. Gish, R.G., Finn, R.S., and Marrero, J.A., Extending survival with the use of targeted therapy in the treatment of hepatocellular carcinoma. Gastroenterol Hepatol (N Y), 9 (4 Suppl 2): 1-24, (2013) 30. Lahoti, T.S., et al., Doxorubicin-induced in vivo nephrotoxicity involves oxidative stress-mediated multiple pro- and anti-apoptotic signaling pathways. Curr Neurovasc Res, 9 (4): 282-95, (2012) 31. Patel, N., et al., Silymarin modulates doxorubicininduced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver. Toxicol Appl Pharmacol, 245 (2): 143-52, (2010) 32. Capone, F., et al., Synergistic antitumor effect of Doxorubicin and tacrolimus (FK506) on hepatocellular carcinoma cell lines. ScientificWorldJournal, 2014 450390, (2014) 33. Wagner, H., Synergy research: approaching a new generation of phytopharmaceuticals. Fitoterapia, 82 (1): 34-7, (2011) 34. Fiume, L., et al., Doxorubicin coupled to lactosaminated albumin inhibits the growth of hepatocellular carcinomas induced in rats by diethylnitrosamine. J. Hepatology, 43 645-652, (2005) 35. Herman, E.H., et al., Comparison of the protective effects of amifostine and dexrazoxane against the toxicity of doxorubicin in spontaneously hypertensive rats. Cancer Chemother Pharmacol, 45 (4): 329-34, (2000) 36. Di Stefano, G., et al., Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats. Eur. J. Pharm. Sci., 33 (2): 191198, (2008) 37. Bansal, A.K., et al., Protective role of Vitamin E pretreatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chem Biol Interact, 156 (2-3): 101-11, (2005) 38. Sayed-Ahmed, M.M., et al., Thymoquinone attenuates diethylnitrosamine induction of hepatic carcinogenesis through antioxidant signaling. Oxid Med Cell Longev, 3 (4): 254-261, (2010)

39. Jain, D., Tissue diagnosis of hepatocellular carcinoma. J Clin Exp Hepatol, 4 (Suppl 3): S67-73, (2014) 40. Lagana, S.M., et al., Utility of an immunohistochemical panel consisting of glypican-3, heat-shock protein-70, and glutamine synthetase in the distinction of low-grade hepatocellular carcinoma from hepatocellular adenoma. Appl Immunohistochem Mol Morphol, 21 (2): 170-6, (2013). 41. Wang, N.Y., et al., Prognostic value of serum AFP, AFPL3, and GP73 in monitoring short-term treatment response and recurrence of hepatocellular carcinoma after radiofrequency ablation. Asian Pac J Cancer Prev, 15 (4): 1539-44, (2014). 42. Enan, E.T., et al., Diagnostic role of glypican 3 and CD34 for differentiating hepatocellular carcinoma from nonmalignant hepatocellular lesions. Ann Diagn Pathol, 17 (6): 490-3, (2013). 43. Hu, J.S., et al., GP73, a resident Golgi glycoprotein, is sensibility and specificity for hepatocellular carcinoma of diagnosis in a hepatitis B-endemic Asian population. Med Oncol, 27 (2): 339-45, (2010) 44. Afzal, M., et al., Preventive effect of Metformin against N-nitrosodiethylamine-initiated hepatocellular carcinoma in rats. Saudi Pharm J, 20 (4): 365-70, (2012). 45. Golla, K., et al., A target-specific oral formulation of Doxorubicin-protein nanoparticles: efficacy and safety in hepatocellular cancer. J Cancer, 4 (8): 644-52, (2013). 46. Sivaramakrishnan, V., et al., Attenuation of Nnitrosodiethylamine-induced hepatocellular carcinogenesis by a novel flavonol-Morin. Chem Biol Interact, 171 (1): 79-88, (2008) 47. Reed, J.C., Apoptosis mechanisms: implications for cancer drug discovery. Oncology, 18 (13 Suppl 10): 1120, (2004). 48. Deavall, D.G., et al., Drug-induced oxidative stress and toxicity. J Toxicol, 2012 645460, (2012) 49. Feng, Y., et al., Hepatoprotective effects of berberine on carbon tetrachloride-induced acute hepatotoxicity in rats. Chin Med, 5 33, (2010). 50. Zhu, T., et al., Berberine Increases Doxorubicin Sensitivity by Suppressing STAT3 in Lung Cancer. Am J Chin Med, 43 (7): 1487-502, (2015).

*Corresponding Author: Bakheet E. M. Elsadek* Email: b_elkot@yahoo.com

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MICROALGAE AS SOURCE FOR POTENTIAL ANTI-ALZHEIMER´S DISEASE DIRECTED COMPOUNDS - SCREENING FOR GLUTAMINYL CYCLASE (QC) INHIBITING METABOLITES S. Krause-Hielscher1, H.-U. Demuth2, L. Wessjohann3, N. Arnold3, C. Griehl1* 1

Anhalt University of Applied Sciences, Department of Applied Biosciences and Process Technology, Group Algae Biotechnology, 06366 Köthen, Germany 2 Fraunhofer Institute for Cell Therapy and Immunology IZI, Department of Drug Design and Target Validation, 06120 Halle/S. Germany 3 Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, 06120 Halle/S., Germany

*Corresponding Author Email: c.griehl@bwp.hs-anhalt.de

ABSTRACT Alzheimer´s disease (AD) is the most common form of dementia affecting predominantly elderly people from developed countries. One aspect of the illness is that patients suffer from an impaired memory due to deposition of aggregated A-peptides forming amyloid plaques. According to the glutaminyl cyclase (QC) hypothesis this enzyme plays a key role in generating neurotoxic amyloid peptides (amyloid-β or Aβ) by modifying the N-terminus of peptides to N-pyroglutamated derivatives. These modified proteins are resistant to degradation and are at the same time “seeds” for the formation of toxic A-oligomers in the brain. In order to screen for natural inhibitors of QC, strains of different species of algae belonging to Chlorophyceae and Eustigmatophyceae were cultivated in 100 L tubular photobioreactors. The resulting crude extracts of algae from exponential and stationary growth phases were tested for their inhibition properties of glutaminyl cyclase (QC). Here 27 of the 72 tested extracts inhibited the QC. Fractions separated by Sephadex G-15 column chromatography also showed QC inhibition activity.

KEY WORDS Alzheimer´s Disease; Chlorophyceae; Eustigmatophyceae; Glutaminyl Cyclase (QC) Inhibitor; Screening.

INTRODUCTION Alzheimer´s disease (AD) is the most common form of dementia characterized by neurodegeneration and neuroinflammation. The Alzheimer’s disease International estimated that there are 46.8 million people living with dementia worldwide in 2014 (70% with AD), increasing to 74.7 million by 2030 and 131.5 million by 2050. The reasons are, firstly, the population growth and other demographic change [1]. Thus, there is a pressing need for new and efficient treatment strategies and more effective drug candidates to treat or prevent Alzheimer's disease. New causal approaches should be provided which halt or delay the progression of the disease which is not possible with today’s available medications. Therefore it is important to understand the underlying molecular basis of Alzheimer's disease to find ways for causal treatment strategies. International Journal of Pharmacy and Biological Sciences

Alzheimer's disease is characterized by neuron loss and the degeneration of synapses as well as neuroinflammation. In the brains of Alzheimer's patients, especially in the regions of the hippocampus and the neocortex protein deposits, the "amyloid plaques" and "neurofibrillary tangles" are formed. Because these areas of the brain are responsible for the higher mental abilities such as language and memory, a progressive loss of brain substance leads to increasing memory loss, confusion, and disorientation. Two other characteristics of this disease are constituted by a reduced acetylcholine level and an increased level of glutamate. These are the targets for previously approved drugs (inhibitors of acetylcholine and butyrylcholine esterase = AChE-I; NMDA-receptor antagonists = NMDA-RA) used in treatment of Alzheimer's disease. AChE-I such as donepezil (Aricept®), galantamine (Razadyne®), rivastigmine (Exelon®) and previously tacrine (Cognex®) typically delay worsening of symptoms for S.Krause-Hielscher* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. an average of 6–12 months for about 50% of patients, and the NMDA-RA memantine (Namenda®) modulates glutamate activity and temporarily delays worsening of symptoms for some patients [2]. They mainly do not influence the formation of "amyloid plaques" and "neurofibrillary tangles". The formation of amyloid deposits is based on a false splitting of the transmembrane amyloid precursor protein (APP = amyloid precursor protein) by the enzymes β-and γ-secretase [3]. The Aβ peptides, the main constituent of amyloid plaques, and various other metabolites are derived from APP by this false proteolytic cleavage [4, 5]. The further processed A peptides possess at their N-terminus a glutamate residues, which can be modified to a pyroglutamyl residue (pE) leading to the A peptides Aβ3 (pE)40/42 and Aβ11 (pE)-40/42. This posttranslational pEmodification causes proteolytic resistance. The formation of the pyroglutamate-ring also increases the hydrophobicity at the N-terminus which in turn induces accelerated aggregation of the pEApeptides. Several studies have shown that the senile plaques possess a prominent proportion of these neurotoxic pGlu-A peptides [6]. Pyroglutamatemodified amyloid-β (Aβ3(pE)) peptides are gaining considerable attention as potential key participants in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity [7]. N-terminally truncated and modified peptides are likely to be important for initiation of pathological cascades leading to AD. The formation of pGlu-peptides is catalyzed by glutaminyl cyclase, thus making QC to a valuable drug target [8]. First potential QC-inhibitors were identified and synthesized by Buchholz and coworkers. The structures are developed by applying a ligand-based optimization approach starting from imidazole [9]. Further classes of QC inhibitors were identified by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the QC active site. The efficacy assessment of the QC inhibitors was performed in cell culture, directly monitoring the inhibition of Aβ3 (pE)-40/42 formation [10]. Oral application of QC-inhibitors in two different transgenic AD mouse models and a Drosophila model reduced the Aβ3 (pE)-42 levels. Furthermore the treatment of mice reduced the production of plaque, gliosis formation and resulted in an improved performance in memory training and spatial learning tests [6, 8, 11-12,].

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Further identification of QC inhibitors from different biological sources can help to broaden the diversity of the active compound classes and to enlarge the chemical space for drug development. Thus, algae were investigated for potential QC-inhibiting secondary metabolites. It is known that these organisms produce a range of bioactive compounds, which are not commonly available from other plants or animals. This enormous potential of algae is proven previously by a multitude of identified secondary metabolites with, for example, cytostatic, antibacterial, antiviral, anti-inflammatory, or antifungal properties, many acting via the specific inhibition of enzymes [13-19]. Thus some algae and secondary metabolites thereof can be expected to be a potential source of new QC inhibiting compounds. Therefore, we analyzed algal strains and crude extracts of different algae species belonging to Chlorophyceae and Eustigmatophyceae.

MATERIALS AND METHODS Cultivation Selected species of microalgae were cultivated fedbatch wise in a 100 L tubular photobioreactor 100 GS/PL at pH 7 using Setlik media (Setlik, 1967). The cultures were illuminated continuously with warmwhite light (90 μmol/m2×s < OD 20; 150 μmol/m2×s >OD 20) at 28°C. Algae species belonging to Chlorophyceae (species 1-5) and Eustigmatophyceae (species 6) were obtained from SAG (Sammlung von Algenkulturen Göttingen, Germany). A disc separator (Westfalia) was used for harvesting the biomass from the exponential growth phase (GP) and stationary growth phase (SP) of each species. Extraction The freeze-dried algal biomass of the exponential growth phase (GP) and stationary growth phase (SP) of each species were extracted by two solid-liquidextraction procedures (Single solvent extraction and three step solvent extraction) using n-hexane, methanol and water. In the single step extraction procedure in each case 10 g of all biomasses were grinded using sea sand and extracted triply repeated with 600 mL of n-hexane, methanol or water. The resulting extracts were labeled as n-hexane extract species 1 sSP/GP ….6 sSP/GP, methanol extract species 1 sSP/GP* ….6 sSP/GP* and water extract species 1 sSP/GP ….6 sSP/GP (Fig. 1a). The successive three step extraction with increasing polarity from the same biomass was carried out for S.Krause-Hielscher* et al

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. each biomass by the extraction (triply repeated) with 600 mL of n-hexane using sea sand for grinding in the first step. After centrifugation (5 min, 2000 rpm) the n-hexane unextractable solid biomass was dried and extracted (triply repeated) with 600 mL of methanol in a second step. In the third step the dried biomass was extracted 3 times with water. The resulting extracts were labeled n-hexane extract species 1 mSP/GP ….6 mSP/GP, methanol extract species

1 mSP/GP* ….6 mSP/GP* and water extract species 1 mSP/GP ….6 mSP/GP (Fig. 1b). The resulting n-hexane and methanol extracts were concentrated to dryness in a rotary evaporator under reduced pressure. For water extracts, an Infrared Vortex Evaporator (Zinsser Analytic, Germany) was employed. The two extraction procedures resulted in a total number of 72 crude extracts.

Fig. 1a Single solvent extraction *methanol extracts: subsequence removal of chlorophyll by Chromabond SA-cartridges

Fig. 1b Successive three step extraction with increasing polarity from the same biomass *subsequence removal of chlorophyll by Chromabond SA-cartridges Sample preparation methanol for assaying. All extracts were prepared The final crude n-hexane extracts were dissolved in with a concentration of 1 mg×mL-1 for assaying. DMSO for testing in QC-assay. Aqueous crude extracts were dissolved in water and insoluble particles were Sephadex G-15 column chromatography removed by centrifugation for assaying. Positive assayed extracts were separated into 96 Due to the high chlorophyll content of the methanol fractions (each fraction from 8 mL eluent) by column crude extracts (methanol extract species 1 sSP/GP* chromatography on Sephadex G-15 (Pharmacia Fine ….6 sSP/GP* and methanol extract species 1 mSP/GP* Chemicals Inc., NJ) column (3.2 × 62 cm) using 70 % ….6 mSP/GP*), Chromabond SA – cartridges methanol as solvent. (Macherey & Nagel) were used to remove this undesired metabolite(s) according to the company Glutaminyl cyclase (QC) inhibition assay method (Macherey & Nagel Application database; The determination of the catalytic QC-enzyme activity http://www.mn-net.com). The resulting pre-cleaned was accomplished by utilization of the fluorogenic extracts were evaporated to dryness and redissolved substrate Gln-AMC (N-glutaminyl-7-amino-4-methylin pure methanol followed by a solid phase extraction coumarin) and the supporting enzyme pyroglutaminyl on Chromabond C18eC – cartridge (Macherey & aminopeptidase (pGAP) as well as purified human QC. Nagel) using methanol as solvent. The obtained For short, QC cyclizes Gln-AMC to pGlu-AMC which extracts were evaporated to dryness and dissolved in serves in a second step as substrate for pGAP. The International Journal of Pharmacy and Biological Sciences

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. removal of AMC from pGlu-AMC is than detected at λex = 380 nm; λem = 460 nm (Fig. 2) [20]. In this coupled optical assay the increase of the free AMC was continuously analyzed for more than 12 min at 30°C. The assay was conducted in microtiter plates by adding 100 µL 0.25 mM substrate, 50 µL 0.2 mg×mL1 extract, 25 µL supporting enzyme pGAP

and 25 µL QC with a final volume of 250 µL per well. Because of the QC pH value dependency, 50 µL Tris buffer (0.1 M; pH 8) was added. The exact procedure was published by [21, 22]. All extracts and fractions were assayed in triplicate and classified as QC-active if inhibition activities were more than 20 % vs. control.

Fig. 2 reaction sequence of the QC assay

RESULTS AND DISCUSSION Algae are chlorophyll rich organisms. For the successful bioassaying the removal of chlorophyll and its degradation products usually is a prerequisite, because the absorption of the dark green methanol crude extracts is interfering in photometric assays like the QC-enzyme assay. Thus a cartridge method to eliminate the chlorophyll was used. This method is fast, reproducible and offers high yields of the desired secondary metabolites. In comparison to a chlorophyll precipitation with citric acid buffer [23, 24], this more advanced method increases the yield of secondary metabolites up to 25-fold. However, certain organic cationic species may be retained too. Altogether 72 crude extracts of 6 microalgae species in either their exponential growth phase GP or stationary growth phase SP were tested for their ability to inhibit human glutaminyl cyclase (QC). The results are given in Fig. 3 (only extracts with a relative inhibition of > 20 % are considered active). Altogether 27 (38 %) of the tested algae extracts with -1 final extract concentrations of 0.2 mg×mL inhibit the glutaminyl-cyclase (QC). Among these, 5 n-hexane extracts show moderate inhibition of QC with activities between 24 % and 37 %. High levels of inhibition (48 % - 64 %) were obtained with three water extracts. In general, the chlorophyll-free methanol crude extracts constitute the most frequent and prominent inhibiton properties: 79 % of tested International Journal of Pharmacy and Biological Sciences

methanol extracts exhibit an activity between 21 % (moderate activity) and 71 % (high activity). In summary, extracts resulting from algae`s growth phase (GP) are slightly more potent than extracts taken from the stationary growth phase (SP). Out of 27 positively tested extracts, 63 % (17 extracts) result from the biomass of the growth phase. Additionally, extracts from the single step solid-liquid-extraction procedure (sSE) proved more potent as extracts from the three-step extraction (mSE). In order to exclude unspecific enzyme inhibition, the 24 methanol extracts were also tested at a higher final extract concentration of 2 mg×mL-1 (Fig. 4) which causes QC inhibition up to 99 % (Species 1 sGP, Species 2 sGP, Species 2 sSP, Species 4 sGP, Species 4 sSP, Species 5 sGP), i. e. the inhibition basically is concentration-dependent as required by theory. To identify QC-inhibition metabolites, the active methanol extracts of species no. 3 were further separated by Sephadex G-15 column chromatography into 96 fractions as described above. This crude separation let to a moderat concentration of activity peaking in fraction 72 with an inhibition activity of 66 % (Fig. 5), vs. an activity of the total extract of 57 %. However, no distinct separation of active constituents is possible by this chromatographic technique alone. In order to identify single active compounds, more advanced separation techniques will have to be applied in the future.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Glutaminyl cyclase (QC) is an emerging new and promising target for the causal treatment of AD. Accordingly, there is an increasing interest in exploring new and potent QC-inhibitors, especially if they are of natural origin. Until now, algae as a potential source of QC-inhibiting compounds have not been investigated. Only compounds acting as inhibitors of acetylcholine esterase (AChE) - a known target to treat Alzheimerâ&#x20AC;&#x2122;s disease - have been isolated [25]. A particularly active substance class of

these are Phlorotannins from macroalgae. The isolated compounds Eckol, Dieckol, 2- Phloroeckol and 7-Phloroeckol from E. stolonifera show a concentration-dependent inhibition of AChE [26, 27]. Phlorotannins isolated from Eisenia bicyclis inhibit the BACE-1 (APP-cleavinge enzyme), which plays an important role in the APP processing [28]. Also a BACE-1 inhibiting linear depsipeptide (Tasiamide B) was isolated from cyanobacteria [14].

Fig. 3 Inhibition activity of crude extracts tested positive against QC: 27 of 72 crude extracts from 6 different -1 species tested with a final extract concentration of 0.2 mgĂ&#x2014;ml .

Fig. 4 Simple concentration dependency of the inhibitory activity of methanol crude extracts against QC.

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Fig. 5 Partial concentration of the active principle(s) in the middle fraction on sephadex G15.

CONCLUSION In toto, 1/3 (27 extracts) of extracts from algal species belonging to Chlorophyceae (species 1 - 5) and Eustigmatophyceae (species 6) contain bioactive material which can be used as a starting point for potential compound development. The screening results demonstrate that algae are also able to produce secondary metabolites with inhibiting activity against glutaminyl cyclase. Methanol extracts of several algae strains seem to be particularly rich in effective compounds. They inhibit the QC-enzyme concentration dependent and will serve as basis for further bio-guided isolation efforts to identify the active principles.

ACKNOWLEDGEMENT We thank the Ministry for Economy and Sciences of Saxony Anhalt for the financial support of this study.

REFERENCES [1] Prince M., Albanese E., Guerchet M., World Alzheimer Report 2014, (2014) [2] Mikulca J.A., Nguyen V., Gajdosik D.A., Teklu S.G., Giunta E.A., Lessa E.A., Raffa R.B., Potential novel targets for Alzheimer pharmacotherapy: II. Update on secretase inhibitors and related approaches. Journal of clinical pharmacy and therapeutics, 39(1): 25-37, (2014) [3] Glenner G.G. and Wong C.W., Alzheimer´s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun 120: 885-890, (1984)

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[4] Citron M., Alzheimer’s disease: strategies for disease modification. Nature Reviews. Drug Discovery, 9(5): 387–98, (2010) [5] Strooper B. De, Vassar R., Golde T., The secretases: enzymes with therapeutic potential in Alzheimer disease. Nature Reviews Neurology, 6(2): 99–107, (2010) [6] Schilling S., Zeitschel U., Hoffmann T., Heiser U., Francke M., Kehlen A., Holzer M., Hutter Paier B., Prokesch M., Windisch M., Jagla W., Schlenzig D., Lindner C., Rudolph T., Reuter G., Cynis H., Montag D., Demuth H.U., Rossner S., Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer´s disease-like pathology. Nat. Med., 14(10): 1106–1111, (2008) [7] Jawhar S., Wirths O., Bayer T., Pyroglutamate amyloid-β (Aβ): a hatchet man in Alzheimer disease. The Journal of biological chemistry, 286:38825–38832, (2011) [8] Demuth H.U., Schilling S., Roßner S., Morawski M., Hartlage-Rübsamen M., Lues I., Glund K., Toxic pGluAbeta is enhanced and Glutaminyl Cyclase (QC) upregulated early in Alzheimer´s disease (AD): Inhibitors of QC blocking pGlu-Abeta formation are in clinical development. Alzheimer's & Dementia: The Journal of the Alzheimer's Association, 10(4): P149, (2014) [9] Buchholz M., Heiser U., Schilling S., Niestroj A.J., Zunkel K., Demuth H.U., The first potent inhibitors for human glutaminyl cyclase: synthesis and structure-activity relationship. J. Med.Chem., 49: 664–677, (2006) [10] Buchholz M., Hamann A., Aust S., Brandt W., Böhme L., Hoffmann T., Schilling S., Demuth H.U., Heiser U., Inhibitors for Human Glutaminyl Cyclase by Structure Based Design and Bioisosteric Replacement. J. Med. Chem., 52: 7069–7080, (2009) [11] Demuth H.U., Cynis H., Alexandru A., Jagla W., Graubner S., v. Hoersten S., Schilling S., Inhibition of

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. Glutaminyl Cyclase: Pharmacology and steps towards clinical development. Alzheimer's & Dementia: The Journal of the Alzheimer's Association, 6 (4): 571-572, (2010) [12] Jawhar S., Wirths O., Schilling S., Graubner S., Demuth H.U., Bayer T., Overexpression of glutaminyl cyclase, the enzyme responsible for pyroglutamate A{beta} formation, induces behavioral deficits, and glutaminyl cyclase knock-out rescues the behavioral phenotype in 5XFAD mice. The Journal of biological chemistry, 286:4454–4460, (2011) [13] Lindequist U. and Schweder T., Marine Biotechnology. In: Rehm, HJ, Reed G (ed) Biotechnology, Bd 10 2. edn. Wiley-VCH, Weinheim, pp 441-484, (2001) [14] Kelecom A., Secondary metabolites from marine microorganisms. Anais Da Academia Brasileira de Ciências, 74(1): 151–170, (2002 [15] Sakai R., Swanson G.T., Recent progress in neuroactive marine natural products. Natural Products Reports, 31: 273–309, (2014) [16] Blunt J.W., Copp B.R., Hu W.-P., Munro M.H.G., Northcote P.T., Prinsep M.R., Marine natural products. Natural Product Reports, 26(2): 170–244, (2009) [17] Blunt J.W., Copp B.R., Munro M.H.G., Northcote P.T., Prinsep M.R., Marine natural products. Natural Product Reports, 28(2): 196–268, (2011) [18] Blunt J.W., Copp B.R., Keyzers R.A., Munro M.H., Prinsep M.R. Marine natural products. Natural Product Reports, 30: 237–323, (2013) [19] Blunt J.W., Copp B.R., Keyzers R.A., Munro M.H.G., Prinsep M.R., Marine natural products. Natural Product Reports, 31(2): 160–258, (2014) [20] Zimmermann M., Yurewicz E., Patel G., A new fluorogenic substrate for chymotrypsin. Anal. Biochem., 70(1): 258-262, (1976)

[21] Schilling S., Hoffmann T., Wermann M., Heiser U., Wasternack C., Demuth H.U., Continuous Spectrometric Assays for Glutaminyl Cyclase Activity. Analytical Biochemistry, 303: 49–56, (2002) [22] Schilling S. and Demuth H.U., Continuous Assays of Glutaminyl Cyclase: from Development to Application. Spectroscopy, 18(2): 363-373, (2004) [23] Gross E.M., Wolk C.P., Jüttner F., Fischerellin a new allelochemical from the freshwater cyanobacterium Fischerella muscicola. J. Phycology, 27: 686-692, (1991) [24] Greilinger D., Charakterisierung bioaktiver Fischerelline in benthischen Cyanobakterien der Gattung Fischerella. Diploma Thesis Universität Konstanz. (2002) [25] Becher P.G., Baumann H.I., Gademann K., Jüttner F., The cyanobacterial alkaloid nostocarboline: an inhibitor of acetylcholinesterase and trypsin. Journal of Applied Phycology, 21(1): 103–110, (2009) [26] Yoon N.Y., Lee S.H., Kim S.-K., Phlorotannins from Ishige okamurae and their acetyl- and butyrylcholinesterase inhibitory effects. Journal of Functional Foods, 1(4): 331–335, (2009) [27] Suganthy N., Karutha Pandian S., Pandima D.K., Neuroprotective effect of seaweeds inhabiting South Indian coastal area (Hare Island, Gulf of Mannar Marine Biosphere Reserve): Cholinesterase inhibitory effect of Hypnea valentiae and Ulva reticulata. Neuroscience Letters, 468(3): 216–9, (2010) [28] Jung H.A., Oh S.H., Choi J.S., Molecular docking studies of phlorotannins from Eisenia bicyclis with BACE1 inhibitory activity. Bioorganic & Medicinal Chemistry Letters, 20(11): 3211–5, (2010)

*Corresponding Author: S. Krause-Hielscher Email: s.krause-hielscher@bwp.hs-anhalt.de

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EFFECT OF ACUTE EXPOSURE OF FORMALDEHYDE ON PULMONARY FUNCTION TESTS OF FIRST YEAR M.B.B.S. STUDENTS Shital Rameshrao Mankar and Amita Rajesh Ranade Department of Physiology, Shri. Bhausaheb Hire Government Medical College, Dhule

*Corresponding Author Email: smankar500@gmail.com

ABSTRACT AIMS: The aim of this study was to assess the effects of acute exposure of formalin on the pulmonary function. Methods: The study group consists of 60 First M.B.B.S. Medical Students of Shri. Bhausaheb Hire GMC Dhule; (Male =30, Female =30) (mean age male = 18.55+0.685 yrs, female = 18.17+0.8481 yrs.) who were regularly exposed to formaldehyde in anatomy department two hours once a day for six days per week. Inclusion criteria was clinically healthy, non-smokers, without any chronic respiratory disease, systemic illness like diabetes, hypertension etc. This study was conducted after obtaining ethical clearance and consent. MEDSPIRORCOMPUTERISED SPIROMETER was used to find out the PFT at start of anatomy dissection (pre-exposure) and after 2 hours of dissection (post-exposure). The pulmonary function tests included were FVC, FEV1, FEV1/FVC, MVV and PEFR. All these parameters helped in evaluating pulmonary functions among medical students exposed to formaldehyde for two hours during their anatomy dissection. Statistical Analysis: The data was entered in the MS Excel spreadsheet. Appropriate statistical analysis was performed using SPSS. Paired students‘t’ test was applied to compare the PFT parameters in Pre and post exposure changes. The data was expressed as mean + standard deviation. Result: In the present study acute exposure to formalin resulted in significant decrease in FEV1, FEV1/FVC, MVV, PEFR but in FVC no significant change seen in male students and decrease in all parameters in female students following acute exposure. Conclusion: Formaldehyde causes obstructive changes in form of bronchoconstriction at some extent due to acute exposure.

KEY WORDS formaldehyde, pulmonary functions, anatomy dissection hall.

INTRODUCTION Formaldehyde is a colorless, flammable gas at room temperature. It has a pungent, distinct odor and may cause a burning sensation to the eyes, nose and lungs at high concentrations. Formaldehyde is combined with methanol and buffers to make embalming fluid. Formaldehyde is also used in many hospitals and laboratories to preserve tissue specimens. Doctors, nurses, dentists, veterinarians, pathologists, embalmers, workers in the clothing industry or in furniture factories, and teachers and students who handle preserved specimens in laboratories also might be exposed to higher amounts of formaldehyde. At medical colleges formaldehyde has been used for years to preserve cadavers. Various complaints from medical students led to study its effects. Thus the study was planned with the aim to see effects of formalin on pulmonary functions in International Journal of Pharmacy and Biological Sciences

medical students who were exposed to formalin vapours for two hours every day for 6 days a week in anatomy dissection hall. AIMS AND OBJECTIVES: The aim of this study was to assess the effects of acute exposure of formalin on the pulmonary function of first year M.B.B.S. students SBH GMC Dhule during dissection hours.

MATERIALS AND METHODS: Selection and Description of Participants: The study was longitudinal study carried out on first M.B.B.S. students. The study group consist of 60 students (Male =30, Female =30) (mean age male = 18.55+0.685 yrs, female = 18.17+0.8481 yrs.) who were regularly exposed to formaldehyde in anatomy department two hours once a day for six days per week. The above students were having daily dissection class of two hours. Dissection hall was Shital Rameshrao Mankar & Amita Rajesh Ranade

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. having 10 dissection tables. One cadaver was given to 10 students, so there was equal exposure to all the subject. The relationship between exposure to formalin and change in pulmonary functions tests was compared prior to exposure and after two hours of dissection class in Department of Anatomy. Approval was taken from the institutional ethical committee. Technical Information: Project Instruments:  Examination proforma for obtaining medical history and for recording clinical examination.  Portable weighing machine was used to record the weight in kg.  Measuring tape was used to measure the standing height in centimeters.  MEDSPIROR - COMPUTERIZED SPIROMETER respiratory analysis system available in the research lab of Department of Physiology, SBH Govt. Medical College Hospital and Research center was used to perform the pulmonary function tests (PFTs). History and Clinical Examination : A thorough history was collected from all the participants including personal history such as name, age, sex, ethnicity, address, habit of smoking and medical history including history of any respiratory and cardiac diseases. All the subjects underwent an anthropometrical assessment including standing height and weight. The subjects for this study were included based on the following criteria. Inclusion criteria will be clinically healthy, nonsmokers, without any chronic respiratory disease, systemic illness like diabetes, hypertension etc. Exclusion criteria: Exclusion criteria were H/o chronic respiratory disease, H/o cardiac disease etc. Examination finding suggestive of respiratory or cardiac disease, abnormal pulmonary function test, extremes of weight and height, continuous 7 days absence from the class. For pulmonary function test medspiror was used. Pulmonary function test was recorded in PFT laboratory at around morning session before the start of dissection so as to avoid any acute effect of formalin. On the previous day, students were told to avoid any physical exertion cm and take proper rest and diet. The data of the subject as regards. name, age, height, weight, sex, date of performing the test,

atmospheric temperature was fed to the Spirometer. The tests were performed in sitting position. The subject was asked to take full inspiration which was followed by as much rapid and forceful expiration as possible in the mouthpiece of medspiror. Three consecutive readings were taken and the best reading amongst the three was selected. We have followed the guidelines of Americal Thoracic Society. [1] PFT was taken at start of anatomy dissection (preexposure) and after 2 hours of dissection. (Postexposure). The pulmonary function tests included were forced vital capacity (FVC) [L], forced expiratory volume 1 s (FEV1) [L], FEV1/FVC, peak expiratory flow (PEFR) [L/S], maximum ventilation volume or maximum voluntary ventilation (MVV) carried out by computerized spirometer. All these parameters helped in evaluating pulmonary functions among medical students exposed to formaldehyde for two hours during their Anatomy dissection. The procedure for doing test parameters for FVC, the subjects were asked to execute fast forceful expiration as much as possible at the end of deep inspiration. This test was repeated 2 or 3 times, and the best value was obtained. For MVV, the subjects were asked to inhale and exhale as deep and the fast as possible over a period of 12s during which recording were done. Statistics: The data collected was entered in the MS Excel spreadsheet. Descriptive table was generated, and appropriate statistical analysis was performed using SPSS (version 10). Student’s t-test was applied to compare the PFT parameters between pre exposed and formalin post exposed group. A significance level of “P” < 0.05 was considered for the student’s t-tests. The data were expressed as mean + standard deviation.

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RESULTS The mean age (years) of the subjects in the present study was (mean ± SD): male=18.55 ± 0.685 yrs, female=18.17 ± 0.8481 yrs. The mean height of these subjects was male=170.13 ± 5.99cm; female=158.62±3.93 and mean weight was male 59.069 ±10.54 kg; female=56.20 ± 11.87 kg.

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. TABLE I: Anthropometric profile (male n=30 and female n=30) Basal Values Male (n=30)mean+SD female (n=30)mean+SD Age (years) 18.55+0.685 18.17+0.8481 Height (cm) 170.13+5.99 158.62+3.93 Weight (kg.) 59.069+10.54 56.20+11.87 TABLE II: Comparison of lung functions in male students before and after exposure for 2 hours to formalin in anatomy dissection hall (n=male 30) Parameters Before Exposure mean±SD After exposure mean+SD P Value Significance ns FVC (L) 3.31+0.43 3.33+0.65 0.84 Not significant FEV1 (L) 3.13+0.40 2.82+0.69** 0.005 Significant FEV1/FVC 94.52+5.13 84.20+14.63** 0.001 Significant PEFR (L/S) 7.33+1.39 5.67+1.87*** 0.000 Significant MVV 107.76+15.71 105.38+21.38 0.000 Significant TABLE III: Comparison of lung functions in female students before and after exposure for 2 hours to formalin in anatomy dissection hall (n=female 30) Parameters Before Exposure mean±SD After exposure mean+SD P Value Significance FVC (L) 2.54+0.46 2.45+0.53* 0.025 Significant FEV1 (L) 2.40+0.42 2.22+0.509** 0.004 Significant FEV1/FVC 95.66+3.76 93.28+5.10* 0.02 Significant PEFR (L/S) 5.33+1.27 3.68+1.20*** 0.000 Significant MVV 95.38+11.32 87.66+15.71 0.000 Significant Values in mean ± S.D. *P Value<0.05, **P<0.001, ***P<0.0001, ns=not specified

Formaldehyde is produced naturally by our bodies [2]. It is found in all cells and is a normal component of human blood. In fact, formaldehyde is an essential chemical in the body and serves as a building block for the biosynthesis of more complicated molecules. [3] Formaldehyde is one of the most studied chemicals in use today. Studies in rats, monkeys, and humans show that inhaled formaldehyde does not change the levels of formaldehyde normally present in the blood. [4] At levels to which humans may be exposed, adverse effects are most likely to be observed primarily following inhalation. It has been experimentally proved that effects on organisms (e.g. mammals) are more closely related to concentration than to the accumulated total dose; this is due to the rapid metabolism and high reactivity and water solubility of formaldehyde. Dermal exposure predominantly affects the skin itself, and little if any formaldehyde reaches the blood stream. There is a relatively large exposure to formaldehyde from ingestion of flood, but most of it is present in a bound form.

Blood exchange is a critical form of exposure but is very rare, even in the very small segment of the population at risk [5]. Inhaled formaldehyde rapidly breaks down in the body from a gas into the soluble form of formaldehyde (methanediol) and then is changed into format in the nose and upper respiratory tract. Format is either used as a building block chemical for the body to make more complicated, larger chemical molecules or broken down into carbon dioxide,[6] which is exhaled in breath. The tiny fraction (i.e. <0.1%) of formaldehyde in the body that can exist in a gaseous form in small amounts (<0.8 ppb to 8 ppb; that is 0.001-0.01 mg/m3) [7] is exhaled in the breath. Consequently, formaldehyde levels in the blood do not increase as a result of inhaled formaldehyde. [8] In the present study acute exposure to formalin for 2 hrs/day for 6days/week resulted in decrease in FEV1, FEV1/FVC, PEFR, and MVV except FVC in male students in post exposure indicating mild broncho constriction. (Table no. II) Exposure to moderate levels of formaldehyde (1-3 ppm) can result in eye and upper respiratory tract irritation [9, 10]. Feihman states that most people cannot tolerate exposures to more than 5 ppm

International Journal of Pharmacy and Biological Sciences

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DISCUSSION

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ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. formaldehyde in air; above 10-20 ppm symptoms become severe and shortness of breath occurs. [11] High concentrations of formaldehyde may result in nasal obstruction, pulmonary edema, choking, dyspnea, and chest tightness [12,13] A medical intern with known atopy and exposure to formaldehyde over a period of 1 week developed dyspnea, chest tightness, and edema, following a final 2 hour exposure to high concentrations of formaldehyde [12]. Five workers exposed to high concentrations of formaldehyde from urea-formaldehyde foam insulation experienced intolerable eye and upper respiratory tract irritation, choking, marked dyspnea, and nasal obstruction [13]. However, the concentrative of formaldehyde and the contribution of other airborne chemicals were unknown in both of the reports. A series of pulmonary function studies has been conducted in healthy non smokers and asthmatics exposed to formaldehyde vapour; generally, lung function was unaltered. Fifteen healthy non smokers and 15 asthmatics were exposed to 2.4 mg/m3 formaldehyde for 40 mintutes to determine whether acute exposures could induce asthmatic symptoms [14,15]. No significant airway obstruction, changes in pulmonary function or bronchial hyperreactivity were noted. Similar observations were made on a group of 15 hospital laboratory workers who had been exposed to formaldehyde. [16] Lung function tests were performed on embalmers [17, 18], medical students [19] and anatomy and histology workers [20]. In most of the studies, formaldehyde alone or in combination with other agents caused transient, reversible declines in lung function, but there was no evidence that formaldehyde induces a chronic decrement in lung function. In BK Binawarastudy, highly significant (p<0.001) decrease in values of FVC, FEV1 and PEFR after acute exposure but reverted back to normal within 24 hrs. But FEV1/FVC ratio and FEF25-75% did not show any significant change [21]. Alexanderson and Hedenstierna, evaluated lung function tests and immunoglogulin levels in 34 wood workers who were exposed to formaldehyde. A significant decrease in FVC, FEV1, FEF25-75 was reported. [22] In the present study there was a decrease in dynamic lung function tests FEV1, FEV1/FVC, PEFR, MVV and FVC in female students following acute exposure (Table No. III) Akbar Khanzadeh et al evaluated acute pulmonary response in group of 34 workers exposed to formalin in gross anatomy dissection hall, also reported decrease in FVC but FEV1/FVC ratio

increased during exposure [23]. A trend towards decrease in values of FEV1 immediately after exposure was observed but it was not statistically significant [24]. In the study of ABHA SHRIVASTAVA AND YOGESH SAXENA there was a sharp decrease in dynamic lung function tests following 1 month of exposure, however the basal values were restored after 11 months of exposure to formalin vapours [25]. The effect of formaldehyde exposure in plywood workers resulted in significantly reduced FEV1, FEV1/FVC ratio, FEF25-75 but not FVC [26]. The exact concentration of formaldehyde to which our subjects were exposed in dissection hall could not be determined, which is the limitation of the study, but it is definitely at a concentration (2-3 ppm) causing severe eyes and nose irritation which was reported by the students following acute exposure [27].

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Shital Rameshrao Mankar & Amita Rajesh Ranade

CONCLUSION: Formaldehyde causes obstructive changes as is evidenced by decrease in FEV1, FVC/FEV1, PEFR, MVV .It can cause broncho constriction at some extent due to acute exposure. It also decreases FVC in female students. Scope of study: Study of use of mask can decreases various acute symptoms and need of further study of chronic effect of formaldehyde. We would like to recommend proper ventilation system in the dissection hall and students should be allowed to use mask so as to reduce personal exposure.

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18.

Guidelines for Indoor Air Quality; Selected Pollutants at pg. 108. Weber â&#x20AC;&#x201C; Tschopp A, Fisher T., Granjean E., Irritating effects of formaldehyde on men. Int occup Environ Health 1977; 39:207-218. Kulle J.T. Sauder L. R. Hebel JR, Green D., Chatham M.D., Formaldehyde dose- response in healthy non smokers. J Air pollution control asso 1987;37:919-924. Feinman SE, Editor, Formaldehyde sensitivity and toxicity, Boca Raton (FL); CRC Press Inc. 1988. Porter JAH, Acute respiratory distress following formalin inhalation, Lancet 1975; 1:603-604. Solomons K, Cochrane JWC, Formaldehyde toxicity; Part 1. Occupational exposure and a report of 5 cases, S Afr. Med. J 1984; 66:101-102. SCHACHTER, E.N. ET. AL A study of respiratory effects from exposure to 2 ppm formaldehyde in healthy subjects. Archieves of environment health, 41:229-239 (1986.) WITEK, T. J. ET. AL. An evaluation of respiratory effects following exposure to 2.0 ppm formaldehyde in asthmatics; lung function, symptoms, and airway reactivity. Archieves of environmental health, 42:230237 (1987.) 13 SCHACHTER, E. N. ET AL. A study of respiratory effects from exposure to 2.0 ppm formaldehyde in occupationally exposed workers, Environmental research, 44:188-205 (1987). LEVINE, R.J. ET AL. The effects of occupational exposure on the respiratory health of west Virginia morticians, Journal of occupational medicine, 26:91-98 (1984.) HOLNESS, D.L. & NETHERCOTT, J.R. Health status of funeral service workers exposed to formaldehyde, Archives of environmental health, 44:222-228 (1989).

19. UBA, G. ET AL. Prosepective study of respiratory effects of formaldehyde among healthy and asthmatic medical students, American Journal of industrial medicine, 1591-101 (1989). 20. KHAMGAONKAR, M.B. & FULARE, M.B. Pulmonary effects of formaldehyde exposure. An environmental epidemiological study. Indian Journal of Chest disease and alliedscicnes, 33:9-13 (1991). 21. BK Binawara, Rajnee, S. Chaudhary, KC Mathur. H Sharma, K. Goyal, Acute effect of formalin on pulmonary function tests in medical students. Pak J Physio 2010; 6(2) : 8-10.15. 22. Alexandersson R, Hedenstierna G. Pulmonary function in wood workers exposed to formaldehyde; a prospective study, Arch Environ Health 1986; 44(1); 511. 23. Akbar-Khanzadeh F., Mlynek JS, Changes in respiratory function after one and three hours of exposure to formaldehyde in non smoking subjects. Occup Environ Med. 1997, 54(5); 296-300. 24. Khaliq Farah, Tripathi Praveen, Acute effect of formalin on Pulmonary function in Gross Anatomy Laboratory, IJPP 2009;53: 93-96. 25. Effect Of Formalin Vapours On Pulmonary Functions Of Medical Students In Anatomy Dissection Hall Over A Period Of One Year Abha Shrivastava* And Yogesh Saxena Indian J Physiol Pharmacol 2013; 57(3) : 255260. 26. Malaka T, Kodama AM, Respiratory health of plywood workers occupationally exposed to formaldehyde, Arch Environ Health 1990: 45(5) : 288-294. 27. Chia SE, Ong CN, Foo SC, Lee HP, Medical students exposure to formaldehyde in a gross anatomy dissection laboratory. J Am Coll Health 1992; 41(3); 115-119.

*Corresponding Author:

Shital Rameshrao Mankar* Email: smankar500@gmail.com

International Journal of Pharmacy and Biological Sciences

Shital Rameshrao Mankar & Amita Rajesh Ranade

www.ijpbs.com or www.ijpbsonline.com

International Journal of Pharmacy and Biological Sciences

INSTRUCTIONS TO AUTHORS

INSTRUCTIONS TO AUTHORS ABOUT JOURNAL International Journal of Pharmacy and Biological Sciences is a peer-reviewed international journal scheduled to appear quarterly, which publishes innovative research works in the field of Pharmaceutical and Biological Sciences includes:                           

Pharmaceutics Polymer sciences Biotechnology Pathology Novel Drug Delivery Systems Pharmaceutical Biotechnology Microbiology Cytology Nanotechnology Biomaterial Sciences Cell Biology Immunobiology Pharmacology Natural Chemisty Biochemistry Pharmacognosy Pharmacoinformatics Bioinformatics AnalyticalChemistry BioPharmaceutics Molecular Biology Medicinal Chemistry Hospital and Clinical Pharmacy Neurobiology Pharmacy Practice Pharmacokinetics Pharmacogenomics

Therefore, the corresponding author is requested to mention the branches which come under either pharmaceutical or biological sciences for their manuscript in their cover letter.

SUBMISSION OF MANUSCRIPT Authors are encouraged to submit their manuscript electronically through online submission or an Email address, juksanthi2000@gmail.com along with a covering letter preferably by the corresponding author or first author. Each manuscript will be provided with a manuscript ID by IJPBS automatic system.

COVER LETTER During submission of the article, a cover letter should be included having

 Authors full address and telephone/fax number.  The type of article (Research or Review) along with the title and the type of branch (for branch kindly see “Aim and Scope” of this journal) under which the article to be published should be mentioned.  The corresponding author should mention the undertaking that if any animal studies carried was in accordance with their country or institutional ethical committee and also state that the manuscript has not been published elsewhere (except in the form of an abstract or as part of a published lecture or academic thesis). Please refer "Model Cover Letter" of this journal to get an idea.

MANUSCRIPT STATUS Track Your Manuscript: through online system You can track the status of your manuscript through the online production process by entering Manuscript ID in that field. For the submission of revised manuscript and queries regarding manuscript status or any other enquiries, please contact us at editor@ijpbsonline.com,juksanthi2000@gmail.co m With referring your manuscript ID

RESEARCH ARTICLES Manuscript should be type written in 12 font size, double-spaced, with margins of at least 2 cm on all sides. Pages should be numbered consecutively, starting with the title page and the matter arranged in the following order: Title page, Abstract, Keywords, Sections (Introduction, Materials and Methods, Results and Discussion, Conclusions), Acknowledgements, References, Tables and Figures along with caption and legends. Research articles should have more than 15 pages and Review articles in the range of 15-30 pages, inclusive of illustrations. Short communications should have not more than 4 Pages inclusive of illustrations. Title Page: Title page contains title of the manuscript in bold face, title case (font size 14), names of the authors in normal face, title case (font size 12) followed by the address of authors in normal face, title case (font size 12). Names of the authors should appear

Ijpbs-INSTRUCTIONS TO AUTHORS as initials followed by surnames. Full names may be given in some instances to avoid confusion. Followed by the author names, please provide the complete postal address or addresses with pin code number of the place(s), where the research work has been carried out. If the publication originates from several institutes, the affiliation of each author should be clearly stated by using superscript Arabic numbers after the name and before the institute. The author to whom correspondence should be directed must be indicated with an asterisk. At the bottom left corner of this page, please mention “*Corresponding Author” and provide telephone number and fax number of the research institution/college and functional Email address of the corresponding author to whom all correspondence (including galley proofs) is to be sent. Sections: Manuscripts should be divided into the following sections: Titles (normal face, upper case) and subtitles in each section (bold face, lower case): Abstract: An abstract not exceeding 250 words (for Short Communications between 60 and 80 words) should be provided typed on a separate sheet. Abstract should include aims, methods, results and conclusion. Keywords: Up to 4-6 keywords must be provided in alphabetical order. These keywords should be typed at the end of the abstract. Introduction: It should be a concise statement of the background to the work presented, including relevant earlier work, suitably referenced. It should be started in a separate page after keywords. Materials and Methods: It shall be started as a continuation to introduction on the same page. All important materials and equipments, the manufacturer’s name and, if possible, the location should be provided. The main methods used shall be briefly described, citing references. New methods or substantially modified methods may be described in sufficient detail. The statistical method and the level of significance chosen shall be clearly stated.

Results and Discussion: The important results of the work should be clearly stated and illustrated where necessary by tables and figures. The statistical treatment of data and significance level of the factors should be stated wherever necessary. The discussion should deals with the interpretation of results, making the readers to understanding of the problem taken and should be logical. The scope of the results, which need to be further explored, could also be dealt. Digital files are recommended for highest quality reproduction and should follow the following guidelines.  300dpi or higher sized to fit journal page  JPEG, GIF, TIFF and PDF formats are preferred)  Acknowledgement (if any) Conclusions: Concisely summarizes the principal conclusions of the work and highlights the wider implications. This section should not merely duplicate the abstract. Acknowledgements: Acknowledgements as well as information regarding funding sources may be provided. References: Citations of literature within the text must be presented in numerical order and should be set in square brackets, thus [1, 12]. The cited literature are also collected in numerical order at the end of the manuscript under the heading “References”. The abbreviated title and the volume number should appear in italics. Only the papers and books that have been published or in press may be cited. Please note that website addresses must not be included as a reference, but should be inserted in the text directly after the information to which they refer. Please note the following examples: Journals: [1]Gregoriadis G., Engineering liposomes for drug delivery: progress and problems. Trends Biotechnol, 13 (12): 527–537, (1995) Books: [1]Joseph R. Robinson and Vincent HL Lee, Ed. Controlled Drug Delivery Fundamentals and applications, 2nd Edn, Vol 29, Lippincott Williams’s publisher:555–561,(1994) [2] Myers, R.H., Montgomery, D., Response Surface Methodology, Wiley, New York 1995. ii

Ijpbs-INSTRUCTIONS TO AUTHORS Chapter in a book: [1] Brown, M.B., Traynor, M.J., Martin, G.P., Akomeah, F.K., in: Jain, K.K., Walker, J.M. (Eds.), Drug Delivery Systems, Humana Press, USA 2008, pp. 119-140. For Patent Reference [1]H. Aviv, D. Friedman, A. Bar-Ilan and M. Vered. Submicron emulsions as ocular drug delivery vehicles, U.S. Patent US 5496811, 1996. Tables: Should each be typed on a separate page, numbered in sequence with the body of the text. Tables should be headed with a short, descriptive caption. They should be formatted with horizontal lines only: vertical ruled lines are not required. Footnotes to tables should be indicated with a), b), c) etc. and typed on the same page as the table. Figures: Should be on separate pages but not inserted within the text. All figures must be referred to in the text and numbered with Arabic numerals in the sequence in which they are cited. Each figure must be accompanied by a legend explaining in detail the contents of the figure and are to be typed under the figures. Graphs and bar graphs should preferably be prepared using Microsoft Excel and submitted as Excel graph pasted in Word. Alternatively photographs can be submitted as JPEG images. Keys to symbols, abbreviations, arrows, numbers or letters used in the illustrations should not be written on the illustration itself but should be clearly explained in the legend. Avoid inserting a box with key to symbols, in the figure or below the figure. All Tables and Figures captions and legends should be typed on a separate page.

pages (Including Tables, Figures and References). Short communication should contain novel experimental or theoretical findings in need of urgent publication.

GALLERY PROOFS Gallery proofs are sent to the designated author through Email. They must be carefully checked and returned the revised manuscript within 48 hours of receipt.

Organization of the review article is at the author’s discretion and must be at a length of 3000 words excluding references and abstract. Abstract and key words are required. Tables, figures, illustrations and references are to be arranged according to research papers.

Authors are asked to sign a warranty and copyright agreement upon acceptance of their manuscript, before the manuscript can be published. The Copyright form can be downloaded from our website. Submission of your paper to this journal implies that the paper is not under submission for publication elsewhere. Material which has been previously copyrighted, published, or accepted for publication will not be considered for publication in this journal. Submission of a manuscript is interpreted as a statement of certification that no part of the manuscript is copyrighted by any other publisher nor is under review by any other formal publication. By submitting your manuscript to us, you agree on IJPBS Publication copyright guidelines. It is your responsibility to ensure that your manuscript does not cause any copyright infringements, defamation, and other problems. Submitted papers are assumed to contain no proprietary material unprotected by patent or patent application; responsibility for technical content and for protection of proprietary material rests solely with the author(s) and their organizations and is not responsibility of the IJPBS Publications or its Editorial Staff. The main author is responsible for ensuring that the article has been seen and approved by all the other authors. It is the responsibility of the author to obtain all necessary copyright release permissions for the use of any copyrighted materials in the manuscript prior to the submission.

SHORT COMMUNICATIONS

ETHICAL MATTERS:

Please add the term “Short Communication” below the title. A short communication contains a summary and should not exceed 4 printed journal

Authors involving in the usage of experimental animals and human subjects in their research article

REVIEW ARTICLE

iii

Ijpbs-INSTRUCTIONS TO AUTHORS should seek approval from the appropriate Institutional Animal Ethics committee in accordance with "Principles of Laboratory Animal Care". The Method section of the manuscript should include a statement to prove that the investigation was approved and that informed consent was obtained.

acknowledged, otherwise, it will be considered as an offense as per international Publications law.

CHECKLIST FOR SUBMISSION 1. 2.

Have you provided a Title Page? Have you provided an Author Information section at the end of the paper? 3. Have you provided an Abstract of not more than 250 words? 4. Have you provide keywords of not more than 3 to 4? 5. Are your Tables denoted by Arabic numerals, and are they in order as cited in the text? 6. Are your Tables submitting at the end of the text file? 7. Are your Figures denoted by Arabic numerals, and are they in order as cited in the text? 8. Have all your Figures been submitted text file or PNG or JPG files? 9. Are your References cited in the required style of the Journal? 10. Have you obtained permission and submitted documentation for all Personal Communications cited?

PUBLICATION POLICY: Papers will normally be published in order of acceptance by the editors, quickly as soon as the corrected electronic manuscripts are received. The details of the published paper (s) will be communicated to the corresponding author.

DECLARATION While submitting the manuscript the corresponding author ( on behalf of all the authors) should declare that the manuscript is original and is not published or communicated for publication elsewhere either in part or full. Without the declaration the manuscripts are not considered for their publication.

DISCLAIMER “No responsibility is assumed by the publisher” for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. The respective individual author(s) are responsible for the facts and views expressed in their articles. “Authors are solely responsible for their manuscripts and data published in the journals” and any mistakes in the published articles. The publisher does not take any such responsibilities. Copying and reproducing any parts of the published articles should be duly

AUTHOR BENEFITS The benefit of publishing in IJPBS includes:  Fast publication times  Free colour in electronic version  Access free on-line issue of IJPBS on www.ijpbs.com  A rigorous, fast and constructive peer review process  Excellent editorial standards  Any-Time, Any-Where access  Faster Review  No need for Hard copy submission  Ease of Contacting the IJPBS: 9912185510.

MODEL COVER LETTER (TEMPLATE)

International Journal of Pharmacy and Biological Sciences Date: Place: From

(Name and Address of the corresponding author)

To The Editor

International Journal of Pharmacy and Biological Sciences (IJPBS) Sir, Ref:

Title

………………….......................................

Type

…………………...................................... (Research/Review/Short communication)

Subject …………………...................................... (Pharmaceutical/Biological Sciences)

Branch …………………...................................... (Branch of subject-Refer author instructions)

In reference to the above title, I as a corresponding author, submit the manuscript for publication in International Journal of Pharmacy and Biological Sciences. I undertake that animal study (if any) was taken after the prior approval of country/institutional ethical committee. This manuscript has not been published or considered for publication by any other journal or elsewhere. Kindly consider the manuscript for publication in your journal.

Thank you

Corresponding author name and Signature

COPY RIGHT FORM

International Journal of Pharmacy and Biological Sciences I certify that I have participated sufficiently in the conception and design of this work entitled“……………………………………………………………………………………………………….……………………………………… ……………………………………………………………………………………………………………………………………………………………… ………..” and the analysis of the data, as well as writing of this manuscript, to take public responsibility for it. I believe the manuscript represents valid work. I have reviewed the final version of manuscript and approve it for publication. The manuscript is not published elsewhere, in any language, and is not under simultaneous consideration by any other journal. All Authors agree that the contents of the manuscript are confidential and will not be copyrighted, submitted, or published elsewhere (including the Internet), in any language, while acceptance by the Journal is under consideration. Furthermore I attest that I shall produce the data upon which the manuscript is based for examination by the editors or their assignees, if requested. I hereby assign and transfer to the juksanthi2000@gmail.com, editor@ijpbsonline.com copyright and all rights under it. I further confirm that this article has not been published elsewhere, nor is it under consideration by any other publisher.

SIGNATURE AUTHOR/AUTHORS 1. Signature: _____________________________Date: ______________________________

2. Signature: _____________________________Date: _____________________________

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5. Signature: _____________________________Date: ______________________________

Agreed- Yes / No (Put Tick) After completion of this form, please email the scanned file of original signed form to juksanthi2000@gmail.com, editor@ijpbsonline.com .

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