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An International, Peer Reviewed, Open access, Monthly E-Journal
ISSN 2277 – 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman
Managing Editor Dr. Shwetha Hari
Administrator & Associate Editor Miss. Shyamala Rupavahini
Advisory Board Prof. Rabinarayan Acharya Dr. Dinesh Katoch Prof. Sanjaya. K.S. Dr. Mathew Dan Mr. Tanay Bose Dr. Nagaraja T.M Dr. Narappa Reddy
Editorial board Dr. Kumaraswamy Dr. Madhu .K.P Dr. Sushrutha .C.K Dr. Ashok B.K. Dr. Janardhana.V.Hebbar Dr. Vidhya Priya Dharshini. K. R. Mr. R. Giridharan
Honorary Members - Editorial Board Dr. Shubha Ganguly Dr Farhad Mirzaei
INDEX Medicinal Plant Research Theoretical & Applied Biology EFFECTS OF COMBINING CRUDE ETHANOLIC EXTRACT OF JATROPHA CURCUS L. LEAF AND SOME ANTIBIOTICS AGAINST SOME SELECTED MICROORGANISMS Akanwariwiak W G, Addo-Fordjour P, Musah A A……………………………..140–148 Biochemistry PROTECTIVE EFFECT OF RUTIN ON ACETAMINOPHEN-INDUCED ACUTE HEPATIC DAMAGE IN RATS Awah Francis M, Chukwumezie Princess U, Ezema Ogechukwu C, Emiliarita Iloakasy, Ubokudom Queen I……………………………………………………………………………149–159 Veterinary Science ASPARAGUS RACEMOSUS WILLD. ROOT EXTRACT AS HERBAL NUTRITIONAL SUPPLEMENT FOR POULTRY Kumari R, Tiwary B K, Prasad A, Ganguly S…………………………………….160–163 Nutrition and Dietetics THE EFFECTS OF VITAMIN C AND GRAPE FRUIT JUICE SUPPLEMENTS ON THE POTENCY AND EFFICACY OF SOME SELECTED ANTI-MALARIAL DRUGS Adumanya O C, Uwakwe A A, Odeghe O B, Okere T O, Akaehi H C………..…164–171 Biochemistry EFFECTS OF AQUEOUS AND ETHANOLIC EXTRACTS OF DANDELION (TARAXACUM OFFICINALE F.H. WIGG.) LEAVES AND ROOTS ON SOME HAEMATOLOGICAL PARAMETERS OF NORMAL AND STZ-INDUCED DIABETIC WISTAR ALBINO RATS. Nnamdi Chinaka C, Uwakwe A A, and Chuku L C…………………………..…..172–180 Pharmacology EVALUATION OF ANTHELMINTIC ACTIVITY OF JUSSIAEA SUFFRUTICOSA LINN. Singh Vijayendra, Panda S K, Choudhary Puneet Ram………………………….181–185
Indigenous Medicine Ayurveda ASTASTHANA PARIKSHA – A DIAGNOSTIC METHOD OF YOGARATNAKARA AND ITS CLINICAL IMPORTANCE Sharma Rohit, Amin Hetal, Galib, Prajapati P K……………………………..186–201
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – TENDER LEAVES OF ZIZIPHUS RUGOSA LAM., RHAMNACEAE PLACE – KOPPA, CHIKMAGALUR DISTRICT, KARNATAKA, INDIA
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Original Research Article EFFECTS OF COMBINING CRUDE ETHANOLIC EXTRACT OF JATROPHA CURCUS L. LEAF AND SOME ANTIBIOTICS AGAINST SOME SELECTED MICROORGANISMS Akanwariwiak W G1*, Addo-Fordjour P1, Musah A A1 1
Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology
(KNUST), Kumasi, Ghana *Corresponding author: Email: garibawa@yahoo.co.uk, Tel: 233-24-571570, Fax: 233-51-60306 Received: 02/04/2012; Revised: 22/04/2012; Accepted: 25/04/2012
ABSTRACT Evidences are mounting concerning the resistance of microorganisms to antibiotics throughout the world. This development has awakened scientists to explore alternative approaches that target and block resistance. One way of accomplishing this has been the combination of plant extracts with antibiotics to increase their activity. The study was therefore, aimed at determining the effects of combining the leaf extract of Jatropha curcas L. with some antibiotics on certain selected microorganisms. The antimicrobial activity of the ethanolic extract of J. curcas leaf and its combination with selected antibiotics was assessed against certain microorganisms using the agar well diffusion method. The diameter of inhibition zone and minimum inhibitory concentration (MIC) were used as indicators of antimicrobial activity. The plant extract alone showed antimicrobial activity against all the test organisms, with diameter of inhibition zone ranging from 2–13.7 mm. The diameter of inhibition zone of the antibiotics alone ranged from 3.7–23 mm. The activity of the antibiotics varied upon combination with the plant extract, but the diameter of inhibition zone was between 6 and 25 mm. The antimicrobial activity of ciprofloxacin was increased significantly (MICs reduced significantly) when combined with the plant extract whereas that of tetracycline was reduced. In all, ciprofloxacin and ciprofloxacin-plant extract were the most effective treatments recording the lowest MICs. The most significant reduction of MICs was observed in the ciprofloxacin-plant extract combination.
Keywords: antimicrobial activity, crude ethanolic extract, Jatropha curcus leaf, diameter of inhibition zone, minimum inhibition concentration (MIC) Global Journal of Research on Medicinal Plants & Indigenous Medicine
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INTRODUCTION Infectious diseases caused by microorganisms are increasing in numbers thereby drawing the attention of researchers. Since the twentieth century, antibiotics have been employed in the treatment of many of these diseases. However, some microorganisms have already become resistant to many antibiotics while more continue to develop resistance to the action of some antibiotics (Lewis et al. 2002). For instance, Candida albicans is now reported to be resistant to a standard drug, clotrimazole, which once used to be very effective in tackling candidiasis (Goff et al. 1995; Nolte et al. 1997; Kieren et al. 1998). The problem of microbial resistance to antibiotics is growing and the outlook for the use of antimicrobial drugs in future is still uncertain, as newly developed antimicrobial agents are also being resisted (Coates et al. 2002). In the midst of increasing resistance of antibiotics to microorganisms, it is imperative to explore alternative approaches that target and block resistance. The use of agents that do not kill pathogenic bacteria but modify them to produce a phenotype that is susceptible to the antibiotic has been suggested as an alternative approach to the treatment of infectious diseases (Taylor et al. 2002). Such agents could render the pathogen susceptible to a previously ineffective antibiotic, and because the modifying agent applies little or no direct selective pressure, this concept could slow down or prevent the emergence of resistant genotypes. One way of accomplishing this has been the combination of plant extracts with antibiotics with the view to reducing the minimum inhibitory concentration (MICs) of the antibiotics significantly, against resistant strains (Darwish et al. 2002; Al-hebshi et al. 2006; Betoni et al. 2006). It is speculated that inhibition of drug efflux and alternative mechanisms of action could be responsible for the interactions between plant extracts and antibiotics (Zhao et al. 2001; Lewis and Ausubel 2006). Jatropha curcas (Figures 1 a. & b.) has played a major role in the treatment of various
diseases including bacterial and fungal infections. The extracts of many Jatropha spp. including J. curcas have displayed potent cytotoxic, antitumor and antimicrobial activities in different assays. For example, the leaves are utilized extensively in West African ethnomedical practice in different forms to cure various ailments like fever, mouth infections, guinea worm sores and joint rheumatism (Irvine 1961; Oliver-Bever 1986). The latex of J. curcas is reported to have antibacterial activity against Staphylococcus aureus (Thomas 1989), while the methanolic extract of the roots has been shown to exhibit antidiarrhoeal activity in mice through the inhibition of prostaglandin biosynthesis and reduction of osmotic pressure (Mujumdar et al. 2001). Although the antimicrobial activity of J. curcas on some microorganisms has been extensively studied, no work has been conducted on the possible interaction effects produced on microorganisms when extracts of the plant are combined with certain antibiotics. The study was therefore, carried out to determine the effects of combining the leaf extract of J. curcas with some antibiotics on certain selected microorganisms. METHODOLOGY Plant extraction Fresh leaves of Jatropha curcas were obtained at Maxima, a suburb of Kumasi. The sample was air-dried at room temperature and ground using a hammer mill. Five-hundred and fifty grams of the ground plant material was soaked in ethanol for 48 h after which extraction was done using the Soxhlet extractor. The solvent was removed from the extract with the Buchi rotary evaporator (R152) and the residue dried to a constant weight in an electric oven at 50°C. The dry plant extract was re-dissolved in methanol to the final graded concentrations of 10, 20, 30 and 40%. Tetracycline, Amoxicillin and Ketoconazole were used as positive control at concentrations of 0.1, 0.05, 0.025 and 0.0125. Ciprofloxacin was also used as a positive control at concentrations of 0.001%, 0.0001%, 0.00001% and 0.000001%.
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Figure: 1 a. Fruits of Jatropha curcus
Preparation of nutrient agar An amount of 24.8 g of nutrient agar was weighed into a conical flask. One thousand milliliters of distilled water was added and the mixture was melted over a Bunsen flame. The mixture was then poured into test tubes, 20 ml each and plugged with cotton wool. The cotton wool was covered with cellophane and the test tubes were autoclaved at 1.1 kg/cm3 steam pressure for 15 min. The nutrient agar was then stabilized in an electric water bath at 45°C for 15 min before use. Test microorganisms Six species of bacteria namely, Salmonella typhi, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumonia and Bacillus subtilis where used for the antimicrobial assay. C. albicans was the only fungal species included in the antimicrobial assay. Pure cultures of these organisms were obtained from the Microbiology laboratory of the Department of Pharmaceutics of the Faculty of Pharmaceutical Sciences, KNUST. The following chemotherapeutic agents were used as positive control: Tetracycline, Amoxicillin and Ciprofloxacin for the bacteria and Ketoconazole for the fungus. Determination of antimicrobial activity
b. Jatropha curcus in its habitat
stabilized nutrient agar was seeded with microorganisms, palmed and poured into a Petri dish to solidify. A cork borer of 9 mm in diameter was used to make wells in the agar. With the aid of a syringe, the wells were filled with different concentrations of the plant extracts. The extract was allowed to diffuse for 30 minutes and the plates were incubated at 37°C for 24 h. The zone of inhibition of the extract, the clear area around the well was measured in millimeters (mm) using a ruler after 24 h of incubation. Determination of minimum concentration (MIC)
inhibitory
A graph of the diameter of inhibition zones of the plant extract and the antibiotics was plotted against the log of concentration. The MIC of the particular treatment was then calculated as the antilog of the X-intercept from the equation of the line obtained. Determination of the combined effects of the plant extract-antibiotics combination on the test organisms The original concentrations of the antibiotics were maintained in combination with a concentration below the lowest MIC of the plant extract against the test organisms. The sub minimum inhibitory concentration of the plant extract, 2% was used as a solvent to dissolve the antibiotics.
The agar well diffusion method was employed in the assay. Twenty milliliters of Global Journal of Research on Medicinal Plants & Indigenous Medicine
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Statistical analysis Analysis of variance (ANOVA) was used to determine differences between the diameter of inhibition zones on one hand and the MICs on the other hand, between the plant extract,
antibiotics and antibiotics-plant extract treatments. The 11th Edition of the GenStat software (VSN International Ltd., Hemel Hempstead, UK) was used for the analysis at a significant level of 5 %.
Table 1: Antimicrobial effect of different concentrations (%) of J. curcas leaf extract on the test organisms Extract
DIZ (mm) at the various concentrations 10
20
30
40
MIC (%)
S. typhi
3.7
5.7
7.7
8.7
3.8
C. albicans
6.7
9
11.3
13.7
2.7
P. mirabilis
2
3
3.7
5
4.4
P. aeruginosa
4
5.3
6.7
7.7
2.3
S. aureus
4.3
6.7
9.3
10.7
4.2
K. pneumonia
2.7
3.7
5.3
7.3
4.8
B. subtilis
6.3
7.3
10.3
11.3
2.1
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone RESULTS Effects of J. curcas leaf extract on the test organisms The crude extract of J. curcas exhibited diverse antimicrobial activity against all the microorganisms used (Table 1). The plant extract inhibited growth of C. albicans and B. subtilis (ranged from 6.3–13.7 mm) more than the other microorganisms (ranged from 2– 10.7 mm). The activity of the plant extracts on all the microorganisms, increased with increasing concentration. There was no significant difference between the activity of the plant extract and that of amoxicillin and ketoconazole (P > 0.05). The MIC of the plant extract against B. subtilis (2.1%) was smaller compared to that of the other organisms. This was followed by the MIC against P. aeruginosa
(2.3 %). The highest MIC of the plant (4.8 %) extract against the microorganisms was recorded for K. pneumoniae. Effect of the antibiotics and antibiotics-plant extract combinations on the test organisms Ketoconazole and ketoconazole-plant extract The ketoconazole-plant extract combination produced significantly greater diameter of inhibition zones compared to those produced by ketoconazole alone (p < 0.001) (Table 2). The MIC of ketoconazole-plant extract combination was lower than that of ketoconazole only. Ciprofloxacin and ciprofloxacin-plant extract Ciprofloxacin showed activity against all the bacteria used (Table 3). The diameter of
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inhibition zone ranged from 7–22.7 mm for ciprofloxacin. The highest inhibition of growth occurred in B. subtilis (ranged from8– 22.7 mm). The activity of ciprofloxacin-plant extract was lower than that of the antibiotics alone although the difference was not
significant (p = 0.563). The MICs of the ciprofloxacin-plant extract combination were significantly higher than those of ciprofloxacin alone (p = 0.01). The best MIC of the ciprofloxacin-plant extract (1.0 × 10-9) was recorded against S. typhi.
Table 2: Antifungal effects of different concentrations of ketoconazole and ketoconazole-J. curcas leaf extract on C. albicans Concentration (%)
DIZ (mm) of ketoconazole
DIZ (mm) of ketoconazoleplant extract
0.1
11
15
0.05
6.7
13
0.025
5
10
0.0125
3.7
8
MIC
5.148 × 10-3
1.296 × 10-3
DIZ: Diameter of inhibition zone
Amoxicillin and amoxicillin-plant extract Amoxicillin alone and amoxicillin-plant extract combination did not show any activity against S. typhi, P.mirabilis and P. aeruginosa (Table 4). The growth of the rest of the microorganisms were however, inhibited by both treatments. The diameter of inhibition zones recorded for amoxicillin against S. aureaus and K. pneumoniae (10–20 mm) were lower than the diameter of inhibition zones of amoxicillin-plant extract combination against these bacteria (15–23 mm). The difference between the treatments with regard to the diameter of inhibition zones were however, not significant (p = 0.192). The MICs of the amoxicillin-plant extract combination were all lower than those of amoxicillin treatment. However, the differences between the MICs of the two treatments were not significant (p = 0.071). The lowest MIC for amoxicillin-
plant extract combination (3.148 × 10-5) was recorded against K. pneumonia. Tetracycline and tetracycline-plant extract The diameter of inhibition zones produced by tetracycline-plant extract combination against P. mirabilis, P. aeruginosa and S. aureus were higher than those produced by tetracycline alone (Table 5), although the differences in diameter of inhibition zones of the two treatments were not significant (p = 0.725). All the MICs produced by tetracycline-plant extract combination were significantly lower than those produced by tetracycline only (p = 0.003).
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Table 3: Antimicrobial effects of different concentrations of ciprofloxacin and ciprofloxacin-J. curcas leaf extract on the test organisms Microorganism
DIZ (mm) of ciprofloxacin
MIC
10-3
10-4
10-5
10-6
15.7
12.3
9.3
6.3
P. mirabilis
18
14.3
11
P. aeruginosa
20
15.3
S. aureus
19
K. pneumonia B. subtilis
S. typhi
DIZ (mm) of ciprofloxacinplant extract
MIC
10-3
10-4
10-5
10-6
1.015 × 10-8
12
10
8
6
1.0 × 10-9
7
1.086 × 10-8
15
11
8.3
7.7
1.71× 10-9
11.3
7.3
1.992 × 10-8
10.3
7.3
5.7
4.7
4.96 × 10-9
12.7
9
6.3
4.887 × 10-8
24.7
19
13.3
11
7.37 × 10-9
21.7
17.3
12.3
9
1.005 × 10-8
20
15.7
11.3
9
5.71 × 10-9
22.7
18.3
13.3
8
2.128 × 10-8
21.7
18
14
11
1.05 × 10-9
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone
Table 4: Antimicrobial effects of different concentrations of amoxicillin and amoxicillin-J. curcas leaf extract on the test organisms Microorganism
DIZ (mm) of amoxicillin
0.1
0.05
0.025
0.0125
S. typhi
0
0
0
0
P. mirabilis
0
0
0
P. aeruginosa
0
0
18.3
K. pneumonia B. subtilis
S. aureus
MIC
DIZ (mm) of amoxicillinplant extract
MIC
0.1
0.05
0.025
0.0125
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
15
12.3
11
6.674 × 10-4
23
20.7
17.7
17
5.623 × 10-5
17
14
12
10
6.643 × 10-4
20.3
18
16.7
15
3.148 × 10-5
20
18
15.7
12.7
3.110 × 10-4
18
15.7
14.7
13
4.701 × 10-5
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone
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Table 5: Antimicrobial effects of different concentrations of tetracycline and tetracycline-J. curcas leaf extract on the test organisms Microorganism
DIZ (mm) of tetracycline
0.1
0.05
0.025
0.0125
S. typhi
21
18
15
12.7
P. mirabilis
13
12
11
P. aeruginosa
15
12
S. aureus
15
K. pneumonia B. subtilis
MIC
DIZ (mm) of tetracyclineplant extract
MIC
0.1
0.05
0.025
0.0125
5.72 × 10-4
17
14
12
10
6.64 × 10-4
10
1.26 × 10-5
18
14.7
13
12
2.26 × 10-4
10
9
6.69 × 10-4
18.7
15.3
12.3
10.3
1.10 × 10-3
13.7
12.7
11.3
1.92 × 10-5
18.7
16.3
14.3
12.3
2.35 × 10-4
22
20
17.7
16.3
4.10 × 10-5
20
19
14
13
4.43 × 10-4
23
21.3
19.7
17
2.82 × 10-5
25
21.3
17.3
15.3
5.78 × 10-4
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone DISCUSSION The leaf extract of J. curcas showed some levels of activity against all the test organisms by inhibiting their growth. This suggests that the extract contained antimicrobial substances which were responsible for its activity (Srinivasan 2001). The effect of the plant extract varied from one microorganism to another. Candia albicans and B. subtilis were more susceptible to the extract than the rest of the microorganisms. The activity of the plant extract was concentration dependent, increasing with increasing concentration. Although the antimicrobial activities of ciprofloxacin and tetracycline were significantly higher than the plant extract (p < 0.001), the plant extract had inhibiting effects that were similar to those of amoxicillin and ketoconazole (p > 0.05). The leaf extract of J. curcas interacted with the antibiotics to produce varying effects on the tested microorganisms. The plant extract and antibiotics contained active ingredients which when combined with each other, resulted in additive, synergistic or antagonistic effects
(Delaquis et al. 2002; Fu et al. 2007). While the activity of some of the antibiotics was improved upon combination with the plant extract, the activity of others was reduced. The improved antimicrobial activity strength (indicated by the zone of inhibition size) of the combined treatments varied across the various treatments and tested organisms. The ketoconazole-plant extract combination produced significantly greater inhibition of growth of C. albicans compared to that produced by ketoconazole alone (p < 0.001). Amoxicillin alone was not able to inhibit the growth of S. typhi, P. aeruginosa and P. mirabilis. Although the plant extract alone was able to inhibit the growth of these organisms, its combination with amoxicillin did not produce any different effect from that of amoxicillin. However, amoxicillin alone showed some levels of activity against the other microorganisms, and this activity became slightly better when combined with the plant extract. Compared to tetracycline, tetracyclineplant extract inhibited the growth of P. mirabilis, P. aeruginosa and S. aureus more. Generally however, the differences in diameter
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of inhibition zones of these treatments were not significant (p = 0.725). These results suggest that there are possibly some phytochemicals in the plant extract which either decreased the resistance of the microorganisms or increased the mechanisms of action of the antibiotics (Alhebshi et al. 2006). There was no significant combining effect (of ciprofloxacin and ciprofloxacin-plant extract) on the inhibitory effect of ciprofloxacin against the microorganisms (p = 0.153). The MICs of the standard drugs were relatively lower than those of the plant extract due to the crude nature of the extract. When the antibiotics were combined with the ethanolic extract of J. curcas leaf at a sub minimum inhibitory concentration, the MICs of ciprofloxacin were decreased substantially (p = 0.01) against the test organisms. This reflects the interaction effects between the treatments (Cha et al., 2009). The MIC of ketoconazole was also slightly decreased when combined with the plant extract. On the contrary, the combination between tetracycline and the sub minimum inhibitory concentration of the plant extract caused a significant increase (p = 0.003) in the MICs of the drug. This may indicate that, some active ingredients in the extract interfered with the mechanism of action by which the antibiotic works. In all, ciprofloxacin and ciprofloxacin-plant extract were the most effective treatments since they had the lowest MICs against all the microorganisms. By far, the best combining REFERENCES Al-hebshi N, Al-haroni M, Skaug N (2006). In vitro antimicrobial and resistancemodifying activities of aqueous crude khat extracts against oral microorganisms. Arch. Oral Biol. 51:183–188. Betoni JEC, Mantovani RP, Barbosa LN, DiStasi LC, Fernandes A (2006). Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem. Inst. Waldo Cruz. 101 No. 4.
effects was observed in the ciprofloxacin-plant extract. The susceptibility of microorganisms to both the antibiotics and antibiotics-plant extract combinations varied tremendously. For instance, S. typhi was most susceptible to both ciprofloxacin alone and ciprofloxacin-plant extract treatments since it required the least dose to be inhibited. On the other hand, S. aureaus was least susceptible to these treatments requiring higher doses for inhibition. CONCLUSION The leaf extract of J. curcas showed antibacterial and antifungal activities against all the micro-organisms. The antimicrobial activity of ciprofloxacin was increased significantly (MICs reduced significantly) when combined with the plant extract. On the other hand, the activity of tetracycline was reduced significantly (increased MICs) when combined with the plant extract. In all, ciprofloxacin and ciprofloxacin-plant extract were the most effective treatments with the lowest MICs. The most significant reduction of MICs was observed in the ciprofloxacin-plant extract combination. ACKNOWLEDGEMENTS Logistical support for the study was provided by the Department of Pharmaceutics, KNUST, Kumasi, Ghana.
Cha JD, Moon SE, Kim JY, Jung EK, Lee YS (2009). Antibacterial activity of sophoraflavanone G isolated from the roots of sophora flavescens against methicillin-resistant staphylococcus aureus. Phytother. Res. 23(9):1326– 1331. Coates A, Hu Y, Bax R (2002). The future challenges facing the development of new antimicrobial drugs. Nat. Rev. Drug Discovery 1:895–910.
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Darwish RM, Aburjai T, Al-Khalil S, Mahafzah A (2002). Screening of antibiotic resistant inhibitors from local plant materials against two different strains of Staphylococcus aureus. J. Ethnopharm. 79:359–364. Delaquis PJ, Stanich K, Girard B, Mazza G (2002). Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food. Microbiol. 74:101–109. Fu Y, Zu Y, Chen L, Shi X, Wang Z, Sun S, Efferth T (2007). Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytother. Res. 21: 989–994. Goff
DA, Koletar SL, Buesching WJ, Barnishan J, Fass RJ (1995). Isolation of fluconazole resistant Candida albicans from human immunodeficiency virus-negative patients never treated withazoles. Clin Infect Dis. 20(1):77–83.
Companies, Inc., 1221 Avenue of the Americas, New York p. 275. Mujumdar AM, Miser AV, Salaskar MV, Upadhye AS (2001). Antidiarrhoeal effect of an isolated fraction of Jatropha curcas roots in mice. J. Nat. Remedies 1: 98–93. Nolte FS, Parkinson T, Falconer DJ, Dix S, Williamsm J, Gilmore C, Geller R, Wingard JR (1997). Isolation and characterization of fluconazole- and amphotericin B- resistant Candida albicans from blood of two patients with leukemia Antimicrob. Agents Chemothe. 41: 196–199. Oliver-Bever B (1986). Medicinal plants in tropical West Africa, Cambridge University Press, London. Srinivasan D, Nathan S, Suresh T, Perumalsamy O (2001). Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine. J. Ethnopharmacol. 74:217–220.
Irvine ER (1961). Woody plants of Ghana with special reference to their uses. 2nd Ed. O.U.P. London p. 233–237.
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Kieren AM, Lyons CN, Rustad T, Bowden RA, White TC (1998). Rapid, Transient Fluconazole Resistance in Candida albicans Is Associated with Increased mRNA Levels of CDR Antimicrob. Agents Chemother. 42: 2584–2589.
Thomas OO (1989). Re-examination of the antimicrobial activities of Xylopia aethiopica, Carica papaya, Ocimium grastissimum and Jatropha curas. Fitoterapia 60:147–161.
Lewis K, Ausubel FM (2006). Prospects for plant-derived antibacterials. Nat. Biotechnology. 24(12): 1504–1507. Lewis R, Gaffin D, Hoefnagels M, Parker B (2002). Life. 4th Ed. McGraw-Hill Source of Support: Nil
Zhao WH, Hu ZQ, Okubo S, Hara Y, Shimamura T (2001). Mechanism of synergy between Epigallochatechin gallate and B-Lactams against methicillin resistant Staphylococcus aureus. Antimicrob. Agents Chemotherapy 45(6): 1737–1742. Conflict of Interest: None Declared
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Original Research Article PROTECTIVE EFFECT OF RUTIN ON ACETAMINOPHEN-INDUCED ACUTE HEPATIC DAMAGE IN RATS
Awah Francis M1*, Chukwumezie Princess U2, Ezema Ogechukwu C3, Emiliarita Iloakasy4, Ubokudom Queen I5 1, 2,3,4,5
Department of Biochemistry, Madonna University, Elele Campus, Rivers State, Nigeria *Corresponding author: E-mail: awambuh@yahoo.com; Tel: (+234) 8057431113
Received: 18/03/2012; Revised: 16/04/2012; Accepted: 25/04/2012;
ABSTRACT Acetaminophen is a widely used analgesic and antipyretic drug; overdose however, can cause acute hepatic and renal damage. In this study, rutin a natural antioxidant belonging to the class of bioflavonoids was investigated for its hepato- and nephro-protective capabilities in acetaminopheninduced damage. Male albino rats were divided into five groups. Group A (control) was given normal saline only, group B was given acetaminophen only (8 g/kg body weight) for seven days, while groups C, D and E were co-administered acetaminophen (8 g/kg body weight) and 100, 200 and 500 mg/kg body weight of rutin respectively for seven days. On the eighth day the rats were killed. Liver and kidney function tests were performed using Randox diagnostic reagent kits. Oxidative stress status was assessed by assaying for catalase, superoxide dismutase, ascorbate and malondialdehyde using standard methods. Oral administration of acetaminophen produced liver damage as rats in group B had significant elevations (p < 0.05) in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared to group A, C, D and E. Creatinine levels were significantly (p < 0.05) maintained to normal levels in group C, D and E rats as compared with group B. Significantly low activity (p < 0.05) of superoxide dismutase (SOD) were observed in group B, relative to groups A, D and E. Co-treatment with 200 and 500 mg/kg body weight rutin also significantly lowered the level of lipid peroxidation while ascorbate was elevated compared to group B. These results suggest that in-vivo, rutin could counteract the deleterious effects caused by acetaminophen metabolic intermediates and could therefore be used as an antidote in combination with acetaminophen to protect the liver in case of an overdose.
Keywords: Rutin, acetaminophen, hepato-protective effect
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INTRODUCTION Acetaminophen (paracetamol) is a widely used analgesic and antipyretic (Cranswick and Coghlan, 2000; Moller et al., 2005; Bertolini et al 2006). Its mechanism of action is considered to be via the inhibition of cyclooxygenase (COX-2) (Hinz et al., 2008). While generally safe for use at recommended doses, acute overdoses of acetaminophen can cause potentially fatal multiple-organ damages, particularly liver damage and acute kidney failure (Jaeschke et al., 2002; Mahadevan et al., 2006; Ryder and Beckingham, 2001). Acetaminophen toxicity is not from the drug itself but from the alklating electrophillic metabolites, N-acetyl-p-benzoquinoneimine (NAPQI) (Mitchell et al., 1973; Cohen et al., 1997). Acetaminophen is metabolized primarily via phase II metabolism in the liver, into non-toxic products before excretion in the kidney (Muldrew et al., 2002). A small, yet significant amount is metabolized via the hepatic cytochrome P450 enzyme system, which is responsible for the formation of NAPQI. At normal doses, NAPQI is quickly detoxified by conjugation with glutathione. Following overdose, this detoxification pathway becomes saturated, and, as a consequence, NAPQI depletes hepatic glutathione (Mitchell et al., 1973). NAPQI is then free to react with cellular membrane molecules, resulting in acute hepatic damage. Animal studies have shown that hepatic glutathione is depleted to less than 70% of normal levels for hepatotoxicity to occur (Richardson, 2000). The increasing liver damage alters biochemical markers of liver function (hepatic transaminases), leading to abnormal rise in serum levels. In some cases, acute kidney failure may be the primary clinical manifestation of toxicity. In these cases, it has been suggested that the toxic metabolite is produced more in the kidneys than in the liver (Boutis and Shannon, 2001). There is evidence pointing to the fact that oxidative stress is involved in acetaminophen toxicity. Free radicals such as superoxide anion may be formed via a number of mechanisms including formation from cytochrome P450
(Puntarulo and Cederbaum, 1996). Superoxide anion rapidly reacts with nitric oxide forming peroxynitrite which is another very toxic radical (Hinson et al., 2002). In addition, during formation of NAPQI by cytochrome P450, the superoxide anion formed, undergoes dismutation leading to the formation of another reactive oxygen species (ROS) hydrogen peroxide (Dai and Cederbaum, 1995). Also, peroxidation of acetaminophen to the semiquinone free radical could lead to increased superoxide anion generation via the redox cycling between the acetaminophen and the semiquinone (de Vries, 1981). Rutin is an antioxidant that belongs to a class of plant secondary metabolites called bioflavonoid that are also known as rutoside, sophorin and quercetin-3-rutinoside (Yang et al., 2008). It is sometimes referred to as Vitamin P, although not strictly a vitamin. Rutin is gotten from natural sources like buckwheat, tomato, orange, carrot, sweet potato, black tea and apple peels (Kreft et al., 1999; Fabjan et al., 2003, Wang et al., 2003). Ingestion of rutin is said to have abundant health benefits. Rutin enhances the effectiveness of vitamin C, lowers blood cholesterol levels as well as works as a very potent antioxidant (Guo et al., 2007; Caillet et al., 2007; Jiang et al., 2007). Rutin is also helpful in treating glaucoma, high blood pressure, heart disease and allergies (Rosane et al., 2006; Sheu et al., 2004). It is reported to possess anti-inflammatory, anticancer, antibacterial, antiviral and antiprotozoal properties (Webster et al., 1996; Guardia et al., 2001; Calabro et al., 2005; Kwon et al., 2005; MartĂnez et al., 2005; Luo et al., 2008). Oxidative stress, hepatotoxicity and nephrotoxicity have been reported to be hallmarks in the toxicity of acetaminophen. Rutin is known to have a potent in vitro antioxidant activity; however, little data is available regarding the in vivo antioxidant potentials. This study was aimed at investigating the in vivo antioxidant potential, hepatoprotective and nephroprotective effects
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of rutin in albino rats administered with high doses of acetaminophen. MATERIALS AND METHODS Chemicals All chemicals and reagents used were of analytical grade. Acetaminophen, acetic acid, L-ascorbic acid, sulfuric acid, potassium dihydrogen phosphate (KH2PO4), potassium hydroxide (KOH), ferric chloride (FeCl3), ethylenediaminetetraacetic acid (EDTA), sodium carbonate (Na2CO3), acetaminophen, methanol, ferrous sulfate (FeSO4.7H2O), hydrogen peroxide (H2O2), thiobarbituric acid (TBA), Folin–Ciocalteu’s reagent (FCR) and trichloroacetic acid (TCA) were all purchased from Sigma Chemical Co. (St. Louis, MO). Animals All animals were cared for in accordance with the principles and guidelines of the ethical committee for conduction of animal studies in Madonna University, Elele, Nigeria. Eightweek old healthy male albino rats of the Wistar strain with an average weight of 200–250 g were used in the study. The experimental animals were housed in aluminum cages in an animal house properly ventilated with good sanitary condition. Experimental design The acetaminophen-induced hepatotoxicity model experiment was employed in this study. A total of 25 rats received water ad labitum and vital feed grower pelletized mash (Grand Cereals and Oil Mills Ltd, Nigeria) and were randomly divided into five groups (n = 5): Group A (control), was given normal saline only; Group B, acetaminophen-only (8 g/kg body weight); Group C, rutin (100 mg/kg body weight) + acetaminophen; Group D, rutin (200 mg/kg body weight) + acetaminophen; and Group E, rutin (500 mg/kg body weight) + acetaminophen. Groups B, C, D and E were intragastrically co-administered 8 g/kg acetaminophen for seven days. All animals were anaesthetized with chloroform and killed
on the eighth day. Blood was collected by cardiac puncture in plain tubes. This was then centrifuged at 5000 rpm for 10 min to obtain the serum for biochemical assays. The liver was removed, weighed and individually homogenized in ice cold phosphate buffer solution (0.1 M, pH 7.4) to give a 10 % (w/v) liver homogenate. Tissue homogenates were prepared and the homogenate was centrifuged at 5000 rpm for 20 min. The supernatant was used for biochemical assays. Assessment of liver function and kidney function Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, and urea levels were assayed in fresh serum using the commercial kits supplied by Randox (UK). These analyses were carried out according to the manufacturer’s protocols. Assessment of Liver homogenate antioxidants and oxidative stress markers The liver homogenate total protein concentration was measured by the method of Lowry et al. (1951). Catalase (E.C.1.11.1.6.) activity was determined according to Aebi (1984) with phosphate buffer pH 7.0, at 240 nm. Total superoxide dismutase (mitochondrial Mn-containing and cytosolic Cu- and Zn-containing forms E.C. 1.15.1.1) activity was determined by the method of Beauchamp and Fridovich (1971) at room temperature. Measurement of the extent of lipid peroxidation in the liver homogenates was determined based on the formation of thiobarbituric acid reactive substance as described by Buege and Aust (1978). Ascorbate levels in homogenates were determined following the method of Tietz (1986). The spectrophotometric readings were performed in a Jenway UV/visible spectrophotometer (Camlab, UK). Statistical analysis The data were analysed using the Statistical Package for Social Sciences (SPSS) version
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10.0 for Windows. Analysis of variance (ANOVA) was used to compare means, and values were considered significant at p < 0.05. All the results are expressed as mean ± standard error of the mean (SEM). RESULTS AND DISCUSSION Acetaminophen overdose is the most frequent cause of drug-induced liver injury in many parts of the world. In the present study, we investigated the protective potential of rutin when co-administered with high doses of acetaminophen to induced hepatotoxicity. Considering the fact that oxidative stress is a major hallmark in hepatotoxicity, an antioxidant like rutin with potent in vitro radical scavenging capabilities could be effectively used to prevent, manage or treat liver damage. Effect of rutin on liver function acetaminophen-intoxicated rats
in
Acetaminophen-induced hepatic damage is a commonly used model for hepatoprotective drug screening and the extent of hepatic damage is assessed by the serum level of AST, ALT and ALP (Sallie et al., 1991). In this study the hepatotoxic effect of acetaminophen overdose was confirmed in accordance with previous reports (Jaeschke et al., 2003; Mahadevan et al., 2006). The acetaminophen metabolite NAPQI caused damage in the hepatocytes leading to a leakage of ALT and AST into the serum. Co-administration of rutin at the doses of 100, 200 and 500 mg/kg with toxic doses of acetaminophen significantly (p < 0.05) protected the liver from damage as shown by the serum transaminases (AST and ALT) compared to the control (96.2 ± 7.8 U/L and 46.5 ± 9.8 U/L respectively) and the
acetaminophen-only groups (534.8 ± 44.2 U/L and 236.9 ± 20.6 U/L respectively) (Table 1). AST is found in cardiac, hepatic, muscle and kidney tissues while ALT is produced principally in the liver where it catalyses transamination reactions. ALT is therefore more specific for hepatocellular damage than AST and remains elevated in the serum for longer periods, due to its longer half-life. AST is found in the cell cytoplasm and mitochondria while ALT is found solely in the cytoplasm, hence in an inflammatory condition, there is simply leakage of cytoplasmic enzymes into circulation and ALT will rise more than AST (Bramstedt, 2006). In this study, the level of AST rose above the ALT, suggesting gross cellular necrosis in acetaminophen poisoning, resulting in both cytosolic and mitochondrial AST. However, ALP levels were not significantly different (p > 0.05) between the control, acetaminophen-only and acetaminophen + rutin co-treated rats. As observed in this study, rutin significantly attenuated the hepatotoxic effects of acetaminophen and assisted in maintaining the normal integrity of the hepatocytes. Since oxidative damage and inflammation play central roles during drug-induced damage, the observed protective effect of rutin could possible be due to its inherent antiinflammatory activity (Guardia et al., 2001) and free radical scavenging and anti-lipid peroxidation capabilities (Gao and Zhou, 2005). Alternatively, inhibition of cytochrome P450 isoenzymes (CYPs) could also have reduced the toxicity of acetaminophen since formation to the toxic metabolite NAPQI will be minimized (Bear and Teel, 2000). These observations suggest that rutin may find clinical application in a variety of conditions where oxidative stress causes cellular damage.
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Table 1: Serum hepatic enzymes levels of control and acetaminophen-intoxicated rats AST
ALT
ALP
(U/L)
(U/L)
(U/L)
Control
96.2 ± 7.8*
46.5 ± 9.8*
296.2 ± 26.5
Acetaminophen Only
534.8 ± 44.2
236.9 ± 20.6
314.8 ± 24.2
Acetaminophen + 100 mg/kg Rutin
280.2 ± 25.4*
166.8 ± 14.4*
290.2 ± 35.4
Acetaminophen + 200 mg/kg Rutin
218.6 ± 23.3*
146.6 ± 21.3*
308.6 ± 23.3
Acetaminophen + 500 mg/kg Rutin
182.4 ± 14.4*
151.3 ± 22.7*
282.4 ± 14.4
Data represented as Mean ± SEM; * p < 0.05 vis-à-vis the Acetaminophen-only group AST-Aspartate Aminotransferase; ALT-Alanine Aminotransferase; ALP-Alkaline Phosphatase
Table 2: Serum urea and creatinine levels of control and acetaminophen-intoxicated rats Urea
Creatinine
(mmol/L)
(mg/dL)
Control
6.32 ± 1.65
0.43 ± 0.06*
Acetaminophen Only
7.82 ± 1.37
1.62 ± 0.07
Acetaminophen + 100 mg/kg Rutin
6.69 ± 1.59
0.97 ± 0.02*
Acetaminophen + 200 mg/kg Rutin
6.63 ± 0.58
0.84 ± 0.04*
Acetaminophen + 500 mg/kg Rutin
6.34 ± 0.50
0.65 ± 0.03*
Data represented as Mean ± SEM; * p < 0.05 compared to the Acetaminophen-only group Effect of rutin on kidney function in acetaminophen-intoxicated rats In addition to liver damage, the acetaminophen metabolite N-acetyl-pbenzoquinoneimine (NAPQI) also induces kidney damage. Increase in serum concentrations of urea and creatinine is prominent in acute nephrotoxicity (Erdem et al., 2000). The results of the present study showed that the acetaminophen-only rats had higher urea level (7.82 ± 1.37 mmol/L)
compared to the control (6.32 ± 1.65 mmol/L) and those co-treated with rutin, though the mean difference was not statitistically significant (p < 0.05) (Table 2). Serum creatinine levels were significantly higher (p < 0.05) in acetaminophen-only rats (1.62 ± 0.07 mg/dL) compared to rats treated with rutin at 100 mg/kg (0.97 ± 0.02 mg/dL), 200 mg/kg (0.84 ± 0.04 mg/dL) and 500 mg/kg (0.65 ± 0.03 mg/dL) and the control (0.43 ±
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0.06 mg/dL) (Table 2). The level of creatinine in the acetaminophen-only rats was very high which could be as a result of the inflammation of the kidney caused by the free radicals generated by the acetaminophen overdose that led to the decreased filtration rate of the nephron. This suggeststhat rutin possesses dose-dependent protective effects against acetaminophen-induced kidney damage. As a potent antioxidant rutin possibly scavenged the free radicals generated during acetaminophenintoxication thereby preventing renal damage by oxidants and stabilizing the renal function. The observed nephroprotective potential of rutin is in accordance with previous reports by Alsaif (2009). Effect of rutin on hepatic antioxidant enzymes activity in acetaminophenintoxicated rats According to the present data, the extent of reactive oxygen species production by the administered of acetaminophen is significantly quenched by rutin in a dose-dependent manner; thereby reducing the extent of liver damages among the rats co-treated with rutin and acetaminophen, relative to normal and acetaminophen-only rats. Superoxide dismutase (SOD) and catalase (CAT) are endogenous antioxidant enzymes responsible for the detoxification of deleterious oxygen radicals. The protective effect(s) of rutin was evident through significantly higher levels (p < 0.05) of total SOD activities among the rutin co-treated rats (10.63 ± 1.58 and 10.34 ± 1.50 U/mg protein for 200 and 500 mg/kg rutin respectively) relative to acetaminophen-only rats (5.82 ± 0.37 U/mg protein) (Table 3). Acetaminophen decreased the liver total SOD activity by about 50% relative to the normal healthy control rats indicating that the high dose of acetaminophen administered to the rats, constituted a stressor agent that lead to depletion of the liver tissue antioxidant enzymes. Table 3 clearly indicates that rutin co-treatment has increased the SOD, but not CAT, activity among the acetaminophentreated rats relative to control rats. This suggests that the antioxidant enzyme CAT is
not very much affected probably because the major free radicals involved in acetaminophen toxicity are superoxide anion and peroxynitrite. The decreased activity in total SOD could be due to exhaustion of the enzyme because of increased generation of free radicals such as superoxide anion during NAPQI metabolism and peroxidation. Rutin co-treatment significant increased (p < 0.05) the hepatic SOD activity (Table 3) by possibly scavenging the free radical generated thereby preventing radical-induced hepatic damage. The increase in total SOD activity in rutin co-treated rats is a definite indication of hepatoprotective action of the drug (Curtis and Mortiz, 1972). Previous studies have revealed another possible mechanism of action of rutin is by upregulating the expression of genes for antioxidant enzymes (Lores-Arnaiz et al., 1995). In this context, treatment with rutin probably increased the activity of enzymatic antioxidants and also levels on non-enzymatic antioxidants in the liver of acetaminophen-intoxicated rats. Effect of rutin on hepatic malondialdehyde (MDA) and ascorbic acid levels of acetaminophen-intoxicated rats Lipid peroxidation causes changes in the properties of biological membranes, thus altering their fluidity and permeability, leading to impairment in membrane signal transduction and ion exchange, resulting in lipid peroxidation, oxidation of proteins and DNA and eventually, cytotoxicity (Fang et al., 2002; Stehbens, 2003; Jaeschke et al., 2003; Teimouri et al., 2006). Generation of free radicals such as superoxide anion and peroxynitrite during acetaminophen metabolism results in the depletion of antioxidants such as glutathione, ascorbate and superoxide dismutase leading to oxidative stress and lipid peroxidation. In our study, an increase in hepatic MDA levels in the acetaminophen-only rats (Table 4) suggests enhanced lipid peroxidation leading to hepatic damage and failure of antioxidant defense mechanisms resulting in oxidative stress. The observed increase in levels of hepatic MDA correlates with the decrease in hepatic total SOD activity (Table 3). The rats co-treated
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with acetaminophen and rutin (200 and 500 mg/kg) showed significantly (p < 0.05) lower levels of MDA (0.331 ± 0.05 and 0.323 ± 0.02 µmol/mg protein respectively) relative to the acetaminophen-only rats (0.471 ± 0.02 µmol/mg protein). Reduction of MDA levels in the groups co-treated with both of acetaminophen and rutin was possibly due to the ability of rutin to quench free radicals by transfer electrons to the free radicals (Ferrali et al., 1997) and possibly by activation of antioxidants enzymes (Elliott et al., 1992). Hepatic ascorbate levels were significantly reduced in the acetaminophen-only rats relative to the healthy control. Co-administration of rutin (200 and 500 mg/kg) significantly (p < 0.05) increased the ascorbate levels (6.6 ± 0.3 and 6.4 ± 0.7 mg/dL respectively) vis-à-vis the acetaminophen-only rats (4.5 ± 0.2 mg/dL) (Table 4). Low levels of ascorbate are associated with increase levels of free radicals
and oxidative stress since much ascorbate would be utilized to quench radical. The present results show that rutin could help protect against the assault of free radical thereby stabilizing the oxidative status of the rats. The pharmacokinetics of rutin in humans is still under investigation. Studies have shown that about 17% of an ingested dose of rutin is absorbed mainly from the colon following the removal of the carbohydrate moiety by bacterial enzymes to form quercetin (Walle, 2004). Quercetin and glucuronide conjugates of quercetin are then transported to the liver via the portal circulation, where they undergo significant metabolism forming metabolites like isorhamnetin, kaempferol and tamarixetin (Walle, 2004). It could therefore be inferred that quercetin and its metabolites are most likely responsible for the in vivo antioxidant and hepatoprotective capabilities of rutin.
Table 3: Hepatic total superoxide dismutase (SOD) and catalase (CAT) activites of control and acetaminophen-intoxicated rats
Total SOD Activity
CAT Activity
(U/mg protein)
(U/mg protein)
Control
11.32 ± 1.50
90.43 ± 3.06
Acetaminophen Only
5.82 ± 0.37
81.62 ± 4.07
Acetaminophen + 100 mg/kg Rutin
8.69 ± 1.05
97.54 ± 5.02
Acetaminophen + 200 mg/kg Rutin
10.63 ± 1.58*
84.32 ± 4.04
Acetaminophen + 500 mg/kg Rutin
10.34 ± 1.50*
85.23 ± 2.03
Data represented as Mean ± SEM; * p < 0.05 relative to the Acetaminophen-only group
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Table 4: Hepatic malondialdehyde (MDA), protein carbonyls and ascorbate levels in control and acetaminophen-intoxicated rats MDA
Ascorbic acid
(µmol/mg protein)
(mg/dL)
Control
0.367 ± 0.08
6.2 ± 0.5
Acetaminophen Only
0.471 ± 0.02
4.5 ± 0.2
Acetaminophen Rutin
+
100 mg/kg 0.409 ± 0.03
5.2 ± 0.4
Acetaminophen Rutin
+
200 mg/kg 0.331 ± 0.05*
6.6 ± 0.3*
Acetaminophen Rutin
+
500 mg/kg 0.323 ± 0.02*
6.4 ± 0.7*
Data represented as Mean ± SEM; * p < 0.05 compared to the Acetaminophen-only group (n = 5)
CONCLUSION Many, if not most, of rutin's possible activities can be accounted for, in part, by rutin's antioxidant activity. The present study shows that rutin has the abilities to preserve the activity of antioxidant enzymes and hepatocyte membrane, which may be referred to its role in modulating the levels of superoxide anion associated with acetaminophen toxicity. Rutin could therefore be used as an antidote in combination with acetaminophen to protect the
liver in case of an overdose. Further investigations are however, warranted to ascertain the feasibility of such combination. ACKNOWLEDGEMENT We are very grateful to Prof. Peter N. Uzoegwu of the Department of Biochemistry, University of Nigeria, Nsukka for encouragement, guidance, and financial support extended to us during the course of the study.
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Muldrew KL, James LP, Coop L, McCullough SS, Hendrickson HP, Hinson JA, Mayeux PR (2002). Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen using high- performance liquid chromatography with electrochemical detection. Drug Metab. Dispos. 30: 446–451. Plaa GL, Hewitt WR (1982). In: Zakim D, Boyer TD, editors, Toxicology of the liver, New York: Raven Press, p103. Puntarulo S, Cederbaum AI (1996). Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin. Biochim. Biophys. Acta. 1289: 238–246. Richardson JA (2000). Management of acetaminophen and ibuprofen toxicoses in dogs and cats. J. Vet. Emerge Crit. Care. 10: 285–291.
antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets. J. Agric. Food Chem. 52(14): 4414–4418. Stehbens WE (2003). Oxidative stress, toxic hepatitis, and antioxidants with particular emphasis on zinc. Exp. Mol. Pathol. 75: 265–276. Teimouri F, Amirkabirian N, Esmaily H, Mohammadirad A, Ali ahmadi A, Abdollahi M (2006). Alteration of hepatic cells glucose metabolism as a non cholinergic detoxification mechanism in counteracting diazinon induced oxidative stress. Hum.Exp.Toxicol. 25: 697–703. Tietz NW (1986). Textbook of Clinical Chemistry. W.B. Saunders Co., Philadelphia, pp.1270–1271. Walle T (2004). Absorption and metabolism of flavonoids. Free Rad. Biol. Med. 36(7): 829–837.
Rosane WI, Oliveira ZD, Fernandes SC, Vieira IC (2006). Development of a biosensor based on gilo peroxidase immobilized on chitosan chemically crosslinked with epichlorohydrin for determination of rutin. J. Pharmaceut. Biomed. Analy. 41: 366–372.
Wang M, Tadmor Y, Wu QL, Chin CK, Garrison SA, Simon JE (2003). Quantification of protodioscin and rutin in asparagus shoots by LC/MS and HPLC methods. J. Agric. Food Chem. 51(21): 6132–6136.
Ryder SD, Beckinghan IJ (2001). ABC of diseases of liver, pancreas and biliary system. Other causes of parenchymal liver diseases. Br. Med. J (Clinical research ed.) 322 (7281): 290–292.
Webster RP, Gawde MD, Bhattacharya RK (1996). Protective effect of rutin, a flavonol glycoside, on the carcinogeninduced DNA damage and repair enzymes in rats. Cancer Lett. 109: 185– 191.
Sallie R, Tredger JM, William R (1991). Drugs and the liver. Biopharmaceut. Drug Dispos. 12:251–253.
Yang J, Guo J, Yuan J (2008). In vitro antioxidant properties of rutin. LWT Food Sci. Technol. 41: 1060–1066.
Sheu JR, Hsiao G, Chou PH, Shen MY, Chou DS (2004). Mechanisms involved in the Source of Support: Nil
Conflict of Interest: None Declared
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Original Research Article ASPARAGUS RACEMOSUS WILLD. ROOT EXTRACT AS HERBAL NUTRITIONAL SUPPLEMENT FOR POULTRY Kumari R1, Tiwary B K2, Prasad A3, Ganguly S4* 1,2,3
Department of Veterinary Microbiology, Faculty of Veterinary Science & Animal Husbandry, Birsa Agricultural University, Ranchi, Jharkhand 834 006 India 4 AICRP-PHT (I.C.A.R.) [Kolkata Centre], Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal 700 094, India *Corresponding Author, e-mail: ganguly38@gmail.com
Received: 16/03/2012; Revised: 10/04/2012; Accepted: 17/04/2012;
ABSTRACT The present study was done to study the average body weight gain and increase in feed conversion efficiency in broiler chicks administered with different preparations of Asparagus racemosus Willd. root extracts orally mixed in their feed. After the trial, marked (P<0.05) overall improvements were evidenced in the form of increase in average body weight gain and feed conversion efficiency of the birds.
Keywords: weight gain, conversion efficiency, Broiler; Asparagus racemosus Willd.
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INTRODUCTION
MATERIALS AND METHODS
An immuno-modulator is a substance which stimulates or suppresses the components of immune system including both innate and adaptive immune responses (Agarwal and Singh, 1999). The modulation of immune system by various medicinal plant products has become a subject for scientific investigations currently worldwide. One such plant, Asparagus racemosus, commonly called ‘Shatavar’ possess anti-diarrheoal, antiulcerative, anti-spasmodic, aphrodisiac, galactogogue and other properties and has therefore gained its importance in Ayurveda, Siddha and Unani systems of medicine (Nadkarni, 1954). It has also been examined for its immuno-modulatory properties.
Fifty (50 No.) day old broiler chicks were procured from a private hatchery and were maintained under standard hygienic conditions of feeding and housing. On the 7th day, they were divided into three groups (Groups 1–3) comprising of fifteen (15 No.) chicks in each group. They were provided with ration as broiler starter (0–2 weeks), broiler grower (3–4 weeks) and broiler finisher (5–6 weeks). A. racemosus root extract was prepared from root juice concentrated into A. racemosus powder at low temperature under experimental conditions. Group 1 consisted of treated chicks fed with A. racemosus root extract treated feed @ 1 g/kg feed standard dose, Group 2 was kept as vaccinated control comprising of chicks administered with ND vaccine as per recommended schedule but without being fed with A. racemosus extract treated feed and Group 3 was the non-vaccinated control which consisted of untreated and unvaccinated chicks respectively.
Presently, poultry farming has gained immense importance in the socio-economic scenario in Indian livestock sector. For enhanced productivity of eggs and meat, it is needed for cheaper feed supplements which improve the overall weight gain of the birds and their feed conversion efficiency within short period of time. So, nowadays research are being carried out by scientists regarding different herbal preparations..These also possess adequate immune-modulatory effects which augment the resistance of the birds against various infectious diseases. The present study has been carried out with the objective of increase in total body weight gain and feed conversion ratio after the oral administration of A. racemosus root extract mixed with their feed mash in different preparations.
The live body weight of chicks was measured at weekly intervals on 1st, 7th, 14th, 21st, 28th, 35th and 42nd days of experiment. The feed efficiency was calculated in terms of feed conversion efficiency (ratio). Feed conversion efficiency was measured at weekly intervals on the basis of total feed intake and total gain in body weight. The feed conversion efficiency was interpreted as given below:
Total feed consumed (g) in particular period Feed conversion efficiency (ratio) = Total body weight gain (g) during same period Statistical analyses for different parameters were done as per the method described by Snedecor and Cochran (1994). RESULTS Non-significant effect was observed in body weight gain due to herbal treatment from 0 to 35th day at weekly intervals. The tendency of body weight gain was more in Group 1 (A. racemosus treated) as compared to both Groups
2 and 3 respectively. The effect of A. racemosus treatment had significant influence (P<0.05) on body weight at 42nd day of age. Critical difference test showed significantly higher body weight in Group 1 (1901.87g ± 40.82 ) than Groups 2 and 3 respectively (Table 1). Better cumulative feed conversion
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efficiency was observed in Group 1 (2.37:1) in the present study than that of the vaccinated
and non-vaccinated control groups respectively (Table 2).
Table 1. Average body weight gain (in gm) of broiler birds of different groups*. Age of chicks Group 1 Group 2 Group 3 ANOVA-value (in day) 48.06 ± 1.23 48.06 ± 1.23 48.06 ± 1.23 1 NS 114.27 ± 1.27 113.86 ± 1.35 113.67 ± 1.26 7 0.056NS 276.40 ± 5.67 286.6 ± 6.39 261.73 ± 4.71 14 1.69NS 599.20 ± 17.35 588.66 ± 12.47 574.60 ±14.642 21 0.68NS 981.33 ± 20.53 967.40 ± 14.45 955.4 ± 14.59 28 0.60NS 1409.47 ± 43.19 1381.47 ± 23.94 1324.6 ± 30.70 35 1.06NS 42 1901.87 ± 0.82a 1809.87 ± 0.44ab 1765.27 ± 4.63c 3.28* Values bearing different superscripts in a row differed significantly, Values bearing same superscript in the column did not differ significantly, NS: Non significant, *P<0.05 Table 2. Cumulative feed conversion efficiency (ratio) in broiler birds of different groups. Treatment group Group 1 Group 2 Group 3
Total feed consumed (kg) 65.95 65.37 64.26
DISCUSSION The findings of increased body weight gain in the present study by feeding A. racemosus root extract to broiler chicks has been supported by the reports of Sarag and Khobragade (2003) in which higher live body weight gain in broiler birds were observed after supplementation with T. cordifolia, another promising herbal feed supplement in poultry ration. The findings in this study are also supported by Thatte et al. (2001) in which he recorded higher body weight gain in mice supplemented with T. cordifolia. Levamisole, a potent anthelmintic, is also reported to induce body weight gain by the studies of Mani et al. (2001) and Panda and Rao (1994) in which they had observed and reported the effects of levamisole in broiler chicks infected with infectious bursal disease virus. A study was carried out to determine the immuno-modulatory effects of ‘Ashwagandha’ (Withania somnifera) and ‘Satavar’ (Asparagus racemosus) extract treated feed and to analyze
Total body weight gain (kg) 27.82 26.43 25.46
Feed conversion efficiency 2.37:1 2.47:1 2.52:1
the role of T and B cells in host defense against Newcastle disease in one week old normal and immuno-compromized boiler chicks. After the treatment significant (P<0.001) positive effects were observed in both humoral and cell mediated immune responses of the birds. However, the bursectomized and thymectomized birds showed a decline in the antibody titer. The variation in skin thickness was significantly (P<0.001) more among the herbal treated groups rather than the nontreated groups which was a clear marker for immuno-stimulation among the birds (Kumari et al., 2011). In another study carried out by Kumari et al. (2012) the immuno-modulatory effects of Asparagus racemosus extract treated feed was determined to analyze the role of T and B cells in host defense against Newcastle disease in one week old normal and immunocompromised boiler chicks. After the treatment significant (P<0.01) positive effects were observed in both humoral and cell mediated immune responses of the birds which was
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found to be evident by increased antibody titer after HI test. The variation in skin thickness was significantly (P<0.01) more among the herbal treated groups rather than the nontreated groups which was a clear marker for immuno-stimulation among the birds.
results in relation to total body weight gain and feed conversion efficiency. It can also be used potentially before mass vaccination of the chicks for its property of immune-modulation like levamisole. ACKNOWLEDGEMENTS
CONCLUSION The present study showed that herbal preparations of A. racemosus root extract can be beneficially used as an effective feed supplement in poultry for its encouraging
The authors are thankful to Hon’ble ViceChancellor, Birsa Agricultural University and Dean, Faculty of Veterinary and Animal Sciences, Ranchi, India for providing necessary facilities to carry out this research work.
REFERENCES Agarwal, S.S. and Singh, V.K. 1999. Immnomodulators: a review of studies on Indian Medicinal Plants and Synthetic Peptides. PINSA. 65 (3-4): 179–204. Kumari, R., Tiwary, B.K., Prasad, A. and Ganguly, S. (2011) Immunomodulatory effect of herbal feed supplement in normal and immunocompromised broiler chicks. Indian J. Anim. Sci., 81(2): 158-161. Kumari, R., Tiwary, B.K., Prasad, A. and Ganguly, S. (2012) Study on the immuno-modulatory effect of herbal extract of Asparagus racemosus Willd. in broiler chicks. Global J. Res. Medi. Pl. & Indigenous Medi. 1(1): 1–6. Mani. K., Sundaresan, K. and Vishwanathan, K. 2001. Effect of immunomodulators on the performance of broilers in aflatoxicosis. Indian Vet. J. 78(12): 1126–1129. Source of Support: Nil
Nadkarni, A.K. 1954. Indian Materia Medica, Bombay, Popular book Depot, 3rd ed., 1: 153–155. Panda, S.K. and Rao, A.T. 1994. Effect of Levamisole on chicken infected with infectious bursal disease (IBD) virus. Indian Vet. J. 71(5): 427–431. Sarag, A.N. and Khobragade, R.S. 2003. Effect of feed supplementation of medicinal plants Tinospora cordifolia and Leptadenia reticulate on performance of broilers. PKV Res. J. 25(2): 114–115. Snedecor, G.W. and Cochran, W.G. 1967. Statistical Methods. 7th ed. Oxford and IBH Publishing Co., New Delhi. Thatte, U.M., Panekar, Addikari, H. and Dhanukar, S.A. 2001. Experimental study with Tinospora cordifolia in malnourished rats. Scientific Programme and Abstract of XXXIII Annual Conference. Indian J. Pharmacol. 33: 132. Conflict of Interest: None Declared
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Original Research Article THE EFFECTS OF VITAMIN C AND GRAPE FRUIT JUICE SUPPLEMENTS ON THE POTENCY AND EFFICACY OF SOME SELECTED ANTI-MALARIAL DRUGS
Adumanya O C+, Uwakwe A A*, Odeghe O B*, Onwuka F C, Akaehi HC+ +
Department of Nutrition and Dietetics, Imo State Polytechnic, Umuagwo, Imo State, Nigeria *Department of Biochemistry, University of Port Harcourt, PMB 5323, Rivers State, Nigeria. Corresponding Author: E-mail: bensandym@yahoo.com
Received: 06/04/2012; Revised: 25/04/2012; Accepted: 30/04/2012
ABSTRACT Combination therapy (CT) is now advocated for the treatment of malaria, especially the artemisinin based CT. Malaria is one of the most serious health challenges facing the world today. It is a disease caused by plasmodium, which could be cured effectively by the use of combination therapeutic drugs called anti-malarial drugs. The effects of vitamin C and grape vine supplements on the potency and efficacy of some selected anti-malarial drugs-combination therapy (Armact, Coartem, Waipa and Fansider) were investigated. A total of 80 patients (adults) infected with malaria parasites were used. The result showed that the concomitant administration of the drugs with grape fruit juices did not alter the efficacy and potency of the drugs, while vitamin C altered the efficacy and potency of the drugs. Therefore, the concomitant administration of these anti-malarial drugs (combination therapies) with vitamin C supplement should be avoided during the period of malaria treatment for the effectiveness of such drugs.
Keywords: Anti-malarial, malaria, Combination therapy, Grape fruit juice and Vitamin C
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INTRODUCTION Anti-malarials also known as Anti-malarial medications are designed to prevent or cure malaria. Such drugs may be used for some treatment of malaria in individuals with suspected or confirmed infection, prevention of infection in individuals visiting a malariaendemic region who have no immunity (Malaria prophylaxis) and Routine intermittent treatment of certain groups in endemic regions (Intermittent preventive therapy) (Bukirwa, 2006). Early diagnosis and prompt treatment is one of the principal technical components of the global strategy to control malaria (WHO 2006).The effectiveness of this intervention is highly dependent on anti-malarial drugs, which should not only be safe and effective, but also available, affordable and acceptable to the population at risk. The rational use of an effective anti-malarial drug not only reduces the risk of severe disease and death and shortens the duration of the illness, but also contributes to slowing down the development of the parasiteâ&#x20AC;&#x2122;s resistance to anti-malarial drugs (Wiseman, 2006). The emergence and rapid spread of P. falciparum resistance to commonly used anti-malarial drugs, poses a serious challenge to the effectiveness of early diagnosis and prompt treatment as a priority strategy within current malaria control efforts (Shanks, 2006). The potential value of malaria therapy using combinations of drugs was identified as a strategic and viable option in improving efficacy and delaying development and selection of resistant parasites (Yeka, 2005). The concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Current practice in treating cases of malaria is based around the concept of combination therapy, since this offers several advantages reduced risk of treatment failure, reduced risk of developing resistance, enhanced
convenience and reduced side-effects. Prompt parasitological confirmation by microscopy or alternatively by RDTs is recommended in all patients suspected of malaria, before the treatment is started (WHO, 2010). Treatment solely on the basis of clinical suspicion should only be considered when a parasitological diagnosis is not accessible (WHO, 2010). Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a non-anti-malarial drug to enhance the anti-malarial effect of a blood schizontocidal drug are not considered combination therapy (Yeka, 2005). The costs of anti-malarial combination therapies are over ten times more expensive than those of the traditional drugs currently used in Africa as monotherapy. Thus a change to and implementation of combination therapy would involve higher direct and indirect costs to health services, necessitating substantial financial support through sustained international public/private support, as these higher costs would be out of reach for many developing nations, especially in sub-Saharan Africa (Russell, 2008). According to WHO guidelines 2010, artemisinin-based combination therapies (ACTs) are the recommended anti-malarial treatments for uncomplicated malaria caused by P. falciparium. MATERIALS AND METHODS Experimental animals A total of 80 persons (40 males and 40 females) infected with malarial parasite residing in Umuguma in Owerri West local government area of Imo state, Nigeria were selected during the experiment after a general malarial test on all the individuals and their body weights were taken before and after drug administration (treatment).
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Collection of blood sample Methanol was used as a disinfectant to swab the thumb and a lancet was used to puncture it for blood collection. Two drops of blood was placed on free grease slide, a thick film was made and allowed to air-dry. The dried thick blood film slide was laid on a staining rack, Giemsa stained and allowed for 30–40 min, washed off with clean water, drained and allowed to dry at room temperature. Then viewed under the microscope using 10x objective for focusing and 40x objective for identifying the Plasmodium involved. Blood samples of subjects were all confirmed to be malarial parasite infected via malarial parasite test as described by (Sibley, 2001). The research had
the approval of the concerned institutional medicinal ethics boards. Drugs and supplements administered The drugs used were Armact (Artesunate and Amodiquine), Coartem (Artemether and Lumefantrine), both purchased from Novartis pharmaceutica. Waipa (Dihydroartemisinin and Piperaquine) and Fansider (Sulfadoxin and Pyrimethamine) both purchased from Swiss Pharma Nigeria limited. The supplements used were Vitamin C and Grape fruit juice. RESULTS The results of the test on the blood samples before and after administration of the antimalarial drugs to the patients are as follows:
TABLE 1: Effects of Armact only On Patients with Malarial Parasite Patients Weight(kg) Before Treatment Female
60
Malaria parasite Before Treatment ++
Female
65
Male Male + ++ +++ −
Drug/ Malarial Weight(kg) Supplements parasite/After After Treatment treatment
Remarks
Armact only
_
58
Cleared
+++
Armact only
_
64
Cleared
70
++
Armact only
_
65
Cleared
70
++
Armact only
_
67
Cleared
= Positive (Malarial parasite present) = Moderately severe parasite present = Severe malarial parasite = Negative (malarial parasite absent)
TABLE 1.1: Effects of Armact and Vitamin C on Patients with Malarial Parasite Patients Weight(kg) Malarial Drug/ Malarial Weight(kg) Remarks Before parasite Supplements parasite/ After Treatment Before After treatment Treatment Treatment Female
80
++
Armact/ Vit. C
+
80
Not cleared
Female
75
++
Armact/ Vit. C
+
75
Not cleared
Male
55
++
Armact/ Vit. C
+
60
Not cleared
Male
60
++
Armact/ Vit. C
+
60
Not cleared
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TABLE1.2 Effects of Armact and Grape Juice on patients with malarial parasite Patients Weight(kg) Malarial Before parasite treatment Before treatment Female 65 ++ Female 55 ++ Male 75 + Male 75 ++ GJ: Grape Juice
Drug/ Malarial supplements parasite/After Treatment Armact/GJ Armact /GJ Armact /GJ Armact /GJ
Weight(kg) Remarks After Treatment
− − − −
63 50 73 70
cleared cleared cleared cleared
TABLE 2: Effects of Coartem Only On Patients with Malarial Parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment Female 65 ++ Female 65 + Male 60 + Male 60 + Coart: Coartem
Drug/ supplements
Malarial parasite/After Treatment
Coart only Coart only Coart only Coart only
− − − _
Weight(kg) Remarks After treatment 60 65 60 60
cleared cleared cleared cleared
TABLE 2.1: Effects of Coartem and Vitamin C on Patients with malarial parasite Patients Weight(kg) Malarial Drug/ Malarial Weight(kg) Remarks Before parasite supplements parasite/After After Treatment Before Treatment treatment treatment Female 55 ++ Coart /Vit. C + 55 Not cleared Female 65 + Coart /Vit. C − 65 cleared Male 65 ++ Coart /Vit. C + 65 Not cleared Coart: Coartem; Vit. C: Vitamin C TABLE 2.2: Effects of Coartem and Grape Juice On Patients with Malarial Parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment Female 60 ++ Female 60 + Male 75 + Male 70 + Coart: Coartem; GJ: Grape Juice
Drug/ supplements
Malarial parasite/After Treatment
Coart /GJ Coart /GJ Coart /GJ Coart /GJ
− − − −
Weight(kg) Remarks After treatment
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58 56 70 69
cleared cleared cleared cleared
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Table 3: Effects of Waipa only on patient with Malarial Parasite Patients
Weight(kg) Before Treatment
Female Female Male Male Wp - Waipa.
60 60 70 75
Malarial parasite Before treatment + ++ ++ +
Drug/ supplements
Malarial parasite/After Treatment
Weight(kg) After treatment
Remarks
Wp only Wp only Wp only Wp only
− − − −
60 58 68 72
Cleared Cleared Cleared Cleared
Table 3.1: Effects of Waipa and vitamin C on patient with Malarial Parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment Female 70 + Female 70 +++ Male 65 + ++ Male 55 + Wp – Waipa; Vit. C – Vitamin C
Drug/ supplements
Malarial parasite/After Treatment
Wp /Vit. c Wp /Vit. c Wp /Vit. c Wp /Vit. c
+ + + −
Weight(kg) Remarks After treatment 70 70 63 52
Not cleared Not cleared Not cleared cleared
Table 3.2: Effects of Waipa and Grape juice on patient with Malarial Parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment Female 60 + Female 60 +++ Male 65 ++ Male 60 + Wp – Waipa; GJ – Grape Juice
Drug/ supplements
Malarial parasite/After Treatment
Wp /GJ Wp /GJ Wp /GJ Wp /GJ
− − − −
Weight(kg) Remarks After treatment 58 59 63 58
cleared cleared cleared cleared
Table 4: Effects of Fansider only on patients with malarial parasite Patients
Weight(kg) Before Treatment
Female Female Male Male Fans: Fansider
75 70 65 60
Malarial parasite Before treatment + + + +
Drug/ supplements
Malarial parasite/After Treatment
Weight(kg) After treatment
Remarks
Fans only Fans only Fans only Fans only
− − − −
70 68 62 58
cleared cleared cleared cleared
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Table 4.1: Effects of Fansider and Vitamin C on patients with malarial parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment Female 78 +++ Female 65 + Male 60 ++ Male 65 + Fans: Fansider; Vit.C: Vitamin C
Drug/ supplements
Malarial parasite/After Treatment
Fans /Vit. C Fans /Vit. C Fans /Vit. C Fans /Vit. C
+ + + −
Weight(kg) Remarks After treatment 78 65 60 65
Not cleared Not cleared Not cleared cleared
Table 4.2 Effects of Fansider and Grape Juice on patients With malarial parasite Patients
Weight(kg) Malarial Before parasite Treatment Before treatment
Female 70 + Female 70 + Male 60 +++ Male 55 + Fans: Fansider; GJ: Grape Juice
Drug/ supplements
Malarial parasite/After Treatment
Fans /GJ Fans /GJ Fans /GJ Fans /GJ
− − − −
DISCUSSION As shown from the result of the effects of Armact only on Patients with Malarial Parasite presented in Table 1, after the administration of the drug, the malarial parasite in all the groups were absent. The absence of death in the oral administration of the Armact observed in the patients suggests that the drug is practically non-toxic acutely (Salawu et al., 2009) and Russell, (2008). This could also explain the safe use of the drug by the local people, who have been using it in the treatment of malaria, in the eastern part of Nigeria. From Table 1.1, after treating with Armact and Vitamin C, it was observed that the malarial parasite was present in all the groups. The administration of Armact and Grape fruit juice (Table 1.2) showed no malarial parasite presence in all the patients. We can infer that Grape fruit juice can play a significant role in anti-malarial activity which is similar to the report of Adesokan and Akanji, 2010.
Weight(kg) Remarks After treatment
68 67 58 53
cleared cleared cleared cleared
Also, from Table 2, the administration of Coartem drug on patients infected with malarial parasite showed no evidence of these patients being infected. This suggests the findings of (Ajaiyeoba et al 2006) that the use of this drug for the treatment of malaria was due to the presence of alkaloids. The treatment of both Coartem and Vitamin C (Table 2.1) on patients infected with the Plasmodium showed the presence of malarial parasite in some patients. From Table 2.2, the administration of both Coartem and Grape fruit Juice on infected patients showed no evidence of the presence of malarial parasite. The treatment with Waipa anti-malarial (Table 3) on infected patients showed no trace of malarial parasite. Also, this is similar to the effect of the extract reported by previous studies on Alstonia boonei (Iyiola et al., 2011). From Table 3.1, when both Waipa and Vitamin C were administered there was presence of malarial parasite unlike when both Waipa and grape fruit juice (Table 3.2) were administered.
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Treatment with Fansider (Table 4) on patients infected with plasmodium showed no sign of malarial parasite. This study is similar to the reports of Idowu et al., (2010), and also may possess health promoting effects, at least under some circumstances (Basu et al., 2007). But, treatment with both Fansider and Vitamin C (Table 4.1) showed the presence of malarial parasite which is contrary to the result of the treatment with both fansider and Grape fruit juice in patients infected with malarial parasite. CONCLUSION Successful malaria control depends greatly on the treatment with efficacious anti-malarial drugs. The ability of the four drugs (Armact, Coartem, Waipa and Fansider) to reduce the presence of malarial parasite may be due to presence of phyto-chemically active components in the drugs which might be REFERENCES Adesokan, A. A. and Akanji, M. A, (2010). Antimalarial Bioactivity of Enantia chlorantha stem bark. Medical Plants: Phytochemistry Pharmacology and Therapeutics 4(1): 441–447. Ajaiyeoba, E., Falade, M., Ogbole, O., Okpako, L, and Akinboye, D. (2006). In vivo antimalarial and Cytotoxic properties of Annona senegalensis extract. African Journal of Traditional, Complementary and alternative Medicine 3(1): 137–141 Basu, S,K., Thomas, J.E. and Acharya, S.N. (2007). Prospects for Growth in Global Nutraceutical and Functional Food Markets: A Canadian Perspective. Aust J Basic Appl Sci, 1(4): 637–649 Bukirwa, H., Yeka, A., Kamya, M.R., Talisuna, A., Banek, K., Bakyaita, N., Rwakimari, J.B.,
responsible for their therapeutic activity as anti-malarial drugs. Also, the use of Grape fruit juice with anti-malarial drugs (Combination therapy) has potential health promoting effects. Multiple-drug therapies that include a non-antimalarial drug like Vitamin C to enhance the anti-malarial effect of a blood schizontocidal drug are not considered combination therapy. This finding supports the use of Grape fruit juice and anti-malarial drugs as a combination therapy which is safe and possess potent antimalarial activity as found in its ability to suppress Plasmodium infection in patients. ACKNOWLEDGEMENT The authors acknowledge the assistance from the World Bank and the federal Republic of Nigeria with the World Bank step B projects.
Rosenthal, P.J., Wabwire-Mangen, F., Dorsey, G. & Staedke, S.G. (2006) Artemisinin Combination Therapies for Treatment of Uncomplicated Malaria in Uganda. 1(1): 7. Idowu, O. A., Soniran, O. T., Ajana, O. and Aworinde, D. O, 2010. Ethnobotanical survey of antimalarial plants used in Ogun State, Southwest Nigeria. Afr. J. Pharmacy Pharmacol., 4: 055 060. Iyiola, O. A., Tijani, A. Y. and Lateef, K. M, (2011). Antimalarial Activity of Ethanolic Stem Bark Extract of Alstonia boonei in Mice. Asian Journal of Biological Sciences, 4: 235–243. Russell, B.(2008) Determinants of in vitro drug susceptibility testing of Plasmodium vivax. Antimicrob. Agents Chemother. 52, 1040–1045 Salawu, O.A., Chindo, B.A., Tijani, A.Y., Obidike I.C. and Akingbasote, J.A. 2009. Acute and
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sub-acute toxicological evaluations of the methanolic stem bark extract of Crossopteryx febrifuga inrats. African Journal of Pharmacy and Pharmacology, 3: 621–626.
Wiseman, V., Kim M, Mutabingwa T. K, Whitty C. J. M. (2006). Costeffectiveness study of three antimalarial drug combinations in Tanzania. 3(10): 373
Shanks, G.D. (2006) Treatment of falciparum malaria in the age of drug resistance. Journal of Postgraduate Medicine 52 (4): 277–80.
WHO (2006). Guidelines for the treatment of malaria. WHO/HTM/MAL/2006.1108.
Sibley, C.H. 2001 Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next? Trends Parasitol. 17:582–588
Source of Support: Nil
Yeka, A., Banek, K, Bakyaita N, Staedke S. G, Kamya M. R. (2005) Artemisinin versus nonartemisinin combination therapy for uncomplicated malaria: Randomized clinical trials from four sites in Uganda. 2(7): 190.
Conflict of Interest: None Declared
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Original Research Article EFFECTS OF AQUEOUS AND ETHANOLIC EXTRACTS OF DANDELION (TARAXACUM OFFICINALE F.H. Wigg.) LEAVES AND ROOTS ON SOME HAEMATOLOGICAL PARAMETERS OF NORMAL AND STZ-INDUCED DIABETIC WISTAR ALBINO RATS. Nnamdi Chinaka C1*, Uwakwe A A2, and Chuku L C3 1,2,3 *
Department of Biochemistry, University of Port Harcourt, Rivers State, Nigeria. Corresponding author: (+234)8039397700, (+234)8027205705. cn_chinaka@yahoo.com Received: 06/04/2012; Revised: 03/05/2012; Accepted: 06/05/2012
ABSTRACT The effects of aqueous and ethanolic extracts of Taraxacum officinale F.H. Wigg leaves and roots on some haematological parameters of Streptozotocin (STZ)-induced diabetic Wistar albino rats (Rattus rattus) were investigated. The parameters investigated include; packed cell volume (PCV), haemoglobin (Hb) levels and white blood cell (WBC) counts. Exactly 75 wistar albino rats weighing between 100–225 g were used for the study, and a total of four groups were created. Two groups were divided into six sub-groups of five rats each for the leaf and root extracts respectively, with the remaining two groups being the normal control rats (NCR) and diabetic control rats (DCR). The two sub-groups were thus; sub I, comprising of sub-groups 1–4 which were for diabetic test rats (DTR) on 6% and 10% of aqueous and ethanolic extracts of leaves respectively, while sub-group 5 and 6 were normal test rats (NTR) on 10% of both extracts of leaves respectively. Same was also the case for sub II which represents the root extracts. Two days after streptozotocin-induction, the administration of T. officinale leaf and root extracts (Aqueous and Ethanolic) commenced and lasted for three weeks. Changes in PCV, haemoglobin levels and WBC counts between the NCR and DCR against normal treated rats (NTR) and diabetic treated rats (DTR) on various doses of the extracts were evaluated using one way Analysis of Variance (ANOVA). Keywords: Taraxacum officinale, Aqueous and Ethanolic extracts, STZ induced Diabetes, Haematological parameters
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1.0 INTRODUCTION The use of indigenous plants in the management of diseases has been a common practice in most developed countries and importantly in developing countries throughout the world. However, few of these plants have received adequate scientific scrutiny. Most researches into medicinal plants help in ascertaining the efficacy of the flora as a potent remedy, extending our frontiers of knowledge concerning their pharmacological activity, active principles, therapeutic value, dosage and administration as well as contraindication and side effects. Medicinal plants are administered to patients either as entire plant or as root, leaf, stem, bark, fruit, seed, juice, flower or as exudates and may be taken in form of an infusion or as a decoction (Omeodu 2006). Traditional healers form the basic core of primary health care delivery in 90% of our rural population in Africa. It is found that 60% of Nigeria’s rural population depends on traditional medicine for their health care needs (WHO 1996). As such World Health Organisation is pursuing a coordinated approach to encourage the official recognition of traditional healers and to encourage Western trained doctors and pharmacists to study the methodology and recipes of traditional healers. China for instance has achieved a great success in blending traditional healing system with modern western medicine to form new Chinese medicine (Omeodu 2006). The Nigerian flora had already and continually made great contribution to the health care of the nation (Omeodu 2006). Infact, indigenous medicinal plants form an important component of the natural wealth of Nigeria. Taraxacum officinale F.H. Wigg. commonly known as Dandelion (from the French dent-de-lion meaning lion’s tooth) is thought to have evolved about thirty million years ago in Eurasia. They have been used by humans as food and as a herb for much of recorded history (Dijk et al. 2003).
It is a herbaceous perennial plant of the family Asteraceae (compositae), and two major species, T. officinale and T. enythrospermum, are found as weed worldwide. Both species are edible in their entirety. Like other members of the Asteraceae family, they have very small flowers which are yellowish to orange yellow in colour collected together into a compositae (flower head). Each single flower in a head is called floret and they number 40 to over 100 per head. They grow generally from unbranched taproots, producing one to more than ten stems that are typically 5–40 cm tall and sometimes up to 70 cm tall. Dandelion leaves are unique as a diuretic, a valuable alkalizer to the body; eaten regularly they assist the body to reduce excess acidity, oxygenate, purify and build blood, cleanse and regenerate cells (Clarke et al. 1997). 2.0
MATERIALS AND METHODS
2.1.0 Plant Source and Identification The plant Dandelion (Taraxacum officinale) leaves and roots were sourced from farm lands at Umuzi and Umudim villages in Umudioka Ancient kingdom, Orlu local government area of Imo State and the species was identified and confirmed by Dr. F. N. Mbagwu of the Department of Plant Science and Biotechnology, Imo State University, Owerri, Imo State. 2.1.1 Chemicals and Reagents All chemicals and reagents used were of analytical standard and were obtained from reputable sources. 2.2.0 Preparation and Administration of Streptozotocin (STZ) The range of diabetogenic dose of STZ is quite narrow and a light overdose may cause death of many animals (Lenzen et al. 1996). Five gram of STZ was dissolved in 100 ml of distilled water to give a 5% stock solution of which a single dose of 70 mg/kg body weight was injected intraperitoneally to the rats.
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2.2.1 Preparation of Plant Extract Aqueous Extract Fresh leaves and roots of the plant (T. officinale) were washed with distilled water to remove debris and contaminants, after which they were dried. The leaves and roots were homogenized into fine powder respectively. The aqueous pulverized plant leaves and roots were respectively prepared by weighing out 100 g of pulverized leaves and roots into 1l of distilled water respectively. The resultant mixture was allowed to stand for 24 h with occasional shaking after which it was filtered. The filtrate was evaporated and dried to powder with the aid of a thermostatic water bath at a temperature of 50°C. An aliquot of the extract was prepared by dissolving 6 g in 50 ml and 10 g in 50 ml of distilled water respectively to form the two concentrations which served as stock crude drug and stored at 4°C. Ethanolic Extract Fresh leaves of the plant (T. officinale) were washed with distilled water to remove debris and contaminants, after which they were dried. The leaves and roots were homogenized into fine powder respectively. 100 g of powdered leaves and roots were soaked respectively in 500 ml of absolute ethanol and the resultant mixture was allowed to stand for 24 h with occasional shaking, after which it
was filtered. The filtrate was evaporated with rotary evaporator and dried to powder with the aid of a thermostatic water bath at 45°C. 2.3 Extract Administration The test rats were administered 300 mg/kg and 500 mg/kg body weight of concentrations of aqueous and ethanolic extracts of leaves and roots respectively twice daily using a gavage via intubation for 21days, according to the experimental plan/grouping. 6% aqueous extract of leaves and roots were prepared respectively by weighing 6 g of the various extracts (aqueous and ethanolic) and dissolved in 50 ml of water. [6000 mg in 50 ml (3000 mg in 25 ml)] Each rat was administered 0.5 ml (e.g. 200 g rat) of the solution via intubation twice daily for 21days. 10% aqueous extract of leaves and roots were prepared respectively by weighing 10 g of the various extracts (aqueous and ethanolic) and dissolving in 50 ml of distilled water. [10,000 mg in 50 ml (5000 mg in 25 ml)] Each rat was administered 0.5 ml (e.g. 200 g rat) of the solution via intubation twice daily for 21days. The mode of administration and treatment of the animals according to their experimental regimen/groups is shown in the Table below:
Figure 1: Leaf of Taraxacum officinale F.H. Wigg. found in wild.
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GJRMI, Volume 1, Issue 5, May 2012, 172–180 Table 2.0 Feeding illustration. GROUPS 1
2
3
4
5
6
7
8
9
10
11
12
13
14
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Feed + water
+
+
+
+
+
+
+
+
+
+
+
+
+
+
STZ(70 mg/kg) 0.2 ml
–
+
+
+
+
+
+
+
+
+
–
–
–
–
Aq. Leaves Extract 300 mg/kg
–
–
+
–
–
–
–
–
–
–
–
–
–
–
Aq. Leaves Extract 500 mg/kg
–
–
–
+
–
–
–
–
–
–
+
–
–
–
Et. Leaves Extract 300 mg/kg
–
–
–
–
+
–
–
–
–
–
–
–
–
–
Et. Leaves Extract 500 mg/kg
–
–
–
–
–
+
–
–
–
–
–
+
–
–
Aq. Roots Extract 300 mg/kg
–
–
–
–
–
–
+
–
–
–
–
–
–
–
Aq. Roots Extract 500 mg/kg
–
–
–
–
–
–
–
+
–
–
–
–
+
–
Et. Roots Extract 300 mg/kg
–
–
–
–
–
–
–
–
+
–
–
–
–
–
Et. Roots Extract 500 mg/kg
–
–
–
–
–
–
–
–
–
+
–
–
–
+
TREATMENT No. of Rats per group
Key: + Indicates that item was administered; – Indicates that item was not administered. 2.4 METHOD OF BLOOD COLLECTION
2.4.3 Haemoglobin (Hb)
Blood used for analysis was collected via the tail vein by dilating the tail veins with methylated spirit and xylene, after which the tip of the tail is cut off and analysis done immediately with the blood collected using an automated blood analyser.
Haemoglobin level was assayed using the method of Baker et al. (1985). Drabkin’s solution (4 ml) was introduced into a test tube and 0.02 ml of blood sample added. The test tube was stopped with a rubber cork and inverted several times for proper mixing. This was allowed to stand for 10 min at room temperature for complete conversion of cyanomethaemoglobin. This was read at 540 nm wavelength against blank (4 ml of Drabkin’s reagent only). The absorbance of known standard was read alongside those of the sample.
2.4.1 ASSAY METHOD 2.4.2 Packed Cell Volume (PCV) Microhaematocrit method was used. The sample was collected into a heparinized capillary tube and spun at 3000 rpm for 10 min. The resultant product consisting of packed cells, buffy coat and plasma was read with the reader and the values expressed in percentage volume.
2.4.4 White Blood Cell (WBC) Turk’s solution of 1.0% glacial acetic acid was used as the diluent. The 1: 20 dilution was then charged on an improved Neuber Chamber and counted. Values were expressed in 0109 mg/dl.
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3.0 RESULTS AND DISCUSSION Table 3.1.1 PVC VALUES (in %vol.) OF NORMAL AND DIABETIC CONTROLS COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES. WEEK 1
2
3
NCR DCR
35.4 ± 2.51b 34.2 ± 1.10a
36.4 ± 2.07b 36.3 ± 2.75b
37.9 ± 0.86b 36.0 ± 1.00b
DTR on 6% Aq. Extract DTR on 10% Aq. Extract
33.4 ±2.19a 33.2 ± 3.10a
32.8 ± 2.39a 37.2 ± 2.05b
35.2 ± 3.10b 36.4 ± 3.80b
DTR on 6% Et. Extract
34.0 ± 2.73a
32.6 ± 2.61a
32.4 ± 3.71a
DTR on 10% Et. Extract
35.8 ± 3.42b
35.8 ± 1.50b
36.5 ± 2.65b
NTR on 10% Aq. Extract
36.6 ± 0.55b
38.6 ± 1.14c
37.9 ± 1.37c
NTR on 10% Et. Extract
35.8 ± 0.84b
36.0 ± 3.46b
36.8 ± 2.28b
GROUP
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. abc given in superscripts expresses the level of significance (changes) between the weeks and the various groups. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are statistically significantly at 95% confidence level (P ≤ 0.05).
Table 3.1.2 PVC VALUES (in %vol.) OF NORMAL AND DIABETIC CONTROLS COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS. WEEK 1
2
3
GROUP NCR 35.4 ± 2.51b 36.4 ± 2.07a 37.9 ± 0.86b a a DCR 34.2 ± 1.10 36.3 ± 2.75 36.0 ± 1.00a DTR on 6% Aq. Extract 35.4 ± 1.34a 37.4 ± 0.89b 35.8 ± 0.84a b b DTR on 10% Aq. Extract 37.4 ± 0.89 37.4 ± 2.41 35.9 ± 3.36a DTR on 6% Et. Extract 32.7 ± 3.88a 35.8 ± 2.39a 34.0 ± 3.54a DTR on 10% Et. Extract 38.2 ± 1.10b 38.0 ± 1.58c 37.6 ± 1.34b a a NTR on 10% Aq. Extract 33.6 ± 3.91 35.2 ± 3.07 35.2 ± 2.95a NTR on 10% Et. Extract 38.8 ± 0.84c 37.4 ± 0.55b 38.0 ± 1.22c NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are statistically significantly at 95% confidence level (P ≤ 0.05).
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Table 3.2.1 Hb VALUES (in g/dl) OF NORMAL AND DIABETIC CONTROLS COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES. WEEK 1
2
3
GROUP NCR 12.2 ± 0.80b 12.8 ± 0.42c 12.9 ± 0.33c DCR 10.9 ± 1.11a 11.3 ± 1.24a 12.2 ± 0.51b b b DTR on 6% Aq. Extract 12.0 ± 0.45 11.5 ± 0.83 11.9 ± 1.12b DTR on 10% Aq. Extract 11.7 ± 1.06a 11.4 ± 1.72a 11.6 ± 1.44a b b DTR on 6% Et. Extract 12.2 ± 0.20 12.5 ± 0.62 11.0 ± 1.78a DTR on 10% Et. Extract 12.0 ± 1.16b 12.3 ± 0.68b 12.3 ± 0.96b NTR on 10% Aq. Extract 12.2 ± 0.14b 12.1 ± 0.46b 11.6 ± 0.83a b b NTR on 10% Et. Extract 12.6 ± 0.17 12.5 ± 0.26 12.4 ± 0.32b NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are not statistically significantly at 95% confidence level (P > 0.05).
Table 3.2.2 Hb VALUES (in g/dl) OF NORMAL AND DIABETIC CONTROLS COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS. WEEK 1
2
3
GROUP NCR 12.2 ± 0.80a 12.8 ± 0.42b 12.9 ± 0.33c DCR 10.9 ± 1.11a 11.3 ± 1.24a 12.2 ± 0.51b a a DTR on 6% Aq. Extract 11.4 ± 0.45 11.0 ± 0.88 10.5 ± 1.30a DTR on 10% Aq. Extract 11.8 ± 0.86b 11.4 ± 1.29a 12.4 ± 0.25b a a DTR on 6% Et. Extract 11.5 ± 1.46 10.4 ± 0.38 10.6 ± 0.94a DTR on 10% Et. Extract 12.7 ± 0.48c 12.7 ± 0.52c 12.2 ± 0.74b NTR on 10% Aq. Extract 11.2 ± 1.30a 11.5 ± 1.16a 12.1 ± 0.44b d c NTR on 10% Et. Extract 13.5 ± 0.56 13.1 ± 0.72 12.1 ± 0.13b NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are not statistically significantly at 95% confidence level (P > 0.05).
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Table 3.3.1 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES. WEEK 1
2
3
GROUP NCR 8.0 ± 0.73b 8.1 ± 0.45b 8.1 ± 0.21b b b DCR 8.7 ± 1.52 8.8 ± 1.01 8.7 ± 0.99b DTR on 6% Aq. Extract 6.8 ± 0.89a 6.8 ± 0.81a 7.0 ± 0.15a b b DTR on 10% Aq. Extract 8.3 ± 1.51 8.1 ± 1.21 8.1 ± 1.31b DTR on 6% Et. Extract 9.2 ± 3.73b 9.0 ± 2.61b 8.5 ± 3.21b c c DTR on 10% Et. Extract 11.3 ± 2.30 10.7 ± 2.14 10.9 ± 1.99c NTR on 10% Aq. Extract 7.8 ± 4.79a 7.9 ± 3.57a 7.9 ± 2.69a NTR on 10% Et. Extract 7.6 ± 3.39a 7.5 ± 2.87a 7.7 ± 2.57a NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are not statistically significantly at 95% confidence level (P > 0.05). Table 3.3.2 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS CMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS. WEEK 1
2
3
GROUP NCR 8.0 ± 0.73b 8.1 ± 0.45b 8.1 ± 0.21b DCR 8.7 ± 1.52b 8.8 ± 1.04b 8.7 ± 1.30b a a DTR on 6% Aq. Extract 4.9 ± 1.30 5.2 ± 1.58 5.1 ± 1.28a DTR on 10% Aq. Extract 7.7 ± 0.81a 7.9 ± 0.18a 7.7 ± 0.43a DTR on 6% Et. Extract 10.0 ± 0.61b 10.1 ± 0.24b 10.4 ± 0.81b a a DTR on 10% Et. Extract 7.8 ± 0.91 7.8 ± 0.64 7.6 ± 0.33a NTR on 10% Aq. Extract 4.9 ± 0.87a 5.0 ± 0.13a 4.9 ± 1.02a a a NTR on 10% Et. Extract 6.7 ± 0.64 6.7 ± 0.64 6.5 ± 0.38a NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats. Results are Means ± Standard Deviation of triplicate determinations. Values in the same column with different superscripts letters are not statistically significantly at 95% confidence level (P > 0.05). DISCUSSION A clear demonstration of the comparison between the effects of T. officinale leaves and roots as well as the mode of extraction on some haematological parameters such as PCV, haemoglobin levels and white blood cell count, can be drawn from the experimental results above.
The effect of the aqueous leaf extract compared to the roots on PCV from the result above, indicates that their was a gradual increase (P ≤ 0.05) in PCV level of both the diabetic treated rats and the normal treated rats when compared to those of the ethanolic leaf extract. This shows that the mode of extraction (especially the aqueous) has a significant effect on the parameter ascertained. See tables 3.1.1 and 3.1.2.
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On the other hand, it was noticed that the effects of the various extracts and plant parts (leaves and root) had no significant (P 2 0.05) changes on the haemoglobin (Hb) levels and white blood cell counts of both the diabetic treated rats and normal treated rats as demonstrated in tables 3.2.1; 3.2.2 and tables 3.3.1 and 3.3.2. Finally the values obtained from the investigation validates that the plant/herb T. officinale has little or no adverse side effects on the haematology of the test animals (diabetic treated rats and normal treated rats) when compared to that of the normal control rats and diabetic control rats which showed no significant change on Hb levels and WBC counts. 4.0
CONCLUSION
herb and extremely versatile, as the whole plant can be used for medicinal as well as culinary purposes. As a medicinal plant, dandelion has been considered to be an aperient, diuretic, stimulant, anti-diabetic and detoxicant. Dandelion leaves are unique as a diuretic, a valuable alkalizer to the body; eaten regularly they assist the body to reduce excess acidity, oxygenate, purify and build blood, cleanse and regenerate cells. Dandelion leaves contain a significant amount of potassium, a mineral generally lost when using conventional medications. Its calcium content helps the bones (which play a role in blood generation), teeth and nerves. ACKNOWLEDGEMENT The authors acknowledge support from the World Bank and the Federal Republic of Nigeria under the World Bank step B project.
The result of this investigation affirms that dandelion (Taraxacum officinale) is a valuable REFERENCES Al-Awadi, F, Fatana, H. and Shamte, U. (1991). The effect of a plant mixture extract on liver gluconeogenesis in STZ-induced rats. Diabetes Res. 18:163–168.
Davidson, J.K., Delcher, H.K., and England, A. (1979). Spin-off cost-benefits of expanded nutritional care; Journal of the American Diabetic Association. 75:250–257.
Arky, R.A. (1983). Prevention and therapy of diabetes mellitus. Nutritional reviews 41:165–173.
Dijk, P. J. Van. (2003). “Ecological and evolutionary opportunities of apomixes: Insights from taraxacum and chondrilla” Philosophical transactions of the Royal Society. Biol. Scie. 358 (1434):1113.
Bailey, C.J. and Day, C. (1999). Traditional treatments of diabetes. Diabetes care. 12:553–564. Bierman, E.L. (1999). Nutritional management of adult and juvenile diabetes. In nutritional management of genetic disorders (ed.) M. Winick. pp 107–117, New York: Wiley. Clarke, C. B. (1997). Edible and useful plants of California. Berkeley: University of California press. pp 191.
George, D. and Pamplona-Roger, M. D. (1999). Encyclopedia of medical plants. pp.246–7, 227. Green, D. R, and Reed, J. C. (1998). Mitochondrion and apoptosis: Science 281:1309–1312. Harborne, J. B. (1998). Phytochemical method. A guide to modern techniques of plant analysis (3rd edition).
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Lenzen, S., Tiedge, M., Jones, A., Munday, R. (1996). Alloxan derivatives a tool for elucidation of the mechanism of the diabetogenic action of alloxan; In lesson for animal diabetes. E. Shafrir (ed.). Boston Birkhauser. pp. 113–122. Loew,
D. and Kaszkin, M. (2002). Approaching the problem of bioequivalence of herbal medicine products. Phytother Res; 16:705–711.
Omeodu, S. I. (2006). “The effects of aqueous extracts of mistletoe and garlic on serum AST, ALT and ALP of wistar albino rats with CCl4-induced liver damage. Department of Biochemistry, University of Port Harcourt, Nigeria. pp. 2–8. Robert, F., Barnes, C., Jerry, N., Kenneth, J.M., and Michael, C. (2007). Forages: The science of grassland agriculture. WileyBlackwell pp.11. Rose, Francis (1981). The wild flower key. Frederick Warne & Co. Pp 388–391.
Source of Support: Nil
Schutz, K., Carle, R. and Schieber, A. (2006). Taraxacum- A review on its phytochemical and pharmacological profile. J. Ethnopharmacol. 10–11, 107(3):313–323. Sofowora, A.O. (1982). Medicinal plants and traditional medicine in Africa. John Wiley and Sons. Pp 204–208. Stearn, W.T. (1992). Botanical Latin: History, grammar, syntax, terminology and vocabulary, 4th edition. David and Charles. Thompson, L.U. (1993). Potential health benefits and problems associated with nutrients in foods. Food Res. Inter. 26:131–149. WHO study group. Diabetes Mellitus (1996). WHO Tech. Report Ser. No 727. Yashpal, P. C. (2004). Chemical constituents of dandelion. pp. 12–14.
Conflict of Interest: None Declared
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Original Research Article EVALUATION OF ANTHELMINTIC ACTIVITY OF JUSSIAEA SUFFRUTICOSA LINN. Singh Vijayendra1*, Panda S K2, Choudhary Puneet Ram3 1
Asst. Professor, Department of Pharmacy, Surguja University. Department of Pharmacognosy, The Pharmaceutical College, Barpali, (Bargarh) Orissa. *Corresponding author e-mail: vijayendra.singh94@yahoo.com, Mob.No.- 09770816465
2,3
Received: 30/03/2012; Revised: 25/03/2012; Accepted: 07/05/2012
ABSTRACT The plant Jussiaea suffruticosa Linn. (Onagraceae) possesses anti- inflammatory, antidiarrhoeal, CNS activity, anti-tussive, anti-pyretic, anti-diabetic and diuretic property. The present study reports anthelimintic activity of various extracts obtained from J. suffruticosa. Adult Earth worms (Pheretima Posthuma) treated with 20 and 25 mg/ml of methanolic extract of J. Suffruticosa showed significantly higher action as an anthelimintic when compared with the standard drug, Albendazole suspension. Observations were made for the paralysis time (PT) & subsequently for death time (DT). The paralysis and death times decreased with increase in concentration of the test solution. Key words: Jussiaea suffruticosa linn., anthelimintic, Pheretima Posthuma Methanolic extract
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INTRODUCTION
MATERIAL AND METHODS
Anthelmintics are the drugs used to eradicate or reduce the number of helmintic parasites (worms) in the intestinal tract or tissues of human and other animals. A large proportion of mankind, particularly those in tropical and subtropical regions harbours worms. Helminthiasis is prevalent globally (1/3rd of worldâ&#x20AC;&#x2122;s population harbours them) Tripathi K D et al., (2008) but is more common in developing countries with poorer personal and environmental hygiene.
Plant Material
Jussiaea suffruticosa Linn. (Onagraceae) exhibits a wide range of pharmacological activities useful to mankind of which anthelmintic activity is one. J. Suffruticosa is a widely growing plant in the central parts of India. The plant has been studied by Saha B.P. et al., (2000, 2010) and Mythreyi R. et al., (2010) for its antipyretic, anti-diarrhoeal, CNS activity, as well as anti-diabetic, antiinflammatory and diuretic properties. However, no work has been done on the anthelmintic properties of this plant. Hence J. Suffruticosa was selected for the study on its anthelmintic properties with earth worms (Pheretima posthuma) as the animal model.
The whole plant of J. suffruticosa was collected from cultivated wet fields of Barpali in Bargarh district in Odisha. The taxonomical identification of the plant was established by the Botanical survey of India, Shibpur, Howarh. The voucher specimen has been deposited at research laboratory for future reference. Prepartion of Methanolic Extract The whole plant of J. suffruticosa were dried under shade, pulverized, sieved through 40-mesh and extracted with 90% methanol in soxhlet apparatus. Then the solvent was completely removed under vacuum distillation. A brownish, semi-solid was obtained (yield 16.8%). This semisolid was taken as the standard extract. This was refrigerated for further use as the test drug for anthelmintic property. For testing anthelmintic property, solution of different strengths of this extract was prepared in normal saline with 2% gum acacia. WORMS USED Earth worms (Pheretima posthuma) Giri, R.K., sahoo, M. K. et al (2009).
Figure 1a: Habit of Jussiaea suffruticosa L
b: flowering branch of Jussiaea suffruticosa L
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METHODS Anthelmintic activity was evaluated on adult Indian earth worms (Pheretima posthuma) due to anatomical and physiological resemblance with the intestinal round worms. The experimental animals were acclimatized by keeping them in the laboratory, in the soil of their original habitat, for 24 h. The worms were then divided into 5 groups with 6 earthworms in each group. Different groups were treated with the aqueous solution of albendazole (10mg/ml) and methanolic extract of the test solution with 15mg, 20mg and 25mg/ml concentration of the semi solid in normal saline, containing 2% gum acacia. Observations were made for the paralysis time (PT) & subsequently for death time (DT). Paralysis was inferred to have set in when the organisms stopped movement and took a circular shape and the worms do not revive. Death was declared when the worms lose their motility followed by fading away of their body colour. Normal saline was taken as control, albendazole (Bendy suspension mankind
pharma Ltd.) was taken as standard and solution of methanolic extract semi-solid was taken as test solution. 5 groups of earthworms were treated in 50 ml. of 5 types of different solutions, as follows: Group I: Placed in albendazole solution (Standard solution) Group II: Placed in normal saline – (Control) Group III: Placed in 15mg/ml of semi solid solution (test solution) Group IV: Placed in 20mg/ml of semi solid solution (test solution) Group V: Placed in 25mg/ml of semi solid solution (test solution) RESULTS AND DISCUSSION Statistical analysis The results were presented as mean ± SEM. “One-way ANOVA with Dunnett’s post test was performed using Graph Pad Prism version 3.00 for windows. Graph Pad Software, San Diego California USA, P < 0.01 implies at 3% level of significance.
Table 1: Anthelmintic activity of Jussiaea suffruticosa. Treatment
Concentration (mg/ml)
Time taken for paralysis (min)
Time taken for death (min)
Control (G-I)
_
_
_
Albendazole suspension (Standard G-2)
10 mg/ml
4.90 ±0.37***
23.8 ±0.37***
15mg/ml
8.80±0.37***
43.4 ±0.51***
20mg/ml
7.80 ±0.45***
41.4 ±0.25***
25mg/ml
5.88 ±0.37***
24.0 ±0.44***
Test Groups (G-III, G-IV, G-V Methanolic extract of J. suffruticosa in different concentrations)
*** = Highly significant at 3% level of significance.
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10
(-) control 10mg/ml (standard) 15mg/ml (Test) 20mg/ml (Test) 25mg/ml (Test)
8 6 4 2 0 (-) 10 co m nt g/ ro m l l( st an da 15 rd m ) g/ m l( Te 20 st m ) g/ m l( Te 25 st m ) g/ m l( Te st )
Time taken for paralysis(min)
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fig1 :-Time taken for paralysis of P. posthuma in different concentrations of Test solution.
Each value represents the mean ± S.E.M.
Time of death (min)
50
(-)control 10mg/ml (standard) 15mg/ml (Test) 20mg/ml (Test) 25mg/ml (Test)
40 30 20 10
) st Te
st l(
Te m
l(
g/
m m
g/ 25
20
m
g/ m 15
)
) Te l( m
an st l(
10
m
g/ m
st
) rd da
(- ) co nt
ro l
0
fig 2:-Time taken for Death of P. posthuma in different concentractions of test solution.
Each value represents the mean ± S.E.M. From the results shown in table no. 1, the predominant effect of albendazole on the worm is to cause a flaccid paralysis that result in expulsion of the worm by peristalsis. Albendazole by increasing chloride ion conductance of worm muscle membrane produces hyperpolarisation and reduced excitability that leads to muscle relaxation and flaccid paralysis. Results obtained indicate that the higher concentration of each plant extract
produced paralytic effect much earlier and the time to death was shorter. The perusal of the data (Table-1, fig-1, fig2) revealed that the methanolic extract at the concentration of 20 mg, and 25 mg/ml showed paralysis time in 7.80, & 5.88 min respectively and death time of 41.4 and 24.0 min respectively. The effect increased with concentration which implies that the paralysis and death time decreased with increase in
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concentration of the test solution. The extract caused paralysis followed by death of the worms at all the tested dose levels. The results of the current investigation indicate that methanolic extracts of Jussiaea suffruticosa, is a potent form and requires less time to the paralysis and death of the worms. Methanolic extract showed a concentration depended anthelmintic property (Table-1,fig1,fig-2) Methanolic extract of J. suffruticosa demonstrated paralysis as well as death of worms especially at higher concentration of 25 mg/ml while 20mg/ml concentration also shown significant activity. CONCLUSION J. suffruticosa used by tribals traditionally to treat intestinal worm infections, showed
significant anthelmintic activity. The experimental evidence obtained in the laboratory model could provide a rationale for the traditional use of this plant as anthelmintic. The plant may be further explored for its phytochemical profile to recognize the active constituent accountable for anthelmintic activity. ACKNOWLEDGEMENTS The author wish to thank Prof. S.K. panda, Principal of The pharmaceutical college, Barpali and Dr. M.L. Nayak director of university teaching department Surguja university, Ambikapur for his tremendous enthusiasm to my research work and helpful comments on the text.
REFERENCES Anonymous (1966) The Wealth of India, Vol.I, publication & information Directorate, New Delhi, CSIR, pp-311.
Linn. Extract in albino mice, phytotherpay research pp- 541–542.
Giri, R.K., sahoo, M. K., 2009.anthelimintic activity of Momordica Dioica, Indian journal of Natural products.
Murgesan T, Rao B, Sinha S, Biswas Swati, Pal M, Saha B.P, (2010) anti-diabetic activity of Jussiaea suffruticosa Linn. extracts in rats pp- 362–365
Kirtikar K R, Basu B D, (1935) in Mhaskar (eds.), Indian Medicinal plants, Dehradun, Bishen singh and Mahendra pal singh, 20Murgesan T, Pal M., Ghosh. L, Mukherjee K, Das.J, Saha B.P. (2000). Evaluation of Antidiarrhoeal profile of Jussiaea suffruticosa, Linn. extract in rats., Phytotherapy Research, pp- 381–83. Murgesan,T., Pal M, Saha B.P.(2000), Evaluation of anti-inflammatory potential of Jussiaea suffruticosa linn. Extract in albino Rat, phytotherapy research, pp-395–398. Murgesan T, Ghosh L, Mukherjee P. K, Pal M, Saha B.P. (2000) Evaluation of antitussive potential of Jussiaea suffruticosa Source of Support: Nil
Murgesan T, Ghosh L, Pal M, Saha B.P. (2010) CNS activity Jussiaea suffruticosa Linn. extracts in rats and mice, pharmacy and pharmacology communication, vol.5, pp- 663–666. Mukhrjee P K, Shah K, Balasubramanian R, Pal M, Saha B P 1996. Journal of Ethnopharmacology, Vol-54, pp-63. Nadkarni K M, Nadkarni A K. (1992) Indian Materia Medica, vol.1 Popular prakasan, Bombay, pp-731. Tripathi K.D. (2008), essential of medical pharmacology, 6th edition, jaypee brothers medical brothers (p) ltd., pp808. Conflict of Interest: None Declared
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Review Article ASTASTHANA PARIKSHA - A DIAGNOSTIC METHOD OF YOGARATNAKARA AND ITS CLINICAL IMPORTANCE Sharma Rohit1*, Amin Hetal2, Galib3, Prajapati P K4 1 2
PG Scholar, Department of Rasashastra and Bhaishajya Kalpana including drug research PG Scholar, Department of Basic Principles including drug research
3
Asst. Professor, Department of Rasashastra and Bhaishajya Kalpana including drug research. Professor and Head, Department of Rasashastra and Bhaishajya Kalpana including drug research., IPGT & RA, Gujarat Ayurveda University, Jamnagar, Gujarat, INDIA *Corresponding Author: Mail: dhanvantari86@gmail.com, Mob: +919408325831 4
Received: 04/04/2012; Revised: 19/04/2012; Accepted: 30/04/2012
ABSTRACT Indian traditional medicine, Ayurveda has a great history. Researchers of India have tried to corroborate ancient wisdom with modern scientific practices. It is necessary to diagnose the disease after proper examination and medicines are to be given. There are many diagnostic tools of examination. Yogaratnakara provides a clear picture of scenery of illness and healthy condition through Astasthana Pariksha. Tailabindu pariksha, one among Ashtasthana pariksha is a diagnostic tool of urine examination developed by the medieval Ayurvedic scholars. It also helps in establishing prognosis of various diseases. In current paper, attempts were made to study the relation of Ashtasthana Pariksha in therapeutics with special emphasis and its applicability in medical practice.
Keywords: Ashtasthana Pariksha, Ayurveda, Nadi, Tailabindu, Yogaratnakara
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INTRODUCTION A physician by treating the persons who are deeply immersed in the sea of disease (Roga) due to their ill fate (Papa) are pulled out from the sea. This humanistic effort under taken by the physician ensures an honourable place for him in the society, even-though he does not perform other routine dharma1. Yogaratnakara stresses on the importance of “Vyadhi Vinischaya” (Diagnosis of ailment). It is essential that physician should examine the
disease thoroughly and arrive at a proper diagnosis (Vyadhi Nirnaya). Afterwards i.e. knowing fully about the nature etc of diseases he should commence the Chikitsa (treatment) by administering suitable “Aushadha” or by employing a procedure e.g. Snehana, lepa etc. Different methods of examination have been explained in classics of Ayurveda, which will be helpful in diagnosis of a disease, estimating the status of Rogibala and Rogabala etc. Following table provides a glimpse on this:
Table-1 Methods of Examination explained in different lexicons Sl. No. 1 2
Methods of Examination
Methods
Dwividha Pariksha2 Trividha Pariksha3
3 4 5 6
Chaturvidha Pariksha4 Sadvidha Pariksha5 Ashtasthana Pariksha6 Navavidha Pariksh7
7
Dashavidha Pariksha8
8
Ekadashavidha Pariksha9
9
Charakokta Dwadashavidha Pariksha10 Sushrutokta Dwadashavidha Pariksha11
Pratyaksha and Anumana Aptopadesha, Pratyaksha and Anumana Darshana, Sparshana and Prashna Aptopadesha, Pratyaksha, Anumana and Yukti Panchendriya pariksha and Prashna Pariksha Nadi, Mala, Mutra, Jihva, Shabda, Sparsha, Drika, Akrti Dosha, Aushadha, Desha, Kala, Satmya, Agni, Satva, Vaya and Bala Prakriti, Vikriti, Sara, Samadhana, Pramana, Satmya, Satva,Aharashakti, Vyayama Shakti and Vaya Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Ahara, Satmya, Satva, Pakriti and Vaya Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Sara, Ahara, Satmya, Satva, Prakriti and Vaya Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Sara, Ahara, Satmya, Satva, Prakriti, Vaya
10
Among all these methods of examination Ashtasthana Pariksha (popularly known as Ashtavidha Pariksha) has its own significance. Asta Sthana Rogi Pariksha12 (Eight- fold examination of patient) (1) Nadi Pariksha (Pulse Study) (2) Mutra Pariksha (Examination of Urine) (3) Mala Pariksha (Stool Examination) (4) Jihwa Pariksha (Tongue Examination) (5) Shabda Pariksha (Voice Examination) (6) Sparsha Pariksha (Skin Examination) (7) Drik Pariksha (Eye Examination)
(8) Akrti Pariksha (General appearance Examination) Nadi Pariksha (Pulse Study) The status of Doshas in diseased as well as in healthy individual can be assessed by Nadi Pariksha13. It illustrates all types of diseases progressions, just as the strings of a veena (a musical instrument) can produce different ragas so the Nadi can speak of different diseases14. Like Prakriti, Nadi also varies in person depending on health and disease condition. (Tables 2–5)
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Paryayas of Nadi Snayu, Nadi, Hansi, Dhamani, Dharani, Dhara, Tantuki, and Jeevan Gyan15. Nadi location Vata, Pitta and Kapha Nadi lies respectively under Tarjini (index), Madhyama (middle) and Anamika (ring) fingers of examining physician16. Tridosha examination Three fingers placed in position over Nadi indicate the condition of the Tridosha and their Gati (i.e. Manda, Madhyama and Tikshna) 17. The index finger denotes Vata, the middle finger Pitta and the ring finger Kapha. Nadi Pariksha offers knowledge about involvement of dosha- Vata, Pitta and Kapha, Dwandaja (any two dosha) and Tridoshaja (all three dosha), and Sadhya Asadhyata (prognosis of disease)18. Jiva sakshini Anatomical position of the Jiva sakshini Nadi at Angushtha moola and its clinical importance as pulse has been stressed19. The pulsation in the Dhamani (artery) reflects the evidence of life and the learned physician through Sparsana Pariksha is able to come to assessment of the person concerned, whether the person is ill or well. In female left hand Nadi should be palpated and vice versa. How to examine Nadi should be examined in mental stability and peace of mind before with his hand pulse (beat) below the right thumb. As regards methodology, the elbow (Kurpara) of the patient should be lightly flexed to the left and the wrist slightly bent to the left with the fingers distended and dispersed. Nadi should be
examining repeatedly for three times by applying and releasing pressure alternately over Nadi to assess the condition of Dosas rightly20. After Nadi Pariksha physician should wash his/her hands because disease disappears from the patient like mud gets washed away21. Method for Arterial pulse examinationAn ideal time for pulse examination is early morning with empty stomach. But in case of emergency, it can be examined at any time of the day or night. It is essential as a routine to feel not only the radial pulse but also the other peripheral pulses. The pulse is usually felt at the wrist and over the radial artery, because of its superficial position and ease of palpability. The radial artery is situated slightly medial to the styloid process of the radius, on the anterior aspect of the wrist, and is best felt with the subjectâ&#x20AC;&#x2122;s forearm slightly pronated and wrist somewhat flexed22. New Findings- in relation to Nadi Pariksha The pulse is a wave, which, after being produced by cardiac systole, travels or advances through the arterial tree in a peripheral direction. It arrives at the wrist long before the column of blood ejected by heart. Characteristics of the pulse i.e. Rate, Rhythm, Volume, Force etc of pulse varies from individual to individual and even from time to time23. The pulse rate is unduly high during fever, infectious diseases etc. and slow pulse rate may be indicative of certain clinical status e.g. Hypotension, etc24. Contraindication for Nadi Pariksha In the following conditions Nadi Pariksha gives no correct information- immediately after bath, immediately after having food, after massaging, hungry, thirsty and while sleeping25.
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Table-2 Nadi Gati26 Vataja Nadi
Pittaja Nadi
Kaphaja Nadi
Snake and leech
Crow, lark and frog
Swan, pigeon and cock.
Vata Kaphaj Nadi Snack and swan
PittaKaphaj Nadi Monkey and swan
Vata Pittaj Nadi
Sannipataja Nadi
Snake and frog
Wood pecker
Table-3 Nadi Gati in different pathological condition27 Sl.No 1 2 3 4 5 6 7 8 9 10
Pathological Conditions Jwara Kama Krodha, Chinta and Bhaya Mandagni Rakta Dosha Ama Deeptagni Kshudhita Tripta Asadhya Vyadhi
Nadi Gati (Pulse movements) Gambheera, Ushna and Vegavati Vegavati (rapid) Kshina (weak) Manda (slow) Ushna, Gurvi (heavy) and Sama Gambheera Laghu and Vegavana Chanchala (unstable) Sthira (stable) Kampana (vibration) and Spandana (pulsation)
Table-4 Nadi Gati in different Jwaravastha28 Sl.No 1 2 3 4 5 6
Jwara Avastha Vata Jwara Pitta Jwara Kapha Jwara Vata pitta Jwara Kapha vataja Pitta Kapha
Nadi condition Vakra, Chapala (unstable), cold on touch Rapid, straight and of long duration Slow, stable, cold and sticky Somewhat Vakra, Chapala and Kathin Manda (Slow) Sukshma, Sheetala and Sthira
Table-5 Arishtha lakshana of Nadi for prognosis of disease29 Sl.No 1 2 3 4 5 6 7 8 9 10
Pulse movement with Physical condition Sthira (Stable) and Rapid like Vidhyut (electrical force) Shigra (very rapid) / Sheeta and passing mala repeatedly Sometime Tivra and sometimes slow with body sweating Tivra Nadi with burning and coldness in the body with dyspnoea No facial pulsation coldness in the body with Klam Very rapid and sometimes thin, sometimes forceful yet cold Vidhyuta unmita (curvilinear motion) Tiryaka, ushna , vegavati (moves like snake) along with Kapha filled throat Chanchalita (unstable), Ativega, Nasikadharsamyuta (felt like cloth wave on the strength of respiration) Tridoshas influence the Nadi simultaneously
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Prognosis May die 2nd day Will die within 2 days May die within 7 days Will die within 15 days May die within 3 days About to die Imminent death May die May die in one yama kala Krichhasadhya or Asadhya
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Asadhya Nadi ‘Sannipata Nadi’ pulsate slowly, intermittently (Vyakula) and is extremely thin. This is mentioned as Asadhya Nadi. It indicates imminent death. When Nadi firstly pulsates like Pitta gati, afterwards it becomes like Vata gati then transforming to Kapha gati and moves like a wheel, sometimes it is rapid and sometimes very thin such Nadi should be considered as Asadhya Nadi and act accordingly. Mrityu Suchaka Nadi- the Nadi which resembles Damru (a musical instrument), means which is strong at opening and ending but very slow in between, is the indicator of death in a day30. New findings- in relation to Asadhya nadi Forceful and jerky rise of the Corrigan pulse is due to the rapid filling of the radial artery caused by an extra large amount of blood pushed by the distended left ventricle during systole into relatively empty arterial vessels. The collapsing character or the sudden down stroke of the pulse may be due to partly to the sudden fall of pressure in the aorta due to regurgitation of blood into the left ventricle through a leaky valve during diastole31. Healthy Pulse: Hamsa gamana (Swan like walk), Gajagamini (elephant like) and who is
having cheerful face is considered to be a healthy32. Mutra Pariksha Importance By Mutra Pariksha (urine examination) one can assess any running pathology inside the body30. Urine is the end product of metabolism by billions of human cells and the body chemistry, blood pressure, fluid balance, nutrient intake, and the state of health are key elements in establishing the characteristic of urine33. Method The wise physician should wake up the patient early in the morning around 4 o’clock, avoid the first stream of early morning urine, then collect the urine of subsequent flows in a clean glass vessel and examine thoroughly to assess the disease process and treat the patient accordingly34-35. New findings- in relation to mutra pariksha For routine urine examination, midstream sample of urine which is the first morning sample, collected in a clean container is preferred since it gives a more constant result36.
Table-6 Urine appearance involving doshas37 Sl.No. 1 2 3 4 5
Dosha Vata Kapha Pitta Dwandaja Sannipataja
Urine colour/appearance Pandu Phenayukta Rakta Mixed / as per predominant dosha Krishna
Table-7 New Findings of probable causative factors and Urine appearance38 Sl.No. Urine colour/appearance 1 Greenish yellow 2 Red 3 4
Black Cloudy appearance/ sedimentation
Probable causative factor Bile pigments Porphyrins, haemoglobin, myoglobin, numerous other drugs. Melanin and homogentisic acid Epithelial cells, W.B.C., red cells, bacteria and fat
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(Tailabindu
Along with the examination of colour, appearance and consistency of urine (Tables 6– 7), a special technique for the examination of the Mutra, Tailabindu Pariksha, was developed to diagnose disease conditions and to find out their prognosis. Both examination of urine sample and questioning of patients are important for assessing Doshic influence. A modification of this is the oil (taila) drop (bindu) test (pariksha) in which the effect of an oil drop on urine sample suggests the curability of disease. Urine should be examined carefully as stipulated. Instil one or two drops of Tila taila into the vessel, where in the patients’ urine is collected37. Type of dosha vikara is assessed by appearance of taila bindu39 (Table 8). According to direction of spread of drop one can assess the curability or non-curability of disease40 (Tables 9–10), prognosis of disease41. By urine appearance doshic predominance42 and disease condition43 can be diagnosed (Tables 11–12). Mala Pariksha Type of dosha vikara and disease condition can be determined by Mala pariksha44 (Tables13–14). If digestion & absorption of food are poor, the stool carries a foul odour and
sinks in water. Vata aggravated, the stool is hard, dry and grey/ash in colour. Excess Pitta makes it green/yellow in colour and liquid in form. And high Kapha lines it with mucus. New Findings- in relation to Mala pariksha Stool examination is one of the simplest, widely applicable and most important tests for the diagnosis of intestinal parasitic infection and other inflammatory condition. In Ayurveda Rashi, Swarupa, Varna, Gandha, Sama-Nirama Lakshana of stool etc are the diagnostic tools for many diseases. In modern era microscopic examination of the stool is important to diagnose Amoebic dysentery etc. Blood in stool indicate gastrointestinal lesion and fat determination is done for seborrhoea45. Jihwa Pariksha46 Detection of the type of disease condition can be made by Jihwa Pariksha (Tables15–16). New findings in relation to Jihwa pariksha Different areas of the tongue correspond to different organs of the body. Hence by correlating the location of the blemishes on the tongue, the Ayurvedic practitioner can determine which organs of the body are out of balance. The colour, size, shape, coating, anomalies, surface, mobility and local lesion are all noted47.
Table-8 Taila bindu appearance in different Dosha Vikara48 Sl. No. 1 2 3
Dosha Vikara Vata Pitta Kapha
Taila bindu appearance Snake Umbrella Pearl
Table-9 Oil position in different diseased condition49 Sl.No. Urine Disease condition 1 If instilled oil spreads quickly over the surface of urine Saadhya (Curable) 2 If the oil does not spread Kashta-saadhya (difficult to treat) 3 If oil sinks and touches the bottom of vessel Asaadhya (incurable)
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Table-10 Prognosis according to the direction drop spread of urine50 Sl.No. 1 2 3 4 5 6 7
Direction of urine drop spread Towards east Towards south Towards northern Towards west Towards Esanya Agneya or Nairuti direction or oil gets split Vayavya direction
Prognosis Patient will get relief Will suffer from Jwara and gradually recover Will be cured and become healthy Will attain Sukha and Arogya Will die in a month Bound to die Going to die anyway
Table-11 Urine appearance in different Doshik aggravation51 Sl.No.
Urine Doshic Indication
1
Vata aggravation
2 3 4
Pitta aggravation Kapha aggravation Rakta aggravation
Urine appearance
Slightly Neela and Ruksha (free from oily appearance) Pitta (yellow) and slightly reddish , looks like oil Snigdha cloudly and watery Snigdha, Ushna and blood coloured
Table-12 Urine appearance in different diseases52 Sl.No. Diseases 1 Ajeerna 2 Naveena jwara (acute fever) 3 Vata pitta Jwara 4 Vata Shleshma Jwara 5 Shleshma pitta Jwara 6 Jeerna Jwara (Chronic) 7 Sannipata Jwara
Urine appearance Rice water Smoky and excessive Smoky, watery and hot Whitish and is like budbuda Polluted and with blood mixed Yellowish and red Mixed shades depending on doshas involved
Table-13 Mala Lakshana in different Dosha Vikara53 Sl.No. 1 2 3 4 5 6 7 8 9
Dosha Vikara Vata vikara Pitta vikara Kapha vikara Tridosha Vata prakopa Vata Kapha Pitta Vata Kapha Pitta Tridoshaja
Mala Lakshana Dridha (hard) and Shushka (dry) Peeta (yellowish) Shweta (white) Sarva lakshana Trutita (broken), fenila (frothy), Ruksha (dry), Dhumala (smoky) Kapisha Badddha (binding), Tritita (broken), Peeta , Shyam Peeta,Sweta, Ishat Sandra, Pichchhila Shyama, Tritita, Pittabha, Baddha Sweta
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Table-14 Mala Swarupa in different diseases54 Sl.No. 1 2 3 4
Mala Swarupa Whitish, bulky with foul smell Shyama Yellowish associated with pain in the Kati Jatharagni passes pandu and dry Mala while in Mandagni state passes Drava and Durgandhita mala
Diseases Jalodara Kshaya Amayukta disorders Asadhya vyadhi
Table-15 Characteristics of tongue in different Doshik condition55 Sl.No. Disease 1 Vataja 2 3 4 5
Pittaja Kaphaja Sannipataja Dwandaja
Tongue Cold, rough and cracked (brown or black) Reddish and blackish Whitish and sticky Blackish, Kantaka (thorny) and dry Mixed symptoms and sign
Table-16 Tongue features in different diseased condition56 Tongue features Colour
Fur coating (consisting of epithelial debris, food particles and micro-organisms)
Diseases condition Anaemic Jaundice, possible liver disorders Blue Heart diseases Posterior part of Toxins in large intestine tongue Middle part of Toxins in stomach and tongue small intestine. Pale Yellow
Table-17 Asya Pariksha57 Sl.No. 1 2 3 4 5 6
Disease Vataja Pittaja Kaphaja Tridoshaja Ajeerna Agnimandhya
Taste of mouth Sweet Katu (pungent) Madhuramla Mixed feeling Ghrita purna Kashaya
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Asya Pariksha Different dosha can change the taste of mouth. This can be including only in Prashna Pariksha. (Table-17) New findings- in relation to Asya Pariksha In Yogaratnakar, Asya pariksha is described as subjective condition. Acharya Charaka described different types of Rasa vishayaka arishta in Indriyasthana but they all are in excessive condition and in abnormal conditions48. According to modern science, oral examination contains tongue, teeth, gums,
buccal mucosa etc examination but in Yogaratnakara it is described as taste examination. Different taste on tongue in abnormal condition is important to know by asking for different Doshik vitiation. In modern science, there is no any direct relation with taste is described for diagnosis. Shabda Pariksha Healthy and natural when the doshas are in balance, the voice will become heavy when aggravated by kapha, cracked under pitta effect and hoarse & rough when afflicted by vata. (Table 18)
Table-18 Shabda Pariksha58 Sl.No. 1 2 3
Dosha Kapha Pittaja Vataja
Swara Guru (heavy) Sphuta vaktra (cracked) Devoid of these two qualities (hoarse or rough)
New Findings- in relation to Shabda pariksha Auscultation can be compared with the Shabda Pariksha of Ayurveda. Four auscultatory areas of the heart facilitate clinical diagnosis. Interscapular area, infrascapular area, cranial area, Abdominal area and peripheral arterial sites may disclose murmers of diagnostic significance59.In Respiratory examination, inspiratory and expiratory sounds with or without an intermediate pause or interval is observed as normal 51 condition .Abnormal breath sounds are heard if they are abnormally generated and if they are abnormally conducted52.Aucultation is an important part of abdominal examination. It is best carried out in deep expiration and with light application of the bell chest piece over all the four abdominal quadrants60. In abnormal condition also Auscultation of abdomen give some clue for diagnosis e.g. Succussion (Gastro intestinal splashing sound) sounds are found in stomach fluid or gas etc61.
New Findings- in relation to Sparsha pariksha Sparsha Pariksha can be compare with palpation and percussion. Palpation is only second to that of auscultation. It is an important clinical method for examination of skin for assessing the state of organs and tissues. The examiner stands or sits on the right of the patient and places the palm of hand which must be warm, on the area under investigation. It was customary to define the apex of the left ventricle57. Sparsha Pariksha Used for assessing the state of organs and tissue, palpation is an important clinical method for examination of skin. Noted for doshic influences, a vata aggravated skin is course & rough with below normal temperature, a pitta influenced one has quite high temperature and kapha affected it becomes cold & wet. (Table19)
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Table-19 Sparsha Pariksha62 Sl.No. 1 2 3
Dosha Kapha Pittaja Vataja
Sparsha (Touch) Wet and cold Hot and moist Cold and rough
Table-20 Apical impulse in relation to different abnormalities Sl.No. Apical impulse Abnormalities 63 1 Hyperdynamic Thrust Dilated Left Ventricle 2 Slapping Apex beat64 Thyrotoxicosis, fever, after exercises etc 3 Tapping Apex beat65 Mitral stenosis In Respiratory system, comparative palpation of both two sides of the chest and Localised swelling, tenderness, Crepitus, Ronchial Fremitus, Palpable rales, friction fremitus, lymph nodes enlargement are observed through palpation66. In abdominal examination also Muscle rigidity, tenderness, oedema, doughy feel, haematoma, lump etc are examined. By percussion normal health condition, area of cardiac dullness (Table 20), liver dullness, Splenic dullness etc are examined67. Abnormal percussional findings in different disease also
give clue for many diseases e.g. Shifting dullness in Hydro or pyo-pneumothorax, Fluid thrill, horse shoe shaped dullness, shifting dullness are found in Ascites68. Drika Pariksha Vata domination makes the eyes sunken, dry and reddish brown in colour. On aggravation of pitta, they turn red or yellow and the patient suffers from photophobia and burning sensations. High kapha makes them wet & watery with heaviness in the eyelids. (Table 21â&#x20AC;&#x201C;23)
Table-21 Drika Pariksha69
Sl.No. 1
2 3
4 5
Doshaja Prakriti Vata
Drika
Dhumra (smoky), Aruna (pink), Nila (blue), Ruksha (dry), Chanchala (unsteady), Antrapravista (sunken), Roudra (trrrifying), Antarjwala (glowy inside) Pitta Aruna (pink), Haridra (yellow), Rakta (red), Malina (dirty), Tikshna (penetrating), Dipa dwesha (dislikes light), Dahayukta (burninig) Kapha Sweta (whitish), Dhavala (glistening), Pluta (watery), Snigdha (greasy), Sthira (steady), Santa (affectionate), Jyotish (lustreless), Kanduyukta (itchy) Dwandaja Mixed lakshana of involved Dosha Sannipataja Rakta (red), Roudra (horrifying), sunken and lustreless
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It can be understood as follows: Table-22 Eye features in relation to Doshik dominancy70 Dominancy Dosha Vata
of Eyeball features
Eyelids
Small, nervous, shrunken
Drooping and dry Moderate in size, shape, lustrous, Reddish sensitive to light, burning sensation Big, beautiful and moist Heavy
Pitta Kapha
Eye lashes
Sclera
Scanty and rough Scanty and oily Long, thick, oily
Muddy Flushed Pale or very white
Some special features of eyes also indicate certain diseases eg. Excessive blinking is a sign of nervousness, anxiety or fear and a drooping upper eyelid indicates a sense of insecurity, fear or lack of confidence.
eyeball rotates; all these are bad prognosis. Acharya Charaka described Arishta vishayaka lakshanas of Chakshu71.
Arishta lakshana One eye opened and the other closed, whose eyes become bright lustrous and red, when patient sees reddish, bluish and terrifying images, when one eye loses vision and other
Different types of eyes features may reflect the personality of the individual and his reaction to disease72.Expression of the eyes may reflect the health and diseased condition of an individual.
New Findings- in relation to drika pariksha
Table-23 Eye features in different diseased condition73 Sl.no. 1 2 3 4
Eye feature Prominent /bulging Yellow conjunctiva Small iris Prominent white ring around iris
Disease condition Thyrotoxicosis Weak liver Weak joints Joint degeneration with potential for arthritis
Table-24 Akriti Pariksha74 Sl.No. Dosha 1 Kapha 2 3
Pittaja Vataja
Akriti (Rupa) Saumya, snighdha, well built body and joints, tolerant to hunger, thirst, hardship, hot sun. Hungry and thirsty, fair in colour, brave, Swabhimani, less hair Vibhu, ashukari, balvana, prone to many diseases, split hair and dry skin with Dhusara Varna, dislikes cold, Pralapa, unstable Dhriti, Smriti, Buddhi, Cheshta etc
Akriti Pariksha
New Findings- in relation to Akriti pariksha
The doshic influences that reflect on the face of the patient enables physicians to gauge the basic constitution and the nature of the disease. (Table-24)
The doshic influences that reflect on the face of the patient enable physicians to gauge the basic constitution and the nature of the disease. The constitution or body type of the individual may have a bearing on the disease
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process75. The regional distribution of eruptions gives an idea of the diagnostic clues. Abnormal dryness of the skin from loss of sweating may be found in dehydration, hypothyroidism, Scurvy etc76. DISCUSSION Yogaratnakara describes movements of Nadi under the influence of Doshas and their combinations. Further to make it more appealing, the author correlates the character of each type of pulse with movement of animals, birds etc (Table-2). The position of index finger denotes Vata Dosha. In Vata predominant constitution, the index finger will feel the pulse strongly. The pulse movement will be like motion of a serpent. This type of pulse is called snake pulse. The middle finger denotes the pulse corresponding to the Pitta Dosha. When the person has a predominant Pitta constitution, the pulse under the middle finger will be stronger. Ayurveda describes this pulse as "active, excited, and move like jumping of a frog." This pulse is called frog pulse. When the throbbing of pulse under the ring finger is most noticeable, it is a sign of Kapha constitution. The pulse feels strong and its movement resembles the floating of a swan. Hence, this pulse is called swan pulse. Acharya Charaka described different Nadi conditions in Indriyasthana for Jwara purvalakshana77. The movements of Nadi according to different pathological conditions are well described by Yogaratnakara78 (Table-3). Not only different status of fever79 but also the prognosis of disease can be made by detecting Nadi gati (Table-4 and Table-5). Examination of mootra is very important in diagnosis. Mutravaha Srotas is affected by various causes like Ahara (excess of Katu, tikshna, Amla, Lavana), Vihara (Trishnanigraha, Atapasevana, Ativyayama),Abhighata ,etc or some diseases affecting Rakta, Hridaya etc. The color, consistency, character, quantity of Mutra varies in different illnesses. Examination of faces gives valuable clues regarding the Annavaha Srotas as well as Purishavaha Srotas. Prakriti, Ahara, Vihara, kala, Satmya, Vyadhi etc influence features of Purisha. Susruta describes the features of Purishakshaya are to be inferred
from complaints of pain on the sides and cardiac regional feeling of vayu crushing upwards, flatus, rumbling sounds in intestine etc. Jihva Pariksha has great importance. Tongue is considered as the index of stomach and its examination produce vital clues to diagnosis. Any abnormality in color, shape, size, presence of fissures or cracks ulcerations, salivation, furr on tongue, tremor, and deviation to one side should be noted. Shabda pariksha has specific role in diagnosis. Pratyaksha is main stream to understand things and Shabda is one of the main Upadhi for that purpose. Different organs like heart, intestine etc produce sound while working. These sounds may be altered in diseases. People use sounds in communicating with others, this can also be altered in various diseases. By percussion and listening to the sounds produced, the position of hard organs, presence or absence of fluid or gas in cavities etc can be determined. Sparsha has great role in diagnosis; it is mentioned by all acharyas and also included in Trividha, Shadavidha and Ashtasthan Pariksha. These all shows the importance of Sparsha pariksha in diagnosis. By examination of eye, one can find some Arishtalakshana like Urdhva Drishti, Bramayuta, etc. Akriti Pariksha has mainly to do with physiognomy it means judging a manâ&#x20AC;&#x2122;s nature by his features. Here, the most obvious external features like appearance, built, height, shape, size, complexion etc are put to evaluator scrutiny. The attitude of affected organs in Dhanustambha, Manyastambha, Ardita etc are also included in Akriti pariksha. CONCLUSION The principles of the treatment vary from patient to patient on the strength of the patients and morbidity of the disease. Hence it is essential to acquire complete knowledge of Ashtasthana Pariksha of Yogaratnakara. Different methods of examinations were adopted with the different times. These examination methods were designed in such a way that these were very much applicable in leading to the diagnosis of a certain disease. These got modified with the advent of time and the additions of things were done according to the requirements.
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REFERENCES 1. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 2. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.274 3. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.247 4. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, 70 5. Trikamji Jadavaji, (2007), Shushruta Samhita, Varanasi, Kavyatiratha Chaukhambha Publication,p.43 6. Bydgi P.S., Rogi Pariksha and Roga Pariksha, (2007), Paramoswarappaâ&#x20AC;&#x2122;s Ayurvediya Vikriti Vijnana, 1st edition, Varanasi, Chaukhambha Sanskrit Saamsthana, p. 376 7. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p. 69 8. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p. 276 9. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.70 10. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p. 4 11. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.4 12. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.4
13. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.4 14. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.4 15. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 16. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.6 17. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 18. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 19. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.6 20. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 21. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.8 22. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.220 23. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.78
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24. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.78 25. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.77 26. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 27. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 28. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 29. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 30. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 31. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.224 32. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.7 33. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.8 34. An Experienced professor, (1996), Notes on Pathology Bacteriology Virology & Parasitology by 10th revised and enlarged edition, Shroff Publishers and Distributors, p.2 35. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda
Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.8 36. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.8 37. An Experienced professor, (1996), Notes on Pathology Bacteriology Virology & th Parasitology by 10 revised and enlarged edition, Shroff Publishers and Distributors, p.2 38. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.8 39. An Experienced professor, (1996), Notes on Pathology Bacteriology Virology & th Parasitology by 10 revised and enlarged edition, Shroff Publishers and Distributors, p.2 40. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.5 41. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.10 42. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.9 43. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.10 44. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.9 45. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.9 46. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11 47. An Experienced professor, (1996), Notes on Pathology Bacteriology Virology &
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Parasitology by 10th revised and enlarged edition, Shroff Publishers and Distributors, p.24 48. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.12 49. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.12 50. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.12 51. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 52. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 53. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 54. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 55. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 56. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 57. 58. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 59. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.150
60. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.13 61. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.224 62. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11 63. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.255 64. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.355 65. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.359 66. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.411 67. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.409 68. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.244 69. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11 70. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media
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Performance and Publishers, 11th Edition, Delhi, p.244 71. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p.224 72. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.244 73. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.244 74. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11 75. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of
Source of Support: Nil
Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.244 76. Aspi F. Golwala and Sharukh A. Golwala, (2006), Physical Diagnosis, A textbook of Symptoms and Physical Signs, by Media Performance and Publishers, 11th Edition, Delhi, p.345 77. Trikamji Jadavaji, (2007), Charaka Samhita (Dridhabala with Chakrapani), Reprint, Varanasi, India, Chaukhambha Prakashana, p. 276 78. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11 79. Tripathi Indradeva and Tripathi Dayashankara, (2007), Yogaratnakara, Krishnadasa Ayurveda Series 54,Varanasi, Chaukhambha Ayurveda Prakashana, p.11
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