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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

www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148

Global Journal of Research on Medicinal Plants & Indigenous Medicine

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 A

1

1Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology

(KNUST), Kumasi, Ghana

*Corresponding author: Email: [email protected], 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)

mailto:[email protected]



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 cyto-

toxic, antitumor and antimicrobial activities in

different assays. For example, the leaves are

utilized extensively in West African ethno-

medical 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 anti-

diarrhoeal 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%.



Figure: 1 a. Fruits of Jatropha curcus b. Jatropha curcus in its habitat

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

The agar well diffusion method was

employed in the assay. Twenty milliliters of

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 inhibitory

concentration (MIC)

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.



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 MIC (%)

10 20 30 40

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



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 ketoconazole-

plant 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).



Table 3: Antimicrobial effects of different concentrations of ciprofloxacin and ciprofloxacin-J.

curcas leaf extract on the test organisms

Table 4: Antimicrobial effects of different concentrations of amoxicillin and amoxicillin-J.


Microorganism DIZ (mm) of amoxicillin MIC DIZ (mm) of amoxicillin-

plant extract

MIC

0.1 0.05 0.025 0.0125 0.1 0.05 0.025 0.0125

S. typhi 0 0 0 0 0 0 0 0 0 0

P. mirabilis 0 0 0 0 0 0 0 0 0 0

P. aeruginosa 0 0 0 0 0 0 0 0 0 0

S. aureus 18.3 15 12.3 11 6.674 × 10-4

23 20.7 17.7 17 5.623 × 10-5

K. pneumonia 17 14 12 10 6.643 × 10-4

20.3 18 16.7 15 3.148 × 10-5

B. subtilis 20 18 15.7 12.7 3.110 × 10-4

18 15.7 14.7 13 4.701 × 10-5


Microorganism DIZ (mm) of

ciprofloxacin

MIC DIZ (mm) of ciprofloxacin-

plant extract

MIC

10-3

10-4

10-5

10-6

10-3

10-4

10-5

10-6

S. typhi 15.7 12.3 9.3 6.3 1.015 × 10-8

12 10 8 6 1.0 × 10-9

P. mirabilis 18 14.3 11 7 1.086 × 10-8

15 11 8.3 7.7 1.71× 10-9

P. aeruginosa 20 15.3 11.3 7.3 1.992 × 10-8

10.3 7.3 5.7 4.7 4.96 × 10-9

S. aureus 19 12.7 9 6.3 4.887 × 10-8

24.7 19 13.3 11 7.37 × 10-9

K. pneumonia 21.7 17.3 12.3 9 1.005 × 10-8

20 15.7 11.3 9 5.71 × 10-9

B. subtilis 22.7 18.3 13.3 8 2.128 × 10-8

21.7 18 14 11 1.05 × 10-9




Table 5: Antimicrobial effects of different concentrations of tetracycline and tetracycline-J.



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, tetracycline-

plant extract inhibited the growth of P.

mirabilis, P. aeruginosa and S. aureus more.

Generally however, the differences in diameter

Microorganism DIZ (mm) of tetracycline MIC DIZ (mm) of tetracycline-

plant extract

MIC

0.1 0.05 0.025 0.0125 0.1 0.05 0.025 0.0125

S. typhi 21 18 15 12.7 5.72 × 10-4

17 14 12 10 6.64 × 10-4

P. mirabilis 13 12 11 10 1.26 × 10-5

18 14.7 13 12 2.26 × 10-4

P. aeruginosa 15 12 10 9 6.69 × 10-4

18.7 15.3 12.3 10.3 1.10 × 10-3

S. aureus 15 13.7 12.7 11.3 1.92 × 10-5

18.7 16.3 14.3 12.3 2.35 × 10-4

K. pneumonia 22 20 17.7 16.3 4.10 × 10-5

20 19 14 13 4.43 × 10-4

B. subtilis 23 21.3 19.7 17 2.82 × 10-5

25 21.3 17.3 15.3 5.78 × 10-4



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 (Al-

hebshi 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

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.

REFERENCES

Al-hebshi N, Al-haroni M, Skaug N (2006). In

vitro antimicrobial and resistance-

modifying activities of aqueous crude

khat extracts against oral

microorganisms. Arch. Oral Biol.

51:183–188.

Betoni JEC, Mantovani RP, Barbosa LN, Di-

Stasi LC, Fernandes A (2006).

Synergism between plant extract and

antimicrobial drugs used on

Staphylococcus aureus diseases. Mem.

Inst. Waldo Cruz. 101 No. 4.

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.



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.

Irvine ER (1961). Woody plants of Ghana with

special reference to their uses. 2nd

Ed.

O.U.P. London p. 233–237.

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.

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

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.

Taylor PW, Stapleton PD, Luzio JP (2002).

New ways to treat bacterial infections.

Drug. Discov. Today 7: 1086–1091.

Thomas OO (1989). Re-examination of the

antimicrobial activities of Xylopia

aethiopica, Carica papaya, Ocimium

grastissimum and Jatropha curas.

Fitoterapia 60:147–161.

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.

Source of Support: Nil Conflict of Interest: None Declared




PROTECTIVE EFFECT OF RUTIN ON ACETAMINOPHEN-INDUCED

ACUTE HEPATIC DAMAGE IN RATS

Awah Francis M1*

, Chukwumezie Princess U2, Ezema Ogechukwu C

3, Emiliarita Iloakasy

4,

Ubokudom Queen I5

1, 2,3,4,5

Department of Biochemistry, Madonna University, Elele Campus, Rivers State, Nigeria

*Corresponding author: E-mail: [email protected]; 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 acetaminophen-

induced 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



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;

Martínez 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



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. Eight-

week 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).


The data were analysed using the Statistical

Package for Social Sciences (SPSS) version



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 in

acetaminophen-intoxicated rats

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 anti-

inflammatory 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.



Table 1: Serum hepatic enzymes levels of control and acetaminophen-intoxicated rats

AST

(U/L)

ALT

(U/L)

ALP

(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



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

(mmol/L)

Creatinine

(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*



Data represented as Mean ± SEM; * p < 0.05 compared to the Acetaminophen-only group

Effect of rutin on kidney function in


In addition to liver damage, the

acetaminophen metabolite N-acetyl-p-

benzoquinoneimine (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 ±



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 acetaminophen-

intoxication 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 acetaminophen-

intoxicated 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 acetaminophen-

treated 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


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



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


Total SOD Activity

(U/mg protein)

CAT Activity

(U/mg protein)

Control 11.32 ± 1.50 90.43 ± 3.06


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



Table 4: Hepatic malondialdehyde (MDA), protein carbonyls and ascorbate levels in control

and acetaminophen-intoxicated rats

MDA

(µmol/mg protein)

Ascorbic acid

(mg/dL)

Control 0.367 ± 0.08 6.2 ± 0.5


Acetaminophen + 100 mg/kg

Rutin

0.409 ± 0.03 5.2 ± 0.4


Rutin

0.331 ± 0.05* 6.6 ± 0.3*


Rutin

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.

REFERENCES

Aebi H (1984). Catalase in vitro. Methods

Enzymol. 105: 121–126.

Alsaif MA (2009). Beneficial Effects of Rutin

and Vitamin C Coadministration in a

Streptozotocin-Induced Diabetes Rat

Model of Kidney Nephrotoxicity.

Pakistan J. Nutri. 8 (6): 745–754.

Bear WL, Teel RW (2000). Effects of citrus

phytochemicals on liver and lung

cytochrome P450 activity and on the in

vitro metabolism of the tobacco-specific

nitrosamine NNK. Anticancer Res. 20:

3323–3329.

Beauchamp C, Fridovich I (1971). Superoxid

dismutase: improved assays and an



assay applicable to acrylamide gels.

Analyt. Biochem. 44: 276–287.

Bertolini A, Ferrari A, Otani A, Guerzoni S,

Tacchi R, Leone S (2006).

Acetaminophen: new visitas of an old

drug. CNS Drug Rev. 12 (3-4): 250–

275.

Boutis K, Shannon M (2001). Nephrotoxicity

after acute severe acetaminophen

poisoning in adolescents. J. Toxicol.

Clin. Toxicol. 39 (5): 441–445.

Bramstedt K (2006). Living liver donor

mortality, where do we stand? Am. J.

Gasrointestinal. 101: 755–775.

Buege JA, Aust SD (1978). Microsomal lipid

peroxidation. Methods Enzymol. 52:

302–310.

Caillet S, Yu H, Lessard S, Lamoureux G,

Ajdukovic D, Lacroix M (2007). Fenton

reaction applied for screening natural

antioxidants. Food Chem. 100: 542–

552.

Calabro` ML, Tommasini S, Donato P,

Stancanelli R, Raneri D, Catania S,

Costa C, Villari V, Ficarra P, Ficarra R

(2005). The rutin/b-cyclodextrin

interactions in fully aqueous solution:

Spectroscopic studies and biological

assays. J. Pharmaceut. Biomed. Anal.

36: 1019–1027.

Cohen SD, Pumford NR, Khairallah EA,

Boekelheide K, Pohl LR, Amouzadeh

HR, Hinson JA (1997). Selective

protein covalent binding and target

organ toxicity. Toxicol. Appl.

Pharmacol. 143: 1–12.

Cranswick N, Coghlan D (2000).

Acetaminophen efficacy and safety in

children: the first 40 years. Am. J. Ther.

7:135–141.

Curtis JJ, Mortiz M (1972). Serum enzymes

derived from liver cell fraction and

response to carbon tetrachloride

intoxication in rats. Gastroenterol. 62:

84–92.

Dai Y, Cederbaum AI (1995). Cytotoxicity of

acetaminophen in human cytochrome

P4502E1-transfected HepG2 cells. J.

Pharmacol. Exp. Ther. 273:1497–1505.

de Vries J (1981) Hepatotoxic metabolic

activation of acetaminophen and its

derivatives phenacetin and benorilate:

oxygenation or electron transfer?

Biochem. Pharmacol. 30:399–402.

Elliott AJ, Scheiber SA, Thomas C, Pardini RS

(1992). Inhibition of glutathione

reductase by flavonoids. Biochem.

Pharmacol. 44:1603–1608.

Erdem A, Gundogan NU, Usubutun A, Kilinc

K, Erdem SR, Kara A, Bozkurt A

(2000). The protective effect of taurine

against gentamicin-induced acute

tubular necrosis in rats. Nephrol.

Dialysis Transplant. 15: 1175–1182.

Fabjan N, Rode J, Kosir IJ, Wang Z, Zhang Z,

Kreft I (2003). Tartary buckwheat

(Fagopyrum tataricum Gaertn.) as a

source of dietary rutin and quercitrin. J.

Agric. Food Chem. 51(22): 6452–6455.

Fang YZ, Yang S, Wu G (2002). Free radicals,

antioxidants and nutrition. Nutrition.

18: 872–879.

Ferrali M, Signofrini C, Caciotti B, Sugherini

L, Ciccoli D, Giachetti D, Comporti M

(1997). Protection against oxidative

damage of erythrocyte membranes by

the flavonoid quercetin and its relation

to iron chelating activity. FEBS Letters.

416: 123–139.

Gao H, Zhou YW (2005). Anti-lipid

peroxidation and protection of liver

mitochondria against injuries by

picroside II. World J. Gastroenterol. 11:

3671–3674.



Guardia T, Rotelli AE, Juarez AO, Pelzer LE

(2001). Anti-inflammatory properties of

plant flavonoids. Effects of rutin,

quercetin and hesperidin on adjuvant

arthritis in rat. Farmaco. 56(9): 683–

687.

Guo R, Wei P, Liu W (2007). Combined

antioxidant effects of rutin and Vitamin

C in Triton X-100 micelles. J.

Pharmaceut. Biomed. Analy. 43: 1580–

1586.

Hinson JA, Bucci TJ, Irwin LK, Michael SL,

Mayeux PR (2002). Effect of inhibitors

of nitric oxide synthase on

acetaminophen-induced hepatotoxicity

in mice. Nitric Oxide. 6:160–167.

Hinz B, Cheremina O, Brune K (2008).

Acetaminophen (acetaminophen) is a

selective cyclooxygenase-2 inhibitor in

man. Fed. Am. Soc. Exp. Biol. 22 (2):

383–390.

Jaeschke H, Knight TR, Bajt ML (2003). The

role of oxidant stress and reactive

nitrogen species in acetaminophen

hepatotoxicity. Toxicol. Lett. 144: 279–

288.

Jaeschke H, Gores GJ, Cederbaum AI, Hinson

JA, Pessayre D, Lemasters JJ (2002).

Mechanisms of Hepatotoxicity. Toxicol.

Sci. 65: 166–176.

Jiang P, Burczynski F, Campbell C, Pierce G,

Austria JA, Briggs CJ (2007). Rutin and

flavonoid contents in three buckwheat

species Fagopyrum esculentum, F.

tataricum, and F. homotropicum and

their protective effects against lipid

peroxidation. Food Res. Int. 40: 356–

364.

Kreft S, Knapp M, Kreft I (1999). Extraction of

rutin from buckwheat (Fagopyrum

esculentum Moench) seeds and

determination by capillary

electrophoresis. J. Agric. Food

Chem. 47 (11): 4649–4652.

Kwon KH, Murakami A, Tanaka T, Ohigashi H

(2005). Dietary rutin, but not its

aglycone quercetin, ameliorates dextran

sulfate sodium-induced experimental

colitis in mice: attenuation of pro-

inflammatory gene expression.

Biochem Pharmacol. 69(3): 395–406.

Lores-Arnaiz S, Llesuy S, Curtin JC, Boveris A

(1995). Oxidative stress by acute

acetaminophen administration in mouse

liver. Free Radic. Biol. Med. 19: 303–

310.

Lowry OH, Rosebrough NL, Farr AL, Randall

RJ (1951). Protein measurement with

the Folin-phenol reagent. J. Biol. Chem.

193: 265–275.

Luo H, Jiang B, King SM, Chen YC (2008).

Inhibition of Cell Growth and VEGF

Expression in Ovarian Cancer Cells by

Flavonoids. Nutr. Cancer 60 (6): 800–

809.

Mahadevan SBK, McKiernan PJ, Davies P,

Kelly DA (2006). Acetaminophen

induced hepatotoxicity. Arch. Dis.

Child. 91: 598–603.

Martínez CC, Vicente OV, Yáñez

GMJ, Alcaraz BM, Canteras

JM, Benavente-García O, Castillo J.

(2005) Treatment of metastatic

melanoma B16F10 by the flavonoids

tangeretin, rutin, and diosmin. J. Agric.

Food Chem. 53(17): 6791–6797.

Mitchell JR, Jollow DJ, Potter WZ, Davis DC,

Gillette JR, Brodie BB (1973).

Acetaminophen-induced hepatic

necrosis. I. Role of drug metabolism. J.

Pharmacol. Exp. Ther. 187: 185–194.

Moller P, Sindet-Pedersen S, Petersen C, Juhl

G, Dillenschneider A, Skoglund L

(2005). Onset of acetaminophen

analgesia: comparison of oral and

intravenous routes after third molar

surgery. British J. Anaesth. 94 (5): 642–

648.



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.

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.

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.

Sallie R, Tredger JM, William R (1991). Drugs

and the liver. Biopharmaceut. Drug

Dispos. 12:251–253.

Sheu JR, Hsiao G, Chou PH, Shen MY, Chou

DS (2004). Mechanisms involved in the

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.

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.

Webster RP, Gawde MD, Bhattacharya RK

(1996). Protective effect of rutin, a

flavonol glycoside, on the carcinogen-

induced DNA damage and repair

enzymes in rats. Cancer Lett. 109: 185–

191.

Yang J, Guo J, Yuan J (2008). In vitro

antioxidant properties of rutin. LWT -

Food Sci. Technol. 41: 1060–1066.





ASPARAGUS RACEMOSUS WILLD. ROOT EXTRACT AS HERBAL

NUTRITIONAL SUPPLEMENT FOR POULTRY

Kumari R

1, Tiwary B K

2, Prasad A

3, Ganguly S

4*

1,2,3 Department of Veterinary Microbiology, Faculty of Veterinary Science & Animal Husbandry, Birsa

Agricultural University, Ranchi, Jharkhand 834 006 India 4AICRP-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: [email protected]

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.



INTRODUCTION

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, anti-

ulcerative, 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.

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.


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.

The live body weight of chicks was

measured at weekly intervals on 1st, 7

th, 14

th,

21st, 28

th, 35

th and 42

nd 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



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

(in day)

Group 1 Group 2 Group 3 ANOVA-value

1 48.06 ± 1.23 48.06 ± 1.23 48.06 ± 1.23 NS

7 114.27 ± 1.27 113.86 ± 1.35 113.67 ± 1.26 0.056NS

14 276.40 ± 5.67 286.6 ± 6.39 261.73 ± 4.71 1.69NS

21 599.20 ± 17.35 588.66 ± 12.47 574.60 ±14.642 0.68NS

28 981.33 ± 20.53 967.40 ± 14.45 955.4 ± 14.59 0.60NS

35 1409.47 ± 43.19 1381.47 ± 23.94 1324.6 ± 30.70 1.06NS

42 1901.87 ± 0.82a 1809.87 ± 0.44

ab 1765.27 ± 4.63

c 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 Total feed consumed

(kg)

Total body weight

gain (kg)

Feed conversion

efficiency

Group 1 65.95 27.82 2.37:1

Group 2 65.37 26.43 2.47:1

Group 3 64.26 25.46 2.52:1

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

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 non-

treated 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 immuno-

compromised 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



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 non-

treated groups which was a clear marker for

immuno-stimulation among the birds.

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

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

The authors are thankful to Hon’ble Vice-

Chancellor, 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.

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.





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: [email protected]


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



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 malaria-

endemic 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 parasite’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 anti-

malarial 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.


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).



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 anti-

malarial drugs to the patients are as follows:

TABLE 1: Effects of Armact only On Patients with Malarial Parasite

Patients Weight(kg)

Before

Treatment

Malaria

parasite

Before

Treatment

Drug/

Supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 60 ++ Armact only _ 58 Cleared

Female 65 +++ Armact only _ 64 Cleared

Male 70 ++ Armact only _ 65 Cleared

Male 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)

Before

Treatment

Malarial

parasite

Before

Treatment

Drug/

Supplements

Malarial

parasite/

After

Treatment

Weight(kg)

After

treatment

Remarks

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

Vit. C: Vitamin C



TABLE1.2 Effects of Armact and Grape Juice on patients with malarial parasite

Patients Weight(kg)

Before

treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

Treatment

Remarks

Female 65 ++ Armact/GJ − 63 cleared

Female 55 ++ Armact /GJ − 50 cleared

Male 75 + Armact /GJ − 73 cleared

Male 75 ++ Armact /GJ − 70 cleared

GJ: Grape Juice

TABLE 2: Effects of Coartem Only On Patients with Malarial Parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 65 ++ Coart only − 60 cleared

Female 65 + Coart only − 65 cleared

Male 60 + Coart only − 60 cleared

Male 60 + Coart only _ 60 cleared

Coart: Coartem

TABLE 2.1: Effects of Coartem and Vitamin C on Patients with malarial parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

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)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 60 + + Coart /GJ − 58 cleared

Female 60 + Coart /GJ − 56 cleared

Male 75 + Coart /GJ − 70 cleared

Male 70 + Coart /GJ − 69 cleared

Coart: Coartem; GJ: Grape Juice



Table 3: Effects of Waipa only on patient with Malarial Parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 60 + Wp only − 60 Cleared

Female 60 ++ Wp only − 58 Cleared

Male 70 ++ Wp only − 68 Cleared

Male 75 + Wp only − 72 Cleared

Wp - Waipa.

Table 3.1: Effects of Waipa and vitamin C on patient with Malarial Parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 70 + Wp /Vit. c + 70 Not cleared

Female 70 +++ Wp /Vit. c + 70 Not cleared

Male 65 + ++ Wp /Vit. c + 63 Not cleared

Male 55 + Wp /Vit. c − 52 cleared

Wp – Waipa; Vit. C – Vitamin C

Table 3.2: Effects of Waipa and Grape juice on patient with Malarial Parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 60 + Wp /GJ − 58 cleared

Female 60 +++ Wp /GJ − 59 cleared

Male 65 + + Wp /GJ − 63 cleared

Male 60 + Wp /GJ − 58 cleared

Wp – Waipa; GJ – Grape Juice

Table 4: Effects of Fansider only on patients with malarial parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 75 + Fans only − 70 cleared

Female 70 + Fans only − 68 cleared

Male 65 + Fans only − 62 cleared

Male 60 + Fans only − 58 cleared

Fans: Fansider



Table 4.1: Effects of Fansider and Vitamin C on patients with malarial parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 78 +++ Fans /Vit. C + 78 Not cleared

Female 65 + Fans /Vit. C + 65 Not cleared

Male 60 ++ Fans /Vit. C + 60 Not cleared

Male 65 + Fans /Vit. C − 65 cleared

Fans: Fansider; Vit.C: Vitamin C

Table 4.2 Effects of Fansider and Grape Juice on patients With malarial parasite

Patients Weight(kg)

Before

Treatment

Malarial

parasite

Before

treatment

Drug/

supplements

Malarial

parasite/After

Treatment

Weight(kg)

After

treatment

Remarks

Female 70 + Fans /GJ − 68 cleared

Female 70 + Fans /GJ − 67 cleared

Male 60 +++ Fans /GJ − 58 cleared

Male 55 + Fans /GJ − 53 cleared

Fans: Fansider; GJ: Grape Juice

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.

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.



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

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-anti-

malarial 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 anti-

malarial 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.

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.,

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



sub-acute toxicological evaluations of

the methanolic stem bark extract of

Crossopteryx febrifuga inrats. African

Journal of Pharmacy and

Pharmacology, 3: 621–626.

Shanks, G.D. (2006) Treatment of falciparum

malaria in the age of drug resistance.

Journal of

Postgraduate Medicine 52 (4): 277–80.

Sibley, C.H. 2001 Pyrimethamine-sulfadoxine

resistance in Plasmodium falciparum:

what next?

Trends Parasitol. 17:582–588

Wiseman, V., Kim M, Mutabingwa T. K,

Whitty C. J. M. (2006). Cost-

effectiveness study of three

antimalarial drug combinations in

Tanzania. 3(10): 373

WHO (2006). Guidelines for the treatment of

malaria. WHO/HTM/MAL/2006.1108.

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.





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 A

2, and Chuku L C

3

1,2,3 Department of Biochemistry, University of Port Harcourt, Rivers State, Nigeria.

*Corresponding author: (+234)8039397700, (+234)8027205705. [email protected]


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



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.



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 1-

l 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.



Table 2.0 Feeding illustration.

GROUPS

TREATMENT 1 2 3 4 5 6 7 8 9 10 11 12 13 14

No. of Rats per group 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 – – – – – – – – – + – – – +

Key: + Indicates that item was administered;

– Indicates that item was not administered.

2.4 METHOD OF BLOOD COLLECTION

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.

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.

2.4.3 Haemoglobin (Hb)

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.

2.4.4 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 �109 mg/dl.



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

GROUP

1 2 3

NCR 35.4 ± 2.51b

36.4 ± 2.07b 37.9 ± 0.86

b

DCR 34.2 ± 1.10a

36.3 ± 2.75b

36.0 ± 1.00

b

DTR on 6% Aq. Extract 33.4 ±2.19a

32.8 ± 2.39a

35.2 ± 3.10b

DTR on 10% Aq. Extract 33.2 ± 3.10a

37.2 ± 2.05b

36.4 ± 3.80

b

DTR on 6% Et. Extract 34.0 ± 2.73a 32.6 ±

2.61

a 32.4 ± 3.71

a

DTR on 10% Et. Extract 35.8 ± 3.42b

35.8 ± 1.50b

36.5 ± 2.65

b

NTR on 10% Aq. Extract 36.6 ± 0.55

b 38.6 ±

1.14

c 37.9 ± 1.37

c

NTR on 10% Et. Extract 35.8 ± 0.84b

36.0 ± 3.46b

36.8 ± 2.28

b

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


AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS.

WEEK

GROUP

1 2 3

NCR 35.4 ± 2.51b

36.4 ± 2.07a

37.9 ± 0.86b

DCR 34.2 ± 1.10a

36.3 ± 2.75a

36.0 ± 1.00a


37.4 ± 0.89b

35.8 ± 0.84a

DTR on 10% Aq. Extract 37.4 ± 0.89b

37.4 ± 2.41b

35.9 ± 3.36a

DTR on 6% Et. Extract 32.7 ± 3.88a

35.8 ± 2.39a

34.0 ± 3.54a


38.0 ± 1.58c

37.6 ± 1.34b

NTR on 10% Aq. Extract 33.6 ± 3.91a

35.2 ± 3.07a

35.2 ± 2.95a

NTR on 10% Et. Extract 38.8 ± 0.84c

37.4 ± 0.55b

38.0 ± 1.22c



Values in the same column with different superscripts letters are statistically significantly at 95%

confidence level (P ≤ 0.05).



Table 3.2.1 Hb VALUES (in g/dl) OF NORMAL AND DIABETIC CONTROLS



WEEK

GROUP

1 2 3

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


11.5 ± 0.83b

11.9 ± 1.12b


11.4 ± 1.72a

11.6 ± 1.44a


12.5 ± 0.62b

11.0 ± 1.78a


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

NTR on 10% Et. Extract 12.6 ± 0.17b

12.5 ± 0.26b

12.4 ± 0.32b



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



WEEK

GROUP

1 2 3

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


11.0 ± 0.88a

10.5 ± 1.30a


11.4 ± 1.29a

12.4 ± 0.25b


10.4 ± 0.38a

10.6 ± 0.94a

DTR on 10% Et. Extract 12.7 ± 0.48c 12.7 ± 0.52c

12.2 ± 0.74b


11.5 ± 1.16a

12.1 ± 0.44b

NTR on 10% Et. Extract 13.5 ± 0.56d

13.1 ± 0.72c

12.1 ± 0.13b







Table 3.3.1 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS



WEEK

GROUP 1 2 3

NCR 8.0 ± 0.73b

8.1 ± 0.45b

8.1 ± 0.21b

DCR 8.7 ± 1.52b

8.8 ± 1.01b

8.7 ± 0.99b


6.8 ± 0.81a

7.0 ± 0.15a


8.1 ± 1.21b

8.1 ± 1.31b


9.0 ± 2.61b

8.5 ± 3.21b

DTR on 10% Et. Extract 11.3 ± 2.30c

10.7 ± 2.14c

10.9 ± 1.99c


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





Table 3.3.2 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS

CMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH


WEEK

GROUP 1 2 3

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


5.2 ± 1.58a

5.1 ± 1.28a


7.9 ± 0.18a

7.7 ± 0.43a


10.1 ± 0.24b

10.4 ± 0.81b


7.8 ± 0.64a

7.6 ± 0.33a


5.0 ± 0.13a

4.9 ± 1.02a

NTR on 10% Et. Extract 6.7 ± 0.64a

6.7 ± 0.64a

6.5 ± 0.38a





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.



On the other hand, it was noticed that the

effects of the various extracts and plant parts

(leaves and root) had no significant (P � 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

The result of this investigation affirms that

dandelion (Taraxacum officinale) is a valuable

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.

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.

Arky, R.A. (1983). Prevention and therapy of

diabetes mellitus. Nutritional reviews

41:165–173.

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.

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.

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.

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).



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. Wiley-

Blackwell pp.11.

Rose, Francis (1981). The wild flower key.

Frederick Warne & Co. Pp 388–391.

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.





EVALUATION OF ANTHELMINTIC ACTIVITY OF JUSSIAEA

SUFFRUTICOSA LINN.

Singh Vijayendra

1*, Panda S K

2, Choudhary Puneet Ram

3

1Asst. Professor, Department of Pharmacy, Surguja University. 2,3 Department of Pharmacognosy, The Pharmaceutical College, Barpali, (Bargarh) Orissa.

*Corresponding author e-mail: [email protected], Mob.No.- 09770816465


ABSTRACT

The plant Jussiaea suffruticosa Linn. (Onagraceae) possesses anti- inflammatory, anti-

diarrhoeal, 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



INTRODUCTION

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 world’s population harbours them)

Tripathi K D et al., (2008) but is more common

in developing countries with poorer personal

and environmental hygiene.

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, anti-

inflammatory 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.

MATERIAL AND METHODS

Plant Material

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



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


Group V: Placed in 25mg/ml of semi solid


RESULTS AND DISCUSSION


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***

Test Groups (G-III, G-IV, G-V

Methanolic extract of

J. suffruticosa in

different

concentrations)

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***

*** = Highly significant at 3% level of significance.



(-) control

10mg/ml (standard)

15mg/ml (Test)

20mg/ml (Test)

25mg/ml (Test)

0

2

4

6

8

10(-) control

10mg/ml (standard)

15mg/ml (Test)

20mg/ml (Test)

25mg/ml (Test)

fig1 :-Time taken for paralysis of P. posthuma in different concentrations of Test solution.

Time taken for paralysis(min)

Each value represents the mean ± S.E.M.

(-)control

10mg/ml (standard)

15mg/ml (Test)

20mg/ml (Test)

25mg/ml (Test)

0

10

20

30

40

50(-)control

10mg/ml (standard)

15mg/ml (Test)

20mg/ml (Test)

25mg/ml (Test)

fig 2:-Time taken for Death of P. posthuma in different concentractions of test solution.

Time of death (min)

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, fig-

2) 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



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,fig-

1,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 phyto-

chemical 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.

Giri, R.K., sahoo, M. K., 2009.anthelimintic

activity of Momordica Dioica, Indian

journal of Natural products.

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 Anti-

diarrhoeal 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 anti-

tussive potential of Jussiaea suffruticosa

Linn. Extract in albino mice,

phytotherpay research pp- 541–542.

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

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., pp-

808.




Review Article

ASTASTHANA PARIKSHA - A DIAGNOSTIC METHOD OF

YOGARATNAKARA AND ITS CLINICAL IMPORTANCE

Sharma Rohit1*, Amin Hetal

2, Galib

3, Prajapati P K

4

1PG Scholar, Department of Rasashastra and Bhaishajya Kalpana including drug research 2 PG Scholar, Department of Basic Principles including drug research

3Asst. Professor, Department of Rasashastra and Bhaishajya Kalpana including drug research.

4Professor and Head, Department of Rasashastra and Bhaishajya Kalpana including drug research., IPGT &

RA, Gujarat Ayurveda University, Jamnagar, Gujarat, INDIA

*Corresponding Author: Mail: [email protected], Mob: +919408325831


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



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.

Methods of Examination Methods

1 Dwividha Pariksha2 Pratyaksha and Anumana

2 Trividha Pariksha3 Aptopadesha, Pratyaksha and Anumana

Darshana, Sparshana and Prashna

3 Chaturvidha Pariksha4 Aptopadesha, Pratyaksha, Anumana and Yukti

4 Sadvidha Pariksha5 Panchendriya pariksha and Prashna Pariksha

5 Ashtasthana Pariksha6 Nadi, Mala, Mutra, Jihva, Shabda, Sparsha, Drika, Akrti

6 Navavidha Pariksh7 Dosha, Aushadha, Desha, Kala, Satmya, Agni, Satva,

Vaya and Bala

7 Dashavidha Pariksha8 Prakriti, Vikriti, Sara, Samadhana, Pramana, Satmya,

Satva,Aharashakti, Vyayama Shakti and Vaya

8 Ekadashavidha Pariksha9 Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Ahara,

Satmya, Satva, Pakriti and Vaya

9 Charakokta

Dwadashavidha Pariksha10

Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Sara,

Ahara, Satmya, Satva, Prakriti and Vaya

10 Sushrutokta

Dwadashavidha Pariksha11

Dosha, Bheshaja, Desha, Kala, Bala, Sharira, Sara,

Ahara, Satmya, Satva, Prakriti, Vaya

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)



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 examination-

An 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

subject’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.



Table-2 Nadi Gati26

Vataja

Nadi

Pittaja

Nadi

Kaphaja

Nadi

Vata

Kaphaj

Nadi

Pitta-

Kaphaj

Nadi

Vata Pittaj

Nadi

Sannipataja

Nadi

Snake and

leech

Crow, lark

and frog

Swan,

pigeon and

cock.

Snack and

swan

Monkey

and swan

Snake and

frog

Wood

pecker

Table-3 Nadi Gati in different pathological condition27

Table-4 Nadi Gati in different Jwaravastha28

Sl.No Jwara Avastha Nadi condition

1 Vata Jwara Vakra, Chapala (unstable), cold on touch

2 Pitta Jwara Rapid, straight and of long duration

3 Kapha Jwara Slow, stable, cold and sticky

4 Vata pitta Jwara Somewhat Vakra, Chapala and Kathin

5 Kapha vataja Manda (Slow)

6 Pitta Kapha Sukshma, Sheetala and Sthira

Table-5 Arishtha lakshana of Nadi for prognosis of disease29

Sl.No Pulse movement with Physical condition Prognosis

1 Sthira (Stable) and Rapid like Vidhyut (electrical force) May die 2nd

day

2 Shigra (very rapid) / Sheeta and passing mala repeatedly Will die within 2 days

3 Sometime Tivra and sometimes slow with body sweating May die within 7 days

4 Tivra Nadi with burning and coldness in the body with dyspnoea Will die within 15 days

5 No facial pulsation coldness in the body with Klam May die within 3 days

6 Very rapid and sometimes thin, sometimes forceful yet cold About to die

7 Vidhyuta unmita (curvilinear motion) Imminent death

8 Tiryaka, ushna , vegavati (moves like snake) along with Kapha

filled throat

May die

9 Chanchalita (unstable), Ativega, Nasikadharsamyuta (felt like

cloth wave on the strength of respiration)

May die in one yama

kala

10 Tridoshas influence the Nadi simultaneously Krichhasadhya or

Asadhya

Sl.No Pathological Conditions Nadi Gati (Pulse movements)

1 Jwara Gambheera, Ushna and Vegavati

2 Kama Krodha, Vegavati (rapid)

3 Chinta and Bhaya Kshina (weak)

4 Mandagni Manda (slow)

5 Rakta Dosha Ushna, Gurvi (heavy) and Sama

6 Ama Gambheera

7 Deeptagni Laghu and Vegavana

8 Kshudhita Chanchala (unstable)

9 Tripta Sthira (stable)

10 Asadhya Vyadhi Kampana (vibration) and Spandana (pulsation)



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. Dosha Urine colour/appearance

1 Vata Pandu

2 Kapha Phenayukta

3 Pitta Rakta

4 Dwandaja Mixed / as per predominant dosha

5 Sannipataja Krishna

Table-7 New Findings of probable causative factors and Urine appearance38

Sl.No. Urine colour/appearance Probable causative factor

1 Greenish yellow Bile pigments

2 Red Porphyrins, haemoglobin, myoglobin, numerous other

drugs.

3 Black Melanin and homogentisic acid

4 Cloudy appearance/

sedimentation

Epithelial cells, W.B.C., red cells, bacteria and fat



Method of Examination (Tailabindu

Pariksha)

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. Dosha Vikara Taila bindu appearance

1 Vata Snake

2 Pitta Umbrella

3 Kapha 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)



Table-10 Prognosis according to the direction drop spread of urine50

Sl.No. Direction of urine drop spread Prognosis

1 Towards east Patient will get relief

2 Towards south Will suffer from Jwara and gradually recover

3 Towards northern Will be cured and become healthy

4 Towards west Will attain Sukha and Arogya

5 Towards Esanya Will die in a month

6 Agneya or Nairuti direction or oil gets split Bound to die

7 Vayavya direction Going to die anyway

Table-11 Urine appearance in different Doshik aggravation51

Sl.No. Urine Doshic

Indication

Urine appearance

1 Vata aggravation Slightly Neela and Ruksha (free from oily

appearance)

2 Pitta aggravation Pitta (yellow) and slightly reddish , looks like oil

3 Kapha aggravation Snigdha cloudly and watery

4 Rakta aggravation Snigdha, Ushna and blood coloured

Table-12 Urine appearance in different diseases52

Sl.No. Diseases Urine appearance

1 Ajeerna Rice water

2 Naveena jwara (acute

fever)

Smoky and excessive

3 Vata pitta Jwara Smoky, watery and hot

4 Vata Shleshma Jwara Whitish and is like budbuda

5 Shleshma pitta Jwara Polluted and with blood mixed

6 Jeerna Jwara (Chronic) Yellowish and red

7 Sannipata Jwara Mixed shades depending on doshas involved

Table-13 Mala Lakshana in different Dosha Vikara53

Sl.No. Dosha Vikara Mala Lakshana

1 Vata vikara Dridha (hard) and Shushka (dry)

2 Pitta vikara Peeta (yellowish)

3 Kapha vikara Shweta (white)

4 Tridosha Sarva lakshana

5 Vata prakopa Trutita (broken), fenila (frothy), Ruksha (dry), Dhumala

(smoky)

6 Vata Kapha Kapisha

7 Pitta Vata Badddha (binding), Tritita (broken), Peeta , Shyam

8 Kapha Pitta Peeta,Sweta, Ishat Sandra, Pichchhila

9 Tridoshaja Shyama, Tritita, Pittabha, Baddha Sweta



Table-14 Mala Swarupa in different diseases54

Sl.No. Mala Swarupa Diseases

1 Whitish, bulky with foul smell Jalodara

2 Shyama Kshaya

3 Yellowish associated with pain in the Kati Amayukta disorders

4 Jatharagni passes pandu and dry Mala while in

Mandagni state passes Drava and Durgandhita mala

Asadhya vyadhi

Table-15 Characteristics of tongue in different Doshik condition55

Sl.No. Disease Tongue

1 Vataja Cold, rough and cracked (brown or

black)

2 Pittaja Reddish and blackish

3 Kaphaja Whitish and sticky

4 Sannipataja Blackish, Kantaka (thorny) and dry

5 Dwandaja Mixed symptoms and sign

Table-16 Tongue features in different diseased condition56

Tongue features Diseases condition

Colour Pale Anaemic

Yellow Jaundice, possible liver

disorders

Blue Heart diseases

Fur coating (consisting of epithelial debris, food

particles and micro-organisms)

Posterior part of

tongue

Toxins in large intestine

Middle part of

tongue

Toxins in stomach and

small intestine.

Table-17 Asya Pariksha57

Sl.No. Disease Taste of mouth

1 Vataja Sweet

2 Pittaja Katu (pungent)

3 Kaphaja Madhuramla

4 Tridoshaja Mixed feeling

5 Ajeerna Ghrita purna

6 Agnimandhya Kashaya



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. Dosha Swara

1 Kapha Guru (heavy)

2 Pittaja Sphuta vaktra (cracked)

3 Vataja 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

condition51.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. (Table-

19)



Table-19 Sparsha Pariksha62

Sl.No. Dosha Sparsha (Touch)

1 Kapha Wet and cold

2 Pittaja Hot and moist

3 Vataja Cold and rough

Table-20 Apical impulse in relation to different abnormalities

Sl.No. Apical impulse Abnormalities

1 Hyperdynamic Thrust63

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 21–23)

Table-21 Drika Pariksha69

Sl.No. Doshaja

Prakriti

Drika

1 Vata Dhumra (smoky), Aruna (pink), Nila (blue), Ruksha (dry), Chanchala

(unsteady), Antrapravista (sunken), Roudra (trrrifying), Antarjwala

(glowy inside)

2 Pitta Aruna (pink), Haridra (yellow), Rakta (red), Malina (dirty), Tikshna

(penetrating), Dipa dwesha (dislikes light), Dahayukta (burninig)

3 Kapha Sweta (whitish), Dhavala (glistening), Pluta (watery), Snigdha

(greasy), Sthira (steady), Santa (affectionate), Jyotish (lustreless),

Kanduyukta (itchy)

4 Dwandaja Mixed lakshana of involved Dosha

5 Sannipataja Rakta (red), Roudra (horrifying), sunken and lustreless



It can be understood as follows:

Table-22 Eye features in relation to Doshik dominancy70

Dominancy of

Dosha

Eyeball features Eyelids Eye lashes Sclera

Vata Small, nervous, shrunken Drooping

and dry

Scanty and

rough

Muddy

Pitta Moderate in size, shape, lustrous,

sensitive to light, burning sensation

Reddish Scanty and

oily

Flushed

Kapha Big, beautiful and moist Heavy Long,

thick, oily

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.

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

eyeball rotates; all these are bad prognosis.

Acharya Charaka described Arishta vishayaka

lakshanas of Chakshu71

.

New Findings- in relation to drika pariksha

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.

Table-23 Eye features in different diseased condition73

Sl.no. Eye feature Disease condition

1 Prominent /bulging Thyrotoxicosis

2 Yellow conjunctiva Weak liver

3 Small iris Weak joints

4 Prominent white ring around iris Joint degeneration with potential for arthritis

Table-24 Akriti Pariksha74

Sl.No. Dosha Akriti (Rupa)

1 Kapha Saumya, snighdha, well built body and joints, tolerant to hunger, thirst,

hardship, hot sun.

2 Pittaja Hungry and thirsty, fair in colour, brave, Swabhimani, less hair

3 Vataja 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

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)

New Findings- in relation to Akriti pariksha

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



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 man’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.



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




p.247



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), Paramoswarappa’s Ayurvediya Vikriti

Vijnana, 1st edition, Varanasi, Chaukhambha

Sanskrit Saamsthana, p. 376



Varanasi, India, Chaukhambha Prakashana, p.

69




276




p.70




4




p.4




Prakashana, p.4




Prakashana, p.4




Prakashana, p.4




Prakashana, p.5




Prakashana, p.6




Prakashana, p.5




Prakashana, p.5




Prakashana, p.6




Prakashana, p.5




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





Edition,

Delhi, p.78







Edition,

Delhi, p.78





Edition,

Delhi, p.77




Prakashana, p.7




Prakashana, p.7




Prakashana, p.7




Prakashana, p.7




Prakashana, p.7





Edition,

Delhi, p.224




Prakashana, p.7




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




Prakashana, p.8




Prakashana, p.8






p.2




Prakashana, p.8






p.2




Prakashana, p.5




Prakashana, p.10




Prakashana, p.9




Prakashana, p.10




Prakashana, p.9




Prakashana, p.9




Prakashana, p.11








p.24




Prakashana, p.12




Prakashana, p.12




Prakashana, p.12




Prakashana, p.13




Prakashana, p.13




Prakashana, p.13




Prakashana, p.13




Prakashana, p.13




Prakashana, p.13

57.




Prakashana, p.13





Edition,

Delhi, p.150




Prakashana, p.13




p.224




Prakashana, p.11





Edition,

Delhi, p.255





Edition,

Delhi, p.355





Edition,

Delhi, p.359





Edition,

Delhi, p.411





Edition,

Delhi, p.409





Edition,

Delhi, p.244




Prakashana, p.11







Edition,

Delhi, p.244




p.224





Edition,

Delhi, p.244





Edition,

Delhi, p.244




Prakashana, p.11





Edition,

Delhi, p.244





Edition,

Delhi, p.345




276




Prakashana, p.11




Prakashana, p.11


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