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Global Journal of Research on Medicinal plants & Indigenous Medicine
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An International, Peer Reviewed, Open access, Monthly E-Journal
ISSN 2277 – 4289 www.gjrmi.com
Editor-in-chief
Dr Hari Venkatesh K Rajaraman
Managing Editor Dr. Shwetha Hari
Administrator & Associate Editor
Miss. Shyamala Rupavahini
Advisory Board
Prof. Rabinarayan Acharya
Dr. Dinesh Katoch
Prof. Sanjaya. K.S.
Dr. Mathew Dan
Mr. Tanay Bose
Dr. Nagaraja T.M
Dr. Narappa Reddy
Editorial board
Dr. Kumaraswamy
Dr. Madhu .K.P
Dr. Sushrutha .C.K
Dr. Ashok B.K.
Dr. Janardhana.V.Hebbar
Dr. Vidhya Priya Dharshini. K. R.
Mr. R. Giridharan
Honorary Members - Editorial Board
Dr. Shubha Ganguly
Dr Farhad Mirzaei
INDEX
Medicinal Plant Research
Theoretical & Applied Biology
EFFECTS OF COMBINING CRUDE ETHANOLIC EXTRACT OF JATROPHA CURCUS L.
LEAF AND SOME ANTIBIOTICS AGAINST SOME SELECTED MICROORGANISMS
Akanwariwiak W G, Addo-Fordjour P, Musah A A……………………………..140–148
Biochemistry
PROTECTIVE EFFECT OF RUTIN ON ACETAMINOPHEN-INDUCED ACUTE HEPATIC
DAMAGE IN RATS
Awah Francis M, Chukwumezie Princess U, Ezema Ogechukwu C, Emiliarita Iloakasy, Ubokudom
Queen I……………………………………………………………………………149–159
Veterinary Science
ASPARAGUS RACEMOSUS WILLD. ROOT EXTRACT AS HERBAL NUTRITIONAL
SUPPLEMENT FOR POULTRY
Kumari R, Tiwary B K, Prasad A, Ganguly S…………………………………….160–163
Nutrition and Dietetics
THE EFFECTS OF VITAMIN C AND GRAPE FRUIT JUICE SUPPLEMENTS ON THE
POTENCY AND EFFICACY OF SOME SELECTED ANTI-MALARIAL DRUGS
Adumanya O C, Uwakwe A A, Odeghe O B, Okere T O, Akaehi H C………..…164–171
Biochemistry
EFFECTS OF AQUEOUS AND ETHANOLIC EXTRACTS OF DANDELION (TARAXACUM
OFFICINALE F.H. WIGG.) LEAVES AND ROOTS ON SOME HAEMATOLOGICAL
PARAMETERS OF NORMAL AND STZ-INDUCED DIABETIC WISTAR ALBINO RATS.
Nnamdi Chinaka C, Uwakwe A A, and Chuku L C…………………………..…..172–180
Pharmacology
EVALUATION OF ANTHELMINTIC ACTIVITY OF JUSSIAEA SUFFRUTICOSA LINN.
Singh Vijayendra, Panda S K, Choudhary Puneet Ram………………………….181–185
Indigenous Medicine
Ayurveda
ASTASTHANA PARIKSHA – A DIAGNOSTIC METHOD OF YOGARATNAKARA AND ITS
CLINICAL IMPORTANCE
Sharma Rohit, Amin Hetal, Galib, Prajapati P K……………………………..186–201
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R,
PLANT ID – TENDER LEAVES OF ZIZIPHUS RUGOSA LAM., RHAMNACEAE
PLACE – KOPPA, CHIKMAGALUR DISTRICT, KARNATAKA, INDIA
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)
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148
Global Journal of Research on Medicinal Plants & Indigenous Medicine
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%.
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148
Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148
Global Journal of Research on Medicinal Plants & Indigenous Medicine
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).
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
curcas leaf extract on the test organisms
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
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone
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
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148
Global Journal of Research on Medicinal Plants & Indigenous Medicine
Table 5: Antimicrobial effects of different concentrations of tetracycline and tetracycline-J.
curcas leaf extract on the test organisms
MIC: Minimum inhibition concentration; DIZ: Diameter of inhibition zone
DISCUSSION
The leaf extract of J. curcas showed some
levels of activity against all the test organisms
by inhibiting their growth. This suggests that
the extract contained antimicrobial substances
which were responsible for its activity
(Srinivasan 2001). The effect of the plant
extract varied from one microorganism to
another. Candia albicans and B. subtilis were
more susceptible to the extract than the rest of
the microorganisms. The activity of the plant
extract was concentration dependent, increasing
with increasing concentration. Although the
antimicrobial activities of ciprofloxacin and
tetracycline were significantly higher than the
plant extract (p < 0.001), the plant extract had
inhibiting effects that were similar to those of
amoxicillin and ketoconazole (p > 0.05).
The leaf extract of J. curcas interacted with
the antibiotics to produce varying effects on the
tested microorganisms. The plant extract and
antibiotics contained active ingredients which
when combined with each other, resulted in
additive, synergistic or antagonistic effects
(Delaquis et al. 2002; Fu et al. 2007). While the
activity of some of the antibiotics was
improved upon combination with the plant
extract, the activity of others was reduced. The
improved antimicrobial activity strength
(indicated by the zone of inhibition size) of the
combined treatments varied across the various
treatments and tested organisms. The
ketoconazole-plant extract combination
produced significantly greater inhibition of
growth of C. albicans compared to that
produced by ketoconazole alone (p < 0.001).
Amoxicillin alone was not able to inhibit the
growth of S. typhi, P. aeruginosa and P.
mirabilis. Although the plant extract alone was
able to inhibit the growth of these organisms,
its combination with amoxicillin did not
produce any different effect from that of
amoxicillin. However, amoxicillin alone
showed some levels of activity against the other
microorganisms, and this activity became
slightly better when combined with the plant
extract. Compared to tetracycline, 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
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 140–148
Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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Source of Support: Nil Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
Original Research Article
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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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).
Statistical analysis
The data were analysed using the Statistical
Package for Social Sciences (SPSS) version
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
Acetaminophen + 200 mg/kg Rutin 218.6 ± 23.3* 146.6 ± 21.3* 308.6 ± 23.3
Acetaminophen + 500 mg/kg Rutin 182.4 ± 14.4* 151.3 ± 22.7* 282.4 ± 14.4
Data represented as Mean ± SEM; * p < 0.05 vis-à-vis the Acetaminophen-only group
AST-Aspartate Aminotransferase; ALT-Alanine Aminotransferase; ALP-Alkaline Phosphatase
Table 2: Serum urea and creatinine levels of control and acetaminophen-intoxicated rats
Urea
(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*
Acetaminophen + 200 mg/kg Rutin 6.63 ± 0.58 0.84 ± 0.04*
Acetaminophen + 500 mg/kg Rutin 6.34 ± 0.50 0.65 ± 0.03*
Data represented as Mean ± SEM; * p < 0.05 compared to the Acetaminophen-only group
Effect of rutin on kidney function in
acetaminophen-intoxicated rats
In addition to liver damage, the
acetaminophen metabolite N-acetyl-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 ±
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
acetaminophen-intoxicated rats
Lipid peroxidation causes changes in the
properties of biological membranes, thus
altering their fluidity and permeability, leading
to impairment in membrane signal transduction
and ion exchange, resulting in lipid
peroxidation, oxidation of proteins and DNA
and eventually, cytotoxicity (Fang et al., 2002;
Stehbens, 2003; Jaeschke et al., 2003; Teimouri
et al., 2006). Generation of free radicals such as
superoxide anion and peroxynitrite during
acetaminophen metabolism results in the
depletion of antioxidants such as glutathione,
ascorbate and superoxide dismutase leading to
oxidative stress and lipid peroxidation. In our
study, an increase in hepatic MDA levels in the
acetaminophen-only rats (Table 4) suggests
enhanced lipid peroxidation leading to hepatic
damage and failure of antioxidant defense
mechanisms resulting in oxidative stress. The
observed increase in levels of hepatic MDA
correlates with the decrease in hepatic total
SOD activity (Table 3). The rats co-treated
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
with acetaminophen and rutin (200 and 500
mg/kg) showed significantly (p < 0.05) lower
levels of MDA (0.331 ± 0.05 and 0.323 ± 0.02
µmol/mg protein respectively) relative to the
acetaminophen-only rats (0.471 ± 0.02
µmol/mg protein). Reduction of MDA levels in
the groups co-treated with both of
acetaminophen and rutin was possibly due to
the ability of rutin to quench free radicals by
transfer electrons to the free radicals (Ferrali et
al., 1997) and possibly by activation of
antioxidants enzymes (Elliott et al., 1992).
Hepatic ascorbate levels were significantly
reduced in the acetaminophen-only rats relative
to the healthy control. Co-administration of
rutin (200 and 500 mg/kg) significantly (p <
0.05) increased the ascorbate levels (6.6 ± 0.3
and 6.4 ± 0.7 mg/dL respectively) vis-à-vis the
acetaminophen-only rats (4.5 ± 0.2 mg/dL)
(Table 4). Low levels of ascorbate are
associated with increase levels of free radicals
and oxidative stress since much ascorbate
would be utilized to quench radical. The
present results show that rutin could help
protect against the assault of free radical
thereby stabilizing the oxidative status of the
rats. The pharmacokinetics of rutin in humans
is still under investigation. Studies have shown
that about 17% of an ingested dose of rutin is
absorbed mainly from the colon following the
removal of the carbohydrate moiety by
bacterial enzymes to form quercetin (Walle,
2004). Quercetin and glucuronide conjugates of
quercetin are then transported to the liver via
the portal circulation, where they undergo
significant metabolism forming metabolites
like isorhamnetin, kaempferol and tamarixetin
(Walle, 2004). It could therefore be inferred
that quercetin and its metabolites are most
likely responsible for the in vivo antioxidant
and hepatoprotective capabilities of rutin.
Table 3: Hepatic total superoxide dismutase (SOD) and catalase (CAT) activites of control and
acetaminophen-intoxicated rats
Total SOD Activity
(U/mg protein)
CAT Activity
(U/mg protein)
Control 11.32 ± 1.50 90.43 ± 3.06
Acetaminophen Only 5.82 ± 0.37 81.62 ± 4.07
Acetaminophen + 100 mg/kg Rutin 8.69 ± 1.05 97.54 ± 5.02
Acetaminophen + 200 mg/kg Rutin 10.63 ± 1.58* 84.32 ± 4.04
Acetaminophen + 500 mg/kg Rutin 10.34 ± 1.50* 85.23 ± 2.03
Data represented as Mean ± SEM; * p < 0.05 relative to the Acetaminophen-only group
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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 Only 0.471 ± 0.02 4.5 ± 0.2
Acetaminophen + 100 mg/kg
Rutin
0.409 ± 0.03 5.2 ± 0.4
Acetaminophen + 200 mg/kg
Rutin
0.331 ± 0.05* 6.6 ± 0.3*
Acetaminophen + 500 mg/kg
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.
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Source of Support: Nil Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
Original Research Article
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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
MATERIALS AND METHODS
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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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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):
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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.
Source of Support: Nil Conflict of Interest: None Declared
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 164–171
Global Journal of Research on Medicinal Plants & Indigenous Medicine
Original Research Article
THE EFFECTS OF VITAMIN C AND GRAPE FRUIT JUICE SUPPLEMENTS ON THE
POTENCY AND EFFICACY OF SOME SELECTED ANTI-MALARIAL DRUGS
Adumanya O C+, Uwakwe A A*, Odeghe O B*, Onwuka F C, Akaehi HC
+
+Department of Nutrition and Dietetics, Imo State Polytechnic, Umuagwo, Imo State, Nigeria
*Department of Biochemistry, University of Port Harcourt, PMB 5323, Rivers State, Nigeria.
Corresponding Author: E-mail: [email protected]
Received: 06/04/2012; Revised: 25/04/2012; Accepted: 30/04/2012
ABSTRACT
Combination therapy (CT) is now advocated for the treatment of malaria, especially the
artemisinin based CT. Malaria is one of the most serious health challenges facing the world today. It
is a disease caused by plasmodium, which could be cured effectively by the use of combination
therapeutic drugs called anti-malarial drugs. The effects of vitamin C and grape vine supplements on
the potency and efficacy of some selected anti-malarial drugs-combination therapy (Armact,
Coartem, Waipa and Fansider) were investigated. A total of 80 patients (adults) infected with malaria
parasites were used. The result showed that the concomitant administration of the drugs with grape
fruit juices did not alter the efficacy and potency of the drugs, while vitamin C altered the efficacy
and potency of the drugs. Therefore, the concomitant administration of these anti-malarial drugs
(combination therapies) with vitamin C supplement should be avoided during the period of malaria
treatment for the effectiveness of such drugs.
Keywords: Anti-malarial, malaria, Combination therapy, Grape fruit juice and Vitamin C
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
MATERIALS AND METHODS
Experimental animals
A total of 80 persons (40 males and 40
females) infected with malarial parasite
residing in Umuguma in Owerri West local
government area of Imo state, Nigeria were
selected during the experiment after a general
malarial test on all the individuals and their
body weights were taken before and after drug
administration (treatment).
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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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
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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
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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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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Okpako, L, and Akinboye, D. (2006). In
vivo antimalarial
and Cytotoxic properties of Annona
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Basu, S,K., Thomas, J.E. and Acharya, S.N.
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Rosenthal, P.J., Wabwire-Mangen, F., Dorsey,
G. & Staedke, S.G. (2006) Artemisinin
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Aworinde, D. O, 2010. Ethnobotanical
survey of
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Iyiola, O. A., Tijani, A. Y. and Lateef, K. M,
(2011). Antimalarial Activity of
Ethanolic Stem Bark
Extract of Alstonia boonei in Mice.
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Russell, B.(2008) Determinants of in vitro
drug susceptibility testing of
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1040–1045
Salawu, O.A., Chindo, B.A., Tijani, A.Y.,
Obidike I.C. and Akingbasote, J.A.
2009. Acute and
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sub-acute toxicological evaluations of
the methanolic stem bark extract of
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resistance in Plasmodium falciparum:
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Whitty C. J. M. (2006). Cost-
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Yeka, A., Banek, K, Bakyaita N, Staedke S. G,
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Source of Support: Nil Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
Original Research Article
EFFECTS OF AQUEOUS AND ETHANOLIC EXTRACTS OF DANDELION
(TARAXACUM OFFICINALE F.H. Wigg.) LEAVES AND ROOTS ON SOME
HAEMATOLOGICAL PARAMETERS OF NORMAL AND STZ-INDUCED DIABETIC
WISTAR ALBINO RATS.
Nnamdi Chinaka C1*, Uwakwe A 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]
Received: 06/04/2012; Revised: 03/05/2012; Accepted: 06/05/2012
ABSTRACT
The effects of aqueous and ethanolic extracts of Taraxacum officinale F.H. Wigg leaves and
roots on some haematological parameters of Streptozotocin (STZ)-induced diabetic Wistar albino
rats (Rattus rattus) were investigated. The parameters investigated include; packed cell volume
(PCV), haemoglobin (Hb) levels and white blood cell (WBC) counts. Exactly 75 wistar albino rats
weighing between 100–225 g were used for the study, and a total of four groups were created. Two
groups were divided into six sub-groups of five rats each for the leaf and root extracts respectively,
with the remaining two groups being the normal control rats (NCR) and diabetic control rats (DCR).
The two sub-groups were thus; sub I, comprising of sub-groups 1–4 which were for diabetic test rats
(DTR) on 6% and 10% of aqueous and ethanolic extracts of leaves respectively, while sub-group 5
and 6 were normal test rats (NTR) on 10% of both extracts of leaves respectively. Same was also the
case for sub II which represents the root extracts. Two days after streptozotocin-induction, the
administration of T. officinale leaf and root extracts (Aqueous and Ethanolic) commenced and lasted
for three weeks. Changes in PCV, haemoglobin levels and WBC counts between the NCR and DCR
against normal treated rats (NTR) and diabetic treated rats (DTR) on various doses of the extracts
were evaluated using one way Analysis of Variance (ANOVA).
Keywords: Taraxacum officinale, Aqueous and Ethanolic extracts, STZ induced Diabetes,
Haematological parameters
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
1.0 INTRODUCTION
The use of indigenous plants in the
management of diseases has been a common
practice in most developed countries and
importantly in developing countries throughout
the world. However, few of these plants have
received adequate scientific scrutiny. Most
researches into medicinal plants help in
ascertaining the efficacy of the flora as a potent
remedy, extending our frontiers of knowledge
concerning their pharmacological activity,
active principles, therapeutic value, dosage and
administration as well as contraindication and
side effects. Medicinal plants are administered
to patients either as entire plant or as root, leaf,
stem, bark, fruit, seed, juice, flower or as
exudates and may be taken in form of an
infusion or as a decoction (Omeodu 2006).
Traditional healers form the basic core of
primary health care delivery in 90% of our
rural population in Africa. It is found that 60%
of Nigeria’s rural population depends on
traditional medicine for their health care needs
(WHO 1996).
As such World Health Organisation is
pursuing a coordinated approach to encourage
the official recognition of traditional healers
and to encourage Western trained doctors and
pharmacists to study the methodology and
recipes of traditional healers. China for instance
has achieved a great success in blending
traditional healing system with modern western
medicine to form new Chinese medicine
(Omeodu 2006). The Nigerian flora had already
and continually made great contribution to the
health care of the nation (Omeodu 2006).
Infact, indigenous medicinal plants form an
important component of the natural wealth of
Nigeria.
Taraxacum officinale F.H. Wigg.
commonly known as Dandelion (from the
French dent-de-lion meaning lion’s tooth) is
thought to have evolved about thirty million
years ago in Eurasia. They have been used by
humans as food and as a herb for much of
recorded history (Dijk et al. 2003).
It is a herbaceous perennial plant of the
family Asteraceae (compositae), and two major
species, T. officinale and T. enythrospermum,
are found as weed worldwide. Both species are
edible in their entirety. Like other members of
the Asteraceae family, they have very small
flowers which are yellowish to orange yellow
in colour collected together into a compositae
(flower head). Each single flower in a head is
called floret and they number 40 to over 100
per head. They grow generally from
unbranched taproots, producing one to more
than ten stems that are typically 5–40 cm tall
and sometimes up to 70 cm tall. Dandelion
leaves are unique as a diuretic, a valuable
alkalizer to the body; eaten regularly they assist
the body to reduce excess acidity, oxygenate,
purify and build blood, cleanse and regenerate
cells (Clarke et al. 1997).
2.0 MATERIALS AND METHODS
2.1.0 Plant Source and Identification
The plant Dandelion (Taraxacum
officinale) leaves and roots were sourced from
farm lands at Umuzi and Umudim villages in
Umudioka Ancient kingdom, Orlu local
government area of Imo State and the species
was identified and confirmed by Dr. F. N.
Mbagwu of the Department of Plant Science
and Biotechnology, Imo State University,
Owerri, Imo State.
2.1.1 Chemicals and Reagents
All chemicals and reagents used were of
analytical standard and were obtained from
reputable sources.
2.2.0 Preparation and Administration of
Streptozotocin (STZ)
The range of diabetogenic dose of STZ is
quite narrow and a light overdose may cause
death of many animals (Lenzen et al. 1996).
Five gram of STZ was dissolved in 100 ml
of distilled water to give a 5% stock solution of
which a single dose of 70 mg/kg body weight
was injected intraperitoneally to the rats.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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3.0 RESULTS AND DISCUSSION
Table 3.1.1 PVC VALUES (in %vol.) OF NORMAL AND DIABETIC CONTROLS
COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES.
WEEK
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
COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
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
DTR on 6% Aq. Extract 35.4 ± 1.34a
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
DTR on 10% Et. Extract 38.2 ± 1.10b
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
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats.
Results are Means ± Standard Deviation of triplicate determinations.
Values in the same column with different superscripts letters are statistically significantly at 95%
confidence level (P ≤ 0.05).
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Table 3.2.1 Hb VALUES (in g/dl) OF NORMAL AND DIABETIC CONTROLS
COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES.
WEEK
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
DTR on 6% Aq. Extract 12.0 ± 0.45b
11.5 ± 0.83b
11.9 ± 1.12b
DTR on 10% Aq. Extract 11.7 ± 1.06a
11.4 ± 1.72a
11.6 ± 1.44a
DTR on 6% Et. Extract 12.2 ± 0.20b
12.5 ± 0.62b
11.0 ± 1.78a
DTR on 10% Et. Extract 12.0 ± 1.16b
12.3 ± 0.68b
12.3 ± 0.96b
NTR on 10% Aq. Extract 12.2 ± 0.14b
12.1 ± 0.46b
11.6 ± 0.83a
NTR on 10% Et. Extract 12.6 ± 0.17b
12.5 ± 0.26b
12.4 ± 0.32b
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats.
Results are Means ± Standard Deviation of triplicate determinations.
Values in the same column with different superscripts letters are not statistically significantly at 95%
confidence level (P > 0.05).
Table 3.2.2 Hb VALUES (in g/dl) OF NORMAL AND DIABETIC CONTROLS
COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS.
WEEK
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
DTR on 6% Aq. Extract 11.4 ± 0.45a
11.0 ± 0.88a
10.5 ± 1.30a
DTR on 10% Aq. Extract 11.8 ± 0.86b
11.4 ± 1.29a
12.4 ± 0.25b
DTR on 6% Et. Extract 11.5 ± 1.46a
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
NTR on 10% Aq. Extract 11.2 ± 1.30a
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
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats.
Results are Means ± Standard Deviation of triplicate determinations.
Values in the same column with different superscripts letters are not statistically significantly at 95%
confidence level (P > 0.05).
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Table 3.3.1 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS
COMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale LEAVES.
WEEK
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
DTR on 6% Aq. Extract 6.8 ± 0.89a
6.8 ± 0.81a
7.0 ± 0.15a
DTR on 10% Aq. Extract 8.3 ± 1.51b
8.1 ± 1.21b
8.1 ± 1.31b
DTR on 6% Et. Extract 9.2 ± 3.73b
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
NTR on 10% Aq. Extract 7.8 ± 4.79a
7.9 ± 3.57a
7.9 ± 2.69a
NTR on 10% Et. Extract 7.6 ± 3.39a
7.5 ± 2.87a
7.7 ± 2.57a
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats.
Results are Means ± Standard Deviation of triplicate determinations.
Values in the same column with different superscripts letters are not statistically significantly at 95%
confidence level (P > 0.05).
Table 3.3.2 WBC VALUES (in X109/µl) OF NORMAL AND DIABETIC CONTROLS
CMPARED TO DIABETIC TEST AND NORMAL TEST RATS TREATED WITH
AQUEOUS AND ETHANOLIC EXTRACTS OF T. officinale ROOTS.
WEEK
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
DTR on 6% Aq. Extract 4.9 ± 1.30a
5.2 ± 1.58a
5.1 ± 1.28a
DTR on 10% Aq. Extract 7.7 ± 0.81a
7.9 ± 0.18a
7.7 ± 0.43a
DTR on 6% Et. Extract 10.0 ± 0.61b
10.1 ± 0.24b
10.4 ± 0.81b
DTR on 10% Et. Extract 7.8 ± 0.91a
7.8 ± 0.64a
7.6 ± 0.33a
NTR on 10% Aq. Extract 4.9 ± 0.87a
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
NCR, normal control rats; DCR, diabetic control rats; DTR, diabetic test rats; NTR, normal test rats.
Results are Means ± Standard Deviation of triplicate determinations.
Values in the same column with different superscripts letters are not statistically significantly at 95%
confidence level (P > 0.05).
DISCUSSION
A clear demonstration of the comparison
between the effects of T. officinale leaves and
roots as well as the mode of extraction on some
haematological parameters such as PCV,
haemoglobin levels and white blood cell count,
can be drawn from the experimental results
above.
The effect of the aqueous leaf extract
compared to the roots on PCV from the result
above, indicates that their was a gradual
increase (P ≤ 0.05) in PCV level of both the
diabetic treated rats and the normal treated rats
when compared to those of the ethanolic leaf
extract. This shows that the mode of extraction
(especially the aqueous) has a significant effect
on the parameter ascertained. See tables 3.1.1
and 3.1.2.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
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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
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of the Royal Society. Biol. Scie. 358
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Encyclopedia of medical plants.
pp.246–7, 227.
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281:1309–1312.
Harborne, J. B. (1998). Phytochemical method.
A guide to modern techniques of plant
analysis (3rd
edition).
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Lenzen, S., Tiedge, M., Jones, A., Munday, R.
(1996). Alloxan derivatives a tool for
elucidation of the mechanism of the
diabetogenic action of alloxan; In lesson
for animal diabetes. E. Shafrir (ed.).
Boston Birkhauser. pp. 113–122.
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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.
Source of Support: Nil Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
Original Research Article
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
Received: 30/03/2012; Revised: 25/03/2012; Accepted: 07/05/2012
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
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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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
METHODS
Anthelmintic activity was evaluated on
adult Indian earth worms (Pheretima
posthuma) due to anatomical and physiological
resemblance with the intestinal round worms.
The experimental animals were acclimatized by
keeping them in the laboratory, in the soil of
their original habitat, for 24 h. The worms were
then divided into 5 groups with 6 earthworms
in each group. Different groups were treated
with the aqueous solution of albendazole
(10mg/ml) and methanolic extract of the test
solution with 15mg, 20mg and 25mg/ml
concentration of the semi solid in normal
saline, containing 2% gum acacia. Observations
were made for the paralysis time (PT) &
subsequently for death time (DT). Paralysis
was inferred to have set in when the organisms
stopped movement and took a circular shape
and the worms do not revive. Death was
declared when the worms lose their motility
followed by fading away of their body colour.
Normal saline was taken as control,
albendazole (Bendy suspension mankind
pharma Ltd.) was taken as standard and
solution of methanolic extract semi-solid was
taken as test solution. 5 groups of earthworms
were treated in 50 ml. of 5 types of different
solutions, as follows:
Group I: Placed in albendazole solution
(Standard solution)
Group II: Placed in normal saline – (Control)
Group III: Placed in 15mg/ml of semi solid
solution (test solution)
Group IV: Placed in 20mg/ml of semi solid
solution (test solution)
Group V: Placed in 25mg/ml of semi solid
solution (test solution)
RESULTS AND DISCUSSION
Statistical analysis
The results were presented as mean ± SEM.
“One-way ANOVA with Dunnett’s post test
was performed using Graph Pad Prism version
3.00 for windows. Graph Pad Software, San
Diego California USA, P < 0.01 implies at 3%
level of significance.
Table 1: Anthelmintic activity of Jussiaea suffruticosa.
Treatment Concentration
(mg/ml)
Time taken for
paralysis (min)
Time taken for death
(min)
Control (G-I) _ _ _
Albendazole
suspension (Standard
G-2)
10 mg/ml 4.90 ±0.37*** 23.8 ±0.37***
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.
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
(-) 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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
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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.
Source of Support: Nil Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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
Received: 04/04/2012; Revised: 19/04/2012; Accepted: 30/04/2012
ABSTRACT
Indian traditional medicine, Ayurveda has a great history. Researchers of India have tried to
corroborate ancient wisdom with modern scientific practices. It is necessary to diagnose the disease
after proper examination and medicines are to be given. There are many diagnostic tools of
examination. Yogaratnakara provides a clear picture of scenery of illness and healthy condition
through Astasthana Pariksha. Tailabindu pariksha, one among Ashtasthana pariksha is a diagnostic
tool of urine examination developed by the medieval Ayurvedic scholars. It also helps in establishing
prognosis of various diseases. In current paper, attempts were made to study the relation of
Ashtasthana Pariksha in therapeutics with special emphasis and its applicability in medical practice.
Keywords: Ashtasthana Pariksha, Ayurveda, Nadi, Tailabindu, Yogaratnakara
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INTRODUCTION
A physician by treating the persons who are
deeply immersed in the sea of disease (Roga)
due to their ill fate (Papa) are pulled out from
the sea. This humanistic effort under taken by
the physician ensures an honourable place for
him in the society, even-though he does not
perform other routine dharma1. Yogaratnakara
stresses on the importance of “Vyadhi
Vinischaya” (Diagnosis of ailment). It is
essential that physician should examine the
disease thoroughly and arrive at a proper
diagnosis (Vyadhi Nirnaya). Afterwards i.e.
knowing fully about the nature etc of diseases
he should commence the Chikitsa (treatment)
by administering suitable “Aushadha” or by
employing a procedure e.g. Snehana, lepa etc.
Different methods of examination have
been explained in classics of Ayurveda, which
will be helpful in diagnosis of a disease,
estimating the status of Rogibala and Rogabala
etc. Following table provides a glimpse on this:
Table-1 Methods of Examination explained in different lexicons
Sl.
No.
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)
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Paryayas of Nadi
Snayu, Nadi, Hansi, Dhamani, Dharani,
Dhara, Tantuki, and Jeevan Gyan15
.
Nadi location
Vata, Pitta and Kapha Nadi lies
respectively under Tarjini (index), Madhyama
(middle) and Anamika (ring) fingers of
examining physician16
.
Tridosha examination
Three fingers placed in position over Nadi
indicate the condition of the Tridosha and their
Gati (i.e. Manda, Madhyama and Tikshna) 17
.
The index finger denotes Vata, the middle
finger Pitta and the ring finger Kapha. Nadi
Pariksha offers knowledge about involvement
of dosha- Vata, Pitta and Kapha, Dwandaja
(any two dosha) and Tridoshaja (all three
dosha), and Sadhya Asadhyata (prognosis of
disease)18
.
Jiva sakshini
Anatomical position of the Jiva sakshini
Nadi at Angushtha moola and its clinical
importance as pulse has been stressed19
. The
pulsation in the Dhamani (artery) reflects the
evidence of life and the learned physician
through Sparsana Pariksha is able to come to
assessment of the person concerned, whether
the person is ill or well. In female left hand
Nadi should be palpated and vice versa.
How to examine
Nadi should be examined in mental stability
and peace of mind before with his hand pulse
(beat) below the right thumb. As regards
methodology, the elbow (Kurpara) of the
patient should be lightly flexed to the left and
the wrist slightly bent to the left with the
fingers distended and dispersed. Nadi should be
examining repeatedly for three times by
applying and releasing pressure alternately over
Nadi to assess the condition of Dosas rightly20
.
After Nadi Pariksha physician should wash
his/her hands because disease disappears from
the patient like mud gets washed away21
.
Method for Arterial pulse 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.
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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)
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Asadhya Nadi
‘Sannipata Nadi’ pulsate slowly,
intermittently (Vyakula) and is extremely thin.
This is mentioned as Asadhya Nadi. It indicates
imminent death. When Nadi firstly pulsates like
Pitta gati, afterwards it becomes like Vata gati
then transforming to Kapha gati and moves
like a wheel, sometimes it is rapid and
sometimes very thin such Nadi should be
considered as Asadhya Nadi and act
accordingly. Mrityu Suchaka Nadi- the Nadi
which resembles Damru (a musical
instrument), means which is strong at opening
and ending but very slow in between, is the
indicator of death in a day30
.
New findings- in relation to Asadhya nadi
Forceful and jerky rise of the Corrigan
pulse is due to the rapid filling of the radial
artery caused by an extra large amount of blood
pushed by the distended left ventricle during
systole into relatively empty arterial vessels.
The collapsing character or the sudden down
stroke of the pulse may be due to partly to the
sudden fall of pressure in the aorta due to
regurgitation of blood into the left ventricle
through a leaky valve during diastole31
.
Healthy Pulse: Hamsa gamana (Swan like
walk), Gajagamini (elephant like) and who is
having cheerful face is considered to be a
healthy32
.
Mutra Pariksha
Importance
By Mutra Pariksha (urine examination) one
can assess any running pathology inside the
body30
. Urine is the end product of metabolism
by billions of human cells and the body
chemistry, blood pressure, fluid balance,
nutrient intake, and the state of health are key
elements in establishing the characteristic of
urine33
.
Method
The wise physician should wake up the
patient early in the morning around 4 o’clock,
avoid the first stream of early morning urine,
then collect the urine of subsequent flows in a
clean glass vessel and examine thoroughly to
assess the disease process and treat the patient
accordingly34-35
.
New findings- in relation to mutra pariksha
For routine urine examination, midstream
sample of urine which is the first morning
sample, collected in a clean container is
preferred since it gives a more constant result36
.
Table-6 Urine appearance involving doshas37
Sl.No. 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
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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)
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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
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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
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Asya Pariksha
Different dosha can change the taste of mouth.
This can be including only in Prashna
Pariksha. (Table-17)
New findings- in relation to Asya Pariksha
In Yogaratnakar, Asya pariksha is
described as subjective condition. Acharya
Charaka described different types of Rasa
vishayaka arishta in Indriyasthana but they all
are in excessive condition and in abnormal
conditions48. According to modern science, oral
examination contains tongue, teeth, gums,
buccal mucosa etc examination but in
Yogaratnakara it is described as taste
examination. Different taste on tongue in
abnormal condition is important to know by
asking for different Doshik vitiation. In modern
science, there is no any direct relation with
taste is described for diagnosis.
Shabda Pariksha
Healthy and natural when the doshas are in
balance, the voice will become heavy when
aggravated by kapha, cracked under pitta effect
and hoarse & rough when afflicted by vata.
(Table 18)
Table-18 Shabda Pariksha58
Sl.No. 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)
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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
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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
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Global Journal of Research on Medicinal Plants & Indigenous Medicine
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.
www.gjrmi.com GJRMI, Volume 1, Issue 5, May 2012, 186–201
Global Journal of Research on Medicinal Plants & Indigenous Medicine
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Delhi, p.244
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