Journal of Complementary and

Integrative Medicine
Volume 7, Issue 1 2010 Article 19

Anxiolytic Activity of Canscora decussata in

Albino Rats

Neeraj K. Sethiya, Dr. Hari Singh Gour Vishwavidyalaya,

Sagar, India
Alok Nahata, Dr. Hari Singh Gour Vishwavidyalaya, Sagar,
India
Vinod K. Dixit, Dr. Hari Singh Gour Vishwavidyalaya,
Sagar, India

Recommended Citation:
Sethiya, Neeraj K.; Nahata, Alok; and Dixit, Vinod K. (2010) "Anxiolytic Activity of Canscora
decussata in Albino Rats," Journal of Complementary and Integrative Medicine: Vol. 7: Iss. 1,
Article 19.
DOI: 10.2202/1553-3840.1263

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 Anxiolytic Activity of Canscora decussata in
Albino Rats
Neeraj K. Sethiya, Alok Nahata, and Vinod K. Dixit

Abstract
Shankhpushpi is a popular medicinal plant in the Ayurvedic system of medicine for treating
mental disorders. The present study was undertaken to investigate the effects of Canscora
decussata Schult. (Gentianaceae) commonly known as shankhpushpi on the central nervous
system of albino rats. Ethanolic extract of the aerial parts of the plant was evaluated in the elevated
plus-maze test, open field exploratory behavior and rotarod performance experiments. In the
elevated plus-maze, ethanolic extract at a dose of 100, 200 and 400 mg/kg p.o. showed an
anxiolytic effect as evidenced by increase in the time spent in open arms and the number of open
arm entries, compared to control group. The open field exploratory behaviour was also increased
on administration of the ethanolic extract. The ethanolic extract at dose of 400 mg/kg p.o.
significantly reduced the neuromuscular coordination indicative of the muscle relaxant activity at a
high dose. Diazepam (1 mg/kg i.p.) was used as a standard in all the animal models studied. A
biomarker viz., mangiferin, was also isolated and characterized and its effects were observed in
the animal models along with the ethanolic extract.

KEYWORDS: Shankhpushpi, Canscora decussata, Gentianaceae, mangiferin, elevated plus-

maze

Author Notes: The authors are affiliated with the Department of Pharmaceutical Sciences, Dr.
Hari Singh Gour Vishwavidyalaya, Sagar, India. The authors would like to express their sincere
thanks to the Director, B.R. Nahata Smriti Sansthan, Contract Research Center, Mandsaur (M.P.),
India for granting permission to carry out the in vivo studies. Authors are grateful to Natural
Remedies Pvt. Ltd., Bangalore, India for providing gift sample of mangiferin. Two of the authors,
Neeraj Sethiya and Alok Nahata, are thankful to the University Grants Commission, New Delhi
for providing junior research fellowship.

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 Sethiya et al.: Anxiolytic Activity of Shankhpushpi

Introduction

Ayurveda is the oldest medical science in the Indian sub-continent and has been
practiced since the 12th Century BC. Its objective is to accomplish physical,
mental, social and spiritual well-being by adopting preventive, health promoting
and holistic approach towards life [Patwardhan et al., 2005].
Shankhpushpi is a drug of ayurvedic ‘Medhya Rasayana’ category which
is used to boost memory and intellect. In India, Convulvulus pluricaulis Choisy.,
Evolvulus alsinoides Linn., Clitorea ternatea Linn. and Canscora decussata
Schult. are used as shankhpushpi by practitioners of ayurveda [Nahata et al.,
2010, Sethiya et al., 2009a].
Canscora decussata Schult. (CD) (Gentianaceae) is one of the plants used
as ‘Shankhpushpi’. Convulvulus pluricaulis Choisy. (Convulvulaceae), Evolvulus
alsinoides Linn. (Convulvulaceae) and Clitorea ternatea Linn. (Fabaceae) are
other plants considered as shankhpushpi. In the Ayurvedic Pharmacopoeia of
India, monograph on shankhpushpi describes it as Convulvulus pluricaulis and
also recognize usage of Evolvulus alsinoides as its substitute in some parts of the
country [Anonymous, 2001]. The botanical source of shankhpushpi thus varies,
warranting scientific exploration for validating the traditional therapeutic benefits.
CD is found throughout India up to an altitude of 1300 meters and is also
grown in Sri Lanka and Myanmar. The entire plant, as well as its fresh juice is
used in medicine. It is used in the popular medicine for the treatment of insanity,
epilepsy and nervous debility. This plant contains bitter substances and an
oleoresin. It is also found to contain triterpenes, alkaloids and xanthones [Kokate
et al., 2002]. Studies on the plant showed hepatoprotective, antidepressant,
antianxiety, antistress [Bose and Gupta, 1999] and antimycobacterium
tuberculosis activity [Madan and Ghosh, 2002]. Mangiferin is one of the active
constituents of this plant which has been isolated and reported to possess
monoamine oxidase inhibiting activity [Ghosal and Chaudhuri, 1975]. Aqueous
and alcoholic extract are reported to possess spermicidal and anticonvulsant
activities [Dikshit et al., 1972].
With a view to explore the scientific basis of use of shankhpushpi, it was
considered worthwhile to investigate various plants, which are used as
shankhpushpi for their action on Central Nervous System. Evolvulus alsinoides
and Convulvulus pluricaulis have been shown to possess sufficient anxiolytic
activity in our previous studies [Nahata et al., 2009]. Hence it was thought
worthwhile to investigate the effects of CD on stress induced disorders as well as
anxiogenic conditions.

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 Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 19

Materials and Methods

PLANT MATERIAL

Aerial parts of CD were collected from the outskirts of Raipur (Chattishgarh,

India.) in January 2007. The plant was identified by Dr S.C. Agrawal (Department
of Botany, Central Drug Research Institute, Lucknow). A voucher specimen has
been deposited in the herbarium of the institute (Voucher Specimen no. NS/ AN/
Cd-2409).

PREPARATION OF EXTRACTS

Aerial parts of CD were shade dried at room temperature and reduced to a coarse
powder. The extraction was performed according to the method described by
Nahata et al. (2008). 200 gm powdered material was first defatted with petroleum
ether (Qualigens Fine Chemicals, boiling range 60-80°C). Then marc was
subjected to extraction with ethanol (95%) and the solvent evaporated under
reduced pressure (yield 4.47% w/w). The extract was suspended in Tween-80
solution (0.1% v/v). This suspension was utilized for the neuropharmacological
investigations.

DRUGS

Diazepam (Calmpose 2mg/10ml injection, Ranbaxy Laboratories, India) was

dissolved in normal saline for i.p. administration. Standard mangiferin was
obtained as a gift from Natural Remedies Pvt. Ltd., Bangalore, India. The purity
of the material procured was 99.97% as per the certificate of analysis supplied
with the sample.

CHROMATOGRAPHIC STUDIES AND ISOLATION OF MARKER COMPOUND

TLC of the ethanolic extract in n-butanol: acetic acid: water (4:1:2) revealed the
presence of four spots in the extract on spraying with 1% ferric chloride solution.
Co-chromatography with the reference compound revealed the presence of
mangiferin (Sethiya et al, 2009b). For isolation of this compound, preparative thin
layer chromatography was performed using n-butanol: acetic acid: water (4:1:2)
as the solvent. A crystalline material melting at 273-276°C was isolated. It gave
apricot green yellow colour with 1% ferric chloride solution and a light yellow
fluorescence in UV at 366 nm. It was characterized as mangiferin by mixed
melting point (Superfit Melting Point Apparatus), UV absorption maxima (GBC-
Cintra, Australia) and superimposable FTIR spectrum with standard mangiferin
(Shimadzu FTIR 8400S) (Fig. 1).

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 Sethiya et al.: Anxiolytic Activity of Shankhpushpi

Chaudhuri and Ghosal (1971) have isolated mangiferin from CD and

reported its complete characterization using melting point (271°C) , IR, UV (
λmax at 316 nm) and mass spectroscopy [significant peaks at m/e 404 (M-18)
(12); 368 (45); 326 (14); 300 (21); 285 (34); 273 (100)].
We have confirmed the previous findings of Chaudhuri and Ghosal (1971)
through our phytochemical investigations which included melting point
determination (range 273-276°C), UV absorption maxima (315 nm) and
superimposable FTIR analysis (Fig. 1) with standard mangiferin. This data
sufficiently confirms the presence of mangiferin in our drug sample. Further we
have also developed a spectrofluorimetric method for the determination of
mangiferin in CD (Sethiya et al., 2008; Nahata and Dixit, 2008).

ANIMALS

Albino rats of wistar strain (150-200 gm) were obtained from pharmacological
department of B.R. Nahata college of Pharmacy, Mandsaur (M.P.), India and
were maintained at controlled room temperature (25±2˚C) on 12 hour light/dark
cycle and allowed free access to food (standard pellet feed purchased from
Brooke Bond-Lipton, India) and water. All experiments were conducted between
0900 to 1400 hrs. Procedures were approved by Institutional Animal Ethical
Committee of B.R. Nahata College of Pharmacy and care of animals was taken as
per CPCSEA guidelines (Reg. No. 918/ac/05/CPCSEA).

ACUTE TOXICITY STUDIES

2000 mg/kg of extract was administered as per OECD guidelines per orally to 7
rats. Effects were observed on behavior for 72 hours. Rats were examined for
behavioral effects 45 minutes post administration of the extracts. No change in
behavior or any abnormality in behavior was observed and no mortality was seen.
Thus it was concluded that ethanolic extract of CD was non toxic upto 2000
mg/kg doses. Then 1/5, 1/10, 1/20th of the administered dose was selected for
future studies as per OECD guidelines.

ELEVATED PLUS-MAZE TEST

The elevated plus-maze (EPM) test has been widely validated for measuring
anxiolytic and anxiogenic-like activities in rodents [Lister, 1990]. This apparatus
consists of two open arms (50×10 cm) crossed with two closed arms
(50×10×40cm). The arm was connected together with a central square (10×10
cm). The apparatus was elevated to a height of 70 cm in a dimly illuminated
room. The animals were divided into six groups containing six animals each. The
ethanolic extract in doses of 100, 200, 400 mg/kg p.o. and isolated mangiferin

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 Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 19

(100mg/kg p.o.) were administered to groups I-IV. Control group received the
vehicle only (0.1% tween-80 solution). Diazepam (1mg/kg i.p.) was used as a
reference standard for comparison. Each rat was placed individually at the center
of the elevated maze, 45 minutes post administration of the extracts, the isolate
and the standard. The number of entries in the open and closed arm of the
elevated maze during a period of 5 minutes and the duration of stay in the open
and closed arm were noted [Nahata et al., 2009; Pellow et al., 1985]. After each
test, the maze was carefully cleaned up with a wet tissue paper (10% ethanol
solution).

OPEN-FIELD TEST

This method is used to evaluate exploratory activity and emotionality of animals

[Carlini et al., 1986]. The open-field consisted of a white painted area measuring
55 cm in diameter with a 100W lamp. The floor of the area was divided into 16
units by black painted lines. The entire room except the open field was kept dark
during experiment. Each animal was centrally placed in the test apparatus for 5
minutes [Arletti et al., 2003] and behavioural aspects noted were: ambulation,
rearings, self grooming, activity in center and faecal droppings [Nahata et al.,
2009; Bronstein, 1972]. For the test, the rats were divided into six groups
containing six animals each. The ethanolic extract in doses of 100, 200, 400
mg/kg p.o. and isolated mangiferin (100mg/kg p.o.) were administered to groups
I-IV. Control group received the vehicle only (0.1% v/v tween-80 solution).
Diazepam (1 mg/kg i.p.) was used as a reference standard for comparison.

NEUROMUSCULAR COORDINATION- ROTA ROD

The effect on motor coordination was assessed using a rotarod apparatus

[Dunham and Miya, 1957]. Rats were trained to remain on the rotating rod (3cm
in diameter, 40 rpm) and tested for their performance a day before the testing of
extracts. On the test day, rats were tested on the rotarod before and 45 min after
the administration of vehicle, the ethanolic extracts, isolated mangiferin
(100mg/kg p.o.) and diazepam (1mg/kg i.p.). The number of seconds each rat
remained on the rotating wheel was recorded before and after the administration.
The percentage reduction in the motor coordination was calculated from the
readings obtained. The formula used for calculating the percentage reduction in
motor coordination is as follows:

% Reduction in performance= (T1 – T2)/T1 × 100

where, T1= Time spent on the rotarod before experiment
T2= Time spent on the rotarod after administration of the test sample

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 Sethiya et al.: Anxiolytic Activity of Shankhpushpi

STATISTICAL ANALYSIS

The data were expressed as mean ± SEM and analyzed by one-way ANOVA
followed by Dunnett’s test. p<0.05 was considered to be statistically significant.

Results and Discussion

In the present study, the ethanolic extract of CD (100, 200 and 400 mg/kg p.o)
and isolated mangiferin (100 mg/kg p.o.) were studied for their effects on the
central nervous system in various animal models of anxiety viz., elevated plus
maze and the open field exploratory behavior tests. Further the effects of test
samples on neuromuscular coordination were studied in the rotarod apparatus.
The question of reliability and validity is of prime importance in
establishing experimental paradigms of practical predictable value. These factors
assume further importance when animal models of human behavior and its
perturbations are being used. The paradigms used in the present study have been
subjected to thorough critical appraisal and validated as animal models of anxiety
[Treit, 1971; File, 1985, 1988; Lister, 1990; Kumar et al., 2000]. Thus, in the open
field and similar tests, when the animals are taken from their home cage, and
placed in a novel environment, they express their anxiety and fear by a decrease
in ambulation, rearings, and other exploratory behaviors. Likewise the elevated
plus maze test is based on the principle that exposure of the maze leads to an
approach conflict which is considerably stronger than that evoked by exposure to
the enclosed part of the maze [Pellow et al., 1985]. All these behaviors are
increased by anxiogenic agents and attenuated by anxiolytics under identical
experimental conditions [Vale et al., 1999].
The findings of the present study indicate that ethanolic extract of CD and
isolated mangiferin caused significant anxiolysis in the elevated plus maze test.
Vehicle treated rats spent 31.05±3.73 s in the open arm. The oral administration
of the ethanolic extract of CD (100, 200 and 400 mg/kg p.o.) produced a
significant increase (p<0.01) in the duration of stay in the open arms as well as the
number of entries in the open arm of the EPM indicating the anxiolytic activity of
the drug. Rats treated with mangiferin (100 mg/kg p.o.) spent 103.91±8.02 s in the
open arm. The number of open arm entries also increased to 3.5±0.42, 5.5±0.56,
9.33±0.71 and 8.33±0.66 for ethanolic extract in doses of 100, 200 and 400 mg/kg
p.o. and isolated mangiferin (100 mg/kg p.o.) respectively as compared to the
vehicle (1.5±0.22). The reference drug diazepam (1.0 mg/kg i.p.) significantly
increased (p<0.01) the number of entries as well as the duration of stay in the
open arms, indicating anxiolytic activity. Thus CD proved to be a good anxiolytic
using EPM. The results are shown in Table 1.

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 Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 19

Anxiolytics have been shown to increase the open field ambulation,

rearings, self groomings and activity in the center and a decrease in the number of
faecal droppings is also apparent as a result of their administration .The anxiety
induced by the open field conditions is attenuated by anxiolytic drugs. The
ethanolic extract of CD in a dose of 400 mg/kg p.o. and isolated mangiferin (100
mg/kg p.o.) have shown sufficient anxiolytic activity. The oral administration of
ethanolic extract and the isolate significantly increased the ambulatory activity
(p<0.01 for both ethanolic extract and isolate), rearings (p<0.01 for both ethanolic
extract and isolate), self groomings (p<0.01 for both) and activity in center
(p<0.01 for both). The open field faecal droppings were highly significant in the
case of both the extract and the isolate. Diazepam also induced significant
anxiolytic activity and the effects were found to be highly significant (p<0.001)
and more than that of the extract and the isolate. Isolate has shown an activity
comparable with the reference standard i.e. diazepam. The results are shown in
Table 2.
Vale et al. (1999) affirmed that the absence of interference with the motor
coordination in the rotative bar discards the possibility of a muscular relaxing
effect [16]. In the present work, the ethanolic extracts of CD were devoid of
significant effect on motor coordination at 100 and 200 mg/kg oral dose. The
ethanolic extract (400 mg/kg p.o.) and isolated mangiferin (100 mg/kg p.o.)
caused significant reduction in the number of seconds spent on the rotarod,
compared to control group (p<0.001). The % reduction in the performance on the
rotarod before and after the experiment was 78.42±0.14 and 86.56±0.47 for
groups treated with ethanolic extract (400 mg/kg p.o.) and isolated mangiferin
(100 mg/kg p.o.) respectively and 86.36±0.47 for diazepam treated group as
compared to the vehicle treated control group (6.23±0.28). These results indicate
the muscle relaxant activity of the ethanolic extract at a dose of 400 mg/kg and
isolated mangiferin (100 mg/kg p.o.). Mangiferin is more potent as compared to
ethanolic extract. Therefore, the observed reduction of the motor activity may be
related to the muscle relaxant effect of the extracts and presence of mangiferin in
them. This was a newer finding for CD. The results are shown in Table 3.
All the analysis has been done keeping the vehicle treated group as the
control group. The activity of all groups was compared with the vehicle to have
an insight into the pharmacological action of the drugs. Diazepam was taken only
for comparing with a standard anxiolytic agent and no correlation has been drawn
on the basis of its action.
Nag and De (2008) reported a comparative study of antioxidant and
acetylcholinesterase inhibitory properties of some of the species of plants used as
shankhpushpi in Indian system of medicine viz., Canscora decussata, Canscora
diffusa, Evolvulus alsinoides, Evolvulus nummularius and Clitorea ternatea and

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 Sethiya et al.: Anxiolytic Activity of Shankhpushpi

concluded that C. decussata has the highest acetylcholinesterase inhibitory

activity. Antioxidant activity in all systems was highest in C. decussata.
Mangiferin itself is reported to possess sufficient antioxidant activity
(Prabhu et al., 2006). Antioxidants are supposed to be free radical scavengers and
stress is a cause of damage to the neurons of the brain because of the production
of free radicals. This correlation between stress and antioxidant activity of the
extracts has also been established in one of our previous publications which
further support our view of the antioxidant mode of action of the drug (Nahata et
al., 2009). Bhatnagar et al, 2000 also reported cytoprotective effects of
shankhpushpi- an E. alsinoides preparation on hippocampal cells in mice and
suggested antioxidant mode of action for the drug.
The presence of mangiferin in CD with significant antioxidant properties
[Prabhu et al., 2006] may potentiate the antistress activity of CD. Free radicals
and other reactive oxygen species are considered to be important causative factors
in the development of diseases of aging such as neurodegenerative diseases,
cancer and cardiovascular diseases [Auddy et al., 2003]. Moreover mangiferin has
been reported to possess monoamine oxidase inhibiting activity [Ghosal and
Chaudhuri, 1975] which further ameliorates the stress induced perturbations and
therefore has the potential to act as a tranquilizer. These inherent properties of
mangiferin support our claims about CD as a potent anxiolytic. Thus the
antioxidant potential of mangiferin present in the drug throws light on the
cytoprotective effects of the drug and its usage in stress induced disorders.

Conclusion

The effects of ethanolic extract of CD and isolated mangiferin were studied in

several behavioral animal models for the evaluation of central exploratory
behavior viz. open- field, elevated plus-maze and rotarod test. These are animal
models of preliminary pharmacological test of activities on central nervous
system, which provide information about action upon psychomotor performance,
motor behavior and anxiolysis.
The isolation of mangiferin and its characterization using melting point,
UV and superimposable FTIR analysis was a crucial part of the study. Mangiferin
can be considered as a biological marker on the basis of our studies. Mangiferin
was administered 100 mg/kg p.o. as it has been reported previously for its
antioxidant activity [Prabhu et al., 2006].
In the present studies Canscora decussata exhibited anxiolytic activity
comparable with diazepam which is a GABA agonist. Involvement of similar
mechanism i.e. GABAergic activity of the extract and the isolate can not be ruled
out. It will be fruitful to investigate blockade of the observed anxiolytic activity
with GABA antagonists which may confirm the speculative mechanism.

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 Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 19

Thus it can be concluded that like other species of Shankhpushpi viz.,

Evolvulus alsinoides and Convulvulus pluricaulis, Canscora decussata can be
considered as a drug with significant CNS activity having prominent anxiolytic
effects.

Figure 1: Overlain FTIR spectra of standard mangiferin and the isolated

compound

Table 1: Effect of ethanolic extract of CD and isolated mangiferin on

exploratory activity of the rats in elevated plus-maze apparatus

Time spent (seconds) Number of entries

Treatments
Open arm Closed arm Open arm Closed arm
Vehicle 31.05±3.73 268.94±3.73 1.50±0.22 3.33±0.49
EE (100 mg/kg p.o.) 46.53±3.38** 253.46±3.38** 3.50±0.42* 3.83±0.47
EE (200 mg/kg p.o.) 86.37±8.79** 180.29±32.67** 5.50±0.56** 3.50±0.34
EE (400 mg/kg p.o.) 110.25±4.64** 189.74±4.64** 9.33±0.71** 5.16±0.87
Mangiferin
103.91±8.02** 196.08±8.07** 8.33±0.66** 3.50±0.56
(100 mg/kg p.o.)
Diazepam
137.27±11.82*** 162.72±11.82** 17.66±0.49** 3.83±0.16
(1mg/kg i.p.)
EE- Ethanolic extract of CD. All values are Mean ± S.E.M. (n=6)
***p<0.001, **p<0.01, *p<0.05 compared to vehicle. One way ANOVA followed
by Dunnett’s test.

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 Sethiya et al.: Anxiolytic Activity of Shankhpushpi

Table 2: Effect of ethanolic extract of CD and isolated mangiferin on open

field exploratory behavior in rats
Activity in Fecal
Treatment Ambulation Rearings Self grooming
center droppings
Vehicle 9.66±0.47 2.33±0.33 4.16±0.33 2.50±0.22 4.67±0.71
EE (100 mg/kg
10.00±0.73 4.66±0.34** 5.16±0.40 3.83±0.70 3.33±0.21
p.o.)
EE (200 mg/kg
11.16±0.47 4.83±0.30** 5.33±0.33 3.16±0.30 2.83±0.60
p.o.)
EE (400 mg/kg
16.66±0.71** 5.16±0.60** 9.66±0.49** 4.66±0.33** 1.00±0.25**
p.o.)
Mangiferin (100
19.33±0.42*** 6.33±0.30*** 11.00±0.51** 5.33±0.33** 0.50±0.34**
mg/kg p.o.)
Diazepam
21.00±0.73*** 7.83±0.30** 2.00±0.68*** 8.83±0.47** 0.16±0.16***
(1 mg/kg i.p.)
EE- Ethanolic extract of CD. All values are Mean ± S.E.M. (n=6)
***p<0.001, **p<0.01 compared to vehicle. One way ANOVA followed by
Dunnett’s test.

Table 3: Effect of ethanolic extract of CD and isolated mangiferin on rota

rod performance

Treatments (mg/kg) % Reduction in Performance

Vehicle 6.23±0.28
EE (100 mg/kg p.o.) 16.90±0.78
EE (200 mg/kg p.o.) 13.12±1.12
EE (400 mg/kg p.o.) 78.42±0.14***
Mangiferin (100 mg/kg p.o.) 86.56±0.47***
Diazepam (1mg/kg i.p.) 86.36±0.12***
EE- Ethanolic extract of CD. All values are Mean ± S.E.M. (n=6)
***p<0.001 compared to vehicle. One way ANOVA followed by Dunnett’s test.

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 Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 19

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DOI: 10.2202/1553-3840.1263 12

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