Orient Pharm Exp Med
DOI 10.1007/s13596-012-0099-6
RESEARCH ARTICLE
Anti-inflammatory and analgesic activities of ethyl acetate
and petroleum ether fractions of Cassia auriculata Linn. leaves
A. A. Mali & D. D. Bandawane & M. G. Hivrale
Received: 10 September 2012 / Accepted: 4 December 2012
# Institute of Oriental Medicine, Kyung Hee University 2012
Abstract The effect of petroleum ether fraction (50, 100 and
200 mg/kg) and ethyl acetate fraction (50, 100 and 200 mg/kg)
of Cassia auriculata Linn. (Caesalpiniaceae) leaves was inves-
tigated in various experimental models of pain and inflamma-
tion. Anti-inflammatory activity of petroleum ether fraction
and ethyl acetate fraction was studied using carrageenan in-
duced paw edema and cotton pellet induced granuloma models
in rats. Analgesic activity was studied in mice using tail im-
mersion and hot plate models. Both petroleum ether fraction
and ethyl acetate fraction significantly (P<0.01) inhibited car-
rageenan induced paw edema and granuloma formation in
cotton pellet induced granuloma model. Petroleum ether frac-
tion and ethyl acetate fraction significantly (P<0.01) increased
latency to flick tail in tail immersion method and elevated the
mean basal reaction time in hot plate method. Phytochemical
analysis of petroleum ether fraction showed presence of ste-
roids and ethyl acetate fraction showed presence of flavonoids
and tannins. The ethyl acetate fraction was found to be more
effective than petroleum ether fraction. Thus the anti-
inflammatory and analgesic potential of C. auriculata leaves
may be due to presence of active constituents like flavonoids
and tannins present in petroleum ether fraction.
Keywords C. auriculata . Anti-inflammatory . Analgesic .
Carrageenan . Cotton pellet
Introduction
Till date, number of synthetic steroidal and non-steroidal anti-
inflammatory drugs (NSAID’s) are discovered to treat
A. A. Mali : D. D. Bandawane (*) : M. G. Hivrale
Department of Pharmacology,
PES’s Modern College of Pharmacy, Nigdi,
Pune, Maharashtra 411044, India
e-mail: rspatil_pharma@yahoo.co.in
inflammation and pain. These drugs are potent in action but
show wide range of adverse effects whereas, herbal drugs bear
comparatively less side effects (Tripathi 2008; Mohan 2010).
Herbal drugs can therefore be considered as a better alterna-
tive to synthetic anti-inflammatory drugs. Cassia auriculata
Linn. (fam. Caesalpiniaceae) is a tall, branched, bushy shrub
growing wild throughout forests, along roadsides and in
wastelands (Gaikwad et al. 2011). Traditionally the plant has
been used in ayurvedic medicine as ‘Avarai Panchaga
Chooram’ and as constituent of ‘Kalpa herbal tea’ (Doshi et
al. 2011a). The leaves are bitter, astringent, acrid, constipating
and expectorant (Balakrishna et al. 2011). The qualitative
phytochemical analysis of extracts from the roots and leaves
of C. auriculata showed the presence of anthraquinone glyco-
sides, alkaloids, flavonoids, phenolic compounds, saponins,
steroids and tannins (Chitravadivu et al. 2009).
Previous studies has proved that the chemical constituents
such as flavonoids, bioflavonoids, alkaloids, tannins and ter-
penoids are promising agents in treatment of inflammation
(Peraman et al. 2011; Singh et al. 2008; Doshi et al. 2011b).
Flavonoids such as hesperidin, apigenin, luteolin and querce-
tin are found to be potent anti-inflammatory constituent and
flavonoids like myricetin, quercetin, naringenin, apigenin and
catechin have been reported to be effective antioxidant agents
(Narayana et al. 2001). High content of flavonoids and bio-
flavonoids in methanolic extract of C. auriculata flowers is
reported for its anti-inflammatory activity (Doshi et al. 2011a).
Preliminary phytochemical analysis of methanolic extract of
C. auriculata leaves shows the presence of tannins, flavo-
noids, steroids, alkaloids saponins and glycosides. Therefore
to separate the active constituents responsible for anti-
inflammatory and analgesic activity fractionation of crude
methanolic extract was carried out using ethyl acetate and
petroleum ether as solvent. Presence of flavonoids and tannins
in ethyl acetate fraction and steroids in petroleum ether frac-
tion led us to undertake the present work to evaluate the anti-
inflammatory and analgesic activity of ethyl acetate and

petroleum ether fraction of C. auriculata leaves and to focus
on its possible mechanism of action.
Materials and methods
Plant material
The leaves of the C. auriculata were obtained from their
natural habitat in the Pune region of Maharashtra in the month
of August. The plant was identified and authenticated by the
Botanical Survey of India, Pune. Voucher specimen (no.
CAAAAM5) has been deposited in the herbarium for future
reference. The leaves were shade dried without exposing them
to direct sunlight. The dried leaves were pulverised in grinder
and stored in air tight container for further use.
Extraction
Air dried leaves of C. auriculata were ground to coarse
powder and 50 gm of powder was extracted in methanol
(95 %, 500 ml) for 72 h at room temperature. The extracts
were filtered and the solvents were evaporated to obtain
powdered residues. The extract was suspended in water
and fractionated with petroleum ether and ethyl acetate
and dried under vacuum. The yield (w/w) of methanolic
extract was 12.5 % whereas yield of petroleum ether and
ethyl acetate fractions were 8 % and 10 % respectively.
Animals
Male as well as female Wistar rats (180-200 gm) and Swiss
albino mice (25-30 gm) of either sex were procured from the
National Institute of Biosciences (NIB), Pune, Maharashtra.
The animals were housed in groups of six at an ambient
temperature of 25±1°C, under a 12:12 h light-dark cycle,
with free access to standard diet and water throughout the
study. Animals were deprived of food but not water 4 h
before the experiment. The study protocol was approved by
Institutional Animal Ethical Committee of Modern College
of Pharmacy in accordance with the regulations of CPCSEA
(884/PO/ac/05/CPCSEA).
Chemicals
Carrageenan was procured from Analab Fine Chemicals,
Mumbai. All other chemicals and reagents used were of
analytical grade procured from SRL Mumbai, E. Merck India.
Sub-acute toxicity study
Sub-acute toxicity study was performed according to
Organization for Economic Co-operation and Development
A.A. Mali et al.
(OECD) - 423 guidelines. Female wistar rats selected by
random sampling technique were employed in this study.
Female rats were used because literature surveys of conven-
tional LD50 tests show that females are generally slightly
more sensitive for the study (Lipnick et al. 1995). The
animals were fasted overnight with free access to water.
Ethyl acetate and petroleum ether fractions of C. auriculata
leaves were administered orally to different groups at the dose
levels of 300, 500, 2000, 5000 mg/kg. The animals were
observed 24 h for mortality with special attention during first
2 h and then intermittently for 14 days (Ecobichon 1997).
During sub-acute toxicity study, no adverse effect or mortality
was observed in animals treated with petroleum ether and
ethyl acetate fractions of C. auriculata leaves upto a high dose
of 5000 mg/kg body weight.
Phytochemical analysis
The preliminary phytochemical analysis of all the plant
extracts was performed as per the standard qualitative meth-
ods. A series of chemical tests were carried out for presence
of alkaloids, flavonoids, carbohydrates, glycosides, steroids
and tannins (Khandelwal 2006). Preliminary phytochemical
analysis of petroleum ether fraction showed presence of
alkaloids, steroids, and saponins. The ethyl acetate fraction
showed presence of flavonoids, tannins and saponins.
Thin layer chromatography
Both the fractions were subjected to thin layer chromatogra-
phy plates of 250 μm thickness (TLC Silica gel 60 F254
Merck, Germany). The spots were developed in two different
solvent systems as follows:
Solvent system A - Ethyl acetate: glacial acetic acid:
formic acid: water (100:11:11:27)
Solvent system B - Butanol: acetic acid: water (4:1:5)
Rf was calculated using following equation, (Koua et al.
2011).
Rf ¼ Distance travelled by the sample=Distance travelled
by the solvent:
Thin layer chromatography of petroleum ether fraction
confirmed the presence of steroids while that of ethyl acetate
fraction confirmed the presence of flavonoids and tannins.
Carrageenan induced rat paw edema
The method of Kumar et al. (2011) was used to study acute
inflammation. Rats were divided in 6 groups, each group
comprising of six rats. Rats in different groups were treated
orally with vehicle (1 ml/kg), ethyl acetate fraction (50, 100 and
200 mg/kg), petroleum ether fraction (50, 100 and 200 mg/kg)
Anti-inflammatory and analgesic activities of C. auriculata
and 10 mg/kg indomethacin. Drug administration volume was
not more than 0.5 ml (Biswas et al. 2011), one hour prior to
carrageenan injection. 0.1 ml of 1 % w/v of carrageenan
suspension in 0.9 % normal saline was injected into sub-
planter region of left hind paw of rat. The paw volume upto
the tibio-tarsal articulation was measured using a plethysmom-
eter (VJ Instruments, Nagpur). The paw volume was deter-
mined at 0, 1, 2, 3 and 4 h. The % inhibition in paw volume was
calculated using following formula (Benni et al. 2011),
% inhibition in paw volume ¼ 100  ð1  Vt=VcÞ
Where,
Vt Mean paw volume in drug treated group.
Vc Mean paw volume in control group.
Cotton pellet induced granuloma in rats
Rats in groups of six each were treated orally with vehicle
(1 ml/kg), ethyl acetate fraction (50, 100 and 200 mg/kg),
petroleum ether fraction (50, 100 and 200 mg/kg) and indo-
methacin (10 mg/kg) Drug administration volume was not
more than 0.5 ml. Sterile cotton pellet weighing 25±1 mg
was implanted subcutaneously in the groin region of rat
under thiopental sodium (25 mg/kg) anesthesia. Rats re-
ceived the respective treatments for 7 consecutive days
starting from the day of cotton pellet implantation. The rats
were sacrificed on eighth day and pellets surrounded by
granuloma tissue were dissected out carefully and dried at
60 °C to a constant weight. The pellets were weighted both
moist and dry. The granuloma tissue formation and exudate
formation was calculated using following formula (Doshi et
al. 2011b).
Measure of granuloma tissue formation ¼ Constant dry weight
 Initial weight of pellet:
Measure of exudate formation ¼ Moist weight
 Constant dry weight of pellet:
Histopathology study of granulomatous tissue
For histopathology study, the granulomatous tissue embed-
ded in paraffin block was trimmed lightly with the help of
microtome so as to expose the transverse section of the
tissue. Staining agent used was hematoxylin eosin stain.
The exposed section is examined under resolution power
of 40X using a digital microscope (Luna 1968).
Eddy’s hot plate method
Mice in groups of six each were treated orally with vehicle
(1 ml/kg), ethyl acetate fraction (50, 100 and 200 mg/kg),
petroleum ether fraction (50, 100 and 200 mg/kg) and 9 mg/kg
of diclofenac. Drug administration volume was not more than
0.5 ml (Nikajoo 2009). One hour after the respective treat-
ment, mice were placed on the hot plate maintained at 55 °C.
The latency to lick the paw or jump from the hot plate was
noted as the reaction time, whichever appeared first (Jude and
Paul 2010). The reaction time was measured at 0, 1, 2, 3, 4 and
5 h.
Tail immersion method
Mice divided in six groups of six each were treated orally
with vehicle (1 ml/kg), ethyl acetate fraction (50, 100 and
200 mg/kg), petroleum ether fraction (50, 100 and 200 mg/
kg) and diclofenac (9mg/kg). Drug administration volume
was not more than 0.5 ml. After 1 h, the tip of tail was
dipped upto 5 cm in hot water maintained at 58 °C. The
reaction time was noted as a sudden withdrawal of the tail
from the hot water. Cut off time of 20 s was maintained to
avoid damage to the tail. The reaction time was measured at
0, 1, 2, 3, 4 and 5 h (Nikajoo 2009; Vijay and Vijayvergia
2010).
Data analysis
All the data is presented as mean±S.E.M. (n=6). Statistical
analyses was performed using one-way analysis of variance
(ANOVA) followed by Dunnet’s multiple test for comparison.
Value of P<0.05 was considered to be statistically significant.
Results
Carrageenan induced paw edema
As shown in Table 1, the petroleum ether fraction (50, 100
and 200 mg/kg) and ethyl acetate fraction (50, 100 and
200 mg/kg) significantly (P<0.01) inhibited carrageenan
induced rat paw edema as compared to control group.
Maximum inhibition of rat paw edema was observed with
ethyl acetate fraction (50 mg/kg) at the end of 4 h when
compared to control group. Indomethacin (10 mg/kg)
inhibited the paw edema by 33.49 %.
Cotton pellet induced granuloma
As shown in Table 2, petroleum ether fraction (50, 100 and
200 mg/kg), ethyl acetate fraction (50, 100 and 200 mg/kg)
and indomethacin (10 mg/kg) showed significant (P<0.01)
decrease in moist as well as dry weight of the cotton pellet.
Ethyl acetate fraction showed more extensive inhibition in
granuloma tissue formation than petroleum ether fraction
and indomethacin.
As shown in Fig. 1, histopathology study of granuloma-
tous tissue in toxic control group showed fibromuscular
 A.A. Mali et al.

Table 1 Effect of CAPE (50, 100 and 200 mg/kg) and CAEA (50, 100 and 200 mg/kg) of C. auriculata leaves on carrageenan-induced rat paw
edema

Treatment (mg/kg) Paw volume (ml) % inhibition at 4 h

0h 1h 2h 3h 4h

Control 1.31±0.02 1.50±0.05 1.74±0.07 1.81±0.06 2.12±0.06 –

Indomethacin (10) 1.13±0.02 1.21±0.06** 1.16±0.05** 1.35±0.12** 1.41±0.11** 33.49
CAPE (50) 1.18±0.02 1.27±0.03** 1.38±0.09** 1.46±0.02** 1.48±0.07** 30.18
CAPE (100) 1.24±0.04 1.19±0.04** 1.26±0.06** 1.57±0.06 1.53±0.04** 27.83
CAPE (200) 1.30±0.04 1.35±0.02 1.57±0.05 1.55±0.05 1.62±0.03* 23.58
CAEA (50) 1.21±0.02 1.29±0.05** 1.36±0.04** 1.25±0.07** 1.34±0.05** 41.03
CAEA (100) 1.16±0.04 1.19±0.05** 1.27±0.04** 1.58±0.08 1.61±0.06* 24.05
CAEA (200) 1.22±0.05 1.30±0.02* 1.56±0.05 1.48±0.06* 1.65±0.03* 22.16

n=6. The observations are mean±S.E.M. *P<0.05, **P<0.01, as compared to control. (ANOVA followed by Dunnett’s test). CAPE=Petroleum
ether fraction of C. auriculata leaves, CAEA=Ethyl acetate fraction of C. auriculata leaves

tissues with necrosis. There was dense infiltration by acute
inflammatory cells comprising of lymphocytes, polymorphs,
macrophages and few plasma cells. Treatment with petroleum
ether fraction and ethyl acetate fraction showed reduced infil-
tration by inflammatory cells with no evidence of necrosis.
Granulomatous tissue of indomethacin treated group showed
presence of diffusely arranged lymphocytes, macrophages and
few plasma cells.
Tail immersion induced pain
As shown in Table 3, petroleum ether fraction (50, 100 and
200 mg/kg) and ethyl acetate fraction (50, 100 and 200 mg/kg)
significantly (P<0.01) increased in the reaction time as com-
pared to control animals. Highest nociception inhibition was
exhibited by petroleum ether fraction (50 mg/kg) at 4 h while
the maximum analgesic effect shown by diclofenac (9 mg/kg)
was observed at 3 h.
Hot plate induced pain
As shown in Fig. 2, oral administration of petroleum ether
fraction (50 and 100 mg/kg) and ethyl acetate fraction (50
and 100 mg/kg) of C. auriculata leaves significantly (P<
0.01) increased the reaction time as compared to control
group. Whereas, both ethyl acetate and petroleum ether
fractions at 200 mg/kg significantly (P<0.05) increased
the reaction time as compared to control group. The maxi-
mum analgesic effect was exhibited by ethyl acetate fraction
(100 mg/kg) at 4 h. The maximum analgesic effect shown
by diclofenac (9 mg/kg) was observed at 3 h.
Discussion
The pharmacological activities of herbal extracts are on the
virtue of the phytoconstituents they possess. It is reported that

Table 2 Effect of CAPE (50, 100 and 200 mg/kg) and CAEA (50, 100 and 200 mg/kg) of C. auriculata leaves on cotton pellet induced granuloma
in rats

Treatment Weight of moist Weight of dried % Inhibition Transudative % Inhibition

cotton pellet (mg) cotton pellet (mg) weight (mg)

Control 281.16±21.24 70.33±3.82 – 210.83±20.1 –

Indomethacin (10) 191.36±5.1** 52.33±2.04** 25.59 139.03±5.70 34.05
CAPE (50) 188±15.10** 55.16±3.68** 21.56 132.83±11.68** 36.99
CAPE (100) 194.16±5.49** 54.91±1.76** 21.92 139.25±5.17** 33.85
CAPE (200) 220.16±10.49* 60.97±3.00 13.31 159.18±11.49* 27.74
CAEA (50) 116.83±7.83** 44.73±2.03** 36.39 72.1±5.9** 65.80
CAEA (100) 166.66±18.11** 47.3±4.69** 32.74 119.36±13.6** 43.38
CAEA (200) 221.00±9.30* 58.33±2.36* 17.06 162.66±6.26* 22.84

n=6. The observations are mean±S.E.M. *P<0.05, **P<0.01, as compared to control. (ANOVA followed by Dunnett’s test). CAPE=Petroleum
ether fraction of C. auriculata leaves, CAEA=ethyl acetate fraction of C. auriculata leaves
Anti-inflammatory and analgesic activities of C. auriculata

Fig. 1 Effect of petroleum ether

fraction (CAPE) and ethyl ace-
tate fraction (CAEA) of C.
auriculata leaves on histopa-
thology of granulumatous tissue.
a Control; b Indomethacin
(10 mg/kg); c CAPE (50 mg/kg);
d CAPE (100 mg/kg); e CAPE
(200 mg/kg); f CAEA (50 mg/kg);
g CAEA (100 mg/kg); h CAEA a b
(200 mg/kg)

c d

e f

g h

flavonoids are responsible for antioxidant, anti-inflammatory, prostaglandins (Arulmozhi et al. 2012; Das et al. 2010). In
analgesic and antiulcer activities (Singh et al. 2008). Tannins the preliminary study, we found significant anti-inflammatory
and flavonoids show anti-inflammatory property by inhibiting and analgesic activity of methanol extract of C. auriculata
arachidonic acid metabolism pathway and synthesis of leaves in different animal models.

Table 3 Effect of CAPE (50, 100 and 200 mg/kg) and CAEA (50, 100 and 200 mg/kg) of C. auriculata leaves on tail immersion method in mice

Treatment Reaction time (s)

0h 1h 2h 3h 4h 5h

Control 3.62±0.37 2.72±0.47 4.24±0.67 4.40±0.12 2.39±0.17 3.88±0.55

Diclofenac (9) 4.36±0.53 7.99±0.68 6.79±1.32 16.67±1.16** 9.37±1.00 12.69±0.82**
CAPE (50) 4.46±0.33 9.70±2.07* 10.54±1.94* 17.41±0.71** 29.38±5.29** 21.50±1.25**
CAPE (100) 5.84±0.68 7.50±1.43 9.93±1.56 17.37±2.32** 25.04±11.7** 23.34±1.09**
CAPE (200) 5.96±0.54 8.16±3.04 10.07±0.74 16.38±1.82** 19.30±1.79 11.02±0.30*
CAEA (50) 5.92±0.39 9.76±1.95* 9.37±1.45 17.63±2.72** 11.68±3.12 23±3.38**
CAEA (100) 5.36±0.80 14.38±1.79** 9.83±2.35 16.19±5.13** 12.71±3.74 23.74±2.33**
CAEA (200) 5.27±0.47 8.37±0.64 10.16±0.96 15.07±0.68* 20.66±0.44 16.40±0.99**

n=6. The observations are mean±S.E.M. *P<0.05, **P<0.01, as compared to control. (ANOVA followed by Dunnett’s test). CAPE=Petroleum
ether fraction of C. auriculata leaves, CAEA=ethyl acetate fraction of C. auriculata leaves

Fig. 2 Effect of petroleum
ether fraction (CAPE) and ethyl
acetate fraction (CAEA) of C.
auriculata leaves on Eddy’s hot
plate method. Each column
represents mean±S.E.M.
(n=6). *P<0.05, **P<0.01,
as compared to control
(ANOVA followed by Dunnet’s
multiple comparison test
Preliminary phytochemical analysis of methanol extract of
C. auriculata leaves showed presence of tannins, flavonoids
and steroids. To separate these active phytoconstituents re-
sponsible for the activity, fractionation of methanol extract of
C. auriculata leaves was performed using two solvents name-
ly petroleum ether and ethyl acetate. Phytochemical analysis
showed the presence of tannins and flavonoids in ethyl acetate
fraction and steroids in petroleum ether fraction of C. auric-
ulata leaves.
The tested fractions of C. auriculata leaves protected rats
against both acute and chronic inflammation, which was
evidenced from both carrageenan-induced paw edema and
cotton pellet induced granuloma models respectively.
Variations in activity for both the fractions in carrageenan-
induced paw edema and cotton pellet induced granuloma
models indicate that the different constituents present in both
the fractions may be responsible for anti-inflammatory activity.
Carrageenan-induced inflammation in the rat paw repre-
sents a classical model of edema formation and hyperalgesia,
which has been extensively used for evaluation of anti-edemal
effect of drugs (Guay et al. 2004). The sub-planter adminis-
tration of carrageenan in rat is responsible for the typical
biphasic edema (Fabiana et al. 2009) in which the first phase
observed around 0-2 h is attributed to the release of histamine
and serotonin. The second phase of swelling which last for 2-
6 h is due to release of prostaglandins- like substances
(Kaushik and Jalalpure 2010; Cuman et al. 2001). It has been
reported that second phase of edema is sensitive to both
clinically useful steroidal agents and NSAIDs (Shukla et al.
2012). The significant activity shown by both the fractions in
the suppression of second phase of carrageenan-induced in-
flammation may be due to inhibition of prostaglandin- like
substances (Kaushik and Jalalpure 2010; Cuman et al. 2001).
Ethyl acetate fraction significantly reduced paw edema to a
more extent than petroleum ether fraction.
The cotton pellet induced granuloma is widely adopted
method to evaluate the transudative and proliferative
A.A. Mali et al.
components of the chronic inflammation. Most of the
NSAIDs possess only slight inhibition on the granuloma
formation while steroidal drugs exhibit profound reduction
of granuloma (Ashok et al. 2010). Chronic inflammation
includes a proliferation of fibroblasts, exudation and the infil-
tration of neutrophils and granuloma formation occurs by
means of development of proliferate cells (Recio et al. 1995).
Both the fractions significantly reduced the size of granuloma
suggesting that the anti-inflammatory activity of the fractions
may be mediated by inhibiting granulocyte infiltration, by
preventing generation of collagen fibres and by suppressing
mucopolysaccharides (Veerappan et al. 2005; Hui et al. 2009;
Muralidhar et al. 2010). Moreover, the histopathology of tissue
granuloma justified anti-inflammatory potential of both the
fractions. Ethyl acetate fraction and petroleum ether fraction
treated rats showed diffused infiltration by inflammatory cells
with no evidence of necrosis.
It is reported that most inflammatory conditions are usually
associated with pain as secondary process (Osadebe and
Okoye 2003). In the present study, we therefore evaluated
analgesic activity of fractions using hot plate method and tail
immersion method. These methods are selective in examining
the analgesic compounds acting through opoid receptors
(Vogel 2002). The significant increase in reaction time in tail
immersion and hot plate induced pain by petroleum ether
fraction and ethyl acetate fraction showed that fractions may
act by centrally mediated mechanism.
In accordance with previous studies, phytoconstituents
like steroids, flavonoids, alkaloids, terpenoids and tannins
have been shown to possess anti-inflammatory and analge-
sic activity (Lilian et al. 1998; Das et al. 2010; Chakraborthy
2009). The phytochemical analysis and thin layer chroma-
tography of petroleum ether fraction confirmed the presence
of steroids while that of ethyl acetate fraction confirmed the
presence of flavonoids and tannins.
The ethyl acetate fraction was found to show more anti-
inflammatory activity when compared with petroleum ether
Anti-inflammatory and analgesic activities of C. auriculata
fraction. Thus the anti-inflammatory and analgesic potential
of C. auriculata leaves may be due to presence of active
constituents like flavonoids and tannins.
Acknowledgement The authors are very grateful to the management
and Principal, PE Society’s Modern College of Pharmacy, Nigdi, Pune,
India for providing required facilities of this work. Authors are also
grateful to Dr. Diwakar, Director, Botanical Survey of India for authen-
tification of plant material
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