Industrial Crops and Products 73 (2015) 134143

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Industrial Crops and Products

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Analgesic and acetylcholinesterase inhibition potential of polyphenols

from Scolopia crenata (Flacourtiaceae): An endemic medicinal
plant of India
Rajkumar Gomathi a , Sellamuthu Manian a,b,
a
b

Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu 641 046, India
Research and Development Division, Manian Laboratories Pvt. Ltd., Peelamedu, Coimbatore, Tamil Nadu 641 004, India

a r t i c l e

i n f o

Article history:
Received 19 November 2014
Received in revised form 30 March 2015
Accepted 31 March 2015
Available online 16 May 2015
Keywords:
Radical scavenging
Acetylcholinesterase
Phenolic aids
Flavonoids
Analgesic

a b s t r a c t
Scolopia crenata (Wight & Arn.) Clos. (Flacourtiaceae), an endemic Indian medicinal tree is used traditionally to alleviate musculo-skeletal pain, to treat wounds and as sedatives in herbal formulations. In
the present study, various solvent extracts from stem bark and leaves of S. crenata were quantied for
bioactive secondary metabolites (total phenolics, tannins, total avonoids, total proanthocyanidins, total
monomeric anthocyanins and vitamin E) and assessed for radical scavenging and acetylcholinesterase
(AChE) inhibitory abilities in vitro. The methanol extracts of stem bark and leaf showing promising activity was further assessed for in vivo analgesic activity employing acetic acid induced writhing and hot
plate tests in mice. From the results, the methanol extracts from stem bark and leaf contained higher

amounts of bioactive secondary metabolites and scavenged DPPH and ABTS + effectively in vitro. They
also showed a remarkable inhibition of AChE in vitro (IC50 9.20 and 5.64 g/ml, respectively). The mode
of AChE inhibition was found to be non-competitive in leaf while competitive for stem bark extract. The
methanol extracts of both leaf and stem bark signicantly (p < 0.05) reduced the number of writhing
in acetic acid induced writhing test and prolonged the latency response in hot plate in a dose dependent manner. The plants extracts also showed a decreased analgesic effect in the presence of antagonists
(atropine and mecamylamine) in the cholinergic system. HPLC analysis revealed the presence of phenolic
acids (caffeic acid, p-coumaric acid, ferulic acid and gallic acid), avonoids (apigenin, catechin, quercetin,
luteolin, kaempferol and rutin) and a pentacyclic triterpeniod, betulinic acid in the methanol extracts of
stem bark and leaf of S. crenata. The ndings of the present study highlight the association of analgesic
activity with AChE enzyme inhibition, involvement in cholinergic system and free radical scavenging
potential of S. crenata as a choice for various pharmaceutical applications.
2015 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: (AChE), acetylcholinesterase; (ATCI), acetylthiocholine iodide;

(AD), Alzheimers disease; (ANOVA), analysis of variance; (ABTS), 2,2 -azinobis
(3-ethylbenzothiozoline-6-sulfonic acid) di ammonium salt; (BHA), butylated
hydroxyanisole; (CMC), carboxymethyl cellulose; (CE), catechin equivalent; (CGE),
cyanidin 3-glucoside equivalent; (GAE), gallic acid equivalent; (HPLC), high
performance liquid chromatography; (PD), Parkinsons disease; (QE), quercetin
equivalent; (TE), -tocopherol equivalent; (DPPH), 2,2-diphenyl-1-picryl-hydrazyl.
Corresponding author at: Department of Botany, School of Life Sciences,
Bharathiar University, Coimbatore, Tamil Nadu 641 046, India. Tel.: +91 422
2428301; fax: +91 422 2422387.
E-mail addresses: gomathiraj.85@gmail.com (R. Gomathi),
manian sm@yahoo.co.in (S. Manian).
http://dx.doi.org/10.1016/j.indcrop.2015.03.090
0926-6690/ 2015 Elsevier B.V. All rights reserved.

India, the land of paradise of medicinal plants harbors home for

several topical and temperate forests which supports a vast reservoir of medicinal plant species. In recent years, herbal medicines
have proven more effective than the synthetic drugs owing to
promising efcacy and lesser side effects. Herbal industries are
receiving greater attention worldwide since the innumerable
potential compounds from plants/plant products serve as starting points for the development of new drugs. The World Health
Organization (WHO) is playing a leadership role in encouraging the
development of scientic approaches to the evaluation and utilization of traditional medicines where among the total of 252 drugs
in WHOs essential medicine list, 11% is exclusively of plant origin
(Rates, 2001).

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

In India, about 80% of the rural population depends on medicinal herbs or indigenous systems of medicinal practises for their
primary healthcare (Mukherjee and Wahile, 2006). Pain is an
unpleasant sensation which greatly affects the quality of humans
life. Consequently analgesic drugs that counteract pain are considered as one among the important health care needs. A potent
analgesia has been documented to mediate action through central
cholinergic mechanisms via (i) increased acetylcholine synthesis, (ii) inhibiting cholinesterase activity, and/or (iii) selectively
blocking the autoreceptor or heteroreceptor feedback mechanisms
(Bartolini et al., 2011). In particular, the inhibition of cholinesterase
besides mediating analgesic effect, also plays a major role in attenuating neurodegenerative diseases such as Alzheimers disease (AD)
and Parkinsons disease (PD) (Enz et al., 1993). The pathology of
AD is associated to the loss of acetylcholine, an organic molecule
acting as a neurotransmitter on peripheral and central nervous
systems. Acetylcholine esterase (AChE) catalyzes the hydrolysis of
acetylcholine to choline and acetic acid resulting in loss of memory and impairment of multiple cognitive functions (Frank and
Gupta, 2005). As such, identication of drugs inhibiting acetylcholine esterase and mediating potent analgesic effects is therefore
of considerable importance. The efcient drugs used till date to alleviate pain such as morphine or its surrogates and AChE inhibitors
namely tacrine, donepezil and rivastigmine are limited due to the
unfavourable side effects and tolerance developed after a longterm treatment (Schulz, 2003; Meert and Vermeirsch, 2005). The
ethnomedicinal knowledge gained over ages has been useful as
a starting point for the discovery of new drugs based on herbal
origin. Several evidences suggest that plants/plant extracts used
to relieve pain have shown analgesic effects, while those used to
treat cognitive disorders were found to act as AChE inhibitors (Lima
et al., 2005; de Mattos et al., 2007; Stafford et al., 2008). Furthermore, polyphenols such as phenolic acids, tannins and avonoids
present in the plant materials have shown a profound analgesic and
cholinesterase inhibitory properties (Bone and Mills, 2013; Zhang
et al., 2009).
Scolopia crenata (Wight & Arn.) Clos. belonging to Flacourtiaceae
is an endemic species to India, found distributed in tropical forest
of Andhra Pradesh, Karnataka, Tamil Nadu and Kerala (Pullaiah and
Sandhya Rani, 1999). It is an evergreen tree, wherein the leaves are
traditionally used to treat musco-skeletal pain (Kadavul and Dixit,
2009). The Chellipale community of Kolli hills, Tamil Nadu uses the
bark of S. crenata to treat cut wounds and the leaves grounded with
jaggery are applied over inammations. Various parts of the tree
are also employed as sedative in herbal formulations with several
other herbal ingredients (Oudhia, 2012). An earlier study on S. crenata revealed the presence of phenols, tannins, alkaloids, cardiac
glycosides and steroids in the stem bark conferring DPPH radical
scavenging ability (Smitha et al., 2012). Besides these reports, the
functional properties of S. crenata have not been fully explored.
Therefore, the present study attempted to investigate the analgesic
and AChE inhibitory effects of S. crenata which remains an untapped
candidature for pharmaceutical applications.

135

(ABTS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 5-5 -thiobis2-nitrobenzoic acid (DTNB) were purchased from SigmaAldrich
(St. Louis, MO, USA). Aluminium chloride, ammonium molybdate,
folin-ciocalteu phenol reagent, glacial acetic acid, hydrochloric
acid, magnesium chloride, potassium chloride, sodium acetate,
sodium chloride, sodium hydroxide, sodium nitrite, sodium phosphate, sulphuric acid, -tocopherol and vanillin reagent were
obtained from Himedia (Mumbai, India). Solvents used were of the
highest purity and analytical grade made in India.
2.2. Plant collection and preparation of extracts
Fresh leaves and stem bark of S. crenata were collected from Kolli
hills, Tamil Nadu in June, 2012. The specimen was authenticated
by the Botanical Survey of India, Southern Regional Centre, Coimbatore (No. BSI/SRC/5/23/2012-13/Tech./637) and the voucher
specimen (No. 006173) deposited at the Herbarium in the Department of Botany, Bharathiar University, Coimbatore. Fresh plant
materials were washed in tap water and shade dried at 25 C. The
powdered leaf and stem bark materials (100 g) were subjected to
soxhlet extraction using petroleum ether to remove the lipophilic
substances. The residues were dried in a hot air oven (30 C) and
further extracted using chloroform, ethyl acetate and methanol
successively. The extracts obtained were concentrated in a rotary
vacuum-evaporator (Yamato B0410, Japan) at 40 C and lyophilized
(Vir Tis Benchtop 4K, USA). The freeze dried extracts were then
stored at 20 C for further investigation. The percent yield in
petroleum ether, chloroform, ethyl acetate and methanol for S. crenata stem bark is, respectively, 1.1, 0.8, 4.8 and 7.3, whereas for leaf
it is, respectively, 1.9, 2.4, 5.9 and 9.1.
2.3. Determination of bioactive secondary metabolites
Standard spectrophotometric methods were employed for the
determination of bioactive compounds in the sample extracts. Total
phenolics and tannins were calculated as gallic acid equivalent
(GAE) via Siddhuraju and Manian (2007). The method of Zhishen
et al. (1999) was followed to obtain the total avonoid content
and the amount is expressed in terms of quercetin equivalent (QE).
Total proanthocyanidin content was determined in terms of catechin equivalent (CE) as reported by Butler et al. (1982). Total
monomeric anthocyanins was estimated as cyanidin 3-glucoside
equivalent (CGE) by the pH differential method proposed by Moyer
et al. (2002). The antioxidant vitamin E was quantied based on
the procedure described by Prieto et al. (1999) and the results are
expressed as -tocopherol equivalent (TE).
2.4. In vitro antioxidant activity
2.4.1. DPPH assay
Radical scavenging activity in bleaching of the purple, stable
DPPH radical by the various solvent extracts of stem bark and leaf
of S. crenata was measured according to the method of Blois (1958).

2. Materials and methods

2.1. Chemicals
Acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE),
acetic acid, apigenin, atropine, betulinic acid, butylated hydroxyanisole (BHA), caffeic acid, catechin, p-coumaric acid, cyanidin
3-glucoside, carboxymethyl cellulose (CMC), ferulic acid,
galanthamine, gallic acid, kaempferol, luteolin, indomethacin,
mecamylamine, morphine, quercetin, rutin, tris-HCl, 2,2 -azinobis
(3-ethylbenzothiozoline-6-sulfonic acid) di ammonium salt

2.4.2. ABTS + scavenging assay

The total antioxidant activity of the various solvent extracts of
stem bark and leaf of S. crenata was measured by ABTS radical cation
decolorization assay according to the method of Re et al. (1999).
2.5. AChE inhibitory activity
The AChE activity was measured according to the method
developed by Ellman et al. (1961) with slight modications. To
various concentrations of sample extracts (25250 g) in 250 l,
25 l of 15 mM acetylthiocholine iodide (ATCI) in water, 125 l of
3 mM DTNB (5-5 -thiobis-2-nitrobenzoic acid) in Buffer C (50 mM

136

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

TrisHCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl2 .6H2 0) and

50 l of Buffer B (50 mM TrisHCl, pH 8, containing 0.1% bovine
serum albumin) were added. Absorbance was measured spectrophotometrically at 405 nm every 45 s, ve times consecutively.
Thereafter, 25 l of AChE (0.2 U/ml) was added and the nal mixture was again read at 405 nm ve times consecutively for every
45 sec. Galanthamine was used as standard AChE inhibitor. The
sample concentration providing 50% inhibition (IC50 ) under the
assay condition was calculated from the graph of inhibition percentage against sample concentration.

2.6.2. Betulinic acid

The quantity of betulinic acid in the sample extracts was
determined through HPLC analysis using Luna-5u C18 column
(Phenomenex 250 4.60 mm, i.d., 5 m) eluted with acetonitrile:
water (85:15, v/v) at a ow rate of 1 ml/min. The presence of
betulinic acid was monitored at 205 nm following the method
described by Jaina et al. (2012). The calibration curve and correlation coefcients (R2 ) of betulinic acid recorded y = 230.18x + 0.001
and 0.9997, respectively.
2.7. Animals

2.5.1. Kinetic study to determine the mode of AChE inhibitory

activity
In order to elucidate the type of inhibition of the effective
extracts, the enzyme activity was measured in the presence of an
increasing concentration of ATCI (525 mM), and in the absence or
presence of two concentrations of each extract (50 and 100 g/ml).
Inhibition mode was determined by LineweaverBurk plot analysis of the data resulting from the enzyme assays. Vmax (maximum
enzyme velocity) and Km (dissociation constant) were obtained
from the plot graph of reciprocal of substrate concentration versus
reciprocal of rate of reaction (Graph pad prism, Version 5).
2.6. HPLC analysis
2.6.1. Phenolic acids and avonoids
The quantity of various phenolic acids and avonoids in the
methanol extracts of stem bark and leaf of S. crenata were determined using HPLC employing the method described by Amarowicz
et al. (2010) with minor modications. The HPLC system (Shimadzu
LC-6AD, Shimadzu Corporation, Japan) comprised a FCV-7AL reciprocating double-plunger type pump, a degasser, an autosampler
and a SPD-20A UVvis detector. Luna-5u C18 (Phenomenex
250 4.60 mm, i.d., 5 m) column was used for all separations.
The binary mobile phase comprised of deionized water: acetic acid
(98:2, v/v) (solvent A) and deionized water: acetonitrile: acetic
acid (78:20:2, v/v/v) (solvent B) that was pumped at a ow rate
of 1 ml/min for a total run time of 120 min, with the column temperature set at 25 C, and the sample injection volume of 20 l
(1 mg/ml). The elution gradient established was 010 min, 50% B;
1120 min, 60% B; 2130 min, 70% B; 3140 min, 80% B; 4150 min,
90% B; 51110 min, 100% B and 111120 min, 50% B. Detection was
employed at 280 nm as preferred wavelengths for the simultaneous monitoring of phenolic acids and avonoids. All the samples
were ltered in the sample ltration apparatus through a 0.45 m
PVDF membrane lter prior to HPLC injection. LC solution 2.1.
software was used for data acquisition, processing and reporting. Ten different standard compounds (gallic acid, caffeic acid,
ferulic acid, p-coumaric acid, catechin, apigenin, quercetin, rutin,
kaempferol and luteolin) were run using the same chromatographic
conditions described above for their detection in the sample
extracts through comparison. The calibration curves dened
for each compound were in the range of 0.54 g. Calibration
curves and correlation coefcients (R2 ) of the reference standard
compounds obtained are as follows: apigenin (y = 322.05x + 16;
R2 = 0.9995), caffeic acid (y = 109.58x + 4; R2 = 0.9980), catechin
(y = 295.14x 0.54; R2 = 0.9986), p-coumaric acid (y = 1050.2x 5.2;
R2 = 0.9979), ferulic acid (y = 177.69x + 0.052; R2 = 0.9992), gallic acid (y = 138.95x + 0.025; R2 = 0.9971), kaempferol (y = 1768.3x;
R2 = 0.9982), luteolin (y = 1279.5x + 1.174; R2 = 0.9985), quercetin
(y = 1899.4x 111.85; R2 = 0.9978) and rutin (y = 265.27x 21.48;
R2 = 0.9996). Compounds present in the plant extracts were identied by comparing their retention time and UVvis spectra with
those of standards and the quantities were calculated by comparing
peak area with those of standards.

Swiss albino mice (2530 g) used for the study were housed
under standard conditions of temperature (25 2 C), relative
humidity (3560%), 12 h/12 h light/dark cycle and fed with standard
diet (M/s. Hindustan Lever Ltd., Mumbai, India) and water ad libitum. Animals describes as fasted were deprived of food for 12 h, but
had free access to water. All animal experiments were conducted
with the permission from Institutional Animal Ethical Committee
of M/s. Manian Laboratories Pvt., Ltd., Coimbatore (Approval No.
ML-EA-CPCSEA/01-2013/01 Dt. 16.01.2013).
2.8. Acute toxicity
Acute oral toxicity studies were performed according to OECD
(Organization for Economic Co-operation and Development) guidelines (Ecobichon, 1997). Swiss albino mice (n = 6/dose) selected
by random sampling technique were employed in this study. The
animals were fasted for 12 h with free access to water only. The
methanol extract of stem bark and leaves of S. crenata suspended
in 1% carboxymethyl cellulose (CMC) were administered orally at
a dose of 5 mg/kg initially to mice and mortality was observed for
24 h. If mortality was observed in 4/66/6 animals, then the dose
administered was considered as toxic dose. However, if no mortality was observed or if the mortality was observed in only one mouse
out of six animals, then the extract treatment was repeated with
higher doses such as 50, 300, 1000 and 2000 mg/kg and mortality
observed. The behavior parameters such as motor activity, tremor,
convulsion, straub reaction, aggressiveness, pilo erection, loss of
lighting reex, sedation, muscle relaxation, hypnosis, analgesia,
ptosis, lacrimation, diarrhea and skin colour were also observed
for the rst hour and after 24 h of test drug administration.
2.9. Evaluation of analgesic activity
2.9.1. Acetic acid induced writhing test
The test was carried out according to the method described
by Siegmund et al. (1957) and Koster et al. (1959). This method
was used to preferentially evaluate possible analgesic effects of
methanol extracts of stem bark and leaf of S. crenata on the peripheral nervous system. Six groups of swiss albino mice (n = 6) were
fasted overnight prior to the start of experiment, and water ad
libitum. The peripheral analgesic drug, indomethacin was used as
positive control. Group 1 received the vehicle 1% CMC (10 ml/kg,
body weight p.o.), and group 2 was treated with indomethacin
(5 mg/kg, p.o.). Groups 3 and 4 were orally administered with
methanol extract of stem bark of S. crenata at doses of 200 and
400 mg/kg p.o., respectively, whereas groups 5 and 6 received
the leaf extract at doses 200 and 400 mg/kg, respectively. Thirty
minutes after treatment, the mice were injected (intraperitoneal)
with 0.1 ml of 1% acetic acid solution to induce the characteristic writhings. Writhing movement was recognized as contraction
of abdominal muscle together with stretching of hind limbs. The
mice were placed in an observation box, and the total number of
writhings occurring between 5 and 15 min after acetic acid injection was recorded. The percentage of protection was calculated

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

137

Table 1
Bioactive secondary metabolites of stem bark and leaf extracts of S. crenata.
Sample

BPE
BC
BEA
BM
BHW
LPE
LC
LEA
LM
LHW

Total phenolics
(mg GAE/g
extract)
25.27 0.4h
49.50 0.4g
218.41 2.7b
344.03 5.9a
89.55 1.3d
42.04 0.9g
60.45 6.5f
179.20 5.0c
172.74 9.6c
78.36 4.1e

Tannins (mg
GAE/g extract)

ND
6.01 2.8g
103.14 5.8b
147.16 4.6a
25.90 2.2e
ND
15.03 5.2f
56.99 2.2d
89.63 3.9c
20.77 1.2f

Total avonoids
(mg QE/g extract)

11.44 2.4g
29.33 0.2f
90.36 6.7c
273.44 3.9a
67.03 4.5d
9.77 6.4g
12.36 1.0g
50.67 4.7e
102.21 0.9b
35.69 1.4f

Total
proanthocyanidins
(mg CE/g extract)
ND
53.16 0.6f
95.58 0.4b
110.32 0.1a
41.68 0.3h
ND
77.05 0.9d
55.16 1.3e
92.63 1.4c
44.21 0.5g

Total monomeric
anthocyanins
(mg CGE/g
extract)
ND
0.70 0.01h
16.40 0.10c
77.71 0.06a
3.32 0.12g
ND
4.81 0.05e
7.46 0.08d
24.48 0.15b
3.90 0.03f

Vitamin E
(mg TE/g extract)

0.20 0.1f
0.48 0.1e
1.25 0.2c
2.98 0.1b
0.81 0.1d
0.41 0.1e,f
0.53 0.1e
2.89 0.1b
8.77 0.2a
0.84 0.1d

Values are means of three independent samples with triplicate determination of each standard deviation (n = 9). Mean values followed by different superscript letters in a
column indicate signicant statistical difference (p < 0.05).
BPE, BC, BEA, BM and BHW are, respectively, petroleum ether, chloroform, ethyl acetate, methanol and hot water extracts of bark of S. crenata.
LPE, LC, LEA, LM and LHW are, respectively, petroleum ether, chloroform, ethyl acetate, methanol and hot water of leaves of S. crenata.
ND not detected; GAE gallic acid equivalent; QE quercetin equivalent; CE catechin equivalent; CGE cyanidin 3-glucoside equivalent; TE -tocopherol equivalent.
Table 2

Scavenging of DPPH and ABTS + and inhibitory activity of acetylcholinesterase (AChE) of stem bark and leaf extracts of S. crenata.
Sample

Scavenging ofDPPH (IC50 g/ml)

Scavenging of ABTS + (mol trolox equivalent/g extract)

AChE inhibition(IC50 g/ml)

BPE
BC
BEA
BM
BHW

NA
111.54 0.4g
10.66 1.4b,c
8.89 0.2b
139.08 1.3h

NA
4.04 0.3h
6.09 0.5g
23.14 2.3c
13.02 0.1e

NA
76.713.2f
22.491.3c
9.203.4b
52.152.4e

LPE
LC
LEA
LM
LHW

NA
62.79 0.3f
11.02 0.1c
15.66 2.4c,d
53.15 5.2e

NA
5.69 0.2g
10.61 1.3f
15.87 0.5d
6.58 0.1g

NA
55.9 5.7e
39.86 1.1d
5.64 2.3b
43.9 2.6d

BHA
Galanthamine

2.52 0.6a

30.93 0.2b

0.58 0.07a

Values are means of three independent samples with triplicate determination of each standard deviation (n = 9). Mean values followed by different superscript letters in a
column indicate signicant statistical difference (p < 0.05).
BPE, BC, BEA, BM and BHW are, respectively, petroleum ether, chloroform, ethyl acetate, methanol and hot water extracts of bark of S. crenata.
LPE, LC, LEA, LM and LHW are respectively petroleum ether, chloroform, ethyl acetate, methanol and hot water extracts of leaves of S. crenata.
NA no activity. BHA (Butylated hydroxyanisole) and Galanthamine are standard reference compounds.

using the following formula: (control mean treated mean/control

mean) 100. The response of the extract and indomethacin treated
groups were compared with those of animals in the control
group.

2.9.2. Hot plate test

The hot plate assay (MacDonald et al., 1946) was employed
for the purpose of preferential assessment of possible centrally
mediated analgesic effects of methanol extracts of stem bark and
leaf of S. crenata. The central analgesic drug morphine, was used
as the positive control. For the experiment, six groups (n = 6) of
swiss albino mice were placed on a plate and maintained for
15 min at room temperature for environmental adaptation. Food
was withdrawn on the proceeding night of the experiment. The
control group received the vehicle (1% CMC, 10 ml/kg, p.o.), while
the reference group (positive control) was treated with the standard analgesic drug morphine (5 mg/kg, i.p.). Groups 3 and 4 were
administered with the methanol extract of stem bark of S. crenata at 200 and 400 mg/kg, p.o. respectively whereas the groups
5 and 6 received the leaf extract at 200 and 400 mg/kg respectively.
Each animal was then individually placed gently on Eddys hot
plate at 55 1.0 C. Latency to exhibit antinociceptive responses,
such as licking paws or jumping off the hot plate, was determined
15, 30, 45, 60, 90 and 120 min after administration of the test
substances.

2.9.3. Role of cholinergic system in the analgesic effect of S.

crenata
Possible participation of the cholinergic system in the analgesic
effect of extracts of S. crenata was assessed by antagonist studies.
Separate groups of mice were pre-treated with muscarinic acetylcholine receptor, atropine (1 mg/kg, i.p.) or nicotinic acetylcholine
receptor, mecamylamine (2 mg/kg, i.p.). After 20 min, the animals
were treated with morphine (5 mg/kg, i.p.) or galanthamine (16 mg/
kg, p.o.) or gallic acid (20 mg/kg, p.o.) or methanol extracts of stem
bark or leaf of S. crenata at a dose of 200 mg/kg, p.o. or vehicle (1%
CMC 10 ml/kg, p.o.). Thirty minutes after treatment, the mice were
injected (intraperitoneal) with 0.1 ml of 1% acetic acid solution to
induce the characteristic writhings. The mice were placed in an
observation box, and the total number of writhing behaviour occurring between 5 and 15 min after acetic acid injection was recorded.

2.10. Statistical analysis

For in vitro studies, the values are expressed as means of
three independent samples with triplicate determinations of each
(n= 9) standard deviation. Analysis of variance (ANOVA) and
signicant difference between means, determined by Duncans
multiple range test (p < 0.05). For in vivo studies, the results were
expressed as mean standard error means (S.E.M.). Statistical
analysis was carried out by ANOVA followed by Dunnett test.

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

p < 0.01 and p < 0.05 were considered as indicative of signicance,

as compared to the control group. All calculations were performed
using: SPSS, version 17.0 (Stat Soft Inc., Tulsa, OK).
3. Results
3.1. Bioactive secondary metabolites

0.7

0.6
0.5
0.4
0.3
0.2
0.1
1E-16

-0.2

-0.1 0

0.2
1/[S]

SBM 50 g/ml

(B)
1/V (mM-1/min)

The compounds of biological importance such as total phenolics,

tannins, total avonoids, total proanthocyanidins, total monomeric
anthocyanins and vitamin E estimated in various solvent extracts
of stem bark and leaf of S. crenata are summarized in Table 1. The
methanol extract of stem bark recorded the highest total phenolics, tannins and total proanthocyanidins (344.03 mg GAE/g extract,
147.16 mg GAE/g extract, 110.32 mg CE/g extract, respectively) followed by the ethyl acetate extract of stem bark. With regard to
total avonoids and total monomeric anthocyanins, the methanol
extract of bark (273.44 mg QE/g extract, 77.71 mg CGE/g extract,
respectively) followed by the methanol extract of leaves (102.21 mg
QE/g extract, 24.48 mg CGE/g extract, respectively) recorded the
maximum contents. Further, the methanol extract of leaf contained
considerable amounts of vitamin E (8.77 mg TE/g extract) compared to the other solvent extracts. However, the petroleum ether
extracts of both bark and leaves were devoid of tannins, total proanthocyanidins and total monomeric anthocyanin contents.

(A)
1/ V (mM-1/min)

138

0.4

(mM-1)
SBM 100 g/ml

Control

1
0.8
0.6
0.4
0.2
0

-0.1

-0.2

0.1

0.2

0.3

0.4

0.5

1/[S] (mM-1)

3.2. In vitro antioxidant activity

LM 50 g/ml

DPPH radical scavenging activity, through the donation of

hydrogen, forming the reduced DPPH-H is an extensively used
method for screening antioxidants. As shown in Table 2, all the
extracts with the exception of petroleum ether extracts were
capable of scavenging DPPH radicals, which increased with increasing concentrations. The methanol extract of stem bark (IC50
8.89 g/ml) and ethyl acetate extracts of both stem bark and leaf
(respectively, IC50 10.66 g/ml and IC50 11.02 g/ml) showed comparable levels (p < 0.05) of DPPH scavenging, with the lower IC50
values indicating higher activity. Similarly, higher scavenging activ
ities of ABTS + depicting the antioxidant capacity of both lipophilic
and hydrophilic substances was seen in the methanol extracts of
stem bark (23.14 mol trolox equivalent/g extract) followed by leaf
(15.87 mol trolox equivalent/g extract) (Table 2). However, the
standard antioxidant BHA recorded the highest antiradical activity.
3.3. AChE inhibitory activity
Results of AChE enzyme inhibitory activity of various solvent
extracts of stem bark and leaf of S. crenata, expressed as IC50
values are presented in Table 2. All the solvent extracts of S.
crenata, except petroleum ether extracts of both stem bark and
leaf registered inhibitory activity towards AChE. The percentage
inhibition exhibited was found to be concentration dependent
which increased with increasing concentrations of the extracts.
Pronounced inhibitory activity was seen in the methanol extracts of
leaf and stem bark (IC50 5.64 and 9.20 g/ml, respectively) followed
by the ethyl acetate extract of stem bark (IC50 22.49 g/ml). The
positive control galanthamine recorded the strongest inhibition of
AChE (IC50 0.58 g/ml).
Enzyme kinetic study was performed using the methanol
extracts of stem bark and leaf of S. crenata, as they showed a marked
inhibition of AChE. The mode of inhibition of these extracts was
elucidated using the LineweaverBurk plot (LB plot) (Fig. 1 and
Table 3). Parameter estimates were obtained from specic velocity
(Vmax ) and MichaelisMenton (Km ) values, from plotting the reciprocal of enzyme velocity versus the reciprocal of acetylthiocholine
iodide concentration in the absence and presence of inhibitors.

LM 100 g/ml

Control

Fig. 1. LineweaverBurk double reciprocal plot of substrate dependent enzyme

kinetics showing inhibition of acetycholinesterase by methanol extracts of (A) stem
bark and (B) leaf of S. crenata.

Table 3
Kinetic constants of AChE activity in the presence/ absence of methanol extracts of
stem bark and leaf of S. crenata.
Sample

Concentration(g/ml)

Vmax (mM/min)

Km (mM)

Control
SBM
SBM
LM
LM

0
50
100
50
100

1.82
1.79
1.66
1.83
1.82

33.82
33.81
33.80
34.08
34.58

SBM and LM are, respectively, methanol extracts of stem bark and leaf of S. crenata.
Control absence of plant extracts.

From the results it is evident that the methanol extract of stem bark
signicantly inhibited AChE activity causing a lower Vmax (1.79 and
1.66 mM/min at concentrations of 50 and 100 g/ml, respectively)
than that of the control group (1.82 mM/min). However, Km in the
presence of sample extracts (33.81 and 33.80 mM at concentrations
of 50 and 100 g/ml, respectively) was not signicantly different
from the control (33.82 mM). As such, the values indicate that the
methanol extract of stem bark exhibited a non-competitive mechanism of AChE inhibition. Whereas, the methanol extracts of leaves
did not change the Vmax (1.83 and 1.82 mM/min at concentrations
of 50 and 100 g/ml, respectively) and were similar to the control group (1.82 mM/min) but increased its Km values (34.08 and
34.58 mM at concentrations of 50 and 100 g/ml, respectively) than
that of control (33.82 mM) suggesting a competitive mechanism of
inhibition.
3.4. HPLC analysis
The methanol extracts of stem bark and leaf of S. crenata were
subjected to HPLC analysis for the identication and quantication of certain polyphenolic compounds present in them. The

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

139

Table 4
Retention time (Rt) and quantication of the phenolic compounds present in the methanol extracts of stem bark and leaf of S. crenata.
Peak

1
2
3
4
5
6
7
8
9
10
11

Compound

Apigenin
Caffeic acid
Catechin
p-Coumaric acid
Ferulic acid
Gallic aid
Kaempferol
Luteolin
Quercetin
Rutin
Betulinic acid

Stem bark

Leaf

Rt

Content
(g/mg extract)

Rt

Content
(g/mg extract)

63.42
13.42

24.06
35.14
3.29
57.23
100.26
86.51
6.05

1.78 0.02
1.98 0.06
ND
2.67 0.03
2.74 0.09
0.19 0.00
1.26 0.20
2.56 0.03
1.29 0.01
0.51 0.07
ND

63.01
13.43
4.92

34.89
3.30
55.03
100.10
86.51
6.00
9.38

3.01 0.17
0.57 0.00
0.82 0.03
ND
0.91 0.01
1.12 0.01
2.61 0.13
2.09 0.02
1.03 0.05
0.68 0.01
1.11 0.07

Contents are expressed as mean standard deviation (n = 3). ND not detected.

3.5. Acute toxicity

As the methanol extract from stem bark and leaf of S. crenata
contained considerable amounts of non-enzymatic antioxidants,
showed higher antioxidant activity and effectively inhibited AChE,
the extracts were chosen for further in vivo investigations.
The methanol extracts of both stem bark and leaf of S. crenata
when evaluated for acute toxicity in swiss albino mice neither
showed any deleterious effect nor altered the general behaviour
during the 24 h period of observation. Mice administered with the
test drug extracts failed to produce any mortality even at the highest dose (2000 mg/kg) studied and were found to be safe. Based on
acute toxicity study, two doses i.e., 200 and 400 mg/kg were used
for analgesic investigations.
3.6. Analgesic activity
3.6.1. Acetic acid induced writhing
The effect of stem bark and leaf extracts of S. crenata on acetic
acid induced writhings are presented in Table 5. Pretreatment with
methanol extracts of stem bark and leaf (200 and 400 mg/kg, p.o.)
signicantly (p < 0.01) reduced the number of writhes in a dose
dependent manner. The oral administration of methanol extract
Fig. 2. HPLC chromatograms of methanol extracts of (A) stem bark and (B) leaf of S.
crenata at 280 nm.

concentrations of the phenolic acids, avonoids and betulinic acid

present in the methanol extracts of stem bark and leaf of S. crenata
are shown in Figs. 23 and Table 4. The HPLC chromatograms of
the stem bark and leaf, obtained under optimum conditions, displayed the presence of 9 and 10 compounds, respectively, which
were detected at 205 and 280 nm. Among the phenolic acids, ferulic
acid (2.74 g/mg extract) followed by p-coumaric acid (2.67 g/mg
extract) and caffeic acid (1.98 g/mg extract) were found to be at
higher concentrations in the extract of stem bark than the leaves.
However, gallic acid recorded its maximum content in the extract of
leaves than stem bark (1.12 and 0.19 g/mg extract, respectively).
Caffeic acid existed in smaller amounts (0.57 g/mg extract) and
p-coumaric acid was not detected in the methanol extract of leaf
of S. crenata. With regard to avonoids, leaves recorded higher
amounts of apigenin and kaempferol than stem bark (respectively,
3.01 and 2.61 g/mg extract for leaves, and 1.78 and 1.26 g/mg
extract for stem bark). Both the stem bark and leaf recorded similar levels of luteolin (respectively, 2.56 and 2.09 g/mg extract),
quercetin (respectively, 1.29 and 1.03 g/mg extract) and rutin
(0.51 and 0.68 g/mg extract, respectively). In the case of betulinic
acid, leaf displayed a concentration of 1.11 g/mg extract but was
not detected in the stem bark of S. crenata.

Fig. 3. HPLC chromatograms of methanol extracts of (A) stem bark and (B) leaf of S.
crenata at 205 nm.

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R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

Table 5
Analgesic effect of methanol extracts of stem bark and leaf of S. crenata in acetic acid
induced writhing model.
Treatment groupb

Dose(p.o.)

No. of writhesa

Induced control (vehicle)

Indomethacin
SCL
SCL
SCB
SCB

10 ml/kg
5 mg/kg
200 mg/kg
400 mg/kg
200 mg/kg
400 mg/kg

65.33
6.50
17.17
12.83
23.50
17.17

2.16
1.05**
1.47**
1.47**
1.38**
2.14**

Inhibition (%)

90.05
73.72
80.36
64.03
73.72

Values are expressed as mean standard error mean (n = 6).

*
p < 0.05.
**
p < 0.01 compared to the induced group.
a
Observed between 5 and 15 min after induction by acetic acid injection.
b
Induced control vehicle 1% CMC; SCL and SCB are, respectively, methanol
extracts of leaf and stem bark of S. crenata.

of leaf and bark at higher dose of 400 mg/kg reduced the number of writhings from 65.33 (induced) to 12.83 (80.36%) and 17.17
(73.72%), respectively. However, the standard drug indomethacin
signicantly diminished the number of writhings by 90.05% (6.50)
when compared to the plant extracts.
3.6.2. Hot plate test
The latency response assessed using hot plate test demonstrated
that the methanol extracts of leaf and stem bark of S. crenata caused
a signicant (p < 0.01) increase in the response latency time at
30 min which persisted till the end of experiment period (120 min)
with the maximum analgesia at 60 min. The plant sample extracts
(200 and 400 mg/kg, p.o.) exhibited a dose dependent activity
which increased the latency of response, however, slightly lower
than morphine (5 mg/kg) without affecting the animals ability to
detect the thermal pain threshold (Fig. 4).
3.6.3. Involvement of cholinergic system in the analgesic effect of
S. crenata
As shown in Fig. 5 pre-treatment of animals with the antagonist of cholinergic system atropine and mecamylamine completely
reversed/ attenuated the analgesic effect caused by morphine when
analysed in terms of number of writhings in acetic acid test model
in mice. The analgesic response was not greatly modied in the
galanthamine treated group, however, they did show writhing to
an extent of 9.27% and 21.38% respectively upon pre-treatment
of atropine and mecamylamine. Interestingly, gallic acid treated
group showed an appreciable modication of analgesic response in
the presence of the muscarinic and nicotinic acetylcholine receptors (54.24% and 50.01%, respectively). Under the same condition,
the methanol extracts of leaf and stem bark of S. crenata at a
dose of 200 mg/kg, p.o. showed a decreased analgesic response,
wherein the extract of leaf exhibited a larger number of writhings
(41.73% and 30.42% respectively upon pre-treatment with atropine
and mecamylamine) when compared to the stem bark (35.29%
and 31.80%, respectively, upon pre-treatment with atropine and
mecamylamine).
4. Discussion
Plants/plant products contain a wide range of bioactive
molecules, such as, phenolic compounds, terpenoids, vitamins,
carotenoids and phytomicronutrients which individually or synergistically have been claimed as natural health protecting agents

(Podsedek,
2007). In the present investigation, various solvent
extracts of the stem bark and leaf of S. crenata were assessed for
the contents of bioactive secondary metabolites. Among them, the
methanol extracts registered higher levels of total phenolics, tannins, total avonoids, total proanthocyanidins, total monomeric
anthocyanins and vitamin E contents (Table 1). HPLC analysis also

indicated the presence of phenolic acids (caffeic acid, ferulic acid,

gallic acid, p-coumaric acid), avonoids (apigenin, catechin, luteolin, quercetin, kaempferol, rutin) and a pentacyclic triterpeniod,
betulinic acid in the methanol extracts of stem bark and leaf of
S. crenata, however in variable amounts (Figs. 23 and Table 4).
In addition to it, the methanol extracts also demonstrated excel
lent scavenging of DPPH and ABTS + in vitro (Table 2). From these
results, it is assumed that the presence of bioactive secondary
metabolites in the extracts might have contributed to the radical scavenging activity of S. crenata. In similar lines, Yokozawa
et al. (1998) have demonstrated that the polyphenolic compounds,
such as phenolic acids, avonoids and tannins reduced and decolorized DPPH by their hydrogen donating ability. Further, according
to Hagerman et al. (1996) the high molecular weight phenolics

(tannins) have more ability to quench free radicals (ABTS + ) and

their effectiveness depends on the molecular weight, the number
of aromatic rings and nature of hydroxyl group substitution than
the specic functional groups. Besides, Smitha et al. (2012) have
reported that the methanol fractions from the stem bark of S. crenata demonstrated high DPPH scavenging activity. In addition to it,
Mosaddik et al., (2004) have shown that the methanol extract from
the stem bark of another species of Scolopia (S. braunii) exhibited

the highest ABTS + scavenging activity. Therefore, the phytochemicals present in S. crenata have the potential to prevent and offer
resistance against free radicals induced disorders.
Inhibition of the enzyme AChE serves as a strategy in several
therapeutic applications such as Alzheimers disease, senile dementia, ataxia and myasthenia gravis. The various solvent extracts from
stem bark and leaves of S. crenata were assessed for AChE inhibitory
activity. Besides conferring antioxidant activity, the methanol
extracts of stem bark and leaf also inhibited AChE effectively
in vitro (Table 2). It is to be noted that both the extracts recorded
remarkable levels of non-enzymatic antioxidants which might have
contributed to the AChE inhibitory activity (Table 1). Recently,
several polyphenols such as phenolic acids and avonoids, anthocyanins and vitamin E with strong antioxidant activity have been
reported to play a pivotal role in preventing the pathophysiology of
neurodegenerative diseases (Lloret et al., 2009; Zhang et al., 2009;
Shih et al., 2010). Substrate dependent kinetic studies carried out to
understand the nature of inhibition by the methanol extracts of S.
crenata showed them to be non-competitive in stem bark (Fig. 1a)
while competitive for leaves (Fig. 1b). A competitive inhibitor is
usually a substrate analog that binds at the active site of enzyme.
The competitive inhibitor and substrate are mutually exclusive
and generally compete for the same active site on an enzyme.
Non-competitive inhibitor combines with an enzyme molecule,
regardless of whether a substrate molecule is bound or not to the
enzymes active sites and alters the conformation of the enzyme
and reduces its catalytic activity. Several AChE inhibitors are found
to be competitive, however non-competitive AChE inhibitors are
also reported (Nakagomi et al., 2000; Wang et al., 2011). In general, the standard drug galantamine has been shown to exhibit
a competitive inhibition pattern whereas, tacrine showed noncompetitive inhibition pattern with a reduction in a maximum
velocity (Vmax ) (Chung et al., 2001; Mukherjee et al., 2007). On considering the chemical prole of active compounds in the methanol
extracts of stem bark and leaf of S. crenata, stem bark recorded
higher amounts of ferulic acid (2.74 g/mg extract) while gallic acid
was found in higher amounts in leaf (1.12 g/mg extract) (Table 4).
Further, the presence of catechin and betulinic acid was found only
in the leaf and p-coumaric acid in the stem bark extracts. It has been
reported that ferulic acid is a competitive inhibitor of AChE (Kumar
et al., 2009), whereas upon connecting ferulic acid to a tacrine template, forming a tacrine-ferulic acid hybrid resulted in a reversible
and non-competitive AChE inhibitory activity (Fang et al., 2008).
Similarly, gallic acid showed a competitive inhibition on AChE (Kim

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

141

Fig. 4. Analgesic activity of the methanol extracts of stem bark and leaf of S. crenata as determined by the hot plate model.

Fig. 5. Analgesic effect of the methanol extracts of stem bark and leaf of S. crenata assessed in the presence of antagonist of cholinergic system (A) atropine and (B)
mecamylamine.

et al., 2011), while upon grafting gallic acid onto chitosan presented
a non-competitive mode of AChE inhibition (Cho et al., 2011). Thus
it is evident that the difference in mode of AChE inhibition exerted
in stem bark and leaf extract is a product of synergistic action of
different compounds present in these plant parts. Therefore, the
result of the present study implies that the methanol extract of
stem bark binds at a different site from the substrate, while the
methanol extract of leaf compete with the substrate, thereby acting
as AChE inhibitors. However, the exact mechanism of interaction of
polyphenols with the cholinergic system is still not clear (Ebrahimi
and Schluesener, 2012).
Analgesic properties of cholinesterase inhibitors have been
known for several decades (Hartvig et al., 1989). Although the
clinical utility of synthetic drugs has been limited due to severe
adverse effects, optimistically the advances in the eld of herbal
medicine have counteracted these problems. The medicinal plant S.
crenata, used as a sedative in herbal formulations and traditionally
employed to treat musco-skeletal pain, was assessed for analgesic
activity employing acetic acid induced writhing test and hot plate
test. As the methanol extracts from stem bark and leaves conferred
remarkable antioxidant and AChE inhibitory activity, they were
selected for further in vivo investigations.
Acetic acid induced writhing test is a widely used model to
study the peripheral analgesic effects of drugs (Koster et al., 1959).
The vicerosomatic pain as a response of increased levels of mediators such as bradykinins, prostaglandins and pro-inammatory
cytokines produced by intraperitoneal injection of acetic acid was
signicantly (p < 0.05) reduced upon administration of leaf and
stem bark extracts of S. crenata in a dose-dependent manner
(Table 5). The reduced number of writhings at a high dose of

400 mg/kg of leaf and stem bark extracts substantiates an effective

peripheral analgesic property probably through the inhibition of
prostaglandin and/or other pain mediators. A promising analgesic
drug is said to act on both peripheral and central nervous systems
limiting the tissue damage. Therefore, to obtain a clear perspective
on the analgesic response of methanol extracts of stem bark and
leaf of S. crenata, hot plate test method was employed to assess the
central thermal analgesic activity. The extracts of S. crenata significantly (p < 0.05) increased the latency response of animals in hot
plate when compared to untreated control (Fig. 4). Therefore, these
results made it explicit that the methanol extracts of stem bark and
leaf of S. crenata possess both peripheral and central analgesic activity, wherein the extract of leaves exhibited a slightly higher activity
than the stem bark. However, the observed activities of the plant
extracts were lower than the standard drugs, indomethacin and
morphine (Table 5, Fig. 4).
The possible mode of analgesic activity of methanol extracts
of leaf and stem bark of S. crenata was assessed using antagonists especially atropine, a muscarinic acetylcholine receptor and
mecamylamine, a nicotinic acetylcholine receptor of cholinergic
system (Fig. 5). In addition to plant extracts, central analgesic drug,
morphine; an effective acetylcholinesterase inhibitor drug, galanthamine; and an antioxidant agent, gallic acid was also evaluated
to ensure their involvement towards the antagonist response in
the cholinergic system (Fig. 5). The results made it evident that
upon pre-treatment with both the antagonists, the effect of morphine was found to be completely attenuated (Chen and Pan,
2001), whereas galanthamine showed a slight presence of analgesic
response. Gallic acid showed an appreciable antagonist response in
both the acetylcholine receptor system tested. However, in the case

142

R. Gomathi, S. Manian / Industrial Crops and Products 73 (2015) 134143

of plant extracts, they did reverse the analgesic response however,

showing a decreased level of analgesic activity. Taking all these
into consideration, it could be anticipated that analgesic activity
involves a diverse array of physiological process, wherein morphine
and gallic acid capable of inhibiting acetylcholinesterase (Bhargava
and Way, 1972; Cho et al., 2011; Kim et al., 2011) are also capable of modication in the presence of antagonist of cholinergic
system (Fig. 5), with an exception being only galanthamine, exhibiting a very slight modication. Therefore, the analgesic responses
mediated by the leaf and stem bark extracts of S. crenata, showing
acetylcholinesterase inhibitory property may partly be associated
to the cholinergic system and to antioxidative property. It is to
be noted that in addition to cholinergic systems, analgesia also
involves several other interactions such as dopaminergic, glutamatergic, GABAergic and tachykinergic pathways (De Souza et al.,
2009). Thus, the mechanism governing the analgesic response of
extracts of S. crenata requires several pharmacological interaction
and several pathways of neurotransmission and neuromodulation
studies. Further, in the present study, the reason behind the leaf and
stem bark extracts of S. crenata exhibiting both acetylcholinesterase
inhibition and involvement at least in part, to the cholinergic system remains yet to be elucidated.
It must be taken into account that the doses (200 and 400 mg/kg,
p.o.) of S. crenata used were crude drug and not a synthetic
compound. Therefore, the synergistic effect of different bioactive
compounds present in the extract should also be taken into account.
Further, as the oral route of drug administration was employed,
the bioavailability of compounds in extracts in reaching blood and
tissues bypassing the gastro-intestinal digestion should also be
considered. A large number of different kinds of natural compounds
in plants with analgesic activity have been reported earlier. As
suggested from a number of studies, proanthocyanidins, vitamin
E, phenolic acids, avonoids and tannins are found to promote
analgesic activity (Edmonds et al., 1997; Bone and Mills, 2013;
Miller et al., 2001; Zhang et al., 2009). In particular, avonoids from
medicinal plants have been reported to bind directly to the opiate
receptors and inhibit neuronal transmitter receptors from activating pain neurons and exhibit analgesic activity (Havsteen, 2002).
Gallic acid has been found to be a potent transient receptor potential ankyrin I antagonist implicating analgesic activity (Trevisan
et al., 2014). Ferulic acid exerts analgesic effect through regulating
monoaminergic system, oxidative/ antioxidant defense, inammatory and apoptotic pathway (Xu et al., 2013). The quantication
of bioactive secondary metabolites (Figs. 23, Table 4) shows the
presence of these compounds in higher amounts in the methanol
extracts of leaf and stem bark of S. crenata which might contribute to
the analgesic activity thereby, validating its traditional usage. Further, the presence of the above mentioned observed compounds
(Figs. 23, Table 4) in both the methanol extracts of stem bark and
leaf suggests that S. crenata may act as a potential target for the
development of novel analgesics. The central and peripheral analgesia mediated by the inhibition of acetylcholinesterase has also
been reported earlier (Buerkle et al., 1998). Precisely, owing to the
observed effective acetylcholinesterase inhibition (Table 2, Fig. 1ab), and involvement in part to the cholinergic system (Fig. 5), S.
crenata extracts could be anticipated to modulating the analgesic
activity at the level of spinal cord (Jones and Dunlop, 2007).
Oxidative stress has been implicated in the pathology of several
diseases. Reactive oxygen species, especially superoxides and its
reaction product peroxynitrite have been shown to be important
in the development of pain of several etiologies (Janes et al., 2012).
Further, the free oxygen radicals damage cellular micro-organelles
directly, in particular damage mitochondria contributing to a deciency of cholinergic neurotransmitters (Lin and Beal, 2006; Cho
et al., 2011). Therefore, augmenting antioxidants could be a promising practical approach in pain management and inhibiting the

degradation of acetylcholine via acetylcholinesterase inhibition

in neurodegenerative disorders. In addition, intrinsic cholinergic
inhibitory pathways also present a key modulating system in pain
perception. In line with all these, the methanol extracts of leaf and
stem bark were found to exhibit a remarkable antioxidant (Table 2),
AChE inhibition (Fig. 1ab) and central and peripheral analgesic
activity (Fig. 4 and Table 5). The extracts also showed remarkable
inhibition of superoxide radicals and metal chelating property (data
not shown). Polyphenols, especially phenolic acids and avonoids
confer effective antioxidant activity owing the number of phenolic hydroxyl groups attached to their ring structures. Furthermore,
this function of number and position of OH groups which can form
hydrogen bonds with specic amino acids at the enzymes active
sites produces the cholinesterase inhibitory effects of polyphenols
(Orhan et al., 2007). Hence, based on the above ndings it may
be proposed that the extracts of S. crenata producing analgesia
could be associated to: (i) acetycholinesterase inhibition resulting
in increased concentrations of acetylcholine along with involvement of cholinergic system, in part and subsequently analgesia
by the action of acetylcholine in the cord; and (ii) counteracting
free radicals, as high levels of reactive oxygen species are involved
in persistent pain, especially neuropathic and inammatory pain
(Yowtak et al., 2011).
5. Conclusion
The results of the present study has demonstrated the radical
scavenging ability, AChE inhibition and analgesic activity in the
leaf and stem bark of S. crenata, providing a scientic basis for its
traditional usage for alleviating muscular pain and employment as
sedative. Analgesic effects may be proposed to be mediated through
the AChE inhibition, involvement of cholinergic system, in part
and free radical scavenging properties. However, additional experiments are necessary to further conrm this hypothesis. The present
work suggests that S. crenata can be a potent source of promising,
inexpensive phytotherapeutic agent in the near future.
Conict of interest
Both the authors declare no conict of interest.
Acknowledgement
This work is funded by Department of Science and Technology (DST), Government of India through the INSPIRE Programme
(Letter No. DST/INSPIRE Fellowship/2011 dated 29 June 2011) to R.
Gomathi (IF110194). The authors are grateful to Dr. K. Kadirvelu and
Dr. P.V.L. Rao DRDO, Bharathiar University, Center for Life Sciences
for rending HPLC facility.
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