Professional Documents
Culture Documents
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
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).
136
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
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
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.
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)
(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)
LM 100 g/ml
Control
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
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
Fig. 3. HPLC chromatograms of methanol extracts of (A) stem bark and (B) leaf of S.
crenata at 205 nm.
140
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
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
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
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
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
142
143
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