Analgesic, anti-inflammatory and anti-platelet activities of the methanolic extract of Acacia...

RESEARCH ARTICLE

Analgesic, anti-inflammatory and anti-platelet activities of themethanolic extract of Acacia modesta leaves

Ishfaq A. Bukhari • Rafeeq A. Khan •

Anwar H. Gilani • Sagheer Ahmed •

Sheikh Arshad Saeed

Received: 11 January 2010 / Accepted: 24 March 2010 / Published online: 15 April 2010

� Springer Basel AG 2010

Abstract The current study was aimed to evaluate Acacia

modesta for analgesic, anti-inflammatory, and anti-platelet

activities. The analgesic and anti-inflammatory effects were

assessed in rodents using acetic acid and formalin-induced

nociception, hot plate and carrageenan-induced rat paw

oedema tests. The intraperitoneal (i.p.) administration of the

methanolic extract (50 and 100 mg/kg) produced significant

inhibition (P \ 0.01) of the acetic acid-induced writhing in

mice and suppressed formalin-induced licking response of

animals in both phases of the test. In the hot plate assay the

plant extract (100 mg/kg) increased pain threshold of

mice. Naloxone (5 mg/kg i.p.) partially reversed the anal-

gesic effect of the extract in formalin and hot plate tests.

A. modesta (100 and 200 mg/kg i.p.) exhibited sedative

effect in barbiturate-induced hypnosis test similar to that

produced by diazepam (10 mg/kg i.p.). The plant extract

(50–200 mg/kg i.p.) produced marked anti-inflammatory

effect in carrageenan-induced rat paw oedema assay com-

parable to diclofenac and produced a dose-dependent

(0.5–2.5 mg/mL) inhibitory effect against arachidonic acid

induced platelet aggregation. These data suggest that

A. modesta possesses peripheral analgesic and anti-

inflammatory properties, with analgesic effects partially

associated with the opioid system.

Keywords Acacia modesta � Analgesic �Anti-inflammatory � Anti-platelet � Writhing test �Formalin test � Carrageenan-induced paw oedema

Introduction

Acacia modesta belongs to family Mimosaceae, commonly

known as Phulahi and locally called palosa. It is a medium-

sized tree that grows on stony grounds, and widely found in

different parts of India and Pakistan including Punjab,

NWFP, and Baluchistan (Baquar 1989). Various parts of

A. modesta such as gum, leaves, flowers, sticks, and wood

are used for several medicinal purposes. The plant is used

in the treatment of dysentery, leprosy, oral toothache, tra-

choma, venereal diseases, and wounds, and its twigs are

used as tooth brush for cleansing teeth (Atta-ur-Rahman

et al. 1986; Chopra et al. 1956; Lewis and Elvin-Lewis

1976; Nadkarni 1976). Aqueous preparation of the fresh

leaves of the plant is used for treating sore eyes and cata-

ract (Author’s personal observations).

Plants belonging to genus Acacia are widely used in the

management of pain and inflammation in folk medicine

system. A. farnesiana roots, bark, and leaves are used in

inflammatory conditions, ulcer, and wounds (Wiart 2002).

Various parts of A. nilotica are utilized for treating gon-

orrhea, leucorrhea, bleeding ulcers, wounds, and dysentery

(Kapoor 1990). A. pennata is employed as a remedy for

headache, rheumatism, and fever (Dongmo et al. 2005). At

least five species of Acacia are used in traditional system of

Kenya in stomachache, boils, swelling, and eye infections

(Geisler et al. 2003).

I. A. Bukhari (&)

Section Pharmacology, Shifa College of Medicine,

Islamabad, Pakistan

e-mail: [email protected]

I. A. Bukhari � R. A. Khan

Department of Pharmacology, Faculty of Pharmacy,

University of Karachi, Karachi, Pakistan

I. A. Bukhari � A. H. Gilani � S. Ahmed � S. A. Saeed

Department of Biological and Biomedical Sciences,

Aga Khan University and Medical College, Karachi, Pakistan

Inflammopharmacol (2010) 18:187–196

DOI 10.1007/s10787-010-0038-4 Inflammopharmacology

Pharmacological investigations of various species of

genus Acacia have revealed hypoglycemic effect of

A. modesta (Singh et al. 1975), anti-inflammatory (Dafallah

and Al-Mustafa 1996) and anti-platelet (Shah et al. 1997)

activities of A. nilotica, analgesic and anti-inflammatory

(Dongmo et al. 2005) effects of A. pennata. Some species

of Acacia such as A. adsurgens, A. ancistrocarpa, and

A. catechu have been shown to exhibit cyclooxygenase

inhibitory effects (Li et al. 2003). Flavans from genus

Acacia are reported for their inhibitory effects against

cyclooxgenase and 5-lipoxygenase (Jia et al. 2003).

The genus Acacia is a rich source of bioactive terpe-

noids and flavonoids (Garai and Mahato 1997; Seigler

2003).

In view of the wide use of the genus Acacia in pain and

inflammation the current study was undertaken to investi-

gate analgesic, anti-inflammatory, and anti-platelet

activities of A. modesta plant extract.

Materials and methods

Animals

NMRI (20–30 g) and Sprague-Dawley (200–270 g) of

either sex were obtained from the animal house facility

of The Aga Khan University, Karachi and Department

of Pharmacology, Faculty of Pharmacy, University of

Karachi. The use of experimental animals complied with

the rulings of the Institute of Laboratory Animal Resources,

Commission on Life Sciences, National Research Council

(NRC, 1996), approved by the Ethical Committee of the

Aga Khan University, Karachi, Pakistan. The animals were

housed in plastic cages under standard condition with 12 h

light: dark cycle with free access to food and water.

Chemicals

The following chemicals were used in the experiments.

Acetic acid, arachidonic acid, carrageenan, diclofenace

sodium, indomethacin, and naloxone were purchased from

Sigma Chemical Co. (St. Louis, MO, USA), Formaline

37% was obtained from Fluka Chemie, Switzerland,

Morphine sulphate (MS Contin) from local pharmacy,

while thiopental sodium and diazepam were obtained from

Abbott Laboratories and Roche Pharma, Karachi, Pakistan.

Plant material

The fresh leaves of A. modesta (family Mimosaceae) were

collected in June 2004 from the village of Mandoori

(Kohat), Pakistan. The plant material was identified by

Taxonomist at Department of Botany, University of

Karachi, Pakistan, and voucher specimen No. 68217 has

been deposited in the herbarium of the same department.

Extraction procedure

The shade-dried leaves of the plant were ground or crushed

into small pieces and soaked separately in 70% methanol for

a week with occasional shaking. The soaked material was

then filtered through muslin cloth. The process of shaking

and filtration was repeated thrice and the filtrates were then

pooled and dried by rotary evaporator. The crude extract

thus obtained, as a thick semisolid mass, was stored in the

refrigerator for use in the various experimental protocols.

Phytochemical analysis

The methanolic extract of A. modesta was screened for the

presence of various phytochemical constituents such as

alkaloids, flavonoids, saponins, and tannins according to

the previously described method (Edeoga et al. 2005;

Sofowora 1993).

Acute toxicity test

Different doses of the methanolic extract of A. modesta

(50, 100, 300, 500, 700, 1,000, 1,500, 1,800 mg/kg) were

injected intraperitoneally (i.p.) to mice (20–25 g) divided

into separate groups, each consisting of six animals. The

animals were observed for 1–2 h after administration of the

extract for any acute toxicity symptoms, e.g., behavioral

symptoms. The number of deaths was counted at 48 h after

treatment. LD50 value (dose of the extract producing

mortality in the 50% of the experimental animals) was

determined by graphical method.

Writhing test

Male adult mice (20–25 g, n = 6–20) were used in this

experiment according to the method of Koster et al. (1959).

Animals were randomly divided into three groups and the

treatment to each group was given as follows:

Group I: Control (vehicle treated).

Group II: Pre-treated with diclofenac sodium (reference

drug).

Group III: Pre-treated with different doses A. modesta

extract (50 and 100 mg/kg i.p.).

Mice were given an i.p. injection of 0.7% v/v acetic acid

solution (volume of injection 0.1 mL/10 g) 30 min after

the administration of the plant extract or diclofenac

sodium, placed individually into glass beakers followed by

a 5-min lapse. The number of writhes produced in these

animals was counted for 20 min. For scoring purposes, a

188 I. A. Bukhari et al.

writhe was indicated by stretching of the abdomen with

simultaneous stretching of at least one hind limb. Control

animals received normal saline (10 ml/kg i.p.), and dic-

lofenac (10 mg/kg i.p.) was used as a reference drug.

Formalin test

The formalin-induced pain test was performed according to

the method described by Hunskaar and Hole (1987); mice

(20–25 g, n = 6–20) were grouped as described above and

injected, 20 ll of 1% formalin in 0.9% saline, subcutane-

ously into the dorsal hind paw and placed immediately in

transparent box for observation. The duration of paw

licking was determined between 0–5 min (first phase) and

15–30 min (second phase) after formalin injection. The

time in seconds spent in licking and biting responses of the

injected paw was noted. Animals were treated (i.p.) with

different doses of the plant extract (50 and 100 mg/kg) or

diclofenac sodium (10 mg/kg i.p.) or morphine (5 mg/kg

i.p.) 30 min prior to administration of formalin. Naloxone

(5 mg/kg i.p.) was administered 20 min prior to the extract

or morphine injections. Control animals received the

vehicle (0.1 mL/10 g). The paw-licking time of the ani-

mals was compared with that of control group and

represented as percent inhibition.

Hot plate test

The hot plate assay of the plant extract was conducted

according to the method as described by Eddy and Leim-

back (1953), with slight modification. The temperature of

the hot plate was maintained at 52 ± 0.8�C. Mice (20–

25 g, n = 6–10) were individually placed on the hot plate

and the latency to a discomfort reaction (licking of the paw

or jumping) was noted. The cut off time was 20 s to avoid

thermal injury of the paw. The animals were selected 24 h

before the experiments on their reaction time to the hot

plate test. Animals were given (i.p.), plant extract (100 mg/

kg) or morphine (5 mg/kg), 30 min before their placement

on the hot plate. Animals were then observed before and

0.5, 1, 2, and 3 h after the administration of plant extract or

morphine. Naloxone (5 mg/kg i.p.) was given 20 min

before the administration of plant extract or morphine;

control animals received same volume of vehicle. The

latency time in second of the control animals was com-

pared to that of treated animals.

Thiopental induced hypnosis test

The experiment was carried out according to the method

described by Hosseinzadeh and Nassiri Asl 2003. Mice

(20–25 g, n = 6–15) were given different doses of plant

extract (100 and 200 mg/kg i.p.) or diazepam (10 mg/kg

i.p.) 30 min prior to inducing hypnosis with thiopental

sodium (40 mg/kg i.p.). Control animals received same

volume of vehicle (10 mL/kg i.p.). The total number of

animals that slept during observation period (50 min) was

counted. The latency of sleep (time lapse between admin-

istrations of thiopental sodium and occurrence of complete

hypnosis in mice) was noted. Total duration of sleep was

measured by observing the recovery of straightening reflex.

The sleep-inducing or potentiating effect of the extract was

studied in mice treated with pre-selected sub-hypnotic dose

(40 mg/kg) of freshly prepared sodium thiopental.

Rat paw oedema assay (anti-inflammatory activity)

The carrageenan-induced hind paw oedema test was con-

ducted according to Winter et al. (1962). Rats divided

randomly into different groups of 5–8 animals were injected

subcutaneously into the plantar surface of the hind paw with

0.05 mL of freshly prepared 1% carrageenan (prepared in

distilled water). Different doses of plant extract (50–200 mg/

kg i.p.) or diclofenac sodium (20 mg/kg) were injected i.p.,

30 min before the administration of carrageenan. The con-

trol animals received the same volume of the vehicle. Rat

paw oedema was assessed by volume displacement method

(plethysmometer Ugo Basile 7150) before and after carra-

geenan injection at 1, 2, 3 and 4 h. Difference in paw volume,

determined before and after injection of the phlogistic agent

indicated the severity of oedema.

The % inhibition of the inflammation was determined

for each animal by comparison with controls and calculated

by the following formula:

%I ¼ 1� dt=dcð Þ � 100

where ‘‘dt’’ is the difference in paw volume in the drug-

treated group and ‘‘dc’’ the difference in paw volume in

control group. ‘‘I’’ stands for inhibition.

Anti-platelet aggregation study

The anti-platelet activity of the plant extract was studied

according to the method as described by Saeed et al. (1995).

Briefly, blood was taken by venepuncture from normal

volunteers reported to be free of medication for 7 days.

Blood samples were mixed with 3.8% (w/v) sodium citrate

solution (9:1) and centrifuged at 260g for 15 min at 20�C to

obtain platelet-rich plasma (PRP). The remaining blood

sample was centrifuged at 1,200g for 10 min to obtain

platelet poor plasma (PPP). The aggregation studies were

carried out at 37�C. The aggregation was monitored with

dual channel Lumi Aggregometer using 450 ll samples of

PRP. The PRP was pre-incubated with 0.5–2.5 mg/mL of

A. modesta extract for 1 min before challenge with aggre-

gating agent (arachidonic acid). Aggregation was induced

Analgesic, anti-inflammatory and anti-platelet activities 189

by arachidonic acid (0.8 mM) which was expressed as

percentage inhibition compared with control at 4–5 min

after challenge. Plant extract was dissolved in 0.9% saline;

the vehicle did not interfere with platelet aggregation.

Statistical analysis

The results are presented as mean ± SEM. Comparison

between experimental and control group was performed by

student t test. One-way analysis of variance (ANOVA)

followed by Tukey–Kramer multiple comparison test was

used for the analysis of some data (hot plate assay).

P \ 0.05 was considered statistically significant.

Results

Phytochemical analysis

The results of the qualitative phytochemical screening of A.

modesta leaves extract are summarized in Table 1. Previ-

ous phytochemical studies on A. modesta have revealed

some commonly known compounds such as quercetin,

kaempherol and b-sitosterol (Khan 2004) and triterpenes,

and steroids (Joshi et al. 1975).

Acute toxicity test

Acacia modeta at 500 mg/kg i.p. produced 10% lethality in

experimental animals and the LD50 value was found to be

1,554 mg/kg. The animals treated with high doses of plant

extract ([1,000 mg/kg) showed severe drowsiness and

immobility.

Writhing test

As shown in Table 2, the intraperitoneal (i.p.) administra-

tion of the methanolic extract of A. modesta (50 and 100 mg/

kg), caused significant inhibition (P \ 0.001) of the noci-

ception induced by acetic acid with maximum effect being

86% at 100 mg/kg. The results were comparable to standard

drug diclofenac sodium that displayed 59% inhibition of the

constrictions at the dose of 20 mg/kg i.p.

Formalin test

In the formalin test the plant extract of A. modesta (50 and

100 mg/kg i.p.) significantly (P \ 0.01, P \ 0.001) inhib-

ited both phases of formalin-induced licking response in

mice (Table 3). As shown in Fig. 1a, pre-treatment of the

animals with naloxone (NLX) (5 mg/kg i.p.) partially

reversed the inhibitory effect of the plant extract and the

paw-licking time of the animal in the first phase increased

from 26.7 ± 3 (Without NLX) to 34.2 ± 3 s (With NLX).

Naloxone caused marked reversal of the analgesic effect of

morphine in both phase of formalin test (Fig. 1a, b).

However, unlike morphine the paw-licking response of the

animals treated with plant extract was partially antagonized

by NLX in the second phase of the test (Fig. 1b). Dic-

lofenac sodium at 20 mg/kg predominantly reduced the

paw-licking time of the animals from 75 ± 5 to 27 ± 5 s

(64% inhibition), in the second phase of the test only.

Similarly A. modesta extract at 50 and 100 mg/kg produced

65 and 74% inhibition of nociception, respectively, in the

second phase of formalin test (Table 3).

Hot plate test

In the hot plate assay A. modesta (100 mg/kg i.p.) showed

significant analgesic activity (P \ 0.01, P \ 0.001), simi-

lar to morphine (Table 4). The maximum effect of the plant

extract was attained at 2 h of the test and the latency of

reaction increased from 7.2 ± 0.2 (control) to 17.5 ± 0.5 s

(extract treated). Pre-treatment of the animals with nalox-

one (5 mg/kg i.p.) abolished the analgesic effect morphine.

The analgesic effect of the plant extract was partly reversed

with naloxone (Table 4).

Thiopental induced hypnosis test

In the control group (vehicle treated), 10% of the experi-

mental animals showed sleeping episode that lasted for

15 ± 5 min. Table 5, shows that the plant extract of

A. modesta (100 and 200 mg/kg i.p.) caused marked

Table 1 Qualitative phytochemical analysis of the methanolic

extract of A. modesta leaves

Test performed Observations Results

Alkaloids

Draggendroff’s test Orange red

precipitates/

turbidity

?

Terpenoids

Decolorized extract

residue ? chloroform ? acetic

anhydride ? conc.:H2SO4

Brown precipitates

formed

?

Flavonoids

Defatted extract ? ethanol and

filter; filterate ? AlCl3

Yellow color ?

Saponins

Extract shaken vigorously

in a test tube for 2 min

No frothing occurred -

Tannins

FeCl3 test Dark greenish

precipitate formed

?

‘‘?’’ present, ‘‘-’’ absent


reduction in sleep latency and significantly prolonged the

sleep time (P \ 0.05) similar to diazepam. The plant

extract (200 mg/kg) induced sleeping episode in 60% of

the animals. Diazepam, used as standard sedative agent,

produced sleep in about 66% of the animals (Table 5).

Anti-inflammatory activity

The subplantar injection of carrageenan produced a local-

ized oedema that reached to its maximum at the third hour

after injection. The localized inflammatory response illus-

trated by increase in paw volume was sustained for 4 h and

gradually declined after this time.

Table 6 demonstrates that methanolic extract of A. mo-

desta (50–200 mg/kg i.p.) reduced the paw oedema at third

hour after carrageenan administration, with maximum

inhibition being 70% at 200 mg/kg. The early phase of the

carrageenan-induced oedema was not affected by the plant

extract (data not shown). The difference between the paw

volume of the control and extract-treated animals was

statistically significant (P \ 0.01) at the third hour of

observation. The standard drugs diclofenac sodium

(20 mg/kg i.p.) produced 69.6% inhibition of the carra-

geenan-induced oedema (Table 6).

Effect on platelet aggregation

The inhibitory effects of plant extract against platelet

aggregation were assessed in the in vitro experiments using

arachidonic acid (AA) as the aggregating agent. Control

aggregation induced by AA was carried out in the begin-

ning of each experiment to check the platelet response to

the aggregating agent. When PRP was pre-incubated with

methanolic extract of A. modesta (0.5–2.5 mg/ml) for

1 min before challenge, the AA-induced platelet aggrega-

tion was inhibited in dose-dependent manner (Fig. 2a).

A. modesta (2.5 mg/ml) caused 82% inhibition of the AA-

induced platelet aggregation with IC50 value of 0.8 mg/ml,

Fig. 2b.

Discussion

Large number of plants belonging to genus Acacia are used

in folk medicine in pain and inflammatory conditions

(Geisler et al. 2003; Dongmo et al. 2005). This prompted us

to investigate an indigenous medicinal plant, Acacia

modesta, for its analgesic, anti-inflammatory, and anti-

platelet activities. For the anti-nociceptive study the widely

used pain models such as acetic acid-induced writhing,

formalin-induced licking, and hot plate tests were

employed. Acetic acid produces nociception by increasing

the level of prostaglandins, serotonin, and histamine in

peritoneal fluids, and this animal model is commonly used

for screening peripheral analgesics (Collier et al. 1968;

Deraedt et al. 1980). In the present investigation the plant

extract of A. modesta inhibited acetic acid-induced writh-

ing in mice similar to diclofenac sodium suggesting that the

Table 2 Effect of the methanolic extract of A. modesta and diclofenac sodium on acetic acid induced writhing in mice

Treatment Dose (mg/kg i.p.) Number of writhing % Protection

Control Saline (10 ml/kg) 73 ± 7 –

Acacia modesta 50 17 ± 3*** 77

100 10 ± 4*** 86

Diclofenac sodium 20 30 ± 6*** 59

Values represent mean ± SEM of 6–20 observations. Animals in the control group received equal volume of saline. A. modesta extract and

diclofenac sodium were dissolved in normal saline and administered 30 min before the acetic acid injection (i.p.). *P \ 0.05, **P \ 0.01 and

***P \ 0.001, compared with control

Table 3 Effect of the methanolic extract of A. modesta and diclofenac sodium on formalin-induced paw-licking time in mice

Treatment Dose (mg/kg) Paw-licking time (s) % Inhibition

1st phase 2nd phase 1st phase 2nd phase

Control – 50 ± 3 75 ± 5 – –

Acacia modesta 50 33 ± 5 ** 26 ± 4*** 34 65

100 27 ± 5** 19 ± 6*** 46 74

Diclofenac sodium 20 47 ± 7 27 ± 5*** – 64

Values represent mean ± SEM of 6–20 observations. Animals in the control group received equal volume of saline. A. modesta extract and

diclofenac sodium were dissolved in normal saline and administered 30 min prior to i.p. administration of formalin. The duration of paw licking

was determined between 0–5 min (first phase) and 15–30 min (second phase) after formalin injection. **P \ 0.01 and ***P \ 0.001, compared

with control


analgesic activity of the plant extract might be related to

inhibition of the prostaglandin function (Ferreira 1972).

Peripherally, analgesic drugs such as diclofenac and aspirin

and medicinal plants with folkloric use in the management

of pain and inflammation such as Asparagus pubescens and

Quasia amara (Okpo et al. 2001; Nwafor and Okwuasaba

2003; Toma et al. 2003) have shown analgesic effect in

acetic acid-induced writhing in mice.

It is known that constriction induced by acetic acid is a

non-selective model because it releases endogenous medi-

ators (prostaglandins), which are capable of stimulating

both the peripheral nociceptor(s) and neurons sensitive to

non-steroidal anti-inflammatory drugs (NSAIDs), opioids,

and other centrally acting drugs (Vaz et al. 1996).

To confirm the mechanism of analgesia, the effect of

plant extract was further investigated in the formalin-

induced pain model. Formalin test is useful for elucidating

the mechanism of pain and analgesia (Tjolsen et al. 1992).

Formalin-induced pain involves two distinct phases: in the

first phase (neurogenic phase) the pain is caused due to

direct stimulation of the sensory nerve fiber by formalin and

in the second or late phase (inflammatory phase) the pain is

due to release of inflammatory mediators such as histamine,

serotonin, prostaglandin, and bradykinin (Hunskaar and

Hole 1987; Murray et al. 1988). In the current investigation,

animals pretreated with various doses of A. modesta extract

caused marked reduction of paw-licking response of mice in

both phases of the test similar to morphine pretreatment. It

is well established that centrally acting drugs such as

morphine (opioids) inhibit both phases equally while

peripherally acting drugs (NSAIDs) inhibit the late phase

(Santos et al. 1994; Shibata et al. 1989) of formalin-induced

nociception. The ability of the plant extract to inhibit both

phases of the formalin test indicates that central mechanism

is involved in its analgesic effect. Naloxone, an opioid

antagonist reversed the analgesic effect of morphine, while

that of plant extract was partially inhibited, suggesting the

analgesic effect of the plant extract is partly mediated

through opioid receptors. Moreover, it is likely that opioid

and NSAIDs like constituents present in A. modesta extract

work synergistically to relieve pain in the aforementioned

animal models of pain and inflammation (Christie et al.

1999). Naloxone pre-treatment caused slight increase in

licking response of the animals in formalin test, suggesting

its antagonist effect against endogenous opioid system. The

central pain inhibition by some species of genus Acacia

such as A. ferruginea and A. nilotica (Dhar et al. 1968;

Almeida et al. 2001) has already been reported and A.

modesta might contain some similar constituents with

central action of analgesia. The analgesic effect of the plant

extract was, however, predominant in the second phase of

the test suggesting additional mechanisms such as inhibi-

tion of prostaglandin synthesis (Tjolsen et al. 1992).

First Phase

Treatments

0

10

20

30

40

50

60

70

ControlNaloxone 5 mg/kg Morphine 5 mg/kg Morphine 5 mg/kg + Naloxone 5 mg/kg Am.Cr 100 mg/kg Am.Cr 100 mg/kg + Naloxone 5 mg/kg

***

**

**

++

Lic

king

tim

e (S

ec)

Treatments

0

10

20

30

40

50

60

70

80

90

ControlNaloxone 5 mg/kg Morphine 5 mg/kg Morphine 5 mg/kg + Naloxone 5mg/kg Am.Cr 100 mg/kg Am.Cr 100 mg/kg + Naloxone 5 mg/kg

***

***

**++

Lic

king

tim

e (S

ec)

Second Phase

a

b

Fig. 1 a Effect of naloxone (5 mg/kg i.p.) on the analgesic effect of

intraperitoneally administered methanolic extract of A. modesta(Am.Cr) and morphine on formalin induced licking response in first

phase of the test. Values shown are mean ± SEM of 6–10 observations;

naloxone was administered 20 min prior to the Am.Cr or morphine

injections. **P \ 0.01, ***P \ 0.001, significantly different com-

pared with control; ??P \ 0.01, compared with naloxone ? morphine.

b Effect of naloxone (5 mg/kg) on the analgesic effect of intraperito-

neally administered crude extract of A. modesta (Am.Cr) and morphine

on formalin-induced licking response in second phase of the test.

Naloxone was administered 20 min prior to Am.Cr or morphine

injections. Values are mean ± SEM of 6–10 observations, **P \ 0.01,

***P \ 0.001, significantly different compared with control;??P \ 0.01, compared with naloxone ? morphine


The central analgesic effect of A. modesta was further

confirmed in the hot plate assay, a test considered suitable

for such study (Turner 1965). In the hot plate test the plant

extract significantly increased the reaction time which was

partially antagonized by naloxone, further confirming the

involvement of opioid system in the observed analgesic

effect of the plant extract.

In the thiopental-induced hypnosis test, pretreatment of

mice with plant extract caused significant increase in

sleep duration and reduction in sleep latency indicating

sedative effect of the extract. These results raise the

possibility that suppression of animal reaction in the pain

models may have occurred due to sedative properties of

the plant extract. However, the sedative action alone may

not be the sole cause of the analgesic effect as the plant

extract produced significant analgesic effect at the doses

that were ineffective in the thiopental induced hypnosis

test.

Table 4 Effects of A. modesta and morphine treatments in the presence and absence of naloxone on latency time of mice in hot plate test

Treatment Dose (mg/kg) i.p. Latency time (s)

0 h 0.5 h 1st hour 2nd hour 3rd hour

Control Saline (10 ml/kg) 6.3 ± 0.2 6.8 ± 0.2 7.2 ± 0.3 7.2 ± 0.2 6.8 ± 0.4

Morphine 10 6.5 ± 0.7 8 ± 0.3 11.2 ± 0.7*** 12.6 ± 0.5*** 11.7 ± 0.3***

Morphine ? naloxone 10 ? 5 5.4 ± 0.2 5.5 ± 0.4 6.3 ± 0.2 6.5 ± 0.3 5.8 ± 0.6

Acacia modesta 100 8.5 ± 0.5 12 ± 0.6*** 14.6 ± 0.3*** 17.5 ± 0.5*** 15.6 ± 0.4***

Acacia modesta ? naloxone 100 ? 5 7.8 ± 0.5 9.0 ± 0.3 9.6 ± 0.5* 11.0 ± 0.1*** 10.3 ± 0.4**

Values represent mean ± SEM (n = 6–10). Mice were individually placed on the hot plate maintained at 52 ± 0.8�C and the latency time

(licking of the paw or jumping) was noted. The cut-off time was 20 s to avoid thermal injury of the paw. Animals were given (i.p.) plant extract

or morphine, 30 min before their placement on the hot plate. Animals were observed individually before and at 0.5, 1, 2, and 3 h after the

treatments. Naloxone (5 mg/kg i.p.) was given 20 min before the administration of plant extract or morphine; control animals received same

volume of vehicle. **P \ 0.01, ***P \ 0.001 compared with control values at 0 h (one way ANOVA followed by Tukey–Kramer multiple

comparison test)

Table 5 Effect of methanolic extract of A. modesta on thiopental-induced sleeping time in mice

Treatment Dose

(mg/kg i.p.)

Sleep latency

(min)

Sleep duration

(min)

% of animals showing

sleeping episode

Control 11 ± 2 15 ± 5 10

Acacia modesta 100 5 ± 1.6 26 ± 5* 40

200 2.5 ± 0.5* 32 ± 3* 60

Diazepam 10 3 ± 1* 36 ± 3* 66

Values represent mean ± SEM of 6–15 observations. Control animals received same volume of vehicle. A. modesta or diazepam was injected

30 min prior to the administration of thiopental sodium (40 mg/kg i.p.). The total number of animals that slept during observation period

(50 min) was counted. The latency of sleep (time lapse between administrations of thiopental sodium and occurrence of complete hypnosis in

mice) was noted *p \ 0.05, compared with control

Table 6 Effect of the methanolic extract of A. modesta and diclofenac sodium on carrageenan-induced paw oedema in rats

Treatment Dose

(mg/kg i.p.)

Initial paw

volume (ml)

Paw volume

at 3rd hour

Increase in

paw volume

% Inhibition

Control Saline (1 ml/kg) 0.90 ± 0.03 1.13 ± 0.01 0.230 –

Acacia modesta 50 0.92 ± 0.01 1.06 ± 0.02 0.140** 39

100 1.0 ± 0.02 1.11 ± 0.03 0.11** 52

200 0.96 ± 0.03 1.03 ± 0.04 0.07** 70

Diclofenac sodium 20 1.09 ± 0.03 1.16 ± 0.02 0.07** 69.6

Values represent mean ± SEM of 5–20 observations. Different doses of A. modesta or diclofenac sodium were injected i.p., 30 min prior to the

administration of carrageenan. Animals were injected subcutaneously into the plantar surface of the hind paw with 0.05 ml of freshly prepared

1% carrageenan (prepared in distilled water). The control animals received same volume of the vehicle. Rat paw oedema was assessed by

plethysmometer (Ugo Basile 7150) before and at third hour after carrageenan injection. Difference in paw volume, determined before and after

carrageenan injection indicated the severity of oedema. **P \ 0.01 compared with control


The analgesic effect of the plant extract was predomi-

nant in the second phase of formalin test suggesting its

peripheral anti-inflammatory activity. The peripherally

acting drugs such as aspirin and phenylbutazone (Shibata

et al. 1989) are believed to attenuate pain by inhibition of

cyclooxygenase in arachidonic acid pathways (Levine and

Taiwo 1994). The observed analgesic effect in chemical-

induced pain models suggests NSAIDs like activity of the

plant extract.

Based on above findings and folkloric use of various

species of genus Acacia in inflammatory conditions, we

decided to investigate its anti-inflammatory effect in car-

rageenan-induced rat paw oedema model. The plant extract

of A. modesta exhibited marked anti-inflammatory effect

similar to that observed with diclofenace sodium used as a

reference compound. The carrageenan-induced acute

inflammation is believed to be biphasic:the early phase in

which the oedema production is mediated by histamine and

serotonin and the late phase in which the vascular perme-

ability is maintained by bradykinin and prostaglandins (Di

Rosa et al. 1971; Burch and DeHaas 1990). It has been

reported that second phase of the oedema is sensitive to

clinically effective anti-inflammatory drugs and has been

frequently used to assess the anti-phlogistic effect of the

natural products (Della Loggia et al. 1986; Saeed et al.

1995). In the present investigation the plant extract pro-

duced anti-inflammatory activity predominantly in the late

phase of carrageenan induced oedema test, indicating that

the effect is possibly mediated via inhibition of the activity

of prostaglandin. Similarly, the standard drug, diclofenac

sodium, produced significant anti-edematous effect in the

test. It is known that NSAIDs such diclofenac sodium

reduce inflammation, swelling, and arthritic pain by

inhibiting prostaglandin synthesis and/or production

(Skoutakis et al. 1988). There are evidences that com-

pounds inhibiting the carrageenan-induced oedema are

effective in inhibiting the enzyme cyclooxygenases

(Selvam and Jachak 2004). Based on these reports, it can

be inferred that the inhibitory effect of the plant extract on

the carrageenan-induced inflammation at the third hour is

possibly mediated via these mechanisms.

It has been shown that cyclooxygenase inhibitors sup-

press platelet aggregation (Siess et al. 1983). Furthermore,

compounds with anti-inflammatory activities have been

found effective against platelet aggregation (Saeed et al.

1995; Jose et al. 2004). In separate experiments the plant

extract of A. modesta inhibited arachidonic acid-induced

platelet aggregation, which further supports its anti-

inflammatory effect.

In conclusion, the methanolic extract of A. modesta

possesses analgesic, anti-inflammatory, and anti-platelet

properties. The findings from our current study support

NSAIDs like activity of the plant extract. The analgesic

effect of A. modesta was partially reversed in the presence

of naloxone, suggesting partial involvement of the opioid

system. The present data indicate that A. modesta might be a

new natural source to be explored for the development of

0 1 2 3 4 5

100

120

140

160

180

a2000

20

40

60

80

100

Time in minutes

% A

ggre

gati

on

Am.Cr [mg/mL] Control 0.5 1.0 2.5

0

25

50

75

100

**

***

***

% A

ggre

gati

on

a

b

Fig. 2 a Typical tracing showing the inhibitory effect of methanolic

extract of A. modesta (Am.Cr) against arachidonic acid (0.8 mM)

induced platelet aggregation. b Inhibitory effect of methanolic extract

of A. modesta (Am.Cr) on arachidonic-acid-induced platelet aggre-

gation. Values represent mean ± SEM of the % aggregation of the

control maximum (n = 3–4). The platelet-rich human plasma was

incubated with different doses of Am.Cr or vehicle (control) for 1 min

before the addition of arachidonic acid. The aggregation was

monitored with dual channel Lumi Aggregometer using 450 ll

samples of platelet-rich plasma obtained from blood sample of the

normal volunteers. **P \ 0.01 and ***P \ 0.001, significantly

different compared with control


novel analgesic drugs. The flavonoid and terpenoid contents

of the A. modesta extract may be responsible for its

observed analgesic and anti-inflammatory effects because

plant extracts rich in these compounds have shown prom-

ising effects in experimental models of pain and

inflammation (Della Loggia et al. 1986; Saeed et al. 2010).

Furthermore, our data rationalize the traditional use of

genus Acacia in management of pain and inflammation.

Further studies will be conducted to identify the constitu-

ents responsible for the observed effects of the plant extract.

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