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Int J Pharm Biomed Res 2010, 1(3), 98-101

International Journal of
PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: 0976-0350

Research article

Anti inflammatory and analgesic effect of methanolic extract of Anogeissus acuminata leaf
K. Hemamalini*, K. Om Prasad Naik, P. Ashok
1

Faculty of Pharmacology, Teegala Ram Reddy College of Pharmacy, #4-202, Meerpet, Saroornagar (M), Hyderabad-500 097, Andhra Pradesh, India

Received: 10 Aug 2010 / Revised: 13 Aug 2010 / Accepted: 15 Aug 2010 / Online publication: 25 Aug 2010

ABSTRACT In the present study, the anti-inflammatory and analgesic effect of the methanol extract of Anogeissus acuminata leaf was investigated. The methanolic extracts of Anogeissus acuminata leaf were ingested orally (p.o.) in the form of suspension in 0.5% Tween 80 in two different doses, 200 and 400 mg/kg body weight). The anti-inflammatory effect of Anogeissus acuminata was tested in: carrageenin-induced paw oedema in wistar albino rats and formalin-induced paw oedema in Swiss albino mice and compared with the standard, indomethacin (5 mg/kg body weight). The analgesic effect was evaluated in Swiss albino mice by Eddys hot plate method and compared with the standard, aspirin (25 mg/kg body weight). The results showed that Anogeissus acuminata has significant reduction (p0.01) in inflammation i.e. 66.67 % (200 mg/kg body weight) and 77.78% (400 mg/kg body weight) as compared to the standard drug, indomethacin, which was 88.89%. In assessing analgesic effects, there is a significant (p<0.01) reduction in the paw licking and paw jumping response for Anogeissus acuminata (400 mg/kg) and aspirin (25 mg/kg) when compared to control. These results indicate that the extracts could possess analgesic and anti-inflammatory properties. All these effects and the changes in the behavioural activities could be suggested as contributory effects to the use of Anogeissus acuminata leaf in the management of inflammation and painful conditions. Key words: Anogeissus acuminata, Anti-inflammatory, Analgesic, Indomethacin, Aspirin, Swiss albino mice

1. INTRODUCTION Plants are one of the most important sources of medicines. India is known as the Emporium of Medicinal plants due to availability of several thousands of medicinal plants in the different bioclimatic zones anti-inflammatory diseases including rheumatoid arthritis are still one of the main health problems of the worlds population. Several modern drugs are used to treat these disorders but, their prolonged use may cause severe adverse side effects [1], the most common being gastrointestinal bleeding and peptic ulcers [2]. Consequently, there is a need to develop new antiinflammatory agents with minimum side effects. The use of natural remedies for the treatment of inflammatory and

*Corresponding Author. Tel: +91 40 24090187, Fax: +91 40 24090368 Email: rkhemamalini@yahoo.com

2010 PharmSciDirect Publications. All rights reserved.

painful conditions have a long history, starting with Ayurvedic treatment, and extending to the European and other systems of traditional medicines. Plant drugs are known to play a vital role in management of inflammatory diseases. Anogeissus acuminata (Fig.1) is a moderate size tree with small leaves, which falls earlier on the dry season [3]. Before falling the foliage of these turns a beautifully yellowish red, growing in South Africa and Arabian Peninsula. Its local names include Button tree, Pasichettu, Peddmanu, Buchakram. Leaves of the plant are used in traditional and tribal medicine of Andhra Pradesh to treat painful inflammatory conditions. A perusal of the literature revealed that although Anogeissus acuminata is widely used in traditional medicine as an anti-inflammatory and analgesic agent, these properties have not been scientifically evaluated. Therefore, the present study is an attempt to investigate the anti-inflammatory and analgesic properties of the methanolic extract of Anogeissus acuminata leaves in experimental animals.

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Fig.1. Photograph of Anogeissus acuminata plant

2. MATERIALS AND METHODS 2.1. Plant material The leaves of the Anogeissus acuminata were collected from the herbal garden of the institute during August 2008. This was authenticated by Dr.K.Madhavachetty, taxonomist of the Institute and the voucher specimen has been preserved for the future reference at the herbarium of the institute (SVUT 0153/dated 11/08/2008). 2.2. Preparation of the plant extract The leaves of this plant were dried under shade at 2730C for 15-30 days, after which the leaves of the plant were chopped and grounded into coarse powder. The powder (100 g) was extracted with methanol (1000 mL) overnight, at room temperature with constant stirring. The extract was filtered and the filtrate was concentrated at 30C under reduced pressure in a rotary evaporator. The yield (w/w) of the crude extract was found to be 12.06%. The crude extract was dissolved in 0.5% Tween 80 to required concentrations and used for the experiments. Anogeissus acuminata leaf methanolic extract was referred to as A.A. 2.3. Experimental animals Wistar albino male rats (150 g) and Swiss albino mice 25-30 g, were grouped and housed in polyacrylic cages (two animals per cage) and maintained under standard laboratory conditions (temperature 24-28C, RH, 60-70% and 12 h light dark cycles). They were fed commercial rat feed (Lipton India Ltd, Mumbai) and boiled water, ad libitum. All experiments involving animals were done according to NIH guidelines, after getting the approval of the institutes animal Ethics committee (No.1330/ac/10/CPCSEA). 2.4. Carragenan - induced paw oedema in rats Anti-inflammatory activity of Anogeissus acuminata was assessed by carrageenin induced paw oedema method [4].

Rats were divided into 4 groups (6 animals in each group). Animals of all the groups were injected with 0.1 mL of 1% carrageenin in 0.9% normal saline, under the plantar aponeurosis of the right hind paw. Group I animals (carrageenin control) received methanolic extract suspension in 0.5% of Tween 80 p.o., 30 min prior to carrageenin injection. Group II, the standard reference group was given p.o., an aqueous solution of indomethacin (5 mg/kg), 30 min prior to carrageenin injection. Group III and Group IV received p.o., 200 and 400 mg/kg of Anogeissus acuminata methanolic extract suspension in 0.5% of Tween 80, 30 min prior to carrageenin injection, respectively. The paw volume of the rats was measured plethismographycally just before and 3 h after carrageenin injection. The anti- inflammatory activity was determined as the percentage of inhibition of inflammation after it was induced by carrageenan by taking volume of inflammation in control group as 100%. The percentage inhibition was calculated by using the formula:
Mean paw inflammation of control Mean paw inflammation of test % Inhibition= 100 Mean paw inflammation of control

2.5. Formalin induced oedema Anti-inflammatory activity was evaluated by formalin induced paw oedema method. Swiss albino mice were divided in to 4 groups (6 animals in each group). Animals of all the groups were injected with 0.1 mL of 1% formalin in 0.9% normal saline, under the plantar aponeurosis of the right hind paw. Group I animals (formalin control) received a plain aqueous suspension in 0.5% of Tween 80 p.o., 30 min prior to formalin injection. Group II, the standard reference group was given p.o., an aqueous solution of indomethacin (5 mg/kg), 30 min prior to formalin injection. Group III and Group IV received p.o., 200 and 400 mg/kg of Anogeissus acuminata methanolic extract suspension in 0.5% of Tween 80, respectively, 30 min prior to formalin injection. The paw volume of the rats was measured plethismographycally just before and 3 h after formalin injection. The percentage inhibition of the oedema was calculated for each which respect to the vehicle treated control group. 2.6. Eddys hot plate method Analgesic effect of Anogeissus acuminata was assessed by the Eddys hot plate in Swiss albino mice. Mice were divided into 4 groups (6 animals in each group). Group I control received a plain aqueous suspension in 0.5% of Tween 80 (0.5 mL) p.o., 20 min prior to placement of the animal in hot plate. Group II, the standard control group received p.o., a single dose of aspirin (acetylsalicylic acid, 25 mg/kg), 20 min prior to hot plate. Group III and Group IV received p.o., a single dose of 200 and 400 mg/kg of Anogeissus acuminata methanolic extract suspension in 0.5% of Tween 80, respectively, 20 min prior to hot plate [5]. The

K. Hemamalini et al., Int J Pharm Biomed Res 2010, 1(3), 98-101 Table 1 Evaluation of anti-inflammatory effect of methanolic extract of Anogeissus acuminata (A.A) on carrageenin induced paw oedema in Wistar albino rats Treatment Groups Carragenan control Indomethacin A.A A.A Dose 5 mg/kg 200 mg/kg 400 mg/kg Difference in paw volume at 3h 0.450.01 0.050.02** 0.150.05** 0.100.01**

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Percentage inhibition of oedema 88.89 66.67 77.78

All values are given in meanSD, (n=6) ANOVA **p0.01, when compared to carragenan control group

Table 2 Evaluation of anti-inflammatory effect of methanolic extract of Anogeissus acuminata (A.A) on formalin induced paw oedema in Swiss albino mice Treatment groups Normal saline Indomethacin A.A A.A Dose 10 mL/kg 5 mg/kg 200 mg/kg 400 mg/kg Oedema diameter (cm) 1h 0.730.16 0.590.04 0.440.04** 0.420.07** 2h 0.80 0.12 0.610.04 0.410.07** 0.350.05*** 3h 0.780.1 0.610.02 0.400.07** 0.350.60** 4h 0.78 0.12 0.630.7 0.400.69** 0.350.6**

All values are given in meanSD, (n=5) ANOVA *p<0.05, **p< 0.01, ***p<0.001, when compared to normal saline group

Table 3 Evaluation of analgesic effect of methanolic extract of Anogeissus acuminata (A.A) by Eddys hot plate method Treatment groups Control Aspirin A.A A.A Dose 25 mg/kg 200 mg/kg 400 mg/kg Reaction time 0 min 2.00.1 3.10.2 2.50.3 2.20.5 30 min 2.20.2 7.50.3 2.80.1 4.70.2* 360 min 2.330.21 10.10.3** 4.330.3333 7.330.3333** 120 min 2.280.1 12.80.80** 4.80.7* 8.80.5**

All values are given in meanSD, (n=6) ANOVA *p<0.05, **p< 0.01, ***p<0.001, when compared to control group

number of paw licking and in paw jumping per animal recorded during the 20 min period. 2.7. Statistical analysis The calculation of the average oedema for the antiinflammation and number of paw licking and in paw jumping were based on the expression of numerical data as mean SD. The statistical significance between control and treated groups were analyzed using analysis of variance (ANOVA), where p 0.01 were taken to be significant [6]. 3. RESULTS AND DISCUSSION Oral administration of A.A significantly inhibited (p0.01) the carragenan induced paw oedema in rat at both doses (200 and 400 mg/kg) studied. At 200 mg/kg dose, 66.67% inhibition and at 400 mg/kg dose, 77.78% inhibition was observed. The group treated with indomethacin (5 mg/kg) showed maximum inhibition of oedema, which was 88.89% as shown in Table 1. Oral administration of A.A significantly inhibited the formalin induced paw oedema in mice at both doses (200 and 400 mg/kg) and indomethacin showed maximum inhibition of oedema in Table 2. Animal produced paw licking and paw jumping in the control group, and A.A at both doses used in the study significantly inhibited the jumping and licking response in mice. Aspirin at 25

mg/kg dose was significantly reduced the paw licking and paw jumping response when compared to the control Table 3. Inflammation is a complex process and various mediators e.g. prostaglandins, leukotrienes and kinins, platelet activating factor, etc. have been reported to be involved in the development if inflammatory diseases. Carragenan assay is well studied for comparative bioassay of anti-inflammatroy agents, since the relative potency estimates obtained from most drugs tend to reflect clinical experience [4]. The time course of oedema development on carragenan induced paw oedema model in rats is generally represented by a biphasic curve [8]. The first phase occurs within an hour of injection and also due to the serotonin component [8]. Prostaglandins play a major role in the development of the second phase of reaction which is measured around 3 h time [9]. The presence of prostaglandin in the inflammatory exudates form the injected foot has been well demonstrated previously by other workers [10]. The carragenan induced paw oedema model in rats is known to be sensitive to cyclooxygenase inhibitors and has been used to evaluate the effect of non-steroidal anti-inflammatory agents which primarily inhibit cyclooxygenase involved in prostaglandin synthesis [11]. Based on these reports, it is inferred that the inhibitory effect of A.A on carragenaninduced inflammation in rats in the present study may be due to inhibition of the enzyme cyclooxygenase, leading to inhibition of prostaglandin synthesis. Based on these reports,

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it is inferred that the inhibitory effects of A.A on carragenan induced inflammation in rats in the present study may be due to inhibition of the enzyme cyclooxygenase, leading to inhibition of prostaglandin synthesis. Basal reaction time is recorded as mentioned in the method using analgesiometer. Here the reaction may be hind paw licking or jump response. Hind paw licking appears within 4-6 sec and after 2-3 sec jumping may start. One has to observe both these response before and after administration of drug like Morphine (5 mg/kg). It is well known the that inhibition of formalin induced paw oedema in rats is one of the most suitable test procedure to screen anti-arthritic and anti-inflammatory agent as it closely resembles human arthritis injection of formalin subcutaneously into hind paw of rats produces localised inflammation and pain. The nociceptive effect of formalin is biphasic, an early neurogenic component followed by a later tissue mediated response. Thus formalin induced arthritis is a model used for the evaluation of an agent with probable antiproliferate activity. This experiment is associated with the proliferate phase of inflammation. It has been reported that the leaves and barks of A.A contains flavonoids and oleo resins. Many oleo resins are used in anti-inflammatory and analgesic agents in modern medicine. Preliminary phytochemical studies indicated the presence of gum resins of A.A. Volatile oils, resins, flavonoids and terpenoids isolated form plant extracts are known to produce anti-inflammatory and analgesic effects [12]. 4. CONCLUSIONS The findings of the present study have demonstrated that A.A has potent anti-inflammatory and analgesic activity and

justify its use in traditional medicine to treat inflammatory and painful conditions. The results also furnish evidence that the beneficial effects of this plant may be due to its free radical scavenging activity. ACKNOWLEDGEMENTS The authors would like to thank Dr.K.Madhavachetty, Teegala Ram Reddy College of Pharmacy, for providing necessary facility for the successful completion of the research work. Further, the authors wish to extend their gratitude to the anonymous reviewers for their valuable comments in improvising the research article. REFERENCES
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