In-vivo screening of Bougainvillea glabra leaves for its Analgesic, Anti-pyretic and Anti-inflammatory activities

A. Elumalai*¹, M. Chinna Eswaraiah¹, Karisha Madhavi Lahari², Hamed Ali Shaik²

¹Department of Pharmacognosy, ²Department of Pharmacology, Anurag Pharmacy College, Ananthagiri (V), Kodad (M), Nalgonda (Dt), Andhra Pradesh, India, 508 206.

*Corresponding Author E-mail: cognosyrocks@gmail.com

ABSTRACT:

Previous phytochemical analysis of methanolic extract of Bougainvillea glabra leaves (MEBG) has indicated the presence of steroids, flavonoids, glycosides and terpenoid types of compounds. Since these compounds are of pharmacological interest, coupled with the use of this plant in traditional medicine, prompted us to check Bougainvillea glabra leaves for possible analgesic anti inflammatory and anti-pyretic activities. The analgesic activity of MEBG was studied using tail immersion method in mice. While the efficacy of the MEBG (150 and 300mg/kg.p.o) was compared with pentazocaine 30 mg/kg. MEBG recorded 79.88 % inhibition with the highest dose and 81.21% with pentazocaine. The antipyretic activity of MEBG was studied in Brewer’s yeast-induced pyrexia in rats. MEBG showed more significant activity. MEBG were also subjected to evaluate anti-inflammatory activity by carrageenan induced rat paw edema. Indomethacin (100mg/kg) was taken as standard drug. MEBG produced dose-dependent of oedema, which was comparable to Indomethacin and found to be 48.32% and 42.32 % inhibition in carrageenan induced rat paw edema model. Findings from the present study showed MEBG to possess central and peripheral analgesic activity, anti-inflammatory property similar to steroidal and non-steroidal agents as well as antipyretic effect.

KEYWORDS: Bougainvillea glabra leaves, analgesic, anti-inflammatory and anti-pyretic activity.

INTRODUCTION:

The genus Bougainvillea in the Nyctaginaceae (4 O’ clock) family of plants has 18 species, with three that are horticulturally important Bougainvillea spectabilis, B.glabra and B.peruviana. Bougainvillea glabra ‘Snow White’ is a cultivar of the B. glabra ‘Choicy’ which has white bracts with the greenish veins¹. Bougainvillea glabra ‘Choicy’ have been used by the traditional practitioner of Mandsaur in variety of disorders like diarrhoea, reduce stomach acidity, cough and sore throught, decoction of dried flowers for blood vessels and leucorrohea and decoction of the stem in hepatitis. The main part used is leaves.

The reported constituents in leaf of Bougainvillea glabra ‘Choicy’ are alkaloids, flavonoids, tannins, saponins and proteins. The leaves of Bougainvillea glabra ‘Choicy’ are reported to have insecticidal activity, anti-inflammatory, anti-diarrhoeal activity, anti hyperglycaemic activity, anti-ulcer and anti-microbial activity^2-5. In spite the numerous uses and pharmacological activity attributed of Bougainvillea glabra Choicy but no pharmacological information regarding the leaves of this plant cultivar Bougainvillea glabra ‘Snow White’. Hence, the present investigation is an attempt in this direction and includes evaluation of analgesic and anti-pyretic activity of aqueous and methanolic extracts.

MATERIALS AND METHODS:

Collection and authentication of plant material

The fresh leaves of Bougainvillea glabra were collected in the medicinal garden of Anurag Pharmacy College and authenticated by Department of Pharmacognosy, Anurag Pharmacy College and the voucher specimen was kept in the Department of Pharmacognosy, Anurag Pharmacy College.

Extraction of plant drug

The collected leaves were washed, shade dried and converted into moderately coarse powder by mechanical grinder. The powdered material was extracted with methanol by using soxhlet apparatus. The solvent was removed under reduced pressure which yields respective extracts in the form of semisolid mass.

Animals:

The study was conducted on male Wister rats (175-200gm) housed in polypropylene cages under standard conditions of temperature (22 ± 2°C), relative humidity (60 ± 5%) and light (12h light/dark cycle) were used. They were fed with standard diet and water. The food was withdrawn 18 hours before the experiment but allowed free access of water. All animal experiments were carried out in accordance with the guidelines of CPCSEA. The experimental protocol is duly approved by the institutional ethical committee (Reg.no.212/2012/apc).

Acute oral toxicity studies:

Acute toxicity was carried out according to Organization of Economic Co-Operation and Development (OECD) no 425 guidelines⁸ and LD₅₀ values was estimated to be >5000mg/kg. Based on the results obtained from this study, the doses of further pharmacological studies were fixed to be 150 and 300mg/kg⁶.

ANALGESIC ACTIVITY

Tail immersion method⁷

Mice were divided into four groups each consisting of six mice. The treatment regimen was as follows:

1. Group I (Control): Vehicle (2ml/kg, p.o), 1% suspension of tween-80.

2. Group II (Standard): Pentazocaine (30mg/kg, p.o)

3. Group III (Test 1): MEBG(150mg/kg, p.o)

4. Group IV (Test 1): MEBG(300mg/kg, p.o)

The distal part of the tails of the animals was immersed in hot water maintained at 55. The time taken to with drawl the tail was noted as the reaction time. A cut-off time of 10s was maintained at 55C to prevent tissue damage. The reaction time was checked at 0, 15, 30, 45 and 60 min respectively after treatment.

Antipyretic activity

Yeast induced pyrexia method⁸

Rats were divided into four groups each consisting of six Rats. The treatment regimen was as follows:

1. Group I (Control): Vehicle (2ml/kg, p.o), 1% suspension of tween-80.

2. Group II (Standard): Paracetamol (20mg/kg, p.o)

3. Group III (Test 1): MEBG (150mg/kg, p.o)

4. Group IV (Test 2): MEBG (300mg/kg, p.o)

The test was performed in rats by injecting 10ml/kg, s.c, of the 10% aqueous solution of brewer’s yeast to induce pyrexia. The rectal temperature of each animal was taken before and 24hr after the yeast injection using a digital clinical thermometer. Animals did not show a minimum increase of 0.7C in the temperature 24h after the yeast injection was discarded. The rectal temperature of each animal was given recorded at 0, 1, 2, 3, 4,5hr after treatment.

Anti-inflammatory activity

Carrageenan induced rat paw edema⁹

Rats were divided into four groups each consisting of six Rats. The treatment regimen was as follows:

1. Group I (Control): Vehicle (2ml/kg, p.o), 1% suspension of tween-80.

2. Group II (Standard): Indomethacin (20mg/kg, p.o)

3. Group III (Test 1): MEBG (150mg/kg, p.o)

4. Group IV (Test 2): MEBG (300mg/kg, p.o)

The drugs were given orally. After one hour, a sub plantar injection of 0.1ml of 1% carrageenan was administered in the right hind paw to all the three groups. The paw volume was measured plethysmographically at 0 min, 60 min, 120 min and 180 min. The average paw of swelling in a group of extract treated rats was compared with control group (treated with vehicle) and the positive control (Indomethacin).

Statistical analysis

The result were express as Mean ± SEM. Statistical analysis was carried out using one way ANOVA followed by Student-t test.

RESULTS AND DISCUSSION:

The results obtained showed that the methanolic extracts possess a significant analgesic effect on tail immersion model. The results were tabulated in table 1. This suggests that the analgesic effect of the extract may be peripherally mediated and it might be centrally acting.

Table 1: Analgesic activity of MEBG by tail immersion model in mice.

Group	Treatment	Reaction time (S)	Latency (%)
I	Control (2ml/kg, p.o)	2.81±0.42	--
II	Standard (Pentazocaine:30mg/kg, p.o)	4.91±0.21	81.21
III	MEBG (150mg/kg, p.o)	3.01±0.51	49.34
IV	MEBG (300mg/kg, p.o)	4.7±0.42	79.88

The methanolic extract caused a better hypothermal activity against yeast-induced pyrexia in rats. The subcutaneous injection of yeast induces pyrexia by increasing the synthesis of prostaglandin and is used to screen agents for an antipyretic effect. The results were tabulated in table 2.

Table 2: Anti-pyretic activity of MEBG in yeast induced pyrexia in rats.

Group	Dose (mg/kg)	Rectal Temperature in °C at time (hr)
Group	Dose (mg/kg)	18 hr after yeast injection	0	1	2	3	4	5
Control	2 ml/kg (p.o)	37.2±0.02	37.1±0.02	36.7±0.04	36.4±0.02	36.0±0.01	35.8±0.03	35.4±0.02
Paracetamol	20 (mg/kg.p.o)	37.1±0.02	36.8±0.08	36.5±0.01	36.2±0.07	36.0±0.03	35.5±0.06	35.0±0.01
MEBG	150 (mg/kg.p.o)	37.5±0.03	37.5±0.08	37.1±0.05	36.7±0.07	36.5±0.04	36.2±0.06	36.1±0.04
MEBG	300 (mg/kg.p.o)	37.3±0.02	73.0±0.01	36.6±0.08	36.1±0.03	35.9±0.06	35.3±0.01	35.1±0.02

Values are expressed as Mean± S.E., n=6 by students ‘t’ test

Table 3: Anti-inflammatory effect of MEBG in carrageenan induced paw edema in rats

Groups	Treatment	% Increase in paw volume				% inhibition in paw volume
Groups	Treatment	0	60	120	180	% inhibition in paw volume
I	Control (2ml/kg, p.o)	29.41±1.21	65.32±1.31	95.24±1.42	102.24±2.1	--
II	Standard (Indomethacin 20mg/kg, p.o)	28.21±1.71	55.22±3.1	74.31±0.31	56.21±1.2	42.32
III	MEBG (150mg/kg, p.o)	25.42±0.62	44.21±2.3	65.32±0.21	49.23±2.1	60.23
IV	MEBG (300mg/kg, p.o)	27.31±2.12	53.21±2.1	71.11±0.21	54.31±1.2	48.32

Values are expressed as Mean± S.E., n=6 by students ‘t’ test

Carrageenan induced paw edema was taken as prototype of exudation phase of inflammation where development of edema being described as biphasic. The initial phase which occurs between 0 and 2h after injection of carrageenan has been attributed to release of histamine, serotonin and bradykinin. Inflammation volume reaches its maximum approximately 3h post treatment after which it begins to decline. A more second phase is related to release of prostaglandin like substances. The knowledge of these mediators involved in different phases is important for interpreting mode of drug action. The carrageenan induced paw edema model in rats is known to be sensitive to cyclooxygenase inhibitors. Based on this report it was concluded that the inhibitory effect of MEBG on carrageenan induced inflammation in rats could be due to inhibition of the enzyme cyclooxygenase leading to inhibition of prostaglandin synthesis. Pre-treatment of rats with the extract (50-200 mg/kg) significantly inhibited the paw oedema induced by carrageenan when compared with the control group and 300 mg/kg produced a better efficacy comparable to Indomethacin-treated group. The results were tabulated in table 3.

CONCLUSION:

Hence the presence of flavonoids in methanolic extract of Bougainvillea glabra leaves; they might suppress the analgesic, pyrexia and inflammatory conditions.

ACKNOWLEDGEMENT:

Authors are heart-warming thanks to Management of Anurag Pharmacy College for providing necessary facilities.

REFERENCE:

1. Anonymous. The Wealth of India. Vol I, National Institute of Science Communications and Information Resources. Council of Scientific & Industrial Research. New Delhi.2000.p.68-72.

2. Heuer S, Richter S, Metzger JW, Wray V, Nimtz M, Strack D. Betacyanins and flavonoids from bracts of Bougainvillea glabra. Phytochem. 37; 1994: 761-767.

3. Sheeja E, Edwin E, Amal Raj A, Gupta VB, Rana AC. Pharmacognostical and preliminary phytochemical studies on Bougainvillea glabra Choisy. Planta Indica. 1; 2005: 33-36.

4. Giri SN, Biswas AK, Saha BP, Pal SP. Studies of the anti-inflammatory action of Bougainvillea glabra leaves. Ind J Pharm Sci. 50; 1998: 42-49.

5. Otshudi AL, Foriers A, Vercruysse A, Van Zeebroeck A, Lauwers S. In vitro antimicrobial activity six medicinal plants traditionally used for treatment of dysentery and diarrhoea in Democratic Republic of Congo. Phytomedicine. 7; 2000: 167-172.

6. Anonymous. Organization for economic Co-operation and development Guidance Document on Acute toxicity testing. Environment Directorate, OECD. Peris: 2001. P.1-24.

7. Hasan, S.M.R., M. Jamila, M.M. Majumder, R. Akter, M.M. Hossain, M.E.H. Mazumder, M.A. Alam, R.Jahangir, M.S. Rana and S. Rahman. Analgesic and antioxidant activity of the Hydromethanolic extract of Mikania scandens Willid Leaves. Am J Pharm Tox. 4; 2009: 1-7.

8. Ghule BV, Murugananthan G, yeole PG. Analgesic and Antipyretic effects of Capparis Zeylanica leaves. Fitoterapia. 78; 2007: 365-9.

9. Aiyelero, OM., Ibrahim ZG and Yaro AH. Analgesic and anti-inflammatory properties of the methanol leaf extract of Ficus ingens (moraceae) in rodents. Nig Journ Pharm Sci. 8(2); 2009: 79-86.

Received on 30.05.2012 Accepted on 25.06.2012

Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 85-87