Studies of Antipyretic and Anti-Inflammatory Activities of Odina woodier Leaves Extracts

Valli G.¹, Vijayalakshmi R.¹, Vasanthi A.¹ and Thanga Thirupathi A.²

¹Department of Chemistry, SFR College for women, Sivakasi.

²Department of Pharmacology, SB College of Pharmacy, Anaikuttam, Sivakasi.

ABSTRACT:

Antipyretic and anti-inflammatory activities of different extracts of Odina woodier leaves were studied by observing decrease in rectal temperature and reduction in paw edema using albino rats of both sexes. Animals were divided into ten groups, each consisting of six animals. Group 1 served as control and Group 2 received the standard drug. Group 3-10 received various extracts of Odina woodier leaves. Group 3 received ethanol extracts of 200mg/kg and Group 4 received 400mg/kg. Group 5 and Group 6 received ethyl acetate extracts of 200mg/kg and 400mg/kg respectively. Similarly Group 7 and Group 8 received chloroform extracts of 200mg/kg and 400mg/kg. Group 9 received 200mg/kg and Group 10 received 400mg/kg petroleum ether extracts of Odina woodier leaves. For the determination of antipyretic activity, pyrexia was induced by 20% yeast suspension and for anti-inflammatory activity, inflammation was induced using 1% carageenan suspension. The results showed that all the extracts were found to have both antipyretic and anti-inflammatory activities. Among these four extracts, ethanol extract of 400 mg/kg have shown same antipyretic activity after four hours of drug administration as that of the standard Paracetamol drug which caused the reduction of temperature of 1.03 and 1.02 for the extracts and Paracetamol drug . 400mg/kg of ethyl acetate extract after two hours have shown higher anti-inflammatory activity (40.43%) with a probability < 0.001 than the chosen standard drug diclofenac sodium(39.75%) available as an anti-inflammatory drug in the market.

KEY-WORDS: antipyretic, anti-inflammatory, carageenan, pyrexia and Odina woodier

INTRODUCTION:

Herbal medicines are the synthesis of remedial experiences and practice of indigenous systems of medicine for over hundreds of years. Despite the tremendous progress in medical research during the past decades, the treatment of many serious diseases including pain and inflammation is still problematic. Herbal medicine showed safety, efficacy, cultural acceptability and lesser side effects than the synthetic drugs. Various parts of Odina woodier plant had been found to be used as medicines in Ayurveda. As per reported data the leaves were found to use in elephantiasis of the legs¹ and the juice of green branches as an emetic in case of coma or insensibility produced by narcotic. The dried and powdered bark was used as tooth powder² and its extract had been used in vaginal trouble, curing ulcer and heart diseases³. In the present study, our investigation aims to find out the antipyretic and anti-inflammatory activities of ethanol, ethyl acetate, chloroform and petroleum ether extracts of Odina woodier leaves in experimental animals. Antipyretic activity by Brewer’s yeast induced pyrexia model and anti-inflammatory activity by carrageenan-induced paw edema model in albino rats were evaluated.

MATERIALS AND METHOD:

Materials used:

Collection of plant part:

The leaves of Odina woodier were collected in Sivakasi Town, virudhunagar district in Tamil Nadu. It was cleaned with running tap water to remove adhering elements, shadow dried and powdered in a domestic mixer.

Drugs and reagent:

Paracetamol(standard for antipyretic), Diclofenac sodium (standard for anti-inflammatory) and various solvents such as ethanol, ethyl acetate, chloroform, petroleum ether and carrageenan of SD fine grade were used. The solvents were distilled above their boiling point for purification and then used. Brewer’s yeast was purchased in the local market in Sivakasi.

Animals used:

For the anti-pyretic and anti-inflammatory activities albino rats of both sexes of weight 150-200g were used. The animals were kept in polypropylene cages in a room maintained under controlled atmospheric conditions. The animals were fed with standard diet (Hindustan lever, Mumbai, India) and kept in dark/light cycle 12hrs/12hrs and drinking water ad libitum.

All the experimental protocols were approved by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), animal ethics committee vide number SPCP/2009-2010/IAEC/CPCSEA/10.

Methods used:

Plant extraction:

The powdered leaves of Odina woodier were extracted by using ethanol, ethyl acetate, chloroform and petroleum ether in soxhlet apparatus by using standard procedure⁴. The distillate were collected and distilled separately to yield the concentrated extracts which were studied for antipyretic and anti-inflammatory activities.

Determination of antipyretic activity:^5-8

Antipyretic activity was evaluated by Brewer’s yeast induced pyrexia model in albino rats. Albino rats were fasted overnight before the experiment. Pyrexia was induced by injecting subcutaneously 20% w/v brewer's yeast suspension (10 ml/kg) into the animals' dorsum region. Eighteen hours after the injection, the rectal temperature of each albino rat was recorded using a digital thermometer (Inco, Chennai). Only albino rats that showed an increase in temperature of at least 0.5°C -1°C were used for the experiment ^9,10. The animals were divided into ten groups each consisting of six animals. Group 1 served as control and received normal saline (1ml/kg, p.o).Group 2 received standard paracetamol (33mg/kg,p.o).Group 3 to group 10 received ethanol, ethyl acetate,chloroform and petroleum ether extracts of Odina woodier leaves of 200 and 400 mg/kg respectively. The rectal temperature was recorded at 0, 1, 2, 3 and 4^th hours after drug administration and the recorded data were listed in Table-1.

Anti-inflammatory activity determination:

Carrageenan-induced hind paw edema is the standard experimental model of acute inflammation ¹¹. Carrageenan is the phlogistic agent of choice for testing anti-inflammatory drugs as it is not known to be antigenic and is devoid of apparent systemic effects. Moreover, the experimental model exhibits a high degree of reproducibility¹². Carrageenan-induced edema is a biphasic response. The first phase is mediated through the release of histamine, serotonin and kinins. Whereas, the second phase is related to the release of prostaglandin.

For the determination of anti-inflammatory activity, the animals were divided into ten groups each consisting of six animals. Group 1 served as control, Group 2 received standard diclofenac sodium (10mg/kg).Group 3 to Group 10 received ethanol, ethyl acetate, chloroform and petroleum ether extracts of Odina woodier leaves of 200 and 400 mg/kg respectively. Acute inflammation was produced by sub plantar injection of 0.1 ml of 1% suspension of carrageenan in normal saline in the right hind paw of the albino rats. One hour after oral administration of the drugs. The paw volume was measured¹³ with the aid of a Plethysmometer at 0, 1, 2, 3and 4 hours after the injection of carrageenan. The difference between the readings at time zero hour and the different time intervals were taken as the thickness of edema. The values were recorded in Table-2.Percentage inhibition of paw edema was calculated by comparing the control. The percentage inhibition of inflammation was calculated for each dose at different hours as given below.

Percentage inhibition = 1- Vt / Vc X 100

Where Vc = volume of paw edema in control animals

Vt = volume of paw edema in drug treated animals

RESULT AND DISCUSSION:

Table

-1: Effect Of Odina woodier leaves Extract on rectal temperature(⁰C) in albino rats

Drug Treatment	Dose (mg /kg)	Rectal temperature(^0C)		Rectal temperature after adminstration of drug(^0C)						Reduction in temperarature (^0C)
		Normal	18h after yeast adminstration	I Hour	2 Hours	3 Hours			4 Hours
Control Saline	1ml/ kg	37.8± 0.1291	38.75± 0.1555	38.85± 0.1555	38.95± 0.1555	39.05± 0.1555			39.1± 0.1581	-
Standard paracetamol	33	37.35± 0.2102	38.4± 0.1472	38.1± 0.1871****	37.83± .2323****	37.6± .2345****			37.38± 0.2287****	1.02
Ethanol extract	200	36.5± 0.1931	37.73± 0.2097	37.48± 0.1931****	37.38± .1931****	37.1± .2041****			37± 0.1937****	0.73
	400	38.13± 0.125	39.23± 0.1797	38.98± 0.1548^ns	38.68± 0.1493*	38.18± 0.3092***			38.2± 0.1871***	1.03
Ethyl acetate extract	200	37.8± 0.1080	38.68± 0.1031	38.45± 0.0878****	38.25± .1041****	37.9± 0.1080***			38± 0.1080****	0.68
	400	37.98± 0.125	38.03± 0.125	37.83± 0.125****	37.55± .1190****	37.45± .1190****			37.25± 0.1190****	0.78
Chloroform extract	200	37.65± 0.0645	38.68± 0.1031	38.48± 0.1031****	38.18± .1031****	38.1± 0.0816***			38.05± 0.1041****	0.65
	400	37.43± 0.1702	38.20± 0.1472	38± 0.1472****	37.78± .1493****	37.58± .1888****			37.43± 0.1702****	0.77
Petroleum ether extract	200	37.9± 0.2789	38.73± 0.3400	38.58± 0.3425****	38.48± .3425****	38.3± 0.3**			38.18± 0.2869***	0.56
	400	37.34± .2599	38.3± 0.2121	38.1± 0.2121****	37.88± .2394****	37.7± .2582****			37.58± 0.2394****	0.74
One way anova
F				6.4112	2.7298		7.0567	5.4775
df				(9,30)	(9,30)		(9,30)	(9,30)
P				<0.01	<0.01		<0.01	<0.01

(i) The values are expressed in Mean ± SEM; (ii)****P<0.001, ***P<0.01, **P<0.02, *P<0.05, ns- non significant vs control

(iii) Six animals were used in each group; (iv) One way Anova followed by Dunnet’s t- test.

Figure-1 Antipyretic activity of Odina woodier leaves Extract

Std…standard Paracetamol; E-1… ethanol extracts 200mg/kgE-2 … ethanol extracts 400mg/kg; EA-1… ethyl acetate extracts 200mg/kg

EA-2 … ethyl acetate extracts 400mg/kg; C-1… chloroform extracts 200mg/kg

C-2 … chloroform extracts 400mg/kg ; P-1… petroleum ether extracts 200mg/kg

P-2 … petroleum ether extracts 400mg/kg

Antipyretic activity:

Figure-1 represented the antipyretic activity of Odina woodier leaves Extract

Ethanol extract:

Our study showed the dose dependent decrease in the rectal temperature range from 37.48 ± 0.1931 to 37 ± 0.1937 for 200 mg/kg and from 38.98 ± 0.1548 to 38.2 ± 0.1871 for 400mg/kg of extracts. Higher activity was observed for 400mg/kg after four hours with the total reduction in rectal temperature of 1.02 with a probability < 0.001.

Ethyl acetate extract:

The dose dependent decrease in the rectal temperature range from 38.18 ± 0.0854 to 37.73 ± 0.125 for 200 mg/kg of extract and from 37.85 ± 0.0645 to 37.25 ± 0.0630 for 400mg/kg from our observed results. The total reduction in rectal temperature of 0.77 was observed with a probability < 0.001 for 400mg/kg after four hours.

Chloroform extract:

The dose dependent decrease in the rectal temperature range from 37.75 ± 0.1190 to 37.28 ± 0.0854 for 200 mg/kg of extract and from 37.45 ± 0.1041 to 36.85 ± 0.1708 for 400mg/kg. Higher activity was observed for 400mg/kg after four hours with the total reduction in rectal temperature of 0.75 with p <0.001

Petroleum ether extract:

The decrease in the rectal temperature range from 38.18 ± 0.1750 to 37.73 ± 0.2016 for 200 mg/kg and from 37.85 ± 0.0645 to 37.35 ± 0.0289 for 400mg/kg were observed for petroleum ether extract. The total reduction in rectal temperature was 0.73 with a probability < 0.001 was observed for 400mg/kg after four hours.

From the above observed results, it was found that ethanol extract of 400 mg/kg have shown higher activity after fourth hours of drug administration.

Table-2: Anti-inflammatory activity of Odina woodier leaves extract on Carrageenan-induced paw edema in albino rats

Drug Treatment	Dose (mg/kg)	Mean Paw volume(ml) ± SD
		I Hour	2 Hours	3 Hours	4 Hours
Control Saline	1ml/kg	0.33±0.024	0.705±0.0171	0.855±0.015	1.07±0.028
Standard Diclofenac Sodium	10	0.24±0.0082^** (27.27)	0.44±0.0082^** (37.59)	0.635±0.0065^** (25.73)	0.73±0.0108^** (31.78)
Ethanol extract	200	0.28±0.0082^* (15.15)	0.47±0.0129^** (33.33)	0.67±0.0129^** (21.64)	0.77±0.01^** (28.04)
	400	0.215±0.0096^** (34.85)	0.435±0.0096^** (38.30)	0.62±0.0082^** (27.48)	0.74±0.01^** (30.84)
Ethyl acetate extract	200	0.31±0.0129^ns (6.06)	0.51±0.0129^** (27.66)	0.70±0.0082^** (18.12)	0.80±0.01^** (25.23)
	400	0.275±0.0096^* (16.67)	0.42±0.0082^** (40.43)	0.63±0.0129^** (26.32)	0.7±0.0082^** (27.10)
Chloroform extract	200	0.32±0.0082^ns (3.03)	0.55±0.0129^** (21.99)	0.76±0.0082^** (11.11)	0.85±0.0129^** (20.56)
	400	0.32±0.0082^** (3.03)	0.52±0.0082^** (26.24)	0.655±0.0096^** (23.39)	0.75±0.01^** (29.91)
Petroleum ether extract	200	0.35±0.0129^ns (6.06)	0.58±0.0082^** (17.73)	0.78±0.0082^** (8.77)	0.86±0.01^** (19.63)
	400	0.34±0.0082 (3.03)	0.52±0.0082^** (26.24)	0.72±0.0082^** (15.99)	0.81±0.0129^** (24.30)
OneWay Anova
F		13.8748	59.1376	57.3171	22.5698
df		(9,30)	(9,30)	(9,30)	(9,30)
P		<0.01	<0.01	<0.01	<0.01

(i) The values are expressed in Mean ± SEM . (ii) ^**p< 0.001, ^*P< 0.01, ns – non significant vs control

(iii) Six animals were used in each group. (iv) One way ANova followed by Dunnet’s t- test.

Figure-2 Anti-inflammatory activity of Odina woodier leaves Extract

Std…standard Paracetamol; E-1… ethanol extracts 200mg/kg

E-2 … ethanol extracts 400mg/kg; EA-1… ethyl acetate extracts 200mg/kg

EA-2 … ethyl acetate extracts 400mg/kg; C-1… chloroform extracts 200mg/kg

C-2 … chloroform extracts 400mg/kg ; P-1… petroleum ether extracts 200mg/kg

P-2 … petroleum ether extracts 400mg/kg

Anti-inflammatory activity:

Figure-2 represented the antipyretic activity of Odina woodier leaves Extract

Ethanol extract:

The decrease in the paw edema range from 0.28 ± 0.0082 to 0 .77 ± 0.01 for 200 mg/kg of extract and from 0 .215 ± 0.0096 to 0.74 ± 0.01 for 400mg/kg. Higher activity was observed for 400mg/kg after two hours of 38.30% with a probability < 0.001 than the standard (37.59).

Ethyl acetate extract:

For 200 mg/kg of extract, the decrease in the paw edema volume range from 0.31±0.0129 to 0.80±0.01 and for 400mg/kg from 0.275±0.0096 to 0.78±0.0082 were observed. For 400mg/kg, after two hours maximum activity of 40.43% and for the standard 37.59 % were observed with a probability <0.001.

Chloroform extract:

Chloroform extract showed the decrease in the paw edema volume range from 0.32 ± 0.0082 to 0.85 ± 0.0129 for 200 mg/kg and from 0.32±0.0082 to 0.75±0.01 for 400mg/kg. The activity observed was less than the standard with a probability <0.001.

Petroleum ether extract:

The decrease in the paw edema volume range from 0.35± 0.0129 to 0.86±0.01 for 200 mg/kg and from 0.34±0.0082 to 0.81±0.0129 for 400mg/kg of extract from our observation. The activity observed for 400mg/kg after two hours of 26.24 % with a probability <0.001 was also less than the standard .

Among all the above extracts, 400mg/kg of Ethyl acetate extract after two hours was found to show higher activity of 40.43% with a probability < 0.001 .

CONCLUSION:

From the above investigation of antipyretic and anti- inflammatory activities of odina woodier leaves extracts using solvents like ethanol, ethyl acetate, chloroform and petroleum ether, we observed that ethanol extract (400mg/kg) can be used as a replacement for the standard paracetamol as an antipyretic drug. In future it may be used as a standard antipyretic drug in the market. Ethyl acetate extract (400mg/kg) was found to show appreciable anti- inflammatory activity.

REFERENCES:

1. Kiritikar, K.R. and Basu, B.D. Indian Medicinal Plants, 1935,2nd Edition, International Book Distributors, Book Sellers and Publishers, Dehradun.

2. Chidanbarathanu, S. (1995) Index of herbs in languages. Siddha Medical Literature Research centre, Madras.

3. Kiritikar, K.R. and Basu, B.D. (1987) Indian Medicinal Plants,International Book Distributors, Book Sellers and Publishers, Dehradun

4. Ramakrishnan,P.N.,Palanichamy,S(2004). Extraction and Galenicals/ Pharmaceutics-1Ed.9,Jai publishers, Madurai.PP.82-85.

5. Niazi J, Vikas Gupta V, Chakarborty P,Kumar P; Anti-inflammatory and antipyretic activity of aleuritis moluccana leaves .Asian Journal of Pharmaceutical and Clinical Research , 2010, Vol.3 Issue 1,January- March 35-37.

6. Vogel H.G. ”Drug discovery and evaluation”,2nd edition,772.

7. Hajare S W, Chandra S, Tandan S. K., Sharma J, Lal J,Telang A G; Analgesic and antipyretic activity of Dalbergia Sissoo Leaves. Indian Journal of Pharmacology 2000, 32, 357-360

8. Cheng L, Ming-liang H, Lars B et al. Is COX-2 a perpetrator or aprotector? Selective COX-2 inhibitors remain controversial. Acta Pharmacologica Sinica 2005, 26, 926-933.

9. Brownlee.G.(1937). A comparison of the antipyretic activity and toxicity of Phenacetin and aspirin. Journal of Pharmacy and Pharmacology.10:609-611.

10. Asha.V.V., Pushpangadhan,P(1999). Antipyretic activity of Cardiospermum halicacabum Linn. Indian journal of Experimental biology.37(4):411-414.s

11. Winter CA, Risley EA, Nuss CW. Carrageenan induced edema in hind paw of rat as an assay for anti-inflammatory drugs. In: Proc. Soc. Exp. Biol. Med. 1962, 11,544-547.

12. Dimartins, M.J., Campbell, J.R.,Wolff,G.K., Hanna.N(1987). The pharmacology of arachidonic acid induced rat paw oedema..Agents actions.21(3-4):303-305.

13. K. M. Basavaraja et al .J. Chem. Pharm. Res., 2010, 2(6),387-392.

Received on 13.07.2011

Accepted on 03.08.2011

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Research J. Pharmacology and Pharmacodynamics. 3(5): Sept –Oct. 2011, 263-267