Antipyretic Activity of Rhizome of Drynaria quercifolia. in Rabbit

Abstract

The antipyretic effect of petroleum ether and ethyl acetate soluble fractions of ethanol extract of the rhizome of Drynaria quercifolia. J. Smith (Polypodiaceae) was investigated. Intraperitoneal administration of boiled milk at a dose of 0.5 mL/kg body weight in albino rabbit leads to pyrexia. Intraperitoneal (i.p.) administration of petroleum ether and ethyl acetate soluble fractions of ethanol extract of the rhizome of D. quercifolia. at a dose of 80 mg/kg body weight were shown to significantly reduce the elevated body temperature of rabbit, which was compared with standard aspirin (market product) and solvent used.

Keywords:

• Antipyretic
• aspirin
• boiled milk
• Drynaria quercifolia
• ethyl acetate fraction
• petroleum ether fraction
• rabbits

Introduction

Drynaria quercifolia. J. Smith (syn. Polypodium quercifolium.) (Polypodiaceae), locally known as “gurar,” is a parasitic fern (Bhattacharya, 1990; Kirtikar & Basu, 1994) that is widely distributed in Bangladesh, India, and Thailand (Hasan & Haque, 1993; Kirtikar & Basu, 1994; ASEAN Centre for Biodiversity, 2004). The rhizomes of the plant are traditionally used for the treatment of tuberculosis, loss of appetite, cough, and fever (Bhattacharya, 1990; Kirtikar & Basu, 1994). The rhizomes have antibacterial and constriction properties (Bhattacharya, 1990). By mixing with the plant Asparagus racemosus. Willd. (Liliaceae) then applied on the head, it causes coldness to the head and reduces alopecia (Hasan & Haque, 1993). The ASEAN Centre for Biodiversity mentions in its “Checklist of Medicinal Plant in Southeast Asia” that rhizome decoction or drink of Drynaria quercifolia. is used as an antipyretic preparation (ASEAN Centre for Biodiversity, 2004). Although the plant is widely used for remission of several ailments related to fever, its antipyretic potential has not been explored yet. Therefore, in the current study an attempt was made to establish the antipyretic effect of petroleum ether and ethyl acetate soluble fractions of ethanol extract of the rhizome of D. quercifolia..

Pyrexia or fever is caused as a secondary impact of infection, malignancy, or other disease states (Chattopadhyay et al., 2005). It is the body's natural defense to create an environment where infectious agent or damaged tissue cannot survive (Chattopadhyay et al., 2005). Normally the infected or damaged tissue initiates the enhanced formation of proinflammatory mediators (cytokines like interleukin 1β, α, β, and TNF- α ), which increase the synthesis of prostaglandin E₂ (PGE₂) near the peptic hypothalamus area and thereby trigger the hypothalamus to elevate the body temperature (Spacer & Breder, 1994). The temperature regulatory system is governed by a nervous feedback mechanism, so when body temperature becomes very high, it dilates the blood vessels and increases sweating to reduce the temperature; but when the body temperature becomes very low, the hypothalamus protects the internal temperature by vasoconstriction. High fever often increases disease progression by increasing tissue catabolism, dehydration, and existing complaints, as found in HIV (Veugelers et al., 1997). Most of the antipyretic drugs inhibit cyclooxygenase (COX)-2 expression to reduce the elevated body temperature by inhibiting PGE₂ biosynthesis (Cheng et al., 2005). Moreover, these synthetic agents irreversibly inhibit COX-2 with high selectivity but are toxic to the hepatic cells, glomeruli, cortex of brain, and heart muscles, whereas natural COX-2 inhibitors have lower selectivity with fewer side effects (Cheng et al., 2005). A natural antipyretic agent with reduced or no toxicity is therefore essential. As rhizome of D. quercifolia. is an old medicament used in ailments that caused fever (Bhattacharya, 1990; Kirtikar & Basu, 1994; ASEAN Centre for Biodiversity, 2004), it will be a cost-effective alternative approach to study this plant for the development of an effective antipyretic agent.

Materials and Methods

Plant material

Rhizome of Drynaria quercifolia. was collected from various parts of Lakshmipur district of Bangladesh and identified by Prof. A. T. M. Naderuzzaman, Department of Botany, University of Rajshahi, Bangladesh, where its voucher specimen (no. 1939) was deposited.

Preparation of petroleum ether and ethyl acetate fractions of ethanol extract

The powder materials (600 g) were extracted with ethanol (3 L) in a Soxhlet apparatus (Quickfit, Staffordshire, England) (Bhal & Bhal, 1990). The extraction was continued for 72 h at 65°C (Bhal & Bhal, 1992). The extract was filtered through filter paper. The filtrate was concentrated under reduced pressure at 50°C in a rotary vacuum evaporator to afford a blackish green mass (52.4 g), which was further extracted with petroleum ether (3 × 50 mL), ethyl acetate (3 × 50 mL), and methanol (3 × 50 mL) to afford petroleum ether, ethyl acetate, and methanol fractions, respectively (Haque & Nawab, 1971; Jeffery et al., 2000). The preliminary phytochemical screening of the different fractions was carried out by chemical tests and thin-layer chromatographic methods (Harborne, 1998).

Preparation of sample and standard solutions

Ethanol 2.5% in distilled water (autoclaved) was used as solvent to prepare sample and standard solutions. The sample solutions of petroleum ether and ethyl acetate fractions were prepared by dissolving each dried fraction in the solvent to obtain 120 mg per 2 mL solution. To facilitate dissolution, a few drops of Tween 80 were added. Then, each fraction was administrated at a dose of 80 mg/kg body weight (Alam, 1997).

Aspirin as Disprin soluble tablet obtained from the local market Reckitt Benckiser Ltd (Dhaka, Bangladesh) was used as the known antipyretic agent. The standard solution was prepared by dissolving the tablet in the solvent to obtain 15 mg aspirin per 2 mL solution. The dose of aspirin was maintained at 10 mg/kg body weight (Grover, 1990).

Animals

The experiment was carried out on albino rabbits. They were 13–15 months old, of both sexes, and weighed between 1.5 and 1.6 kg (Nammi, 2003). They were collected from the International Center for Diarrhoeal Diseases and Research, Bangladesh (ICDDR,B). The rabbits were kept in iron cages (BVAAWF, 1993) and were fed with cauliflower, cabbage, banana, and tap water for 40 days before the experiment to acclimate to the environment. Food and water were withdrawn 6 h prior to the experiment (Grover, 1990). The animals were grouped as:

1. Experimental groups: Four groups: two groups receiving two petroleum ether sample doses and the other two groups receiving two ethyl acetate sample doses.

2. Control groups:

   1. Aspirin group (+ ve Control): receiving standard antipyretic agent aspirin.

   2. Solvent group (− ve Control): receiving solvent (that used to prepare sample and standard solutions).

Number of rabbits in each group was six.

Acute toxicity study

Acute toxicity study was carried out by using graded doses of each fraction in albino mice. Both petroleum ether and ethyl acetate fractions were administered intraperitoneally in graded doses (200 to 1000 mg/kg body weight). Animals were observed continuously for the first 2 h for toxic symptoms and up to 24 h for mortality (Mutalik et al., 2003).

Treatment protocol

Before experimentation, rectal temperatures of rabbits were recorded by inserting a well lubricated bulb of a thermometer in the rectum. Care was taken to insert it to the same depth each time (about 6 cm) (Grover, 1990). Milk was collected from a local cow and was boiled. When the temperature of the boiled milk equilibrated to room temperature, then rabbits were injected with boiled milk at the dose of 0.5 mL/kg body weight to induce pyrexia. Induction of fever took about 1 to 2 h (Taran et al., 1984; Grover, 1990).

Then, solvent (2 mL) was given to negative control group, known antipyretic agent aspirin solution (2 mL) was given to positive control group, and each sample solution (2 mL) was given to the experimental group. Intraperitoneal route was used to administer boiled milk, aspirin solution, solvent, and sample solution. Finally, rectal temperatures were recorded at 1-h intervals up to 3 h.

Results

The preliminary phytochemical screening of the petroleum ether and ethyl acetate fraction showed the presence of steroids, tannins, and flavonoids. In acute toxicity study, both fractions were found to be safe, and no mortality was observed at a dose as high as 800 mg/kg. The resultant effects of both fractions of rhizome of D. quercifolia. on boiled milk–induced pyrexia in rabbits are depicted in Table 1. At a dose of 40 mg/kg body weight, petroleum ether and ethyl acetate fractions reduced elevated rectal temperature (38.5 ± 0.15% and 37.03 ± 0.16%, respectively) compared with aspirin (96.3 ± 0.10%) after 3 h. At a dose of 80 mg/kg body weight, petroleum ether and ethyl acetate fractions reduced elevated rectal temperature (88.9 ± 0.23% and 84.6 ± 0.20%, respectively) compared with aspirin (96.3 ± 0.10%) after 3 h.

Table 1.. Effect of petroleum ether and ethyl acetate fractions of rhizome of Drynaria quercifolia. J. Smith on boiled milk–induced pyrexia in rabbit.

		Rectal temperature (°F)		Rectal temperature after administration of fraction (°F)
Groups	Dose	Normal (A.)	3 h after boiled milk administration (B.)	1 h (C.1)	2 h (C.2)	3 h (C.3)
Solvent	2 mL/rabbit	101.4 ± 0.2	104.0 ± 0.23	103.9 ± 0.23	103.9 ± 0.23	103.7 ± 0.31
				(3.84 ± 0.10)	(11.5 ± 0.26)
Aspirin	10 mg/kg	101.4 ± 0.1	104.1 ± 0.11	103.1 ± 0.23	101.5 ± 0.10	101.5 ± 0.10
				(37.03 ± 0.2)*	(96.3 ± 0.10)*	(96.3 ± 0.10)*
Petroleum ether	80 mg/kg	101.5 ± 0.1	104.2 ± 0.08	103.3 ± 0.19	101.9 ± 0.1	101.8 ± 0.1
				(33.3 ± 0.18)*	(85.2 ± 0.20)*	(88.9 ± 0.23)*
Petroleum ether	40 mg/kg	101.6 ± 0.09	104.2 ± 0.08	104.0 ± 0.19	103.3 ± 0.12	103.2 ± 0.11
				(7.7 ± 0.06)	(34.6 ± 0.13)*	(38.5 ± 0.11)*
Ethyl acetate	80 mg/kg	101.5 ± 0.1	104.1 ± 0.20	103.5 ± 0.29	102.0 ± 0.23	101.9 ± 0.23
				(23.1 ± 0.10)	(80.7 ± 0.12)*	(84.6 ± 0.20)*
Ethyl acetate	40 mg/kg	101.3 ± 0.2	104.0 ± 0.10	103.8 ± 0.19	103.1 ± 0.08	103.0 ± 0.08
				(7.4 ± 0.18)	(33.3 ± 0.16)*	(37.03 ± 0.16)*

All values are expressed as mean ± SE (n = 6), percentage reduction in rectal temperature is given within parentheses.

% Reduction = (B. − C._n./B. − A. × 100); where n. = 1, 2 and 3.

Thus, both petroleum ether and ethyl acetate fractions produced significant (p < 0.05) antipyretic effects. It was also observed that solvent has no effect on the reduction of pyrexia in rabbit.

Discussion

The search for safe herbal remedies with potent antipyretics activity received momentum recently as the available antipyretics, such as paracetamol, aspirin, nimusulide, and so forth, have a toxic effect to the various organs of the body (Guyton & Hall, 1998). This acute toxicity result suggests that D. quercifolia. might be considered a broad nontoxic alternative. The antipyretic activity demonstrated that the petroleum ether and ethyl acetate fractions of ethanol extract of rhizome of D. quercifolia. possess a significant antipyretic effect in maintaining normal body temperature and reducing boiled milk–induced elevated rectal temperature in rabbits, and their effects, are comparable with that of standard antipyretic drug aspirin. Such reduction of rectal temperature of tested animals by both fractions at 80 mg/kgappears to be due to the presence of a single bioactive principle or mixture of compounds in the fractions. The phytochemical analysis of the petroleum ether and ethyl acetate fractions showed the presence of steroids, tannins, and flavonoids. The isolation of beta-sitosterol from rhizome of D. quercifolia. was reported in literature (Ramesh et al., 2001). Beta-sitosterol is a plasminogen activator and promotes the formation of essential polyunsaturated fatty acids from linoleic acid (linoleic acid is required for prostaglandin and leukotriene synthesis) (Kinsella et al., 1990), and thus beta-sitosterol reduces prostaglandin and leukotriene synthesis. Beta-sitosterol possesses potent anti-inflammatory and antipyretic activity (Gupta et al., 1996) by reducing the secretion of proinflammatory cytokines and alpha-TNF (Gupta et al., 1996; Bouic, 1999). These phytosterols can enhance adaptive immunity through the stimulation of the innate immune system and are termed adaptogens., which promote overall health without side effects (Wagner, 1995). Moreover, in many studies, flavonoids have been reported to exhibit antipyretic effects (Brasseur, 1989; Vimala et al., 1997). It was also evident from the study that the antipyretic activity of petroleum ether fraction at 80 mg/kg body weight is almost similar to the standard aspirin group and is more than that of the ethyl acetate fraction. The current study, therefore, supports the claims of traditional medicine practitioners of D. quercifolia. as an antipyretic remedy. However, to know the exact mechanism of action of D. quercifolia. rhizome extract, further study with purified fractions/bioactive compounds is warranted.

Acknowledgment

The authors wish to thank to Professor A.T.M. Naderuzzaman, Department of Botany, University of Rajshahi, for identification the plant.

Notes

*p < 0.05 significant compared with control (solvent).