1. INTRODUCTION:

The plant Feronia limonia Corr. (Rutaceae) commonly known as wood apple, elephant apple (English), kaitha, Kavitha, kathbel (Hindi). It is a deciduous, slow-growing, erect tree with a few upward-reaching branches bending outward near the summit where they are subdivided into slender branchlets drooping at the tips. Bark ridged, fissured and scaly, spines sharp, 2-5 cm long on some of the zig-zag twigs. Leaves alternate, 7.5-12.5 cm long, dark-green, leathery, often minutely toothed, blunt or notched at the apex, dotted with oil glands and slightly lemon-scented when crushed. Flowers dull-red or greenish, to 1.25 cm wide, borne in small, loose, terminal or lateral panicles. Fruit round to oval, 5-12.5 cm wide, with a hard, woody, greyish-white, scurfy rind about 6 mm thick, pulp brown, mealy, odorous, resinous, astringent, acid or sweetish, with numerous small, white seeds scattered through it. There are 2 forms, one with large, sweet fruits and the other with small, acid fruits (Gupta et al 1997).

The plant Feronia limonia has been used since ancient days in Ayurvedic medical practice for the treatment of cough, dysentery, heart diseases, asthma, constipation, tumors, ophthalmia, leucorrhoea, snake bite (Kirtikar and Basu 1995). However there is not enough scientific data to support the claims made in the ancient literature.

During a routine screening of Indian medicinal plants for biological activity, Feronia limonia dried leaves afforded a potent mosquito larvicide, identified as n-hexadecanoic acid and found to be effective against fourth instar larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti with lc₅₀of 129.24, 79.58 and 57.23 ppm respectively (Rahuman et al 2000). The plant extracts exerted inhibition activity against the bacteria Staphylococcus aureus, Bacillus subtillis and klebsiella pneumoniae (Jebanesan and Venkatachalam 2002). Oral administration of 250 mg/kg body weight of 95% ethanolic extract of unripe fruits significantly lowerd the blood glucose levels of fasted, fed and streptozocin-induced diabetic male albino rats (Gupta et al 2009). The aqueous extract of leaves of Feronia limonia showed hepatoprotective effect in carbon tetrachloride-induced liver damage model in rats (Kamat et al 2003). The stem bark of Feronia limonia yielded (-)-(2s)-5,3’-dihydroxy-4’-methoxy-6”,6”-dimethylchromeno-(7,8,2”,3”)-flavanone along with several known compounds including an alkaloid, five coumarin, a flavanone, a lignan, three sterols and a triterpene. The antimicrobial screening of compounds by a microdilution technique resulted in MICs in range 25-100 µg/ml (Rahman and Gray 2002). The extract (500 mg/kg p.o.) inhibited indomethacin-induced gastric ulceration by by decreasing acid concentration of gastric fluids (Mishra et al 2009).

A literature survey on the plant Feronia limonia revealed that there is no scientific evidence of its usefulness in the treatment of pain. The need for safer and effective analgesic drug and the lack of enough scientific data to support the claims made in ancient literature promoted the present study.

2. MATERIALS AND METHODS:

2.1 Plant materials:

The leaves of Feronia limonia belonging to family Rutaceae was collected during the month of December 2009, from Marwahi Forest Division, Pendra Road, Bilaspur (C.G). The leaves of Feronia limonia was taxonomically identified and authenticated by Dr. H. P. Agrawal, Senior Scientist, T.C.B. College of Agriculture and Research Station, I.G.K.V, Bilaspur (C.G) and was deposited in the department of pharmacology, Slt. Inst. of pharm. Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G) for future reference. After authentication, leaves were cleaned and the material was dried in shade and milled into coarse powder by a mechanical grinder and store in air tight containers for further studies.

2.2 Drugs and Chemicals:

Drug Ibuprofen (obtained as gift sample from ARBRO Pharmaceuticals, New Delhi), Chemicals like ethanol (CDH, Mumbai), chloroform (Ranbaxy Fine Chemicals Ltd., New Delhi) and acetic acid (CDH, Mumbai) were purchased through local dealers. Other chemicals and reagents used for phytochemical analysis were of LR grade obtained locally.

2.3 Preparation of Plant Extracts:

The successive solvent extraction procedure was adopted for the preparation of various extracts of leaves of Feronia limonia. The material was subjected to successive extraction with solvents in their ascending order of polarity. In this process the substance, which is soluble in a solvent with particular range of polarity, is extracted in the solvent and remaining marc further extracted with next solvent. The constituents, which are soluble in both polar and non-polar solvents, can be extracted separately by adopting this approach (Kokate et al 1996).

2.4 Animals:

Swiss albino mice weighing 20-30 g of either sex were purchased from Biological farms, Bhopal was maintained in the animal house of Oriental College of Pharmacy, Bhopal for experimental purpose. Then all the animals were acclimatized for seven days under standard husbandry conditions, i.e. room temperature of 25±1 ⁰C; relative humidity 45-55% and a 12:12 hr. light/dark cycle. The animals had free access to standard rat pellet (Pranav Agro Industries Ltd, Baroda, India), with water supplied ad libitum under strict hygienic conditions. Each experimental group had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of Oriental College of Pharmacy, Bhopal (M.P) and approval no is OCP/2010/2271/ac/10/CPCSEA/1349 and Proposal no. 12 was taken prior to the experiments. All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

2.5 Acute toxicity studies:

The acute oral toxicity (AOT) of ethanolic and chloroform extract of leaves of Feronia limonia were determined by using female Swiss albino mice (Wistar strains) weighing between 25-30 g those are maintained under standard animal husbandry conditions. The animals were fasted overnight prior to the experiment. The method used for acute oral toxicity (AOT) test according to method proposed by OECD 423 guidelines, 2001. Animals were administered with alcoholic and chloroform extract at doses of 30, 100, 300 mg/kg, 1000 mg/kg, and 2000 mg/kg body weight to separate groups of mice. Each group containing 6 numbers of mice.

During the first 4h after drug extracts administration, animals were observed closely for any toxic manifestations like hyperactivity, grooming, hypothermia, change in fur color, sedation, clonic convulsions, coma, and death were observed, and then observation is continued for 24 hrs. and 72 hrs. in regular intervals for 7 days.

2.6 Analgesic activity:

2.6.1 Eddy’s hot plate method:

Swiss albino mice weighing (25-30g) were divided into 6 groups consisting of 6 animals in each group. Group-I received normal saline (1 ml p.o.) which served as control. Group-II received Ibuprofen (100 mg/kg) and served as the standard. Group-III and Group-IV received ethanolic extract of Feronia limonia at doses of 100 mg/kg and 200 mg/kg respectively. Group-V and Group-VI received chloroform extract of Feronia limonia at doses of 100 mg/kg and 200 mg/kg respectively. All the extracts were administered orally. 60 minutes after oral administration of extracts and 30 minutes after i.p. injection of ibuprofen, animals were individually placed on the hot plate (maintained at 55°C) and the responses such as paw licking or jump response, whichever appeared first were noted. Cut off period of 15 second was maintained (Madhavan et al 2007).

2.6.2 Acetic acid-induced writhing method:

Swiss albino mice weighing (25-30g) were divided into 6 groups consisting of 6 animals in each group. Group 1 received normal saline (1 ml p.o.) which served as control. Group-II received Ibuprofen (100 mg/kg) and served as the standard. Group-III and Group-IV received ethanolic extract of Feronia limonia at doses of 100 mg/kg and 200 mg/kg respectively. Group-V and Group-VI received chloroform extract of Feronia limonia doses of 100 mg/kg and 200 mg/kg respectively. All the extracts were administered orally. 60 minutes before oral administration of extracts and 30 minutes before i.p. injection of ibuprofen, 0.9% aqueous solution of acetic acid in the dose of 10ml/kg body weight was injected and the number of writhing was observed. The percent inhibition of writhing was calculated (Ishfaq et al 2004).

2.7 Statistical analysis:

The statistical analysis of all the results was carried out using one-way ANOVA followed by Dunnet’s multiple comparisons test. P values <0.001 were considered statistically significant.

3. RESULTS:

3.1 Acute toxicity:

Different doses of chloroform and ethanolic extracts were screened for their acute oral toxicity. No mortality was recorded till 2000 mg/kg with chloroform and ethanolic extracts, hence the extracts were found to be safe upto the dose levels of 2000 mg/kg. Thus the safe dose for experimental work is 1/10^th of limit dose i.e. is 200 mg/kg body weight. The dose selected for pharmacological studies were 100 mg/kg and 200 mg/kg of body weight (Ishfaq et al 2004).

3.2 Analgesic activity

3.2.1 Eddy’s hot plate method

When Group-I (Control) is compare with standard (Ibuprofen) and test extracts of Feronia limonia leaves. It was showed that chloroform extract of Feronia limonia leaves treated groups showed significant analgesic activity as evidenced by the increase in reaction time to the pain stimulus. The results were significant in both the dose of chloroform extract of Feronia limonia leaves i.e. 100 mg/kg and 200 mg/kg body weight at p < 0.001. High dose of chloroform extract of Feronia limonia leaves (Group-IV) showed more analgesia as compared to low dose (Group-III). The analgesic activity is presented in table 1 and Histogram 1.

Table No. 1 Analgesic activities of different extracts of Feronia limonia leaves by Eddy’s Hot-plate test in mice

Groups	Dose	Reaction time in seconds
G-I (Control)	Normal saline (1 ml p.o.)	2.83±0.30
G-II (Standard)	Ibuprofen(100mg/kg I.p)	9.33±0.42* * *
G-III (Test)	CEFL 100 mg/kg P.o	4.83±0.3* * *
G-IV (Test)	CEFL 200 mg/kg p.o	5±0.25* * *
G-V (Test)	EEFL 100 mg/kg p.o	3.16±0.30
G-VI (Test)	EEFL 200 mg/kg p.o	2.33±0.21

Values are expressed as mean ± SEM; n=6; ^{* * *}p<0.001.

Histogram-1: Analgesic activity by Eddy’s Hot plate method A-Control, B-Ibuprofen (100mg/kg i.p.), C- CEFL 100 mg/kg p.o., D- CEFL 200 mg/kg p.o., E- EEFL 100 mg/kg p.o., F- EEFL 200 mg/kg p.o.

3.2.2 Acetic acid-induced writhing method:

When Group-I (Control) is compare with standard (Ibuprofen) and test extracts of leaves of Feronia limonia. It was showed that chloroform extract of leaves of Feronia limonia treated groups showed significant analgesic activity as evidenced by the decrease in no of writhes. The results were significant in all the dose of chloroform and ethanolic extract of leaves of Feronia limonia i.e. 100 mg/kg and 200 mg/kg body weight at p < 0.001. High dose of chloroform extract of leaves of Feronia limonia (Group-IV) showed more analgesia as compared to other. The percentage of inhibition was calculated by following formula. The analgesic activity is presented in table 2 and Histogram 2.

Percentage inhibition of writhing was calculated using the formula:

Mean no. of writhes (control) - Mean no. of writhes (test)

Inhibition (%) = --------------------------------------------------X 100

Mean Number of writhes (control)

Table No. 2 Analgesic activities of different extracts of Feronia limonia leaves by Acetic acid-induced writhing in mice

Groups	Dose	No. of Writhes	% of inhibition
G-I (Control)	Normal saline (1 ml p.o.)	72.33±1.054	_
G-II (Standard)	Ibuprofen (100mg/kg i.p)	22.33±0.843***	69.12%
G-III (Test)	CEFL 100 mg/kg p.o	27.5±0.670***	61.97%
G-IV (Test)	CEFL 200 mg/kg p.o	16.83±1.046***	76.73%
G-V (Test)	EEFL 100 mg/kg p.o	23.66±0.881***	67.28%
G-VI (Test)	EEFL 200 mg/kg p.o	17.16±0.703***	76.27%

Values are expressed as mean ± SEM; n=6; ***p<0.001

Histogram-2: Analgesic activity by acetic acid induced writhing in mice. A-Control, B-Ibuprofen (100mg/kg i.p.), C- CEFL 100 mg/kg p.o., D- CEFL 200 mg/kg p.o., E- EEFL 100 mg/kg p.o., F- EEFL 200 mg/kg p.o.

4. DISCUSSION:

The relatively high oral median lethal dose (LD50) in mice suggests that the extract is relatively non toxic when taken orally(Lorke,1983). The acetic acid–induced writhing test is very sensitive and able to detect anti-nociceptive effects of compounds at dose levels that may appear inactive in other methods like tail flick test (Benteley et al., 1981). However, the test is not specific as it does not indicate whether the activity was central and/or peripheral(Chan et al.,1985) The intraperitoneal injection of acetic acid produces an abdominal writhing response due to sensitization chemo-sensitive nociceptors by prostaglandins(Sustharson et al., 2007).

Increased level of prostanoids, particularly PGE2 and PGF2α (Derardt et al., 1980) as well as lipoxygenase products (Dhara et al., 2000) have been found in the peritoneal fluid after intraperitoneal injection of acetic acid. The analgesic effect of the extract may therefore be due either to its action on visceral receptors sensitive to acetic acid, to the inhibition of the production of algogenic substances or the inhibition at the central level of the transmission of painful messages. However, this model may not be able to indicate the mechanism of analgesic effect of the extract because other agents such as antihistamines (Naik et al., 2000) and myorelaxant (Koyama et al., 1997) are able to reduce the pain induced by acetic acid. The formalin test has a distinctive biphasic nociceptive responses termed early and late phases. Drugs that act primarily on the CNS inhibit both phases equally while peripherally acting drugs inhibit the late phase (Chan et al., 1995). The first phase is probably a direct result of stimulation of nociceptors in the paw. Stimulation of opioid receptors has also been suggested as a possible mechanism of action against neurogenic pain (Gaertner et al., 1999). This phase, therefore reflects centrally mediated pain while the late phase is due inflammation with the release of serotonin, histamine, bradykinin and prostaglandins and at least to some degree, the sensitization of central nociceptive neurons (Tjølsen et al., 1992). The suppression of both phases as observed in the highest dose tested (25 mg_kg-1) in this study suggests the presence of both central and peripheral effects. However, the extract has greater inhibitory effect on the neurogenic pain.

Hot plate tests is the most common test of nociception that are based on a phasic stimulus of high intensity (Mandegary et al., 2004). Pain induced by thermal stimulus of the hot plate is specific for centrally mediated nociception (Parkhouse and Pleuvry, 1979). The ability of the extract to prolong the reaction latency to pain thermally-induced in mice by the hot plate further suggests central analgesic activity. Analgesic and anti-inflammatory effects have been observed in flavonoids as well as tannins (Ahmadiani et al., 2000). There are also reports on the analgesic effects of alkaloids, essential oils and saponins (Reanmongkol et al., 2005; de Araujo et al., 2005; Choi et al., 2005). The analgesic and anti-inflammatory effect of the extract may be due to the presence of flavonoids, tannins, alkaloid and saponins, either singly or in combination. However, further studies are needed to isolate the active constituents responsible for the observed effect and to reveal the possible mechanisms of action responsible for the analgesic and anti-inflammatory activities of Feronia limonia leaves.

CONCLUSION:

These findings suggest that the methanol root bark extract of S. virosa (SV) contain bioactive constituents with analgesic and anti-inflammatory activities, and further support the ethnomedical claim of the use of the plant in the management of painful and inflammatory conditions.

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Received on 10.02.2011 Modified on 23.02.2011

Research J. Pharm. and Tech. 4(5): May 2011; Page 710-714