Int J Pharm Pharm Sci, Vol 6, Issue 11, 156-159Original Article

PHARMACOLOGICAL EVALUATION OF ANTI-INFLAMMATORY, ANALGESIC AND ANTIPYRETIC EFFECTS OF GYNOCARDIA ODORATA ROXB IN ANIMAL MODELS

MANI RUPESHKUMAR1, KUNCHU KAVITHA1, PALLAB KANTI HALDAR2,*

1East Point College of Pharmacy, Bidarahalli, Bangalore 49, Karnataka, India, 2Department of Pharmaceutical Technology, Jadavpur University, Kolkata - 32, West Bengal, India.
Email: pallab_haldar@rediffmail.com

Received: 20 Aug 2014 Revised and Accepted: 22 Sep 2014

ABSTRACT

Objective: The purpose of this investigation was to study the anti-inflammatory, analgesic, and antipyretic properties of methanol extract of Gynocardia odorata roxb (MEGO) in rats and mice.

Methods: The plant material was extracted with methanol. Dose of 200, 400 mg/kg of each extracts were used in carrageenan-induced paw oedema, cotton-pellet granuloma in rats, writhing nociception in mice, and yeast induced hyperpyrexia in rats.

Results: All compounds reduced paw oedema into the control group at 5 h post carrageenan injection. The methanol extract of G. odorata roxb were similar to phenylbutazone in reduction of paw oedema and cotton-pellet granuloma. The extract as well as paracetamol induced antinociception in writhing test in comparison to control. Positive results for flavanoids and phenolic compounds were investigated by phytochemical analysis of the extract. The higher antinociception effects of the extract might be due to the presence of flavanoids, and phenol compounds. The methanol extract produced a significant dose dependent inhibition of temperature elevation.

Conclusion: These data suggest that the extract of G. odorata roxb produce antinociceptive, anti-inflammatory, and anti-pyretic activities that could be due to the effect of one or a combination of the bio active components in each extract.

Keywords: Gynocardia odorata roxb leaves, Anti-inflammatory, Antinociception, Anti-pyretic activity.

INTRODUCTION

Traditional medicaments chiefly obtained from plants have played a vital role in sustaining disease free human existence in the planet. Over the centuries, phytopharmaceuticals have been utilized by different communities of the world [1,2]. Pain, inflammation and fever are very common complications in human beings. Several plants and their products are claimed and proved to possess antipyretic property [3]. As a result of adverse effects such as gastric lesions caused by non-steroidal anti-inflammatory drugs (NSAID), tolerance and dependence induced by opiates the use of these drugs as anti-inflammatory and analgesic agents have not been successful in all cases. Attention is being focused on the investigation of the efficacy of plant-based drugs used in the traditional medicine because they are cheap, have few side effects and according to WHO, about 80% of the world population still rely mainly on herbal remedies [4-6]. In recent times, focus on plant research has increased and non-steroidal antiinflammatory drugs (NSAID) constitute one of the most widely used classes of drugs. Herbal drugs are being proved as effective as synthetic drugs with lesser side effects. Herbal medicines are in line with nature, with less hazardous reactions [7].

Gynocardia odorata roxb is also known as Chaulmoogra plant belongs to the family of Flacourtiaceae, which is indigenous to parts of India, Malaysia and tropical countries of the world contain fatty acids chaulmoogric acid, hydnocarpic acid. Chaulmoogra oil is an important therapeutic agent in certain medical traditions [8]. The seeds of G. odorata roxb are most commonly used. The fruits are hot anthelmintic and used in bronchitis, skin diseases, small tumor’s leprosy, and as an analgesic. G. odorata roxb is reported to contain antioxidant properties [9]. G. odorata roxb may have its antiulcer activity because of its active constituents like flavonoids and especially quercetin [10-11]. It was reported that G. odorata roxb could be a natural medication alternative of thrombolytic agents as well as the source of potent bioactive compounds [12].7 Among several traditional claims, the usefulness of G. odorata roxb in fever, inflammation and pain have been emphasized more in literature. Hence it was considered that investigations for these medicinal properties might give scientific authentication to the traditional claims. Thus the aim of this present study was to carry out a pharmacological evaluation of methanol extract of G. odorata roxb for its anti-inflammatory, analgesic, and antipyretic properties.

MATERIALS AND METHODS

Collection and extraction

The fresh leaves of Gynocardia odorata roxb were collected from the Authenticated crude drug supplier in Delhi and authentication of the plant was carried in Botanical Survey of India, Coimbatore, India. A voucher specimen has been deposited in the laboratory for future reference (BSI/SC/7/46/13-14/TECH.785). The leaves of the plant were shade dried and pulverized. The powder was defatted with petroleum ether. Later, it had been subjected to continuous hot extraction with 95% aqueous methanol in a Soxhlet apparatus. The extract (MEGO) was concentrated under vacuum and dried in desiccators (yield 69 gm, 6.9%w/w). The dry extract was kept in vacuum desiccators until use.

Animals

Adult male Wistar albino rats (150–200 g) and Swiss albino mice (20-25 g) were procured from Venkateshwara Enterprises, Bangalore, Karnataka, India and used throughout the study. All the animals were under the age of 8–12 weeks. They were housed in a very clean polypropylene cage and maintained under standard laboratory conditions (temperature 25 ± 2°C with dark/light cycle 12/12 h). They were fed with standard pellet diet and water ad libitum. The animals were acclimatized to laboratory conditions for one week before experiment. Experiments were performed complied with the rulings of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) New Delhi, India under the registration No: 1135/a/07/CPCSEA.

Phytochemical screening

The preliminary phytochemical investigations were performed for various phytoconstituents such as alkaloids, flavonoids, tannins, terpenoids, saponin, and steroids etc which are probably responsible for the activity of the extract [13].

Acute toxicity

Acute toxicity studies were performed as per OECD-423 guidelines [14]. Male Wistar albino rats selected by random sampling technique were utilized during this study. The animals were fasted for 4h with free access to water only. The plant extract was administered orally at a dose of 5mg/kg initially and mortality if any was observed for 3 days. If mortality was ascertained in two out of three animals, then the dose administered was considered as toxic dose. However, if the mortality was ascertained in only one animal out of three animals then the identical dose was repeated again to confirm the toxic effect. If no mortality was ascertained, then higher (50,300 and 2000 mg/kg) doses of extract were utilized for further toxicity studies.

Drugs and chemicals

The drugs and fine chemicals were purchased from Sigma Chemical Co., St. Louis, USA. All other chemicals and solvents were obtained from local firms (India) and were of highest purity and analytical grade.

Studies on inflammation

Acute inflammation study

Carrageenan-induced paw oedema in rats

Pedal inflammation in male Waster rats (150-200 g) was produced according to the method described earlier [15]. An injection (s. c.) was made of 0.1 ml of 1% carrageenan into the right paw of each rat under the sub plantar aponeurosis. The test groups of rats were administered intraperitonially with 200 and 400 mg/kg of the methanol extract of G. odorata roxb1 h before carrageenan injection. At the same time, the control group received 5 ml/kg of 5% gum acacia and the reference group received 100 mg/kg phenyl butanone (i. p.). The paw value was measured immediately after carrageenan injection and at 1,2,3,4 and 5 h intervals after the administration of the edematogenic agent using a plethysmograph-apparatus up to the anatomical hairline on lateral malleolus [16], and compared with the control animals, which received only the vehicle. The inhibitory activity was calculated according to the following formula [17].

Fig. 1: Effect of G. odorata roxb on Carrageenan induced Paw oedema in rats. *P<0.001 vs control.

Sub-acute inflammation study

Cotton-pellet granuloma in rats

This study was carried out by cotton-pellet implantation method in rats [18]. This method used here was adopted from sheth et al., [19], with a slight modification of using only male rats [20]. Under light ether anaesthesia, sterile cotton- pellets (10 mg) were implanted subcutaneously in the axilla and groin regions of the rats. The animals were treated orally with methanol extract at different doses of G. odorata roxb (200 and 400 mg/kg) daily for 7 consecutive days. Animals in the control group received either normal saline or the vehicle gum acacia. Phenyl butazone (100 mg/kg, orally.) was given to animals in the reference groups. They were sacrificed on day 8, the cotton-pellet removed, freed from extraneous tissue and dried overnight at 60°c and weighed. The percent inhibition of the dry weight of the granuloma were calculated and compared.

Antinociceptive activity

Effect on acetic acid-induced writhing in mice

Analgesic activity was evaluated on the acetic acid-induced writhing according to Koster et al., [21]. The writhes were induced by intra-peritoneal injection of 0.6% acetic acid (v/v) (10 ml/kg). Two different doses (200 and 400 mg/kg) of the extract of G. odorata roxb were administered orally to different groups of six animals each, 30 min before chemical stimulus. The numbers of writhing movements were counted 10, 20, 30, and 40 min after acetic acid injection. Antinociception (analgesia) expressed as the reduction of the number of abdominal constrictions between control animals (acetic acid treated mice) and mice pre-treated with the extract.

Antipyretic activity

Yeast induced hyperthermia

Four groups of six rats each were injected subcutaneously with 10 ml/kg body weight. Yeast suspension (15% aqueous suspension) to induce pyrexia, after measuring the basal rectal temperature (0°c) of each animal. Nineteen hours after yeast injection, the rectal temperature was recorded again and animals showing a rise in temperature of <0.6°c were discarded [22]. Thereafter, treatment was carried out as follows:

Group I: Distilled water (10 ml/kg; p. o.),

Group II-Methanol extract of G. odorata roxb (200 mg/kg: p. o)

Group III- Methanol extract of G. odorata roxb (400 mg/kg: p. o)

Group 1V: Paracetamol (100 mg/kg: p. o.).

Rectal temperatures were then recorded at 20, 21, 22, 23 and 24h (T °c) after yeast injection.

Statistical analysis

The results were expressed as mean ± S. E. M. The data obtained from various groups were statistically analyzed using one-way ANOVA followed by Tukey’s test. The p-value < 0.05 was considered to be statistically significant.

Fig. 2: Effect of G. odorata roxb on Cotton-pellet granuloma in rats. *P<0.001 vs control.

RESULTS

Phytochemical Screening

Preliminary phytochemical analysis revealed the presence of flavonoids, alkaloids, and steroids in MEGO Plant material.

Studies on inflammation

Acute inflammation study

Carrageenan – induced paw oedema in rats

Carrageenan – induced rat paw oedema was markedly inhibited by intraperitonial treatment with either the extract (200, 400 mg/kg) or phenyl butazone (100 mg/kg). The extract of G. odorata roxb showed highly significant (p < 0.001) acute inflammatory effect in a dose related manner, more or fewer equals to the effect were produced by phenyl butazone. The results were shown in Fig. 1.

Sub- acute inflammation study

Cotton Pellet granuloma in rats

In sub acute studies Methanol extract of G. odorata roxb shows highly significant sub acute anti-inflammatory effect [Fig. 2].

Antinociceptive activity

Acetic acid induced writhing in mice

The Methanol extract of G. odorata roxb (200-400 mg/kg, i. p.) were significantly reduced (p < 0.001) acetic acid-induced abdominal constrictions and stretching of hind limbs in a dose dependent manner [Fig. 3].

Antipyretic activity

As shown in Fig. 4 subcutaneous injection of yeast caused elevation of rectal temperature in control rats 19h after administration. Oral administration of the extract produced a significant (P<0.001) dose dependent inhibition of temperature elevation. Peak inhibitory effect was observed at 1 h post-therapy, i. e., 20 h post-yeast injection (p<0.001).

Fig. 3: Effect of G. odorata roxb on Acetic acid induced writhing in mice

DISCUSSION

In the present study, the anti-inflammatory, analgesic and anti-pyretic activity of the methanol extract of G. odorata roxb has been established. The test extract at two different doses (200-400 mg/kg) were found to significantly inhibit the carrageenan-induced rat paw oedema, a test, which has significant predictive value for anti-inflammatory agents acting by inhibiting the mediators of acute inflammation [23]. Oedema formation due to carrageenan in the rat paw is the biphasic event [24]. The initial phase is attributed to the release of histamine and serotonin. The second phase of oedema is due to release of prostaglandins, protease and lysosome [25].

Fig. 4: Effect of G. odorata roxb on yeast induced Hyperpyrexia in rats

The second phase is sensitive to most clinically effective anti-inflammatory drugs [26]. Besides in the carrageenan-induced rat paw oedema model the production of protanoides has been through the serum expression of COX-2 by a positive feedback mechanism [27]. Therefore, it is suggested that the mechanism of action of test extract may be related to prostaglandin synthesis inhibition, as described for the anti-inflammatory mechanism of nonsteroidal anti-inflammatory drugs in the inhibition of an inflammatory process induced by carrageenan.

Likewise, the granulomatous tissue formation is related to the chronic inflammatory process, which is characterized by several phases [28]. In this regard, the oral treatment with 200, 400 mg/kg of the extract of G. odorata roxb and 100 mg/kg of phenyl butazone lead to 54.78 and 68.69% reduction of the granulomatous tissue formation, respectively (P<0.001).

In addition, the classification of antinociceptive drugs is usually based on their mechanism of action either on the central nervous system or on the peripheral nervous system [29]. With respect to the writhing test the research group of Deraedt et al., [30], described the quantification of prostaglandins by radio immuno assay in the peritoneal exudates of rats, obtained after intra peritoneal injection of acetic acid. They found high levels of prostaglandins, PGE2 and PGF2 alpha during the first 30 min after acetic acid injection. Nevertheless, it was found that the intra peritoneal administration of acetic acid-induces the liberation not only of prostaglandins, but also of the sympathetic nervous system mediators [31, 32]. Thus, the results obtained for the writhing test using acetic acid are similar to those obtained for the oedematogenic test using carrageenan. Therefore anti-inflammatory substances may also be involved in the peripheral analgesic activity.

The pain induction caused by liberating endogenous substances as well as some other pain mediators such as arachidonic acid via cyclooxygenase, and prostaglandin biosynthesis. This pain paradigm is widely used for the assessment of peripheral analgesic activity due to its sensitivity and response to the compounds at a dose which is not effective in other methods. The local peritoneal receptor could be the cause of abdominal writhings. Pain sensation in acetic acid induced writhing paradigm is elicited by producing localized inflammatory response due to release of free arachidonic acid from tissue phospholipids via cyclo-oxygenase (COX), and producing prostaglandin specifically PGE2 and PGF2α, the level of lipoxygenase products may also increases in peritoneal fluids [33-34].

Indeed, non-steroidal anti-inflammatory drugs (NSAIDS), like paracetamol, exert their antipyretic action by largely inhibiting prostaglandin (E-type) protection in the hypothalamus [35]. Consequently, elevated plasma prostaglandin level, as observed in fever, is suppressed. Acetyl salicylic acid, another reference anti-pyretic drug (not used in this study), also brings about the same effect by a selective action on a specific cyclo-oxygenase (COX) isoenzyme in the CNS. The Methanol extract of G. odorata roxb demonstrated effective anti pyretic activity as evident in the inhibition of the temperature elevation in the yeast model. The antipyretic action of the extract may possibly be through inhibition of prostaglandin production, leading to suppression of elevated plasma level, especially since the extract had been shown to possess analgesic and anti-inflammatory activities.

CONCLUSION

From these investigations, it may be concluded that the Methanol extract of G. odorata roxb showed analgesic, anti-inflammatory and antipyretic effects, similar to those observed for non-steroidal drugs such as, phenyl butazone and paracetamol. It is important to point out that the phytochemical analysis showed the presence of flavonoids and this might be responsible for anti-inflammatory and analgesic activity. Further investigations are under process in our laboratory to isolate and characterize the specific active components of the plant extract which is responsible for observed pharmacological actions.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

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