MATERIALS AND METHODS

Plant material: The fresh aerial parts of Andrographis echioides were collected during the flowering stage from Siddha and Ayurvedic Medicines India Pvt. Ltd., Erode Dist., Tamilnadu, in Aug 2006 and identified by G.V.S. Murthy, Botanical Survey of India, Coimbatore, Tamil Nadu, voucher specimen (No. BSI/SC/5/23/06-07/TECH.835) has been deposited at the Herbarium of the Department of Pharmacology, Bharathi College of Pharmacy, Karnataka. The plant material was air dried, powdered and extracted twice with methanol (80%) in percolator. The combined methanol extracts were evaporated to dryness under reduced pressure. In order to estimate the polarity of the active compounds, the total extract was successively partitioned between ether, chloroform and ethyl acetate (yields 0.94.1.19 and 3.14% w/w, respectively).

Animals: In breed Albino male Wistar rats (150-200 g) and Swiss Albino mice (20-25 g) were used for the experiments. All the animals were obtained from the laboratory animal centre, Bharathi College of Pharmacy, Karnataka. The animals were maintained under standard environmental conditions and fed with standard diet and water ad libitum. The experimental was approved by the Experimentation Ethics Committee (1135/a/07/CPCSEA).

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 Wistar rats (150-200 g) was produced according to the method described earlier (Winter et al., 1962). 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^-1 of the various serial extract of A. echioides 1 h before carrageenan injection.

At the same time, the control group received 5 mL kg^-1 of 5% gum acacia and the reference group received 100 mg kg^-1 phenyl butazone (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 (Goldenberg and IIse, 1977) and compared with the control animals, which received only the vehicle. The inhibitory activity was calculated according to the following formula (Chu and Kovacs, 1977).

Sub-acute inflammation study
Cotton-pellet granuloma in rats: This study was carried out by cotton-pellet implantation method in rats (Meier et al., 1950). This method used here was adopted from Sheth et al. (1972), with a slight modification of using only male rats (Lassman et al., 1977). 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 various serial extracts at different doses (200 and 400 mg kg^-1) daily for 7 consecutive days. Animals in the control group received either normal saline or the vehicle gum acacia. Phenyl butazone (100 mg kg^-1, 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. (1959). The writhes were induced by intra-peritoneal injection of 0.6% acetic acid (v/v) (10 mL kg^-1). Two different doses (200 and 400 mg kg^-1) of the various serial extracts 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 pretreated with the extract.

Antipyretic activity
Yeast induced hyperthermia: Ten groups of six rats each were injected subcutaneously with 10 mL kg^-1 b.wt. 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 (Mukherjee et al., 2002). Thereafter, treatment was carried out as follows:

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

Statistical analysis: The data were analyzed for significance using the unpaired two-tailed student’s t-test.

RESULTS

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 extracts (200 and 400 mg kg^-1) or phenyl butazone (100 mg kg^-1). Various serial extracts of A. echioides 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 the Table 1.

Sub-acute inflammation study
Cotton pellet granuloma in rats: In sub acute studies various serial extracts of A. echioides shows highly significant sub acute anti-inflammatory effect (Table 2).

Antinociceptive activity
Acetic acid induced writhing in mice: The various serial extracts (200-400 mg kg^-1, i.p.) were significantly reduced (p<0.001) acetic acid-induced abdominal constrictions and stretching of hind limbs in a dose-dependent manner (Table 3).

Antipyretic activity: As shown in Table 4 subcutaneous injection of yeast caused elevation of rectal temperature in control rats 19 h after administration. Oral administration of the extracts 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).

Table 1:	Effect of A. echioides on carrageenan induced paw edema in rats
Values are expressed in Mean±SEM. n = 6. *p<0.001 vs. control (Student t-test)

Table 2:	Effect of A. echioides on cotton-pellet granuloma in rats
Values are Mean±SEM. n = 6. *p<0.001 vs. control (Student t-test)

Table 3:	Effect of A. echioides on acetic acid induced writhing in mice
Values are expressed in Mean±SEM. n = 6. *p<0.001 vs. control (Student t-test), **p<0.01, ***p<0.05. Values in parenthesis indicate percentage of inhibition

Table 4:	Effect of A. echioides on yeast induced hyperpyrexia in rats
Values are Mean±SEM, *p<0.001 vs. Control (Students t-test) in respect of writhing response. **p<0.01, ***p<0.05

DISCUSSION

Among several traditional claims, the usefulness of A. echioides 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. Moreover, this plant has not been subjected to above mentioned systemic pharmacological screening so far.

In the present study, the anti-inflammatory, analgesic and anti-pyretic activity of the various serial extracts of A. echioides has been established. The test extracts at two different doses (200-400 mg kg^-1) 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 (Mossa et al., 1995). Oedema formation due to carrageenan in the rat paw is the biphasic event (Vinegar et al., 1969). The initial phase is attributed to the release of histamine and serotonin (Crunkhorn and Meacock, 1971). The second phase of oedema is due to release of prostaglandins, protease and lysosome (Vinegar et al., 1969; Crunkon and Meacock, 1971). The second phase is sensitive to most clinically effective anti-inflammatory drugs (Vineger et al., 1969; Di Rosa et al., 1971). 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 (Nantel et al., 1999). Therefore, it is suggested that the mechanism of action of test extracts may be related to prostaglandin synthesis inhibition, as described for the anti-inflammatory mechanism of non-steroidal anti-inflammatory drugs in the inhibition of inflammatory process induced by carrageenan (Di Rosa et al., 1971).

Like wise, the granulomatous tissue formation is related to the chronic inflammatory process, which is characterized by several phases (Swingle and Shideman, 1972). In this regard, the oral treatment with 200 and 400 mg kg^-1 of the various serial extracts of A. echioides and 100 mg kg^-1 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 (Planas et al., 2000). With respect to the writhing test the research group of Derardt et al. (1980), 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 (Hokansan, 1978; Duarte et al., 1988). Thus, the results obtained for the writhing test using acetic acid are similar to those obtained for the odematogenic test using carrageenan. Therefore, anti-inflammatory substances may also be involved in the peripheral analgesic activity.

Indeed, Non-Steroidal Anti-Inflammatory Drugs (NSAIDP), like paracetamol, exert their antipyretic action by largely inhibiting prostaglandin (E-type) protection in the hypothalamus (Rang et al., 1999). 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 various serial extracts of A. echioides 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 various serial extracts of A. echioides 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.