MATERIALS AND METHODS

Plant material: The plant was collected from Old Elephant Road, Eskaton Garden, Dhaka in April 2008 when weed beds were in their maximum densities. The whole plant with leaves, stems and roots was collected and identified by experts in Bangladesh National Herbarium, Mirpur, Dhaka (Accession No. 32784). The plant was thoroughly washed with water; roots were discarded and the aerial parts were dried in hot air woven at 50°C for 3 days and at 40°C for the next 4 days.

Extraction and solvent-solvent partitioning: The dried aerial parts were coarsely powdered from which 83 g was extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50°C. The solvent was completely removed and obtained 18 g (yield 21.7%) dried crude extract. Solvent-solvent partitioning was done using the protocol. The crude extract was dissolved in 10% aqueous methanol to make the mother solution which was partitioned off successively by three solvents namely pet ether (3x100 mL), chloroform (3x100 mL), n-butanol (3x100 mL). All the three fractions and the residual hydromethanol fraction were subjected to dryness under reduced pressure. The dried extracts thus obtained were used for investigation.

Animal: Swiss albino mice of either sex, 3-4 weeks of age, weighing between 20-25 g were used for the analgesic screening. The mice were collected from the animal research branch of the International Center for Diarrheal Disease and Research, Bangladesh (ICDDR, B). Animals were maintained under standard environmental conditions and had free access to feed and water ad libitum. The newly collected mice were acclimatized to the new environment for one week prior to the experiment.

Analgesic activity
Acetic acid-induced writhing test: The antinociceptive activity of the samples was studied using acetic acid-induced writhing model in mice (Ahmed et al., 2004). The animals were divided into control, positive control and test groups with five mice in each group. The animals of test groups received test samples at the dose of 200 and 400 mg kg^-1 b.wt. Positive control group received standard drug Diclofenac-Na at the dose of 10 mg kg^-1 b.wt. and vehicle control group was treated with 1% Tween 80 in water at the dose of 10 mL kg^-1 b.wt. Test samples and vehicle were administered orally 30 min before intraperitoneal administration of 0.7% acetic acid but Diclofenac-Na was administered intraperitonially 15 min before injection of acetic acid. After an interval of 5 min, the mice were observed for specific contraction of body referred to as writhing for the next 10 min.

Hot plate method: The paws of mice are very sensitive to temperature at 55±0.5°C, which are not damaging to the skin. The animals were placed on Eddy’s hot plate kept at a temperature of 55±0.5°C. A cut off period of 15 sec (Franzotti et al., 2000), was observed to avoid damage to the paw. Reaction time was recorded when animals licked their fore or hind paws, or jumped prior to and 0, 30, 60 and 90 min after oral administration of the samples (Eddy and Leimbach, 1953; Kulkarni, 1999; Toma et al., 2003). The animals of test groups received test samples at the dose of 200 and 400 mg kg^-1 b.wt. Positive control group and vehicle control group were treated with Nalbuphine (10 mg kg^-1 b.wt.) and 1% Tween 80 in water (10 mL kg^-1 b.wt.), respectively.

Tail immersion test: The procedure is based on the observation that morphine like drugs selectively prolongs the reaction time of the typical tail withdrawal reflex in mice (Toma et al., 2003). The animals of the control, positive control and test groups were treated with Nalbuphine (10 mg kg^-1 b.wt.), 1% Tween 80 in water (10 mL kg^-1 b.wt.) and test samples at the dose of 200 and 400 mg kg^-1 b.wt., respectively. 1 to 2 cm of the tail of mice was immersed in warm water kept constant at 55°C. The reaction time was the time taken by the mice to deflect their tails. The first reading was discarded and the reaction time was recorded as a mean of the next three readings. A latency period of 20 sec was defined as complete analgesia and the measurement was stopped when the latency period exceeded to avoid injury to mice. The latent period of the tail-flick response was taken as the index of antinociception and was determined before and 0, 30, 60 and 90 min after the administration of drugs.

Statistical analysis: Statistical analysis was carried out using one-way ANOVA followed by Dunnet’s multiple comparisons. The results obtained were compared with the vehicle control group. The p<0.05 were considered to be statistically significant.

RESULTS AND DISCUSSION

Acetic acid-induced writhing in mice: The results showed that the different fractions of C. benghalensis at all doses produced significant (p<0.05) inhibition of writhing reaction in a dose dependent manner. The reference drug diclofenac sodium was more potent than the plant extracts at all dose levels (Table 1).

Hot plate test: The extracts were found to dose-dependently cause a prolongation of the hot plate latency. The response produced by the fractions was significant and comparable to the reference drug Nalbuphine (Table 2).

Table 1:	Effect of C. benghalensis fractions on acetic acid-induced writhing in mice
Values are Mean±SEM, (n = 5); *p<0.05, Dunnet test as compared to control. PECB: Pet ether fraction, CCB: Chloroform fraction, NBCB: n-butanol fraction and HMCB: Hydromethanol fraction of C. benghalensis; 1 = 200 mg kg-1 b.wt., 2 = 400 mg kg-1 b.wt.

Tail immersion test: The tail withdrawal reflex time after administration of the plant extracts was found to increase with increasing dose of the fractions. The result was statistically significant (p<0.001) (Table 3).

Commelina benghalensis has not been subjected to pharmacological investigations so far for analgesic screening to provide scientific justification to its traditional claim in various pains. All the fractions of C. benghalensis displayed significant analgesic action in a dose dependent manner in the tested models. Therefore, the present study has shown to establish remarkable analgesic potential of C. benghalensis using acetic acid-induced writhing test for visceral pain and hot plate plus tail immersion test for pain mediated by central activity.

Acetic acid-induced writhing in mice is a model of visceral pain which is highly sensitive and useful for screening analgesic drugs. C. benghalensis plant extracts caused dose-dependent antinociception against chemical induced pain in mice. Chloroform extract of the plant (CCB) at 400 mg kg^-1 b.wt. was found to exhibit the highest (68.8 %) writhing response inhibitory effect.

Acetic acid-induced writhing model represents pain sensation by triggering localized inflammatory response.

Table 2:	Effect of C. benghalensis fractions on hotplate latency in mice
Values are Mean±SEM, (n = 5); *p<0.05, Dunnet test as compared to control; PECB: Pet ether fraction, CCB: Chloroform fraction, NBCB: n-butanol fraction and HMCB: Hydromethanol fraction of C. benghalensis; 1 = 200 mg kg-1 b.wt., 2 = 400 mg kg-1 b.wt.

Table 3:	Effect of C. benghalensis fractions on tail withdrawal reflux in mice
Values are Mean±SEM, (n = 5); **p<0.001, Dunnet test as compared to control. PECB: Pet ether fraction, CCB: Chloroform fraction, NBCB: n-butanol fraction and HMCB: Hydromethanol fraction of C. benghalensis; 1 = 200 mg kg-1 b.wt., 2 = 400 mg kg-1 b.wt.

Such pain stimulus leads to the release of free arachidonic acid from tissue phospholipid by the action of phospholipase A2 and other acyl hydrolases (Ahmed et al., 2006). The prostaglandins, mainly prostacyclin and prostaglandin-E have been reported to be responsible for pain sensation by exciting the A-fibres. Activities in the A ∂-fibres cause a sensation of sharp well localized pain. Any agent that lowers the number of writhing will demonstrate analgesia preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition (Ferdous et al., 2008).

Commelina benghalensis is reported to contain sterols, alkaloids, caffeine, anthocyanins (water-soluble flavonoid pigments) and carotenoids (Samant and Pant, 2006; Raju et al., 2007). These compounds may be responsible for the analgesic activity. Flavonoids being powerful antioxidants (Brown and Rice-Evans, 1998; Vinson et al., 1995) are reported to play role in analgesic activity primarily by targeting prostaglandins (Rajnarayana et al., 2001; Ramesh et al., 1998). Carotenoids are also reported to possess antioxidant action (Krinsky, 2001). So it can be assumed that their Cyclooxygenase (COX) inhibitory activity and antioxidant activity may reduce the production of free arachidonic acid from phospholipid or may inhibit the enzyme system, which is responsible for the synthesis of prostaglandins and ultimately relive pain-sensation.

The extracts of C. benghalensis also produced dose dependent prolongation of hot plate latency. At 90 min, hydro methanol fraction of the plant (400 mg kg^-1) produced the maximum pain inhibition (80.2%) which was almost equal to the standard drug nalbuphine that produced 80.4% pain inhibition. The hotplate test is supraspinally mediated and therefore a test of central activity. It is an established fact that any agent that causes a prolongation of the hot plate latency using this test must be acting centrally (Ibironke and Ajiboye, 2007).

The thermal stimulation in tail immersion test is also considered to be selective to screen out centrally acting analgesic activity. Tail withdrawal reflex data shows that at 90 min, the pet ether fraction produced maximum pain inhibition (58.2%) while the standard Nalbuphine produced 73.3% pain inhibition. Narcotic analgesics inhibit both peripheral and central mechanism of pain, while NSAIDs inhibit only peripheral pain (Elisabetsky et al., 1995; Pal et al., 1999). The fractions inhibited both types of pain, suggesting that the plant may act as a narcotic analgesic. However in hotplate test, the response produced was very strong and was comparable to the reference drug nalbuphine, which indicates that the plant is likely to have prominent central analgesic action. Overall, the analgesic action of C. benghalensis is assumed to be due to inhibition of prostaglandin synthesis, antioxidant action and its role on central analgesic mechanism.

CONCLUSION

In conclusion, it can be claimed that C. benghalensis possesses significant analgesic action which provides a scientific basis to the folkloric claim of the plant in the management of pain and similar ailments. However, a lot remains to find out the exact mechanism of analgesic action and to isolate the active phytoconstituent(s) responsible for such activity.