Analgesic screening:
Hot plate method: Result of hotplate test is shown in Table 2. Both doses of the extract produced a dose dependent increase in latency time when compared with the vehicle. The result was found to be statistically significant (p<0.05-0.001).

Tail immersion test: The tail withdrawal reflex time following administration of the extract of M. scandens was found to increase with increasing dose of the sample. The result was statistically significant (p<0.05-0.001) and was comparable to the reference drug Nalbuphine (Fig. 1).

Acetic acid-induced writhing test: Table 3 shows the effects of the extract of on acetic acid-induced writhing in mice. The oral administration of both doses of M. scandens extract significantly (p<0.001) inhibited writhing response induced by acetic acid in a dose dependent manner.

Table 1:	Result of chemical group tests of the methanol extract of Mikania scandens
ME: Methanolic extract; (+): Present; (-): Absent

Table 2:	Effects of the hydromethanol extract of Mikania scandens on latency to hotplate test
Group I animals received vehicle (1% Tween 80 in water), Group II received Nalbuphine 10 mg kg-1 body weight, Group III and Group IV were treated with 250 and 500 mg kg-1 body weight (p.o.) of the crude extract of M. scandens. Values are mean ±SEM, (n = 5); *: p<0.05, **: p<0.001dunnet test as compared to control

Table 3:	Effects of the hydromethanol extract of Mikania scandens on acetic acid-induced writhing in mice
Values are mean ± SEM, (n = 5); **: p<0.001, Dunnet test as compared to control. Group I animals received vehicle (1% Tween 80 in water), Group II received Nalbuphine 10 mg kg-1 body weight, Group III and Group IV were treated with 250 and 500 mg kg-1 body weight (p.o.) of the crude extract of M. scandens

Fig. 1:

Effects of the hydromethanol extract of Mikania scandens on tail withdrawal reflex of mice induced by tail immersion method. Values are mean ±SEM, (n = 5); *: p<0.05, **: p<0.001, Dunnet test as compared to control. Group I animals received vehicle (1% Tween 80 in water), Group II received Nalbuphine 10 mg kg-1 body weight, Group III and Group IV were treated with 250 and 500 mg kg-1 body weight (p.o.) of the crude extract of M. scandens

DPPH radical scavenging activity: The DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity of M. scandens is shown in Fig. 2. This activity was found to increase with increasing concentration of the extract. DPPH antioxidant assay is based on the ability of 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 515-517 nm and also for a visible deep purple color.

Fig. 2:	DPPH radical scavenging activity of the hydromethanol extract Mikania scandens. Values are the average of duplicate experiments and represented as mean ±SD

Fig. 3:	Nitric oxide scavenging activity of the hydromethanol extract Mikania scandens. Values are the average of duplicate experiments and represented as mean ±SD

When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the changes in absorbance. The IC₅₀ value of the extract was 375.40 μg mL^-1, as opposed to that of ascorbic acid (IC₅₀ 55.89 μg mL^-1), which is a well known antioxidant.

Nitric oxide scavenging assay: The hydromethanol extract of the M. scandens exhibited dose dependent scavenging of nitric oxide (Fig. 3) with an IC₅₀ value of 220.43 μg mL^-1 compared to 125.10 μg mL^-1 which was the IC₅₀ value for the reference ascorbic acid.

Fig. 4:	Reducing power of the hydromethanol extract Mikania scandens. Values are the average of duplicate experiments and represented as mean ±SD

Reducing power: The hydromethanol extract displayed remarkable reducing power which was found to rise with increasing concentration as shown in Fig. 4.

DISCUSSION

The hotplate method and tail immersion test are considered to be selective to examine compounds acting through opoid receptor; the extract increased mean basal latency which indicates that it may act via centrally mediated analgesic mechanism. Narcotic analgesics inhibit both peripheral and central mechanism of pain, while non steroidal anti-inflammatory drugs inhibit only peripheral pain^[20,21]. The extract inhibited both mechanisms of pain, suggesting that the plant extract may act as a narcotic analgesic.

Acetic acid-induced writhing model represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from tissue phospholipid^[22]. The acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics The response is thought to be mediated by peritoneal mast cells^[23], acid sensing ion channels^[24] and the prostaglandin pathways^[25].

Preliminary phytochemical screening reveals the presence of flavonoid steroid, alkaloid, tannin, gum and saponin in the plant extract. So, the observed analgesic activity may be attributed to these compounds. Moreover, recent studies suggest that the inflammatory tissue damage is due to the liberation of reactive oxygen species form phagocytes invading the inflammation sites^[26] .There are also reports on the role of flavonoid, a powerful antioxidant^[27,28] , in analgesic activity primarily by targeting prostaglandins^[29,30]. There are also reports on the role of tannins in antinociceptive activity^[31]. Again the plant extract demonstrated good antioxidant action in the tested models. So it can be assumed that cyclooxygenase (COX) inhibitory activity together with antioxidant activity may reduce the production of free arachidonic acid from phospholipid or may inhibit the enzyme system responsible for the synthesis of prostaglandins and ultimately relive pain-sensation.

Polyphenolic compounds, like flavonoids, tannins and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity^{[27,28,32,33]}. Flavonoids and tannins present in the plant extract, as evident from phytochemical screening, may be responsible for the antioxidant action. NO scavenging capacity of the extract may help to arrest the chain of reactions initiated by excess generation of NO that are detrimental to the human health. Nitric oxide is also implicated for inflammation, cancer and other pathological conditions^[34]. A direct correlation between antioxidant capacity and reducing power of certain plant extracts has been reported^[35]. The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom^[36]. Therefore, antioxidants with free radical scavenging activities may have great relevance in the prevention and treatment of diseases associated with oxidants or free radicals^[37].

CONCLUSION

Based on the results of the present study, we conclude that the plant extract possesses strong analgesic and antioxidant potential. However, further studies are necessary to examine underlying mechanisms of analgesic and antioxidant effects and to isolate the active compound (s) responsible for these pharmacological activities.

ACKNOWLEDGEMENT

We express our sincere thanks and gratitude to Professor Dr. Abdul Ghani, Chairman, Department of Pharmacy, Stamford University, Bangladesh for providing laboratory facilities and necessary reagents support while doing the study.