MATERIALS AND METHODS

Plant materials: The bark of the plant of Dillenia indica Linn. was collected from the botanical garden of Pharmacy department, Jahangirnagar University, Bangladesh during January 2009. The plant material was taxonomically identified by the National Herbarium of Bangladesh whose voucher specimen No. JU/32234 is maintained in our laboratory for future reference.

Chemicals: Ammonium molybdate, Folin-Chiocaltu phenol reagent, were purchased from E. Merck (Germany). 1,1-diphenyl-2-picryl-hydrazyl (DPPH), ascorbic acid, quercetin and potassium ferric cyanide and 2',7'-dichlorfluorescein-diacetate (DCFH-DA) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Nalbuphine, Diclofenac-Na was collected from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals and reagents were of analytical grade.

Preparation of plant extract: The plant material was shade-dried with occasional shifting and then powdered with a mechanical grinder, passing through sieve No. 40 and stored in a tight container. The dried powder material (1.5 kg) was refluxed with MeOH for 3 h. The total filtrate was concentrated to dryness, in vacuo at 40°C to render the MeOH extract (490 g).

In vivo analgesic activity
Animal: Albino mice (25-30 g) of both sexes were used for assessing biological activity. The animals were maintained under standard laboratory conditions and had free access to food and water ad libitum. The animals were allowed to acclimatize to the environment for 7 days prior to experimental session. The animals were divided into different groups, each consisting of five animals which were fasted overnight prior to the experiments. Experiments on animals were performed in accordance with guidelines of the Institutional Animal Ethics Committee, Atish Dipankar University of Science and Technology, Dhaka, Bangladesh. Animal treatment and maintenance for acute toxicity and analgesic effects were conducted in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Atish Dipankar University of Science and Technology, Dhaka, Bangladesh.

Acute toxicity study: Acute oral toxicity assay was performed in healthy nulliparous and non pregnant adult female albino Swiss mice (25-30 g) divided into different groups. The test was performed using increasing oral dose of the MDI in water (50, 100, 200, 500, 1000 mg kg^-1 b.wt.), in 20 mL kg^-1 volume to different test groups. Normal group received water. The mice were allowed to feed ad libitum, kept under regular observation for 48 h, for any mortality or behavioral changes (Sanmugapriya and Venkataraman, 2006).

Hot plate method: The animals were divided into four groups with five mice in each group. Group I animals received vehicle (1% Tween 80 in water, 10 mL kg^-1 b.wt.), animals of Group II received nalbuphine at 10 mg kg^-1 b.wt. while animals of Group III and Group IV were treated with 200 and 400 mg kg^-1 b.wt. (p.o.) of the MDI. 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, was observed to avoid damage to the paw (Franzotti et al., 2000). Reaction time was recorded when animals licked their fore or hind paws or jumped prior to 0, 30, 60 and 90 min after oral administration of the samples (Eddy and Leimback, 1953; Malairajan et al., 2006; Toma, 2003).

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, 2003). The animals were treated as discussed above. From 1-2 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 then stopped to avoid injury to mice. The latent period of the tail-flick response was determined before and 0, 30, 60 and 90 min after the administration of drugs.

Acetic acid-induced writhing test: The analgesic activity of the samples was also studied using acetic acid-induced writhing model in mice. Test samples and vehicle were administered orally 30 min before intraperitoneal administration of 0.7% v/v acetic acid but Diclofenac-Na was administered intraperitoneally 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 (Ahmed et al., 2004).

Formalin test: The antinociceptive activity of the drugs was determined using the formalin test described by Dubuisson and Dennis (1977). Control group received 5% formalin. Twenty microliter of 5% formalin was injected into the dorsal surface of the right hind paw 60 min after administration of MDI (200 and 400 mg kg^-1, p.o.) and 30 min after administration of diclofenac Na (10 mg kg^-1, i.p.). The mice were observed for 30 min after the injection of formalin and the amount of time spent licking the injected hind paw was recorded. The first 5 min post formalin injection is referred to as the early phase and the period between 15 and 30 min as the late phase. The total time spent licking or biting the injured paw (pain behavior) was measured with a stop watch.

In vitro antioxidant activity
Determination of total antioxidant capacity: The antioxidant activity of the MDI was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al. (1999).

Free radical scavenging activity measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH): The free radical scavenging activity of MDI, based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was determined by the method described by Braca et al. (2001).

Measurement of the inhibition of the total ROS generation: Mice kidney homogenates, prepared from the kidneys of freshly killed male Swiss albino mice, weighing 30-39 g, were mixed with or without a suspension of extracts and then incubated with 12.5 μM DCFH-DA, at 37°C for 30 min. Phosphate buffer (50 mM, pH 7.4) was used. DCFH-DA is a stable compound which easily diffuses into cells and is hydrolyzed by intracellular esterase to yield a reduced non-fluorescent compound, DCFH which is trapped within the cells. The ROS produced by cells oxidize the DCFH to the highly fluorescent 2',7'-dichlorodihydrofluorescein (DCF). The fluorescence intensity of the oxidized DCF was monitored on a microplate fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT), with excitation and emission wavelengths of 460 and 530 nm, respectively (Label and Bondy, 1990). IC₅₀ value was calculated from the equation of line obtained by plotting a graph of concentration versus % inhibition.

Reducing power activity: The reducing power of MDI was determined according to the method previously described (Oyaizu, 1986). Extracts at different concentrations in 1 mL of 10% DMSO were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide [K₃Fe (CN)₆] (1%) and then the mixture was incubated at 50°C for 30 min. Afterwards, 2.5 mL of trichloroacetic acid (10%) was added to the mixture which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 mL of upper layer solution was mixed with 2.5 mL distilled water and 0.5 mL FeCl₃ (0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Statistical analysis: All values were expressed as the Mean±SEM of three replicate experiments. The analysis was performed by using SPSS statistical package for WINDOWS (version 16.0; SPSS Inc, Chicago). Results related to the reducing power activities were statistically analyzed by applying the Student t-test and p<0.001 were considered to be statistically significant. All in vivo data are subjected to ANOVA followed by Dunnett’s test and p<0.05 were considered to be statistically significant.

RESULTS

In vivo analgesic activity
Hot plate method: Both doses of the extract produced a dose dependent increase in latency time when compared with the vehicle (Fig. 1). The result was found to be statistically significant (p<0.05-0.001).

Fig. 1:	Effects of the MDI on latency to hotplate test. Values are Mean±SEM (n = 5); *p<0.05, Dunnett test as compared to control. Group I: Vehicle (1% Tween 80 in water), Group II: Nalbuphine 10 mg kg-1 b.wt., Group III and Group IV : 200 and 400 mg kg-1 b.wt. (p.o.) of the crude extract of D. indica

Table 1:	Effects of the MDI on acetic acid-induced writhing in mice
Values are Mean±SEM (n = 5), *p<0.05, Dunnett test as compared to vehicle control. Group I: Vehicle (1% Tween 80 in water), Group II: Diclofenac Na 10 mg kg-1 b.wt., Group III and Group IV: 200 and 400 mg kg-1 b.wt. (p.o.) of the MDI

Table 2:	Effect of MDI in hindpaw licking in the formalin test in mice
Values are Mean±SEM (n = 5), * p<0.05, Dunnett test as compared to vehicle control. Group I: Vehicle (1% Tween 80 in water), Group II: Diclofenac Na 10 mg kg-1 b.wt., Group III and Group IV: 200 and 400 mg kg-1 b.wt. (p.o.) of the MDI

Tail immersion test: The tail withdrawal reflex time following administration of the MDI 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. 2).

Acetic acid-induced writhing test: Table 1 shows the effects of the extract of on acetic acid-induced writhing in mice. The oral administration of both doses of MDI significantly (p<0.001) inhibited writhing response induced by acetic acid in a dose dependent manner.

Formalin test: MDI (200 and 400 mg kg^-1, p.o.) significantly (p<0.001) suppressed the licking activity in either phase of the formalin-induced pain in mice in a dose dependant manner (Table 2).

Fig. 2:	Effects of the MDI on tail withdrawal reflex of mice induced by tail immersion method. Values are Mean±SEM (n = 5); *p<0.05, Dunnett test as compared to control. Group I: Vehicle (1% Tween 80 in water), Group II: Nalbuphine 10 mg kg-1 b.wt., Group III and Group IV: 200 and 400 mg kg-1 b.wt. (p.o.) of the crude extract of D. indica

Table 3:	Antioxidant activities of the MDI on total antioxidant capacity, DPPH and total ROS assay methods
Ascorbic acid equivalents (ASC) (mg g-1 of each extract) for the total antioxidant capacity, Values are expressed as Means±SEM of triplicate experiments

MDI, at the dose of 400 mg kg^-1 b.wt., showed the more licking activity against both phases of formalin-induced pain than that of the standard drug diclofenac Na.

In vitro antioxidant activity
Total antioxidant capacity: Total antioxidant capacity of D. indica is expressed as the number of equivalents of ascorbic acid. Total antioxidant capacity of MDI was found to be 213.9±0.69 mg g^-1 m equivalent of ascorbic acid (Table 3).

DPPH radical scavenging activity: The percentage (%) scavenging of DPPH radical was found to be concentration dependent with the IC₅₀ value of 12.32±0.16 μg mL^-1, while IC₅₀ value of standard ascorbic acid was found to be 12.76±0.11 μg mL^-1 (Table 3).

Inhibition of total ROS generation: The percentage inhibition of ROS generation was illustrated in Table 3 and it is observed that scavenging of ROS by the extract is also concentration dependent with the IC₅₀ value of 34.72±0.48 μg mL^-1, while IC₅₀ value of standard trolox was found to be 8.66±0.11 μg mL^-1.

Reducing power ability: For the measurement of the reductive ability, we investigated the Fe³⁺ to Fe²⁺ transformation in the presence of MDI and compared with standards (galic acid, quercetin and ascorbic acid) (Fig. 3).

Fig. 3:	Reducing power of MDI, quercetin, ascorbic acid and galic acid by spectrophotometric detection of Fe3+ to Fe2+ transformation

Like the antioxidant activity, the reducing power of MDI was found to be concentration dependent and statistically significant (p<0.001).

DISCUSSION

The hotplate and tail immersion methods are commonly used for assessing central antinociceptive response. Both methods are further distinguished by their tendency to respond to the pain stimuli conducting through neuronal pathways as tail immersion mediates a spinal reflex to nociceptive stimuli, while the hot plate involves higher brain functions and is a supraspinally organized response (Chapman et al., 1985). Narcotic analgesics inhibit both peripheral and central mechanism of pain, while non steroidal anti-inflammatory drugs inhibit only peripheral pain (Elisabetsky et al., 1995; Pal et al., 1999). As noted, nalbuphine, the reference narcotic analgesic drug (5 mg kg^-1, p.o.) exhibited significant and paramount analgesic effects in both the hot plate (supra spinal) as well as the tail immersion (spinal) test; whereas, MDI (200 and 400 mg kg^-1, p.o.) also produced a statistically significant but lesser in degree antinociceptive response to that of nalbuphine in both test suggesting that the plant extract may act as a narcotic analgesic. However, the mechanism(s) behind the central analgesic response of MDI in both tested methods is not completely understood and may need further investigation.

On the otherhand, acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics and represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from the tissue phospholipid (Ahmed et al., 2006). The response is thought to be mediated by peritoneal mast cells (Ribeiro et al., 2000), acid sensing ion channels (Voilley, 2004) and the prostaglandin pathways (Hossain et al., 2006). The organic acid has also been postulated to act indirectly by inducing the release of endogenous mediators which stimulates the nociceptive neurons that are sensitive to NSAIDs and narcotics (Adzu et al., 2003). It is well known that non-steroidal anti-inflammatory and analgesic drugs mitigate the inflammatory pain by inhibiting the formation of pain mediators at the peripheral target sites where prostaglandins and bradykinin are proposed to play a significant role in the pain process (Hirose et al., 1984). In addition, it was suggested that non narcotic analgesics produce their action by interfering with the local reaction to peritoneal irritation thereby reducing the intensity of afferent nervous stimulation in the acetic acid induced writhing test, a model of visceral pain (Vogel and Vogel, 1997). Therefore, it is likely that MDI might have exerted its peripheral antinociceptive action by interfering with the local reaction caused by the irritant or by inhibiting the synthesis, release and/or antagonizing the action of pain mediators at the target sites and this response in agreement with the previous studies with other parts of D. indica, leaves (Bose et al., 2010). The above findings clearly demonstrated that both central and peripheral mechanisms are involved in the antinociceptive action of MDI. The analgesic activity of MDI could also be linked to the mechanism of action either on central nervous system or peripheral nervous system. Interestingly, compounds like flavonoids (Kim et al., 2004a) and steroids, triterpenes in part, have been shown to possess anti-inflammatory, analgesic activity and the claim made by Pritam et al. (2011). Based on the classes of compounds detected in MDI extract, several mechanisms of action could be used to explain the observed activities of MDI extract.

The formalin model normally postulates the site and the mechanism of action of the analgesic (Chau, 1989). This biphasic model is represented by neurogenic (0-5 min) and inflammatory pain (15-30 min), respectively (Hunskaar and Hole, 1987). Drugs that act primarily on the central nervous system such as narcotics inhibit both as steroids and NSAIDs suppress mainly the late phase (Adzu et al., 2003). The suppression of neurogenic and inflammatory pains by the extract might imply that it contains active analgesic principle that may be acting both centrally and peripherally. This is an indication that the extract can be used to manage acute as well as chronic pain. The mechanism by which formalin triggers C-fibers activation remained unknown for a relatively long time. Recently, however, McNamara et al. (2007) demonstrated that formalin activates primary afferent neurons through a specific and direct on TRPA1, a member of the transient receptor potential family of cation channels, expressed by a subset of C-fiber nociceptors and this effect is accompanied by increased influx of Ca²⁺ ions. TRPA1 cation channels at primary sensory terminals were also reported to mediate noxious mechanical stimuli (Kerstein et al., 2009). These experiments suggest that Ca²⁺ mobilization through TRPA1 cation channels is concomitant with noxious chemicals and mechanical stimuli as they produce their analgesic action. It is likely that the inhibitory effect of MDI to pain response is due to inhibit the increase of the intracellular Ca²⁺ through TRPA1, presumably evoked by formalin. So, the bark extract of D. indica may contain substances that affect the metabolism of Ca²⁺. Literature survey revealed that tannins, triterpenoids and flavonoid are the major phytoconstituents of D. indica (Abdille et al., 2005; Parvin et al., 2009; Muhit et al., 2010). Flovonoids, for example, have been found to suppress the intracellular Ca²⁺ ion elevation in a dose dependent manner, as well as the release of proinflammatory mediators such as TNFα (Kempuraj et al., 2005).

To determine the efficacy of natural antioxidants either as pure compounds or as plant extract, a great number of in vitro methods have been developed in which antioxidant compounds act by several mechanisms. Determination of specific antioxidant species might be less useful than the knowledge of the total antioxidant capacity of a sample. The knowledge of total antioxidant activity can be useful in the analysis of changes in plasma antioxidant activity related to oxidative stress or the understanding of structure–activity relationships of pure antioxidant species. Because of its simplicity and the cheap reagents it uses, the phosphomolybdenum method is an alternative to the methods already available for the evaluation of total antioxidant capacity. The phosphomolybdenum method was based on the reduction of Mo(VI) to Mo(V) by the compounds having antioxidant property and is successfully used to quantify vitamin E in seeds (Prieto et al., 1999). DPPH• is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule (Nakayama, 1994) and is usually used as a substrate to evaluate the antioxidant activity of a compound (Chang et al., 2002). Based on the data obtained from this study, DPPH radical scavenging activity of MDI (IC₅₀ 12.32±0.16 μg mL^-1) was similar to the standard ascorbic acid (IC₅₀ 12.76±0.11 μg mL^-1). These findings agree with previous reports on scavenging of free radicals with other parts of D. indica, leaves and fruits. (Abdille et al., 2005; Parvin et al., 2009). Moreover, it was revealed that MDI did show the proton donating ability and could serve as free radical inhibitor or scavenger. A direct correlation between antioxidant capacity and reducing power of certain plant extracts has been reported (Tanaka et al., 1988). 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 (Duh et al., 1999). Because a substance may act as an antioxidant due to its ability to reduce ROS by donating hydrogen atom (Jayaprakasha et al., 2001; Khanam et al., 2004), the ferric reducing property of plant extracts (Fig. 3) implies that they are capable of donating hydrogen atom in a dose dependent manner. Polyphenolic compounds, like flavonoids, tannins and phenolic acids, commonly found in plants have been reported to have multiple biological effects, including antioxidant activity (Kahkonen et al., 1999). Phenolic compounds are understood to induce the cellular antioxidant system; increase approximately 50% cellular glutathione concentration. Flavonoids are important in the modulation of γ-glutamylcysteine synthase in both cellular antioxidant defenses and detoxification of xenobiotics (Muchuweti et al., 2007). A flavonoid dillenetin have been isolated from the MDI (Muhit et al., 2010) and the total phenol and flavonoid content was found to be the highest in methanolic extract (Abdille et al., 2005) and this may be the cause for the highest antioxidant activity in different model.

Flavonoids may increase the amount of endogenous serotonin or may interact with 5-HT₂A and 5-HT₃ receptors which may be involved in the mechanism of central analgesic activity (Annegowda et al., 2010). Previous researchers reported the presence of several therapeutically valued flavonoids from the D. indica (Arbianti et al., 2007; Bose et al., 2010). Moreover, MDI showed significant analgesic activity in the entire experimental model which may be due to its high flavonoid content as well as free radical scavenging activity, as these free radicals are involved during pain stimulation and antioxidants showed reduction in such pain (Kim et al., 2004b). It is well established that sensitization dorsal horn cells in the spinal cord (central sensitization) plays a fundamentally important role in neuropathic pain. Excessive ROS affects central sensitization and triggers second messengers system that involved in sensitization of dorsal horn neurons and also activates spinal glial cells which in turn play an important role in chronic pain (Raghavendra et al., 2003). In addition, recent studies suggest that the inflammatory tissue damage is due to the liberation of reactive oxygen species form phagocytes invading the inflammation sites (Parke and Sapota, 1996). There are also reports on the role of flavonoid, a powerful antioxidant (Vinson et al., 1995; Brown and Rice-Evans, 1998), in analgesic activity primarily by targeting prostaglandins (Ramesh et al., 1998; Rajnarayana et al., 2001). Moreover, flavonoids have the ability to inhibits eicosanoid biosynthesis. Eicosanoids, such as prostaglandins are involved in various immunological responses and are the end products of the cyclooxygenase and lipoxygenase pathways (Jothimanivannan et al., 2010). Tannins are also found to have a contribution in antinociceptive activity (Ramprasath et al., 2006). Again the plant extract demonstrated good antioxidant action in the tested models. So it can be assumed that cyclooxygenase (COX) inhibitory activity along 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 relieve pain-sensation.

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.