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Antioxidant, Analgesic and Anti-inflammatory Activities of the Herb Eclipta prostrata



M.S. Hossain, M.B. Alam, N.S. Chowdhury, M. Asadujjaman, R. Zahan, M.M. Islam, M.E.H. Mazumder, M.E. Haque and A. Islam
 
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ABSTRACT

The present study was designed to investigate the antioxidant, analgesic and anti-inflammatory potential of the methanolic extract along with its organic soluble fractions of the herb Eclipta prostrata. In addition, total phenolic and flavonoid content and total antioxidant capacity were also determined. Antioxidant potential of the extract/fractions was evaluated by DPPH (1,1diphenyl-2-picrylhydrazyl), NO (nitric oxide) and ONOO¯ (peroxynitrite) scavenging assay method. Ethyl acetate fractions (EtOAc) showed highest scavenging activity in all the methods with IC50 value of 12.98±0.08, 45.98±0.07 and 14.45±0.18 μg mL-1 for DPPH, NO and ONOO- assay method, respectively. In reducing power assay, EtOAc fraction also showed significant (p<0.001) activity. Further, the extract/fractions were studied for their analgesic (hot plate, tail immersion and acetic acid induced writhing test) and anti-inflammatory (carrageenan induced paw edema in rats) activities at a dose level of 200 and 400 mg kg-1 body weight. Among all the extract/fractions, EtOAc fraction showed a dose dependent and significant (p<0.005, p<0.05) analgesic activity in all the tested method. EtOAc fractions also reduced the paw edema considerably (86.80% inhibition after 3 h, p<0.005, p<0.05) in dose dependent manner compared to carrageenan induced rat. Altogether, these results suggest that the MeOH extract and its organic soluble fractions could be used as a potential antioxidant, analgesic and anti-inflammatory agent.

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M.S. Hossain, M.B. Alam, N.S. Chowdhury, M. Asadujjaman, R. Zahan, M.M. Islam, M.E.H. Mazumder, M.E. Haque and A. Islam, 2011. Antioxidant, Analgesic and Anti-inflammatory Activities of the Herb Eclipta prostrata. Journal of Pharmacology and Toxicology, 6: 468-480.

DOI: 10.3923/jpt.2011.468.480

URL: https://scialert.net/abstract/?doi=jpt.2011.468.480
 
Received: November 01, 2010; Accepted: March 14, 2011; Published: May 31, 2011



INTRODUCTION

Endogenous free radicals such as superoxide, nitric oxide and hydroxyl free radicals are produced in the human body during the bio-chemical reactions inside the body everyday. In recent years, oxygen derived free radicals have been reported to be closely involved in many biological symptoms such as inflammation, cancer, atherosclerosis and coronary heart disease (Kourounakis et al., 1999; Gulcin et al., 2002). Various free radicals are also responsible for the induction of short term algesia (Chung, 2004). Moreover, free radicals play an important role in the pathogenesis of inflammation (Winrow et al., 1993). Inflammation is the response to injury of cells and body tissues through different factors such as infections, chemicals, thermal and mechanical injuries (Oyedapo et al., 2008). Various endogenous mediators like histamine, serotonin, bradykinin, prostaglandins etc are most abundant in inflammatory cell and among them prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. These mediators even in small quantities can elicit pain response. Pain results in dropped muscular activities. Most of the anti-inflammatory drugs now available are potential inhibitors of cyclooxygenase (COX) pathway of arachidonic acid metabolism which produces prostaglandins. Prostaglandins are hyperalgesic, potent vasodialators and also contribute to erythema, edema and pain. Hence, for treating inflammatory diseases analgesic and anti-inflammatory agents are required (Anilkumar, 2010). Non steroidal anti-inflammatory drugs (NSAIDs) are the most clinically important medicine used for the treatment of inflammation related diseases like arthritis, asthma and cardiovascular disease (Conforti et al., 2009). Having various and severe adverse effects like gastric lesions for NSAIDs, adverse cardiovascular thrombotic effects for selective cyclooxygenase-2 (COX-2) inhibitors (Chowdhury et al., 2009) and tolerance and dependence induced by opiates, use of these drugs as anti-inflammatory and analgesic agents have not been successful in all the cases. Therefore, new anti-inflammatory and analgesic drugs lacking those effects are being searched all over the world as alternatives to NSAIDs and opiates. Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects. The research into plants with alleged folkloric use as pain relievers, anti-inflammatory agents, should therefore be viewed as a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs (Gupta et al., 2006).

Eclipta prostrata belongs to Astraceae family and very common in tropical and subtropical regions. The herb has been used in the treatment of infective hepatitis in India (Wagner et al., 1986) and snake venom poisoning in Brazil (Melo et al., 1994) and hyperlipidemia in traditional medicine (Kumari et al., 2006). It has been reported that the leaves of this herb are used in case of gastritis and respiratory disorders like cough and asthma (Kobori et al., 2004), hepatic and spleen enlargement and jaundice, antibacterial, antifungal, antimyotoxic and antihemorrhagic (Wiart et al., 2004). In addition, the crude form of the herb is reported to have anti hepatotoxic (Wong et al., 1988) and anti-inflammatory activity and also used in skin diseases (Chandra et al., 1987).

In the previous study, the crude hexane, ethanol and aqueous extract of the leaves of Eclipta prostrata was evaluated for its antioxidant and antibacterial activity (Karthikumar et al., 2007). Therefore, the present study was planned to evaluate the possible analgesic, anti-inflammatory and antioxidant potential of methanolic extract along with its organic soluble fractions (dichloromethane and ethylacetate) of the aerial part of Eclipta prostrata in different experimental models.

MATERIALS AND METHODS

Plant materials: The aerial parts of the Eclipta prostrata were collected from the village Kachuria under Mollahat thana of Khulna district, Bangladesh during the month of October 2009. The sample was identified by Mrs. Mahmuda Begum, Senior Scientific Officer, Bangladesh National Herbarium, Dhaka, where the voucher specimen has been deposited. Its DACB Accession Number is BD/2889.

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 carraggenan, and DL-penicillamine (DL- 2- amino- 3- mercapto- 3- methylbutanoic acid) were purchased from Sigma Chemical Company (St. Louis, MO, USA). The high quality DCFH-DA and DHR 123 (dihydrorhodamine 123) and ONOO¯ were purchased from Molecular Probes (Eugene, Oregon, USA) and Cayman (Ann Arbor, MI, USA), respectively. Diclofenac-Na, and Indomethacin were collected from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals and reagents were of analytical grade.

Preparation of plant extract: The aerial parts of the Eclipta prostrata were dried in an oven at 37°C and then powdered with a mechanical grinder, passing through sieve No. 40 and stored in an air tight container. The dried powdered material (1.0 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 (300 g). This extract was suspended in H2O and then successively partitioned with dicholoromethane (CH2Cl2) and ethylacetate (EtOAc), to afford the CH2Cl2 (120 g) and EtOAC (40 g), fractions along with a residue (120 g) present in aqueous phase.

Animal: Albino mice (25-30 g) and Wistar rats (175-250 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.

Acute toxicity study: Acute oral toxicity assay was performed in healthy nulliparous and non pregnant adult female albino Swiss mice (25-30 g) and albino Wistar rats (175-250 g) divided into different groups. The test was performed using increasing oral dose of the MeOH extract and different organic fractions in water (250, 500, 1000 and 1500 mg kg-1 body weight), in 20 mL kg-1 volume to different test groups. Normal group received water. The mice and rats were allowed to feed ad libitum, kept under regular observation for 48 h, for any mortality or behavioral changes.

In vitro antioxidant activity
The amount of phenolic compounds and flavonoids:
The total phenolic and flavonoid content of methanolic extract and several organic fractions were determined using Folin-ciocalteu reagent (Yu et al., 2002) and aluminium chloride colorimetric method (Chang et al., 2002), respectively. The content of total phenolics in the extract and fractions of T. dioica was calculated from regression equation of the calibration curve (y = 0.013x+0.127, r2 = 0.988) and is expressed as galic acid equivalents (GAE) and the flavonoid contents of the extract and fractions in terms of quercetin equivalent (the standard curve equation: y = 0.009x-0.036).

Determination of total antioxidant capacity: The antioxidant activity of the MeOH extract and several other organic fractions were 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 MeOH extract and other fractions, 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). The percentage inhibition activity was calculated from:

% Inhibition = [(A0–A1)/A0]x100

where, A0 is the absorbance of the control and A1 is the absorbance of the extract/ standard. IC50 value was calculated from the equation of line obtained by plotting a graph of concentration (μg mL-1) versus % inhibition.

Nitric oxide radical scavenging assay: The procedure is based on the method (Sreejayan and Rao, 1997), where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. IC50 value was calculated from the equation of line obtained by plotting a graph of concentration (μg mL-1) versus % inhibition.

Measurement of the ONOO¯ scavenging activity: The ONOO¯ scavenging activity was measured by monitoring the oxidation of DHR 123, by modifying the method of Kooy et al. (1994).

Reducing power activity: The reducing power of E. prostrata was determined according to the method previously described by Oyaizu (1986). Increased absorbance of the reaction mixture indicated increased reducing power.

Analgesic activity
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 body weight), animals of Group II received Diclofenac-Na at 10 mg kg-1 body weight while animals of Group III and Group IV were treated with 200 and 400 mg kg-1 body weight (p.o.) of the crude extract/fractions of E. prostate. The animals were placed on Eddy’s hot plate kept at a temperature of (55±0.5)°C. A cut off period of 15 second, 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 et al., 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 et al., 2003).

Acetic acid-induced writhing test: The analgesic activity of the samples was also studied using acetic acid-induced writhing model in mice (Ahmed et al., 2004).

Anti-inflammatory activity
Carrageenan induced paw edema test in rats: Male Wistar rats (175-250 g) were divided into four groups of five animals each. The test groups received 200 and 400 mg kg-1, p.o. of the extract. The reference group received indomethacin (10 mg kg-1, p.o.) while the control group received 3 mL kg-1 of distilled water. After 1 h, 0.1 mL, 1% w/v carrageenan suspension in normal saline was injected into the subplantar tissue of the right hind paw (Winter et al., 1962). The paw volume was measured at 1, 2 and 3 h after carrageenan injection using a micrometer screw gauge. The percentage inhibition of the inflammation was calculated from the formula:

% inhibition = (1 – Dt/D0)x100

where, as D0 was the average inflammation (hind paw edema) of the control group of rats at a given time, Dt was the average inflammation of the drug treated (i.e., extract/fractions or reference indomethacin) rats at the same time (Gupta et al., 2005).

Statistical analysis: All values were expressed as the mean±standard error of three replicate experiments. The analysis was performed by using student’s t test. p<0.001, p<0.005 and p<0.05 were considered to be statistically significant.

RESULTS

Acute toxicity study: The extract/fractions of E. prostrata were safe up to a dose of 1500 mg kg-1 (p.o.) body weight. Behavior of the animals was closely observed for the first 3 h then at an interval of every 4 h during the next 48 h. All extract/fractions did not cause mortality in mice and rats during 48 h observation but little behavioral changes, locomotor ataxia, diarrhea and weight loss were observed. Food and water intake had no significant difference among the group studied.

In vitro antioxidant activity
Total phenolic and flavonoid contents: Table 1 represents the content of both groups in the extract and different organic soluble fractions. The content of total phenolics in the extract and fractions of E. prostrata was determined using the Folin-ciocalteu assay, calculated from regression equation of the calibration curve (y = 0.013x±0.127, r2 = 0.988) and is expressed as Galic Acid Equivalents (GAE) and the flavonoid contents of the extract and fractions in terms of quercetin equivalent (the standard curve eqation: y = 0.009x-0.036). EtOAc fractions showed the highest total phenolic and flavonoid content and was found to be 99.43±1.01 mg g-1 plant extract (in GAE) and 200.43±0.72 mg g-1 plant extract (in quercetin equivalent), respectively.

Total antioxidant capacity: Percentage yield of methanol extract and different organic fractions of E. prostrata and their total antioxidant capacity are given in Table 1. Total antioxidant capacity of E. prostrata is expressed as the number of equivalents of ascorbic acid.

Table 1: Yield, total amount of plant phenolic compounds, flavonoids and total antioxidant capacity of methanolic extract and soluble organic fraction of Eclipta prostrata
aGallic acid equivalents (GAE, mg g-1 of each extract) for the total phenolic content.b Quercetin equivalents ( mg g-1 of each extract) for the total flavonoid content.c Ascorbic acid equivalents ( mg g-1 of each extract) for the total antioxidant capacity. The GAE, QA and ASC values are expressed as Means±SEM of triplicate experiments

Total antioxidant capacity of EtOAc fractions showed the highest and was found to be 589±0.95 mg g-1 equivalent of ascorbic acid.

DPPH radical scavenging activity: Table 2 represents the DPPH radical scavenging activity. EtOAc fractions showed the highest DPPH scavenging activity with the IC50 value of 12.98±0.08 μg mL-1, followed by MeOH and CH2Cl2 fractions with the IC50 value of 45.32±0.16 and 89.45±0.23 μg mL-1, respectively. EtOAc fractions showed the similar activity as standard ascorbic acid (IC50 12.76±0.11 μg mL-1).

Nitric oxide (NO•) scavenging activity: Table 2 describes that all the extracts are likely to have the nitric oxide scavenging activity and statistically significant (p<0.001). The range of IC50 values were 45.98±0.07 to 149.34±0.72 μg mL-1.

Peroxynitrite (ONOO¯) scavenging activity: The ONOO¯ scavenging activity was measured by monitoring the oxidation of DHR 123. The MeOH extract and its organic soluble fractions exhibited significant ONOO¯ scavenging effects in a dose-dependent manner, with IC50 values of 14.45±0.18 μg mL-1 for EtOAc fraction and 34.97±0.21 for MeOH extract, whereas, penicillamine a well known ONOO¯ scavenger, with an IC50 value of 6.98±0.32 μg mL-1 (Table 2).

Reducing power ability: For the measurement of the reductive ability, we investigated the Fe3+ to Fe2+ transformation in the presence of extract and organic fractions. Like the antioxidant activity, the reducing power of E. prostrata increased with increasing concentration of the sample. Figure 1 shows the reductive capabilities of the E. prostrata compared with quercetin, galic acid and ascorbic acid. All E. prostate extract/fractions concentrations tested showed higher activities and these differences were statistically significant (p<0.001).

Analgesic activity
Hot plate test: In the hot plate test, the results presented in Table 3 showed that all extract/fractions significantly (p<0.05) increased pain threshold of mice in a dose dependent manner. The analgesic effect of EtOAc fraction become stronger in 90 min after drug administration, with similar pain inhibition intensity of Diclofenac-Na at 10 mg kg-1, body weight while, the MeOH and CH2Cl2, extract/fractions at dose of 400 mg kg-1 b.wt. exhibited prominent analgesic activity.

Table 2: Antioxidant activities of the E. prostrata extract on DPPH, ONOO- and NO
aDPPH is the free radical scavenging activity (IC50: μg mL-1), bONOO¯ is the inhibitory activity of authentic peroxynitrite (IC50: μg mL-1), cNO is the inhibition of NO production (IC50: μg mL-1), *p<0.001 by student’s test for values between the sample and the control. **p<0.005 by student’s test for values between the sample and the control. # Not significant

Fig. 1: Reducing power of MeOH extract and fractions of E. prostrata and standards (ascorbic acid, quercetin and galic acid) by spectrophotometric detection of Fe3+ to Fe2+ transformation. Results are mean±SEM of three parallel measurements

Table 3: Effect of E. prostrata extract/fractions on hotplate latency in mice
Values are Mean±SEM (n = 5); *p<0.05 by student’s test for values between the sample and the vehicle treated group. MEP: Methanolic extract, CEP: Dichloromethane fraction, EEP: Ethylacetate fraction of E. prostrata. 1: 200 mg kg-1 b.wt, 2: 400 mg kg-1 b.wt

Tail immersion test: Table 4 depicts the analgesic activity shown by extract/fractions by tail flick method. The extract/fractions showed dose dependent analgesic activity against radiant heat induced algesia. High doses (400 mg kg-1) showed significant difference in the analgesic activity when compared with control group. Maximum analgesic effect was observed at 90 min interval. The result was statistically significant (p<0.005).

Acetic acid induced writhing in mice: The extract/fractions effectively reduced the number of abdominal muscle contractions induced by 0.7% acetic acid solution. EtOAc fractions had a dose dependent protection ranging from 45.69 to 68.78% at 200 and 400 mg kg-1 b.wt., respectively against vehicle treated control group and the result were statistically significant (p<0.05) in chemically induced algesia (Fig. 2).

Anti-inflammatory activity
Carrageenan induced paw edema test: Table 5 shows the results of the anti edematous effect of orally administered MeOH extract and its organic fractions of the herb of E. prostrate on carrageenan induced paw edema in rats. All extract/fractions showed dose dependent anti-inflammatory activity and statistically significant (p<0.05; <0.005). EtOAc fractions showed 86.80% inhibition of edema at a dose of 400 mg kg-1 b. wt. in 3h after the administration of carrageenan.

Fig. 2: Effect of E. prostrata extract/fractions on acetic acid induced writhings in mice. Observations were taken 30 min after oral administration of test agents or 15 min after diclofenac Na. Values are Mean±SEM (n=5); *p<0.05 by student’s test for values between the sample and the vehicle treated group

Table 4: Effect of E. prostrata extract/fractions on tail withdrawal reflux in mice
Values are Mean±SEM (n=5); *p<0.005 by student’s test for values between the sample and the vehicle treated group. MEP: Methanolic extract, CEP: Dichloromethane fraction, EEP: Ethylacetate fraction of E. prostrata. 1: 200 mg kg-1 b.wt, 2:400 mg kg-1 b.wt.

Table 5: Effect of E. prostrata extract/fractions on carrageenan induced paw edema in rats
Values are Mean±SEM (n = 5); *p<0.05 by student=s test for values between the sample and the vehicle treated group. MEP: Methanolic extract, CEP: Dichloromethane fraction, EEP: Ethylacetate fraction of E. prostrata. 1: 200 mg kg-1. b.wt, 2: 400 mg kg-1 b.wt

DISCUSSION

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. 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 EtOAc fractions (IC50 12.98±0.08 μg mL-1) of E. prostrata was similar to the standard (IC50 12.76±0.11μg mL-1). These findings agree with previous reports on scavenging of free radicals (Rao et al., 2009). Moreover, it was revealed that organic soluble fraction of E. prostrata 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. 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). 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 diosmetin, two isoflavonoids 3’-hydroxybiochanin A and 3’-O-methylorobol have been isolated from the E. prostrata (Lee et al., 2009) and the total phenol and flavonoid content was found to be the highest in EtOAc fractions and this may be the cause for the highest antioxidant activity in different model.

The brain and spinal cord play a major role in central pain mechanisms. The dorsal horn of the spinal cord is endowed with several neurotransmitters and receptors including: substance P, somatostatin, neuropeptide Y, inhibitory amino acid, nitric oxide, endogenous opioids and the monoamines which are the major targets for pain and inflammation (McCurdy and Scully, 2005).The hotplate method and tail immersion test were considered to be selective to examine compounds acting through opoid receptor; all the extract/fractions increased pain threshold which 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 (Elisabetsky et al., 1995; Pal et al., 1999). The extract inhibits pain with both mechanisms, suggesting that the plant extract may act as a narcotic analgesic. On the otherhand, 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 the tissue phospholipid (Ahmed et al., 2006). 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 (Ribeiro et al., 2000), acid sensing ion channels (Voilley, 2004) and the prostaglandin pathways (Hossain et al., 2006). 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 E. prostrata (Lee et al., 2009). Moreover, EtOAc extract showed highest analgesic activity in all the experimental model which may be due to its high flavonid 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., 2004).

Carrageenan induced oedema has been commonly used as an experimental animal model for acute inflammation and is believed to be biphasic. The early phase (1-2 h) of the carrageenan model is mainly mediated by histamine, serotonin and increased synthesis of prostaglandins in the damaged tissue surroundings. The late phase is sustained by prostaglandin release and mediated by bradykinin, leukotrienes, polymorphonuclear cells and prostaglandins produced by tissue macrophages (Antonio and Brito, 1998; Gupta et al., 2006). Since the extract/fractions significantly inhibited paw edema induced by carrageenan in the second phase and this finding suggests a possible inhibition of cyclooxygenase synthesis by the extract and this effect is similar to that produced by non-steroidal anti-inflammatory drugs such as indomethacin, whose mechanism of action is inhibition of the cyclooxygenase enzyme. Flavonoids and saponins are well known for their ability to inhibit pain perception as well as anti-inflammatory properties due to their inhibitory effects on enzymes involved in the production of the chemical mediator of inflammation (Sawadogo et al., 2006). This hypothesis is strongly supported by the previous study, which has shown that Eclipta prostrata possess anti-inflammatory activity due to the presence of saponins and high flavonoids contents (Arunachalam et al., 2009). In addition, the release of several ROS (Koblyakov, 2001) and excessive nitric oxide (NO) due to the activation of neutrophils during tissue damage and inflammation, which is responsible a variety of disease states like cerebral ischemia, atherosclerosis, cancers, migraine parkinsonism etc. (Bhandare et al., 2010). Recent findings (Srivastava et al., 2000; Viana et al., 2003) suggest that tannic acid and polyphenols are potent inhibitors of NO synthase activity and NO production. As EtOAc fraction showed significant free radical as well as NO and ONOO¯ scavenging activity, so this can be responsible for the reduction of inflammation in the carrageenan induced paw edema in rats.

The ability of flavonoids to inhibit eicosanoid biosynthesis has been documented. 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). Further flavonoids are able to inhibit neutorophils degranulation and thereby decrease the release of arachidonic acid (Hoult et al., 1994). Thus the presence of flavonoids in the extract/fractions of E. prostrate might be responsible for the anti-inflammatory and analgesic activity in Swiss albino mice and rats as well as in vitro antioxidant activity.

CONCLUSION

In conclusion, the results of the present study, indicate that the MeOH extract and its various organic soluble fractions exhibits interesting antioxidant properties, as well as significant analgesic and anti-inflammatory effect which may be due to the presence of phenolic compounds and flavonoids in the extract/fractions. Now our next aim is to explore the isolation and characterization of lead compound liable for aforementioned activity from this plant.

REFERENCES
1:  Ahmed, F., M.S.T. Selim, A.K. Das and M.S.K. Choudhuri, 2004. Anti-inflammatory and antinociceptive activities of Lippia nodiflora Linn. Pharmazie, 59: 329-330.
Direct Link  |  

2:  Ahmed, F., M.H. Hossain, A.A. Rahman and I.Z. Shahid, 2006. Antinociceptive and sedative effects of the bark of Cerbera odollam Gaertn. J. Oriental Pharmacy Exp. Med., 6: 344-348.

3:  Anilkumar, M., 2010. Ethnomedicinal Plants as Anti-Inflammatory and Analgesic Agents. In: Ethnomedicine: A Source of Complementary Therapeutics, Chattopadhyay, D. (Ed.). Research Signpost, India, ISBN-13: 978-81-308-0390-6, pp: 267-293.

4:  Annegowda, H.V., M.N. Mordi, S. Ramanathan and S.M. Mansor, 2010. Analgesic and antioxidant properties of ethanolic extract of Terminalia catappa L. Leaves. Int. J. Pharmacol., 6: 910-915.
CrossRef  |  Direct Link  |  

5:  Antonio, M.A. and A.R.M.S. Brito, 1998. Oral anti-inflammatory and anti-ulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae). J. Ethnopharmacol., 61: 215-228.
CrossRef  |  PubMed  |  Direct Link  |  

6:  Arunachalam, G., N. Subramanian, G.P. Pazhani and V. Ravichandran, 2009. Anti-inflammatory activity of methanolic extract of Eclipta prostrate L. (Astearaceae). Af. J. Pharm. Pharmacol., 3: 97-100.
Direct Link  |  

7:  Bhandare, A.M., A.D. Kshirsagar, N.S. Vyawahare, A.A. Hadambar and V.S. Thorve, 2010. Potential analgesic, anti-inflammatory and antioxidant activities of hydroalcoholic extract of Areca catechu L. nut. Food Chem. Toxicol., 48: 3412-3417.
CrossRef  |  

8:  Braca, A., N. De Tommasi, L. Di Bari, C. Pizza, M. Politi and I. Morelli, 2001. Antioxidant principles from Bauhinia tarapotensis. J. Nat. Prod., 64: 892-895.
PubMed  |  

9:  Chandra, T., J. Sadique and S. Somasundaram, 1987. Effect of Eclipta alba on inflammation and liver injury. Fitoterapia, 58: 23-32.

10:  Chang, C.C., M.H. Yang, H.M. Wen and J.C. Chern, 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug Anal., 10: 178-182.
Direct Link  |  

11:  Chowdhury, M.A., K.R.A. Abdellatif, Y. Dong, D. Das, M.R. Suresh and E.E. Knaus, 2009. Synthesis of celecoxib analogues possessing a N-difluoromethyl-1,2-dihydropyrid-2-one 5-lipoxygenase pharmacophore: biological evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity. J. Med. Chem., 52: 1525-1529.
PubMed  |  Direct Link  |  

12:  Chung, J.M., 2004. The role of reactive oxygen species (ROS) in persistent pain. Mol. Interv., 4: 248-250.
CrossRef  |  PubMed  |  Direct Link  |  

13:  Conforti, F., S. Sosa, M. Marrelli, F. Menichini and G.A. Statti et al., 2009. The protective ability of Mediterranean dietary plants against the oxidative damage: The role of radical oxygen species in inflammation and the polyphenol, flavonoid and sterol contents. Food Chem., 112: 587-594.
CrossRef  |  

14:  Duh, P.D., Y.Y. Tu and G.C. Yen, 1999. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat). LWT-Food Sci. Technol., 32: 269-277.
CrossRef  |  Direct Link  |  

15:  Eddy, N.B. and D. Leimback, 1953. Synthetic analgesic. II. Dithienyl butenylanddithienyl butylamines. J. Pharmacol. Exp. Ther., 107: 385-393.

16:  Elisabetsky, E., T.A. Amador, R.R. Albuquerque, D.S. Nunes and A.C.T. Carvalho, 1995. Analgesic activity of Psychotria colorata (Willd. ex R. and S.) Muell. Arg. alkaloids. J. Ethnopharmacol., 48: 77-83.
CrossRef  |  

17:  Franzotti, E.M., C.V.F. Santos, H.M.S.L. Rodrigues, R.H.V. Mourao, M.R. Andrade and A.R. Antoniolli, 2000. Anti-inflammatory, analgesic activity and acute toxicity of Sida cordifolia L. (Malva-branca). J. Ethnopharmacol., 72: 273-277.
CrossRef  |  PubMed  |  Direct Link  |  

18:  Gulcin, I., M.E. Buyukokuroglu, M. Oktay and O.I. Kufrevioglu, 2002. On the in vitro antioxidative properties of melatonin. J. Pineal Res., 33: 167-171.
CrossRef  |  Direct Link  |  

19:  Gupta, M., U.K. Mazumder, R.S. Kumar, P. Gomathi, Y. Rajeshwar, B.B. Kakoti and V.T. Selven, 2005. Anti-inflammatory, analgesic and antipyretic effects of methanol extract from Bauhinia racemosa stem bark in animal models. J. Ethnopharmacol., 98: 267-273.
CrossRef  |  PubMed  |  Direct Link  |  

20:  Gupta, M., U.K. Mazumder, P. Gomathi and V.T. Selvan, 2006. Antiinflammatory evaluation of leaves of Plumeria acuminate, 2006. BMC Complementary Alternative Med., 6: 36-36.
CrossRef  |  Direct Link  |  

21:  Hossain, M.M., M.S. Ali, A. Saha and M. Alimuzzaman, 2006. Antinociceptive activity of whole plant extracts of Paederia foetida. Dhaka Univ. J. Pharm. Sci., 5: 67-69.
Direct Link  |  

22:  Hoult, J.R., M.A. Moroney and M. Paya, 1994. Actions of flavonoids and coumarins on lipoxigenase and cyclooxygenase. Methods Enzymol., 234: 443-454.
PubMed  |  

23:  Jothimanivannan, C., R.S. Kumar and N. Subramanian, 2010. Anti-inflammatory and analgesic activities of ethanol extract of aerial parts of Justicia gendarussa Burm. Int. J. Pharmacol., 6: 278-283.
CrossRef  |  Direct Link  |  

24:  Kahkonen, M.P., A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T.S. Kujala and M. Heinonen, 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem., 47: 3954-3962.
CrossRef  |  PubMed  |  Direct Link  |  

25:  Karthikumar, S., K. Vigneswari and K. Jegatheesan, 2007. Screening of antibacterial and antioxidant activities of leaves of Eclipta prostrata (L). Sci. Res. Essay, 2: 101-104.
Direct Link  |  

26:  Kim, H.K., S.K. Park, J.L. Zhou, G. Taglialatela, K. Chung, R.E. Coggeshall and J.M. Chung, 2004. Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain, 111: 116-124.
CrossRef  |  PubMed  |  

27:  Kobori, M., Z. Yang, D. Gong, V. Heissmeyer and H. Zhu et al., 2004. Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex. Cell Death Differ., 11: 123-130.
CrossRef  |  PubMed  |  Direct Link  |  

28:  Koblyakov, V.A., 2001. Free radicals and inflammation (progress in inflammation research series, 1999, 260 p.). Biochemistry, 66: 937-938.
CrossRef  |  

29:  Kooy, N.W., J.A. Royall, H. Ischiropoulos and J.S. Beckman, 1994. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radical Biol. Med., 16: 149-156.
CrossRef  |  PubMed  |  

30:  Kourounakis, A.P., D. Galanakis, K. Tsiakitzis, E.A. Rekka and P.N. Kourounakis, 1999. Synthesis and pharmacological evaluation of novel derivatives of anti-inflammatory drugs with increased antioxidant and anti-inflammatory activities. Drug Dev. Res., 47: 9-16.
CrossRef  |  Direct Link  |  

31:  Kumari, C.S., S. Govindasamy and E. Sukumar, 2006. Lipid lowering activity of Eclipta prostrate in experimental hyperlipidemia. J. Ethnopharmacol., 105: 332-335.
CrossRef  |  PubMed  |  

32:  Lee, M.K., N.R. Ha, H. Yang, S.H. Sung and Y.C. Kim, 2009. Stimulatory constituents of Eclipta prostrata on mouse osteoblast differentiation. Phytother. Res., 23: 129-131.
CrossRef  |  PubMed  |  Direct Link  |  

33:  Malairajan, P., G. Gopalakrishnan, S. Narasimhan and K.J.K. Veni, 2006. Analgesic activity of some Indian medicinal plants. J. Ethnopharmacol., 106: 425-428.
CrossRef  |  PubMed  |  Direct Link  |  

34:  Melo, P.A., M.C. Nascimento, W.B. Mors and G. Surez-Kurtz, 1994. Inhibition of the mytotoxic and hemorrhagic activities of crotalid venoms by Eclipta prostrata (Astraceae) extracts and constituents. Toxicon, 32: 595-603.

35:  McCurdy, C.R. and S.S. Scully, 2005. Analgesic substances derived from natural products (natureceuticals). Life Sci., 78: 476-484.
CrossRef  |  PubMed  |  

36:  Muchuweti, M., E. Kativu, C.H. Mupure, C. Chidewe, A.R. Ndhlala and M.A.N. Benhura, 2007. Phenolic composition and antioxidant properties of some spices. Am. J. Food Technol., 2: 414-420.
CrossRef  |  Direct Link  |  

37:  Nakayama, T., 1994. Suppression of hydroperoxide-induced cytotoxicity by polyphenols. Cancer Res., 54: 1991s-1993s.
PubMed  |  Direct Link  |  

38:  Oyedapo, O.A., C.O. Adewunmi, E.O. Iwalewa and V.O. Makanju, 2008. Analgesic, antioxidant and anti-inflammatory related activities of 21-hydroxy-2,41-dimethoxychalcone and 4-hydroxychalcone in mice. J. Boil. Sci., 8: 131-136.
CrossRef  |  Direct Link  |  

39:  Oyaizu, M., 1986. Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutr. Dietetics, 44: 307-315.
Direct Link  |  

40:  Pal, S., T. Sen and A.K.N. Chaudhuri, 1999. Neuropsychopharmacological profile of the methanolic fraction of Bryophyllum pinnatum leaf extract. J. Pharm. Pharmacol., 51: 313-318.
CrossRef  |  PubMed  |  Direct Link  |  

41:  Prieto, P., M. Pineda and M. Aguilar, 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal. Biochem., 269: 337-341.
CrossRef  |  Direct Link  |  

42:  Rao, D.B., C.R. Kiran, Y. Madhavi, P.K. Rao and T.R. Rao, 2009. Evaluation of antioxidant potential of Clitoria ternate L. and Eclipta prostrate L. Indian J. Biochem. Biophy., 46: 247-252.

43:  Ribeiro, R.A., M.L. Vale, S.M. Thomazzi, A.B.P. Paschoalato, S. Poole, S.H. Ferreira and F.Q. Cunha, 2000. Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice. Eur. J. Pharmacol., 387: 111-118.
CrossRef  |  PubMed  |  Direct Link  |  

44:  Sawadogo, W.R., R. Boly, M. Lompo, N. Some, C.E. Lamien, I.P. Guissou and O.G. Nacoulma, 2006. Anti-inflammatory, analgesic and antipyretic activities of Dicliptera verticillata. Int. J. Pharmacol., 2: 435-438.
CrossRef  |  Direct Link  |  

45:  Sreejayan and M.N.A. Rao, 1997. Nitric oxide scavenging by curcuminoids. J. Pharm. Pharmacol., 49: 105-107.
CrossRef  |  PubMed  |  Direct Link  |  

46:  Srivastava, R.C., M.M. Husain, S.K. Hasan and M. Athar, 2000. Green tea polyphenols and tannic acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett., 153: 1-5.
CrossRef  |  Direct Link  |  

47:  Toma, W., J.S. Graciosa, C.A. Hiruma-Lima, F.D.P. Andrade, W. Vilegas and A.R.M.S. Brita, 2003. Evaluation of the analgesic and antiedematogenic activities of Quassia amara bark extract. J. Ethnopharmacol., 85: 19-23.
CrossRef  |  Direct Link  |  

48:  Viana, G.S.B., M.A.M. Bandeira and F.J.A. Matos, 2003. Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva allemao. Phytomedicine, 10: 189-195.
Direct Link  |  

49:  Voilley, N., 2004. Acid-Sensing Ion Channels (ASICs): New targets for the analgesic effects of Non-Steroid Anti-Inflammatory Drugs (NSAIDs). Curr. Drug Targets Inflamm. Allergy, 3: 71-79.
PubMed  |  Direct Link  |  

50:  Wagner, H., B. Geyer, K. Yoshinobu and S.R. Govind, 1986. Coumestans as the main active principles of the liver drugs Eclipta alba and Wedelia calendulacea. Planta Med., 5: 370-374.
PubMed  |  

51:  Wiart, C., S. Mogana, S. Khalifah, M. Mahan and S. Ismail et al., 2004. Antimicrobial screening of plants used for traditional medicine in the state of Perak, Peninsular Malaysia. J. Fitotera, 75: 68-73.
CrossRef  |  PubMed  |  Direct Link  |  

52:  Winrow, V.R., P.G. Winyard, C.J. Morris and D.R. Blake, 1993. Free radicals in inflammation: Second messengers and mediators of tissue destruction. Br. Med. Bull., 49: 506-522.
Direct Link  |  

53:  Winter, C.A., E.A. Risley and G.W. Nuss, 1962. Carrageenin-induced edema in hind paw of the rat as an assay for anti-inflammatory drugs. Exp. Biol. Med., 111: 544-547.
CrossRef  |  PubMed  |  Direct Link  |  

54:  Wong, S.M., S. Antus, A. Gottsegen, B. Fessler, G.S. Rao, J. Sonnenbichler and H. Wagner, 1988. Wedelolactone and coumestan derivatives as new antihepatotoxic and antiphlogistic principles. Arzneimittelforschung, 38: 661-665.
PubMed  |  

55:  Yu, L., S. Haley, J. Perret, M. Harris, J. Wilson and M. Qian, 2002. Free radical scavenging properties of wheat extracts. J. Agric. Food Chem., 50: 1619-1624.
CrossRef  |  PubMed  |  Direct Link  |  

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