The analgesic and acute anti-inflammatory effect of the ethanolic extract of the leaves of Paederia foetida ( EEPF ) on experimental animal models

Objective:To study the analgesic and acute anti-inflammatory effect of the ethanolic extract of the leaves of Paederia foetida (EEPF) on experimental animal models. Materials and Methods: Fresh leaves were collected, air-dried, powdered, and percolated in 95% ethanol. Acute toxicity test was done according to OECD guidelines. Healthy albino rats (150-200 gm) of either sex were taken and divided into five groups with six animals in each group for central analgesic activity by tail flick method. Peripheral analgesic activity by 1% glacial acetic acid induced writhing test by taking albino mice (20-30 gm) of either sex which were divided into three groups with six animals in each. EEPF was used in a dose of 500 mg/kg bodyweight subcutaneously for tail-flick method and orally for writhing test. In tail-flick method the basal reaction time was taken and a cut-off period of 10 sec was observed. Reaction time were recorded at predrug, 15, 30, 60, 90, 120, 150 and 180 minutes after administration of drug . The standard drug used for central analgesic activity was pethidine 5 mg/kg bw, naloxone 1mg/kg as antagonist and naloxone 1mg/kg bw with 500mg/kg bw of EEPF to study the central mechanism of action. The standard drug used for peripheral analgesic activity was aspirin 100mg/kg bw orally. A control group was maintained in all the models. For anti-inflammatory study, three groups of animals of either sex (n = 6), weighing 150-200g of the species Rattus norvegicus were taken for the study. Group A was taken as control (Normal salie, 10 mL/kg body weight), Group B as test group (EEPF 500 mg/kg body weight), and Group C as standard (Aspirin 100 mg/kg body weight). The animals were studied for acute inflammation by Carrageenan-induced rat paw edema. Statistical analysis was done by one-way analysis of variance followed by multiple comparison tests. Results: EEPF significantly increased the reaction time in tail-flick method (p<0.05) whereas the combination of naloxone and EEPF decreased the reaction time indicating that naloxone inhibits the analgesic effect of EEPF. In 1% glacial acetic acid induced writhing EEPF reduced writhing significantly In acute inflammation, there was significant inhibition of paw edema in Groups B, C in comparison with Group A (P < 0.05). Conclusion:The ethanolic extract of Paederia foetida has significant analgesic and anti-inflammatory activity.

of Assamese cuisine.It is believed to be a laxative or bowel-function regulator.It is traditionally used in Rheumatic disease, as emetic, as astringent. 4.We have found that its leaves contain flavonoids which was confirmed by test on our own laboratory.There is not much information regarding its anti-inflammatory and analgesic activity.Considering this, the present study has been undertaken to evaluate the anti-inflammatory and analgesic activity of the leaves of Paederia foetida on experimental animal models.

Collection, identification, and extraction of plant materials
Fresh tender leaves of Paederia foetida, approximately 1 kg collected during April-May, 2010, were used for the study.The plant was authenticated by Dr. M. Islam, Professor of Life Science, Dibrugarh University, Assam, India.The plant material was airdried at room temperature.The dried leaves were ground to fine powder and stored in air tight container.

Preparation of the extract
An amount of 250 g of the dry powder obtained was soaked in 95% ethanol for 24 h in percolator.After 24 h it was allowed to percolate slowly and the extract was collected in Petri dishes5.The extract was concentrated in vacuum using rotary flash evaporator.There was a net yield of 22.6 g of concentrated extract (9.12%).

Animals
The experiments were carried out in healthy albino rats of the species Rattus norvegicus of either sex weighing 150-200 gm and healthy albino mice of the species Mus Musculus of either sex weighing 20-30 gm.The animals were procured from the Central Animal House, Assam Medical College & Hospital, Dibrugarh, Assam.The study was conducted in accordance with CPCSEA (Committee for the Purpose of Control and Supervision of Experiment on Animals) guidelines and the study was approved by the Institutional Animal Ethics Committee.They were fed with standard diet and water ad libitum was provided.

Acute toxicity studies
Acute oral toxicity test for the ethanolic extract of leaves of Paederia foetida was carried out as per OECD guidelines 425.6One arbitrary dose 500 mg/kg was selected for the study, as the extract was found safe even at doses more than 2000 mg/kg without any sign of toxicity or mortality.

Method
For Analgesic studies: THE FOLLOWING TWO METHODS WERE USED FOR THE EXPERIMENT---Analgesia by central action:--The central analgesic activity was tested by tail flick method in Albino rats 7 .Healthy albino rats (150-200 gm) of either sex and were taken and divided into five groups with six animals in each group for central analgesic activity.The tail flick latencies (reaction time) of the animals were assessed by analgesiometer (Elite).The basal reaction time was taken by placing the tip (last 2cm) of the tail on the radiant heat source and a cut-off period of 10 sec was observed to prevent damage to the tail.Reaction time were recorded at predrug , 15, 30, 60, 90, 120, 150 and 180 minutes after administration of drug .EEPF was used in a dose of 500 mg/kg bw subcutaneously .The standard drug used for central analgesic activity was pethidine 5 mg/kg bw i.p 8, naloxone 1mg/kg as antagonist and naloxone 1mg/kg bw with 500mg/kg bw of EEPF to study the central mechanism of action.Group A-Control, vehicle normal saline (NS) 10 ml/kg s.c Group B-EEPF 500 mg/kg s.c Group C-Naloxone 1 mg/kg s.c Group D-EEPF 500 mg/kg s.c + Naloxone 1 mg/kg Group E-Pethidine 5 mg/kg i.p Analgesia for peripheral action-The peripheral analgesic activity was tested by glacial acetic acidinduced writhing test in Albino mice 9. Healthy albino mice(20-30 gm) of either sex which were divided into three groups .One hour after administration of drugs, induction of writhing was done in mice by giving intraperitoneal injection of acetic acid at the dose of 10ml/kg bw.The number of writhing responses were counted and recorded for 20 min.EEPF was used in a dose of 500 mg/kg bw orally The standard drug used for peripheral analgesic activity was aspirin 100mg/kg bw orally.Aspirin was taken as standard drug at the dose of 100mg/kg p.o 10 Group A-Control, normal saline 10 ml/kg per orally.Group B-.EEPF 500 mg/kg per orally Group C-Aspirin 100 mg/kg per orally To study the analgesic and acute anti-inflammatory effect of the ethanolic extract of the leaves of Paederia foetida (EEPF) on experimental animal models For Anti-inflammatory studies: Healthy albino rats of both sex weighing 150-200gm were taken.The animals were fasted overnight and water was given ad libitum during the experiment.The animals were divided into 3groups 6 animals each and treated as follows: Group A: Control -Normal saline-10 ml/kg body weight, orally, Group B: EEPF -500mg/kg body weight, orally.Group C: Aspirin -100mg/kg body weight , orally All the drugs were administered orally and the volume of medicaments kept constant at 10 mL/kg body weight of the animals.
The anti-inflammatory activity of ethanolic extract of leaves of Paederia foetida(EEPF) against acute inflammation was tested by carrageenan-induced rat paw edema method.Acute inflammation was produced by sub plantar injection of 0.1 mL of freshly prepared 1% carrageenan suspension in normal saline in the left hind paw of rats in each group.The animals were treated with normal saline, EEPF and aspirin in the respective groups, 1 h before carrageenan injection.The paw volume was measured plethysmometrically as described by Kulkarni.11just before carrageenan injection, that is, at "0" h and then at 1st, 2nd, 3rd, and 4th h after carrageenan injection.Increase in paw edema was measured as the difference between the paw volume at "0" h and paw volume at the respective hour.The percentage inhibition of the rat paw edema was calculated after each hour of carrageenan injection up to 4 h by the formula described by Sudjarwo Agus. 12.

Statistical analysis
For all the above methods, the results were expressed as mean ± standard error of mean.Statistical analysis was done using one-way ANOVA followed by Dunnet's multiple comparison test.P values < 0.05 was considered significant.

Results
Acute oral toxicity tests found the LD-50 of the leaves extract of P. foetida (EEPF) to be more than 2000 mg/kg.
The ethanolic extract of Paederia foetida showed significant central analgesic activity as compared to control (p<0.01;Table I) as evidenced by significant increase in the latency time.Significant peripheral analgesic action was also observed with EEPF and aspirin as compared to control (p<0.01;Table II) as evidenced by inhibition of abdominal writhes produced by acetic acid.There was also significant (p<0.05;TableIII) reduction in carrageenan induced paw edema by EEPG and aspirin.

Discussion
Our study showed that ethanolic extracts of the leaves of Paederia foetida produced significant analgesia, both centrally and peripherally.The extract (500 mg/kg s.c) and pethidine showed significant increase in the reaction time.Pre-treatment with naloxone significantly decreased the reaction time producing hyperalgesia while combined treatment of EEPF (500 mg/kg s.c) and naloxone (1 mg/kg s.c) produced significant decrease in the reaction time as compared to EEPF alone.Naloxone is a competitive antagonist at all types of opioid receptors.It also blocks the actions of endogenous opioid peptides 13 .In the face of a variety of physical (pain) or psychological stressors, an increased release of a variety of opioid peptides occurs 14 N=3, a---p<0.01 when compared to the control, b---p<0.05when compared to the standard, ANOVA followed by Dunnets test.

Table III: Anti-inflammatory activity of the ethanolic extract of Parderia foetida(EEPF) leaves on carrageenan induced rat paw edema in albino rats
Mean increase in paw volume (Mean ± SEM) (mL)(% Inhibition within parentheses) Local peripheral receptors are postulated to be partly involved in the abdominal constriction (writhing response).The abdominal constriction is related to the sensitization of nociceptive receptors to prostaglandins 15 .Standard NSAIDs like aspirin offers relief from inflammatory pain by suppressing the formation of pain substances in peripheral tissues, where prostaglandins and bradykinin were suggested to play an important role in the pain process.Prostaglandins elicit pain by direct stimulation of sensory nerve endings to other pain provoking stimuli 16 .Therefore, it is likely that EEPF suppresses the formation of these substances or antagonize the action of these substances which may serve as it peripheral mechanism of analgesic activity.Carrageenan induced paw edema is a suitable test for evaluating anti-inflammatory drugs which has frequently been used to assess the anti-edematous effect of natural products.Development of edema in the paw of the rat after injection of carrageenan is a biphasic event.The initial phase observed during the first hour is attributed to the release of histamine and serotonin.The second phase of edema is due to the release of prostaglandins, protease and lysosome 17 .
Our study showed that the EEPF (500 mg/kg) and aspirin (100 mg/kg) produced significant reduction of the carrageenan induced paw edema suggesting its anti-inflammatory activity.Flavonoids are known to target prostaglandins which are involved in the late phase of acute inflammation and pain perception 18 .Therefore, flavonoids present in the leaves of Paederia foetida may be responsible for its analgesic and anti-inflammatory activities.
The present study demonstrated that the ethanolic extracts of Paederia foetida showed significant analgesic and anti-inflammatory activity thereby establishing its traditional use in inflammatory and painful conditions.However further studies and development of more purified product of leaves of Paederia foetida are required for proper clinical use.

Conclusion:
The present study demonstrated that the ethanolic extract of the leaves of Paederia foetida showed significant anti-inflammatory activity against acute inflammation and both central and peripheral analgesic activity.However, further studies are required to establish and elaborate the molecular mechanism for proper clinical utility.Also, the development of more purified products of Paederia foetida for the treatment of various inflammatory diseases should be encouraged.

Table II : Assessment of peripheral analgesic action of ethanolic extract of P.foetida by glacial acetic acid induced writhing test in Albino mice.
To study the analgesic and acute anti-inflammatory effect of the ethanolic extract of the leaves of Paederia foetida (EEPF) on experimental animal models .This indicates the involovement of endogenous opioid peptides in mediation of analgesic activity of Paederia foetida