Analgesic and Anti-Inflammatory Activity Pericarps of Sapindus emarginatus Vahl

¹Srikanth J and²Muralidharan P

¹Department of Pharmacology, Sri Ramachandra College of Pharmacy, Sri Ramachandra University, Porur, Chennai-600116, Tamilnadu,

²Department of Pharmacology and Toxicology, C.L.Baid Metha College of Pharmacy, Jyothi Nager, Thoraippakkam, Chennai-600 097, Tamilnadu,

ABSTRACT

The aim of the present study was to evaluate the analgesic and anti-inflammatory activity of the methanolic extract of pericarps of Sapindus emarginatus. The preliminary phytochemical screening of the pericarps revealed the presence of saponins, terpenoids, tannins, flavonoids, glycosides and sugars. The central analgesic activity of the extract was evaluated using eddy’s hot plate method and formalin test whereas peripheral analgesic activity using acetic acid induced writhing test. The extract was studied for anti-inflammatory activity in carrageenan-induced hind paw edema in rats and the paw volume was measured plethysmometrically. The study was carried out using dose (200 and 400 mg/kg, p.o.) of the extract. Pentazocin (10mg/kg, i.p.) is the standard drug for the centrally acting analgesic activity whereas indomethacin (10mg/kg, i.p.) is the standard for peripheral acting analgesics and anti-inflammatory activity. The statistical analysis was carried out using one-way ANOVA followed by Dunnet’s test. P value less than 0.5 were considered significant. The methanolic extract of Sapindus emarginatus significantly (p<0.05) reduced carrageenan-induced paw edema in rats and analgesic activity evidenced by increase in the reaction time by eddy’s hot plate method. It also significantly inhibited the neurogenic and inflammatory pain in formalin test as well as the writhing reaction induced by acetic acid. The methanolic extract of Sapindus emarginatus showed significant anti-inflammatory and analgesic effect comparative to the standard drugs. The pharmacological screening of the extract showed significant antinociceptive activity with anti-inflammatory profile.

KEY WORDS: Sapindus emarginatus, carrageenan, inflammation, paw edema.

INTRODUCTION

Inflammation and pain are local response of living mammalian tissue to injury. It is a body defense reaction in order to eliminate or limit the spread of injurious agent. There are various components to an inflammatory reaction that can contribute to the associate symptoms and tissue injury. Edema formation, leukocyte infiltration and granuloma formation represents such components of inflammation¹. Edema formation in the paw is the result of a synergism between various inflammatory mediators that increase vascular permeability/or the mediators that increase blood flow ².Carrageenan- induced paw edema is widely used for determining the acute phase of inflammation. Histamine, 5-hytroxy tryptamine and bradykinin are the first detectable mediators in the early phase of carrageenan induced inflammation ³.Whereas prostaglandins are detectable in the late phase of inflammation ⁴. It is believed that current analgesia inducing drugs such as opiates and non steroidal inflammatory drugs are not useful in all cases, because of their side effects and potency ⁵. As a result a search for other alternatives seems necessary and beneficial.

Sapindus emarginatus Vahl family Sapindaceae is a medium-sized deciduous tree found in South India. It is commonly called as soap nut tree. Native to South India Sapindus emarginatus is found wild or introduced in tropical and sub-tropical regions, particularly the Indo-Malayan region.

Table 1: Antinociceptive effect of oral administration of S.emarginatus extract on pain induced by intraperitoneal injection of acetic acid in mice.

Groups	Dose (mg/kg)	Number of Contractions	Percentage inhibition
Control	CMC	33 ± 0.54	0.00
S.emarginatus	200	17.2 ± 0.66*	47.87*
S.emarginatus	400	13± 0.4*	60.60**
Indomethacin	10	9 ± 0.7*	72.72**
n=5 the values are expressed in Mean ±SEM; ** = p<0.001, * = p < 0.05 when compared with control group.

Traditionally, Sapindus emarginatus is used as anti-inflammatory and antiprurutic. It is used to purify the blood. The seed is in intoxicant and the fruit rind has oxytropic action. Its powder is used as nasal insufflations. S.emarginatus also showed strong anti-bacterial activity against the tested bacterial strains ⁶. Antifertility and antiandrogenic activities of S.emarginatus extract have been reported ⁷. High content of saponins has been reported in the pericarps ⁸. Two Pisicidal triterpenoid saponins, acetylated triterpene saponins, hederagenin, sweet acyclic sesquiterpene glycoside, Mukurozioside IIb have been isolated from the Pericarps of S. emarginatus ^9,
10. Flavonoids have been isolated from the pericarps of Sapindus emarginatus ¹¹. In the previous studies we reported Antihyperlipidemic activity of Sapindus emarginatus in Triton WR-1339 induced albino rats ¹² and Antihyperglycemic, antidiabetic activity of leaves extracts of Sapindus emarginatus¹³.

However, there are no reports on the Antinociceptive and Anti-inflammatory activity of this plant, the present study was undertaken for the first time to investigate Antinociceptive and Anti inflammatory activity of the methanol extract of pericarps of Sapindus emarginatus.

MATERIALS AND METHODS:

Plant materials and extraction:

The plant S.emarginatus fruit was collected in March 2007 from the Thiagarajar college campus, Madurai, Tamilnadu, India. The plant material was taxonomically identified by the Botanical survey of India, Coimbatore, Tamilnadu, India and the voucher specimen BSI/SC/5/23/08-09/Tech 895 was retained in our laboratory for future reference. The dried powder material (500 g) of the pericarps of Sapindus emarginatus was extracted with 2000 ml of methanol in a soxhelt apparatus. The methanol extract was then distilled, evaporated and dried in vacuum. The resulted extract yield was 7.45%, and the appearance of the extract was dried gum resin in nature.

Experimental Animals:

Adult male mice (20-25 g) were used for the antinociceptive experiments. Adult male wistar rats 150-200 g were used to study the anti inflammatory activity. They were obtained from the animal house, C. L. Baid Metha College of Pharmacy, Thoraippakkam, India. The animals were grouped and housed in polyacrylic cages (38x 23 x 10 cm) with not more than five animals per cage and maintained under standard laboratory conditions (temperature 25+20^oC) with dark and light cycle (14/10 hour). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The mice were acclimatized to laboratory condition for 10 days before commencement of experiment. Institutional Animal Ethical Committee (IAEC) constituted under CPCSEA approved the experimental protocol. (IAEC ref no: IAEC/XIII/04/ CLBMCP/2008-2009 dt/16-6-2008)

Preliminary Phytochemical analysis:

The Methanol extract of the Pericarps of S.emarginatus was subjected to preliminary phytochemical screening ¹⁴.

Acute toxicity studies:

A group of 3 swiss albino mice weighing 22-25 g selected by random sampling technique were used in the study. Acute oral toxicity was performed as per OECD- 423 guidelines (acute class method) ¹⁵. The animals were fasted overnight, provided only water after which extract was administered to the groups orally at the dose level of 2000 mg/kg body weight by gastric intubation and the groups were observed for 14 days. If mortality was observed in 2 or 3 animals among 3 animals, then the dose administered was assigned as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsions and mortality for 72 hours.

Determination of Analgesic activity:

Analgesic activity by acetic acid induced writhing test:

Animals were divided in into four groups containing five animals in each group. Group one served as a control and treated with vehicle (1% Carboxy methyl cellulose (CMC), 1 ml/100g of body weight, p.o.). The second and third groups received the extract of S. emarginatus at doses of 200 and 400 mg/kg, p.o. The fourth group received indomethacin 10 mg/kg, i.p. One hour after administration of the standard and test, each animal intraperitoneally received 1% acetic acid injection, with a volume of 1ml/100 g body weight. After the administration of acetic acid injection, the number of stretching or writhing response per animal was recorded during the subsequent 10 min ¹⁶.

Analgesic activity by Eddy’s Hot plate Method:

The paws of mice and rat are very sensitive to temperature at 55 + 0.5^oC, which are not damaging to the skin. The responses were recorded in the form of jumping, withdrawal or the licking of the paws ¹⁷. Male Swiss albino mice were divided in to four groups of five in each. The animals were removed from the holding room and randomly assigned to treatment groups. Animals received the vehicle (1 ml of 1% CMC, p.o.), S.emarginatus (200 & 400 mg/kg, p.o.) and Pentazocine (10mg/kg, i.p.). Following the 30 min of habituation period, the animals were placed in the eddy’s hot plate maintained at a temperature of 55 + 0.5^oC.A Cut of period of 15 seconds was observed to avoid damage to the paw. Reaction time and the type of response were noted using a stop watch. Response latency was recorded at 30, 60 & 90 minutes after administration of the test drugs.

Analgesic activity by Formalin Test:

Male swiss albino mice were divided in to four groups of five in each. The animals were removed from the holding room and randomly assigned to treatment groups. Animals received the vehicle (1 ml of 1% CMC, p.o.), S. emarginatus (200 & 400 mg/kg, p.o.) and Pentazocine (10mg/kg, i.p.). Half an hour after treatment all the groups received 0.05ml of 10% formalin in to the right hind paw. The duration of paw licking, which is an index of nociception, was recorded in two different time series: the first 5 min (neurogenic pain) and the duration between the fifteenth and the thirteenth minute (inflammatory pain) after formalin administration ^{18, 19}.

Determination of Anti-inflammatory activity:

S. emarginatus was evaluated for anti inflammatory activity by carrageenan-induced rat paw edema method ^{20, 21}. Male Wistar rats (150-200g) were randomly distributed in four groups of five animals each. The first group served as a control, second group served as the standard (Indomethacin 10mg/kg, i.p.) while the third and fourth group received (200mg/kg and 400 mg/kg, p.o.) of S. emarginatus. One hour after the administration of the test drugs, 0.1ml of 1% w/v suspension was injected in to the sub-plantar region of the right hind paw to all the four groups. The paw volumes were measured using plethysmometer. Every hour till 3 h after carrageenan injection and mean increase in paw volumes were noted. Thus oedema volumes in control (Vc) and in groups treated (Vt) with test compounds were calculated. The percentage inhibition was calculated by using the formula²².

% Inhibition = (Vc-Vt)/ Vc X 100

Where,

Vc = oedema volume of control

Vt = oedema volume of test.

Statistical analysis:

The results were expressed as mean ± S.E.M. Statistical analysis was carried out by using ANOVA followed by Dunnet’s multiple comparison tests using Graph pad PRISM software version 4.03 (2005). P < 0.5 was considered significant.

RESULTS:

The methanol extract of S. emarginatus was found to be non toxic up to the dose of 2000 mg/kg and did not cause any death of the tested animals. The phytochemical tests with the methanol extract of S. emarginatus indicated the presence of carbohydrates, flavanoids, glycosides, terpenes, saponins & alkaloids.

The oral administration of S. emarginatus extract significantly inhibited the writhing reaction induced by acetic acid. Both the doses of the extract inhibit acetic acid induced writhing; however the dose of 400 mg/ kg significantly reduced the writhing reaction. The data of writhing test is presented in table 1.

The extract of S. emarginatus administered at a dose of 200 and 400mg/kg in mice has shown significant analgesic activity in hot plate method as supported by increase in latency time. The increase in latency time is dose dependant. The results are tabulated in table 2.

The extract of S. emarginatus significantly reduced the licking time for both the doses. The extract inhibited the first phase (neurogenic pain) and as well as the second phase (inflammatory pain). The data of formalin test and the percentage protection is shown in table 3.

The results of carrageenan induced paw edema are showed in table 4. Methanolic extract of S. emarginatus at 200mg/kg body weight per day when given orally as a suspension the paw volume were reduced by 55.03% whereas in case of 400 mg/ kg body weight per day shows 65.5% inhibition after 3 h which indicate that the effect of S. emarginatus is reflected in dose dependent manner.

DISCUSSION:

Pain is a subjective experience which is difficult to define exactly even though we all experience it. Pain is distinguished as two types, peripheral or neurogenic pain may involve the following pathological states: peripheral nociceptive afferent neurons which are activated by noxious stimuli and central mechanism which is activated by afferent inputs stain sensation ²³. The hot plate method is considered to be selective for screening of the compound acting through the opoid receptor; the extract of S. emarginatus increased the mean basal latency which shows that extract act through centrally acting analgesic. The intraperitoneal injection of acetic acid produces pain through the activation of chemo sensitive nocireceptor or irritation of the visceral surface, thereby leading to the liberation of bradykinin, histamine, prostaglandin and serotonin. Thus the extract has inhibited the pain induced by acetic acid which indicates that plants act through both mechanisms, i.e. central as well as peripheral analgesic ²⁴.

Formalin produces pain through two phases: Neurogenic pain by releasing substance P and inflammatory pain with the release of serotonin, histamine bradykinin and prostaglandin. Narcotic analgesic inhibits both the types of pain, while NSAIDS such as paracetamol, aspirin inhibit only the peripheral pain ²⁵.The extract of S. emarginatus has inhibited both the phase of pain induced by formalin which suggests that the extract may act as a narcotic analgesic. From the obtained pharmacological data, it is evident that the plant is having potential of analgesic property. The analgesic activity is found to be dose dependant.

Carrageenan-induced paw edema was taken as a proto type of exudative phase of acute inflammation. Inflammatory stimuli microbes, chemicals and necrosed cells activate the different mediator systems through a common trigger mechanism. The development of carrageenan-induced edema is believed to be biphasic. The early phases are attributed to the release of histamine and serotonin and the delayed phase is sustained by the leukotrienes and prostaglandins ²⁶.Flavanoids and tannins are reported to inhibit PG synthesis ²⁷. Most of the non steroidal anti inflammatory drugs (NSAID’S) have well balanced anti inflammatory and ulcerogenic activities, which are considered to be due to PG synthetase inhibitor activity. We observed that S. emarginatus showed significant inhibition of carrageenan induced paw edema in the dose dependent manner.

It has been reported that a number of flavanoids possess anti-inflammatory and analgesic activities ²⁸.Flavanoids are known to inhibit the enzyme prostaglandin synthetase, more specifically the endoperoxidase and reported to produce anti-inflammatory effects ²⁸ since prostaglandins are involved in the pain perception; inhibition of their synthesis might be the possible reason for the analgesic activity of the extract.

From the above discussion the methanolic extract from the leaves of S. emarginatus exhibited significant analgesic and anti inflammatory activity. Further detailed investigation is underway to determine the exact phytoconstituents that are responsible for these activities.

Table 2: Effect of S.emarginatus extract on the latency of mice exposed to hot plate

Groups	Dose (mg/kg)	Mean latency before and after drug administration(s) + SEM
Groups	Dose (mg/kg)	0 min	30 min	60 min	90 min
Control	CMC	3.7 ± 0.38	4.1 ± 0.3	4.3 ± 0.5	3.8 ± 0.48
S.emarginatus	200	4.5 ± 0.5	7.4 ± 0.4*	10.4 ± 0.56*	12.2 ± 0.44*
S.emarginatus	400	4.1 ± 0.6	9±0.32**	12.4±0.6*	13.2±0.37**
Pentazocine	10	4± 0.31	10±0.6**	13.8±0.3**	14.2±0.37**

n=5 the values are expressed in Mean ± SEM, ** = p<0.01, * = p < 0.05 when compared with control group.

Table 3: Anti-nociceptive effect of S.emarginatus extract on formalin test in mice

Groups	Dose (mg/kg)	First phase (0-5 min)		Second phase (15-30 min)
Groups	Dose (mg/kg)	Mean licking time SEM	Percentage inhibition	Mean licking time SEM	Percentage inhibition
Control	CMC	60 ± 0.89	0.00	25.6±1.9	0.00
S.emarginatus	200	25.4±1.4*	57.66	4.8±0.58*	81.25
S.emarginatus	400	17.2±0.82**	71.33	4.2±0.68**	83.59
Pentazocine	10	14±0.3**	76.66	3.8±0.38**	85.15

n=5 the values are expressed in Mean ± SEM, ** = p<0.01, * = p < 0.05 when compared with control group.

Table 4: Anti-inflammatory effects of S.emarginatus on carrageenan-induced paw oedema

Groups	Dose (mg/kg)	Mean paw volume in ml						Percentage inhibition
Groups	Dose (mg/kg)	0 min	15 min	30 min	60 min	120 min	180 min	Percentage inhibition
Control	CMC	0.77±0.11	1.04±0.09	1.34±0.14	1.68±0.11	1.76±0.12	1.47±0.04	-
S.emarginatus	200	0.76±0.4	1.16±0.16	1.45±0.11	1.34±0.09	0.82±0.14	0.70±0.20*	55.03
S.emarginatus	400	0.73±0.10	0.84±0.16	0.90±0.10	0.82±0.14	0.66±0.17	0.50±0.16*	65.51
Indomethacin	10	0.71±0.06	0.76±0.11	0.84±0.10	0.74±0.12	0.55±0.16	0.27±0.003**	81.08

n=5 the values are expressed in Mean ± SEM, ** = p<0.01, * = p < 0.05 when compared with control group.

CONCLUSION:

The phytochemical screening of the methanol extract of S. emarginatus indicated the presence of carbohydrates, flavanoids, glycosides, terpenes, saponins & alkaloids. The Methanolic extract showed significant anti-inflammatory and analgesic effect comparative to the standard drugs. It is concluded that the pharmacological screening of the extract showed significant antinociceptive activity with significant anti-inflammatory profile. Further studies are anticipated to extract the particular active constituents responsible for the action.

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Received on 17.08.2009

Accepted on 25.08.2009

Research J. Pharmacology and Pharmacodynamics 2009; 1(1): 21-24