Evaluation of Anti-inflammatory and Analgesic potency of Whole Plant Extract of Solanum surattense in Experimental Animals

K. Sravanthi¹*, K.V. Ramana², G. Sumalatha¹, K. Neeraja³

¹Department of Pharmacology, Hindu College of Pharmacy, Guntur, (A.P), India.

²Department of Pharmacognosy, A. S. N College of Pharmacy, Guntur, (A.P), India.

³Department of Pharmacology, Calcutta institute of pharmaceutical t

A.H.S, Kolkata, India.

ABSTRACT:

The anti-inflammatory effect of the alcoholic extract of Solanum surattense whole plant was assessed in the carrageenan-induced rat paw edema, the antinociceptive effect of the alcoholic extract of Solanum surattense, plant was studied using thermal and chemical-induced pain models using animals, at the doses of 100 and 200 mg/kg body weight. The acute toxicity and the phytochemical constituents of the extract were also determined. The estimated LD₅₀ of the extract was 2gm/kg body weight. Extract significantly (P<0.05), dose-dependently demonstrated anti-inflammatory activity against acute inflammation induced by carrageenan and also the extract significantly (P<0.05) prolonged the pain reaction times in tail flick pain model, and reduced acetic acid –induced writhing. Phytochemical analysis revealed the presence of alkaloids, saponins, flavanoids and carbohydrates in the plant

KEYWORDS: Analgesic, anti-inflammatory, Carrageenan, acute toxicity, Solanum surattense

INTRODUCTION:

Non-steroidal anti-inflammatory drugs are still widely employed as analgesics and to relief from inflammatory conditions, while the opiates remain effective in intense unrelenting pains ¹. The adverse effects of these drugs pose some problems and placed limitations in their use. Hence there is a continuous search for effective and safer alternatives. These drugs have their origin in natural products. Medicinal plants are important sources of new chemical substances with potential therapeutic effects ², and literature has documented plants with putative analgesic and ant- inflammatory activities ^{3, 4, 5}. Solanum surattense is known as Indian night shade or yellow berried night shade plant. The Common name is Kantakari, synonym Solanum xanthocarpum and it belongs to family Solanaceae. It plays an important place among medicinal herbs, (especially, for the treatment of cough) especially in India since ancient times. Kantakari is useful in wide range of diseases. It is more commonly used in the diseases like bronchial asthma, cough, worms etc. The fruits facilitate the seminal ejaculation, alleviate worms, itching, and fever and reduce fats. The fruits and stems are known for several medicinal uses like anthelmintic, antipyretic, laxative, anti-inflammatory, antiasthmatic and aphrodisiac activities⁶. The literature survey shows that no reports were found on the anti-inflammatory and antinociceptive effects of the whole plant extract of Solanum surattense. Hence, this study has been undertaken to evaluate the analgesic and anti-inflammatory activities of the alcoholic extract of Solanum surattense whole plant.

MATERIALS AND METHODS:

Plant material:

The fresh whole plants of Solanum surattense were collected from the medicinal garden of Hindu college of pharmacy, Guntur. The plant was identified and authenticated by Dr. Vijaya lakshmi, Department of Botany, Hindu college, Guntur.

Preparation of extract:

The collected fruits were made in to small pieces separately, shade-dried, and coarsely powdered. The coarse powders were subjected to maceration with absolute ethanol with intermittent shaking. After that the extract was filtered and subjected to distillation to remove the solvent and the last trace of the solvents was removed in vacuo. The prepared extract was used for screening.

Animals:

Swiss albino mice of both sexes weighing between 25-30 g were used for acute toxicity study and analgesic experiments. Male Sprague-Dawley rats weighing 150- 200g were used for anti-inflammatory activity. They were housed in standard environmental condition like, ambient temperature (25°C ± 1°C), relative humidity (55±5%), and 12/12h light dark cycle. Animals had free access to standard pellet diet and water ad libitum. The animals were acclimatized for 3 weeks and were fasted over night with free access to water prior to experiments. All animal experiments were carried out in accordance with the guidelines of CPCSEA. The oral acute toxicity study was performed using the up & down procedure (OPPTS guidelines) ⁷.

Drugs and Chemicals:

Carrageenan was procured from Sigma Chemicals, Pentozocin (Ranbaxy), Ibuprofen (Biochem), acetic acid was obtained from Merck. All other chemicals were used of analytical grade.

Phytochemical Analysis: ⁸

The alcoholic extract of the plant was screened for chemical constituents like alkaloids, tannins, phenol, anthraquinones, saponins, volatile oil, carbohydrates, steroids and glycosides.

Effect of acetic acid induced writhing:⁹

Twenty four mice were fasted for 6 hours and divided into four groups of six animals per group. Animals in group I were given orally normal saline (10 ml/kg) [control], group II Aspirin (150 mg/kg) [standard], groups III and IV respectively given the extract at the dose of 100 mg/kg and 200 mg/kg. Animals in all the groups were injected with acetic acid (0.6%) intraperitoneally after 1 hour of drug administration; the number of writhings was recorded for 20 min. Writhing movement was accepted as contraction of abdominal muscle accompanied by stretching of hind limbs. A significant reduction in number of writhes by drug treated as compared to vehicle treated animal which was considered as a positive analgesic response.

Tail flick method: ¹⁰

The animals were divided into 4 groups: Control, Standard and Test, 6 animals in each group. Pentazocine (30 mg/kg) acted as the standard drug. The drugs were administered intraperitoneally. The tail flick latency was assessed by the analgesiometer. The strength of the current passing through the naked nicrome wire was kept at 2 - 3 amps. The distance between the heat source and the tail skin was 1.5 cm. The site of application of the radiant heat in the tail was maintained at 2.5 cm, measured from the root of the tail. The cut-off reaction time was fixed at 12 s to avoid tissue damage.

Anti-inflammatory Activity: ¹¹

Anti-inflammatory activity of Solanum surattense was evaluated by carrageenan-induced rat paw edema method. Twenty albino rats of either sex were taken and divided into 4 groups, each group contained 5 rats. Group I – received Tween 80 solution (0.1%) in a volume of 10 ml/kg body weight, Group II- received Ibuprofen (10 mg/kg body wt) and served as standard, Group III- received ethanolic extract of (100mg/kg body wt), Group IV - received ethanolic extract of (200mg/kg body wt) , half hour after after the administration (as per the experimental protocol), 0.1 ml of 1% carrageenan solution was injected in the sub plantar region of left hind paw of each rat. The measurement of the paw volume was done on the principle of volume displacement using Plethysmometer. The readings were taken before and at 1 hour intervals after the injection of carrageenan for a period of 4 hrs. The edema at each time was calculated in relation to the paw volume before the injection of the carrageenan.

The anti- inflammatory activity was determined as the percentage of inhibition of inflammation after it was induced by carrageenan by taking volume of inflammation in control group as 100%. The percentage inhibition was calculated by using the formula:

(A-B)

% Inhibition = ------------------- x100

Where, A= Mean paw inflammation of control

B= Mean paw inflammation of test

Statistical analysis:

Data for analgesic and anti-inflammatory activity are expressed as Mean ± SEM from six animals in each group. The significance of difference among the groups was assessed using one way analysis of variance (ANOVA) followed by Dunnet’s t-test, with the help of graph pad prism 4.0 soft ware. P value of < 0.05 was considered as statistically significant.

RESULTS:

Phytochemical Studies:

Phytochemical screening of aqueous and methanol extracts revealed the presence of alkaloids, flavonoids, saponins and carbohydrates.

Acute toxicity studies:

In LD₅₀ studies, it was found that the animals were safe up to a maximum dose of 2 gm/kg body weight. There were no changes in normal behaviour pattern and no signs and symptoms of toxicity and mortality were observed. The biological evaluation was carried out at doses of 100 and 200 mg/kg body weight.

Acetic acid induced writhing:

Pretreatment of mice with extract of Solanum surattense at a dose of 200, 100 mg/kg body weight exhibited a significant and dose dependent reduction in writhing induced by acetic acid. The extract of Solanum surattense at a dose of (200, 100 mg/kg body weight) showed number of writhings 14.94±0.9687, 23.41±1.259 respectively, the control group has shown 35.60± 1.274, where as the aspirin group (150mg/kg) showed 07.34±1.392 writhings. The results of analgesic activity of ethanolic extract of Solanum surattense are shown in (Table 1, Fig 1).

Tail flick method:

Animals treated with extract of Solanum surattense 200, 100 mg/kg body weight showed significant increase in tail flick latency compared to control. The tail flick latency at a dose of 200mg/kg body weight for extract of Solanum surattense was found to be 7.33 sec after 60 min of drug treatment where as the standard drug Pentazocin showed the tail flick latency 8.67 sec (Table 2). The activity was found to be dose dependent.

Anti-inflammatory Activity:

Solanum surattense at 200, 100 mg/kg administered 30 min before the injection of carrageenan inhibited the formation of edema by 56.02, 48.69% respectively, 4 h after injection of the inflammatory stimulus (Table. 3). The result for ibuprofen at 10mg/kg, which inhibited the edema by 70.2%. Both results were statistically significant (Table. 3, Fig.-2, p < 0.05). Moreover, there was a dose response correlation for the tested concentrations for the paw.

Table 1: Effect of Solanum surattense extract on Acetic acid induced writhing on mice

Groups	Treatment Design	Dose	Number of writhings	% inhibition of writhing
I	Control	(10 ml/kg)	35.60± 1.274	-
II	Aspirin	(150mg/kg)	07.34±1.392*	79.38%
III	Solanum surattense extract	(200mg/kg)	14.94±0.9687*	58.03%
IV	Solanum surattense extract	(100mg/kg)	23.41±1.259*	34.24%

Values are expressed as mean + SEM, n=6. ^* p< 0.05 when compared with control group

Table 2: Effect of Solanum surattense extract on tail flick method on mice

Groups	Treatment design	Dose	Basal reaction	Reaction time (seconds)
Groups	Treatment design	Dose	Basal reaction	15 min	30 min	45 min	60 min
I	Control	(10 ml/kg)	2.17	2.5±0.12	2.67±0.06	2.67±0.092	2.67±0.02
II	Pentazocine	(30 mg/kg)	2.33	3.0± 0.09	4.67±0.03	6.33±0.066	8.67±0.02
III	Solanum surattense extract	(200mg/kg)	2.0	3.17±0.012	4.5±0.02	5.83±0.032	7.33±0.037
IV	Solanum surattense extract	(100mg/kg)	1.83	2.33±0.013	4.0±0.057	5.00±0.09	6.67±0.023

Values are expressed as mean + SEM, n=6. ^* p < 0.05 when compared with control group

Table 3: Effect of the Solanum surattense extract on Carrageenan induced oedema in rats

Group	Treatment	Dose	Paw Volume (ml)				% inhibition of paw oedema at 4hours
Group	Treatment	Dose	0 h	1h	2h	4h	% inhibition of paw oedema at 4hours
I	Control	(10 ml/kg)	0.967±0.02^*	1.277±0.022^*	1.46±0.019^*	1.61±0.02^*
II	Ibuprofen	(10mg/kg)	0.986±0.017^*	0.701±0.024^*	0.576±0.01^*	0.475±0.019^*	70.2%
III	Solanum surattense extract	(200mg/kg)	0.995±0.027^*	0.892±0.024^*	0.716±0.016^*	0.708±0.012^*	56.02%
IV	Solanum surattense extract	(100mg/kg)	0.985±0.004^*	0.967±0.051^*	0.915±0.015^*	0.826±0.012^*	48.69%

Values are expressed as mean + SEM, n=6. ^* p < 0.05 when compared with control group

Fig. 1. Effect of oral administration of Solanum surattense extract at 200 mg/kg (3), 100 mg/kg (4) on the writhing number-induced by intraperitoneal administration of a 0.6% acetic acid solution in mice (n = 6). Negative control was treated with normal saline solution (1) and positive control was treated with aspirin (150 mg/kg) (2). Results are shown as mean±S.E.M. inhibition of writhing movements.

DISCUSSION:

The potential analgesic effects of Solanum surattense extract was investigated. The analgesic test used in the present study was chosen in order to different nociceptive stimuli, namely thermic (tail flick) and chemical visceral (writhing) stimuli. The results indicate that the Solanum surattense extract exhibit peripheral, central analgesic properties because of its significant effect exerted on chemical (acetic acid induced) and thermal (tail flick) painful stimuli at higher dose.

The mouse-writhing model is well known for the antinociceptive activity bioassay. It involves different nociceptive mechanisms, such as the sympathetic system (biogenic amines release), cyclooxygenases (COX) and their metabolites and opioid mechanisms. In the chemical pain model employed here, besides the direct stimulation of chemical receptors in the peritoneum, the i.p,

administration of acetic acid acts indirectly by inducing the release of endogenous mediators, which then excite the pain nerve ending, locally increases prostaglandin levels, and these prostaglandins are responsible for the induction of contractions. ¹²

In present study, writhing was induced by acetic acid and the number of writhes was counted and it was observed that the extract of Solanum surattense significantly reduced the number of writhes. Aspirin, the prototype shows more significant result as compare with the extract of Solanum surattense

Thus, extract of Solanum surattense can exert antinociception by mechanisms similar to NSAIDs, perhaps by blocking the receptor or the release of endogenous substances that excite pain nerve endings. NSAIDs such as Aspirin inhibit cyclooxygenases (COX) in peripheral tissues, thereby reducing PGE₂ (prostaglandin E₂) synthesis and interfering with the mechanism of transduction in primary afferent nociceptors. The presence of the phytoconstituents like alkaloids, saponins, flavanoids may be responsible for observed activity. However, the tested extract has also been effective in tail flick test. The tail flick test has been found to be suitable for evaluation of centrally acting analgesic; it is possible that Solanum surattense extract exerts its effect through central mechanism.

Carrageenan-induced hind paw edema is the standard experimental model of acute inflammation. Carrageenan is the phlogistic agent of choice for testing anti-inflammatory drugs. Carrageenan-induced edema is a biphasic response. The first phase is mediate through the release of histamine, serotonin and kinins whereas the second phase is related to the release of prostaglandin and slow reacting substances ¹³. Anti-inflammatory agents generally act by inhibition of synthesis or release of inflammatory mediators. In the present study, carrageenan induced paw edema was chosen where paw edema was induced by the subplanter injection of carrageenan in the right hind paw of the rats, for every one hour after oral administration of the drugs, the paw edema was measured plethysmomerically for four hours after carrageenan injection and the percentage anti-inflammatory activity was calculated. The up-regulation of COX-2 with the increased local production of PGE₂is the key event of carrageenan induced inflammation and the secretion of other mediators is the secondary event that amplified the inflammatory response. Therefore, in this present study the Solanum surattense extract showed significant anti inflammatory activity might partly by suppressing the up-regulation of COX-2 at the inflammatory site or inhibition of cytokine release¹⁴. This suggests that Solanum surattense extract is useful in acute inflammation.

Phytochemcial analysis revealed the presence of tannins, alkaloids, saponins and flavonoids. The pharmacological activities of medicinal plants are usually due to their secondary metabolites. Some of the constituents of the extract have been documented to possess analgesic and anti-inflammatory activities.

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Received on 24.03.2011

Modified on 05.04.2011

Accepted on 10.04.2011

Research J. Pharmacology and Pharmacodynamics. 5(2): March–April 2013, 101-105

Phytochemical Analysis: 8

Phytochemical Analysis: ⁸