INTRODUCTION:

The genus Silybum, one of the smallest genus in the family Asteraceae comprises only two species. The plant is annual herb native to southern Europe, Asia minor and Egypt. In India it is distributed from N.W. Himalayas up to an altitude of 2400m from Kashmir to Uttranchal_.¹Earlier reports indicated the presence of Hepatoprotective² and Antioxidant property.³ In addition plant was found to possess anti-ulcerogenic,⁴ neurotropic and neuroprotective activities.⁵ Flavonolignans such as Silymarin⁶; Flavonoids such as taxifolin,⁷ quercetin,⁷ kaempferol⁸; Fixed oils (20 to 30%) and proteins⁸ are previous isolated components of the plant. In present study an attempt was made to investigate ethylacetate and ethanol extracts of Silybum marianum for its analgesic effects as no work has been done for analgesic activity.

MATERIAL AND METHODS:

Collection and extraction

The fruits of Silybum marianum were purchased from Punjab Agricultural University Ludhiana (Punjab), India in September 2007 and were authenticated by the authority of Botany department, Punjab Agricultural University Ludhiana. A voucher specimen no (SM-1) is deposited in the departmental herbarium of G.H.G Khalsa College of Pharmacy, Gurusar Sadhar, Ludhiana.

The fruits were dried in shade, finely powdered and extracted with ethylacetate and ethanol by cold maceration method in an aspirated bottle for 3-7days. The extracts after maceration were filtered though Whattman filter paper no. 41 was evaporated on water bath and finally dried in vacuum to get powder.

Animals

Swiss mice (18-20g) of either sex were used and were maintained at 25 ± 3ºC. They were kept in a well ventilated animal under natural photoperiodic condition in large polypropylene cages and were fed standard rat chow and water ad libitum. The animal experiment was approved by Animal Ethical Committee of Institute.

Evaluation of Analgesic activity

Mouse writhing assay

The method of Koster et al.⁹ was used. The ethylacetate and ethanol extracts (100-200mg/kg, oral) or acetylsalicylic acid (100mg/kg s.c.) was administered to mice 60 and 30 min, respectively, before intraperitoneal injection of acetic acid (0.6% v/v in saline, 10ml/kg). 10% v/v propylene glycol was used as the control. The number of writhes was counted for 15 min.

Tail immersion test

Mice were divided into six groups containing five animals. The lower 5cm portion of the tail was immersed in a beaker of water maintained at 55 ± 0.5ºC.¹⁰ The time in second for tail withdrawl from the water was taken as the reaction time, with a cut-off time of immersion set at 10 sec. The reaction time was measured 1h before and 1h after oral administration of ethylacetate and ethanol extracts (100-200mg/kg) for 10% v/v propylene glycol (10ml/kg). Morphine (10mg/kg) was administered subcutaneously, 30 min before the test.

Formalin test

The method used was similar to that described previously by shibata et al.¹¹ Twenty microlitres of 1% v/v formalin was injected subcutaneously into the right hind paw of mice. The time in sec spent in licking and biting responses of the injected paw was taken as an indicator of pain response. Reponses were measured for 5 min after formalin injection (first phase) and 15-30 min after formalin injection (second phase).

Table1: Effects of the fruits of S. marianum ethylacetate and ethanol extracts on acetic acid–induced writhing in mice.

Material	Dose (mg/kg)	No. of writhes (´15min)	Inhibition (%)
Control	–	37.0 ± 0.70	–
Ethylacetate extract	100	32.6 ± 0.54	11.90
	200	27.6 ± 0.89^b	25.41
Ethanol extract	100	27.6 ± 0.54^b	25.41
	200	25.2 ± 0.83^b	31.90
Acetylsalicylate	100	118. ± 0.83^a	68.11

Values are expressed as mean ± S.D. ^aP <0.05 significantly different from control. Student’s t – test. N = 6.

Table 2: Effects of the fruits of S. marianum ethylacetate and ethanol extract on tail-immersion test in mice.

Material	Dose (mg/kg)	Reaction time (S)	Increase (%)
Control	-	2.6 ± 0.54	-
Ethylacetate extract	100	3.4 ± 0.54	30.76
	200	4.8 ± 0.83^b	45.83
Ethanol extract	100	3.8 ± 0.83^a	46.15
	200	5.2 ± 0.44^b	100.00
Morphine	10	7.0 ± 0.70^b	169.00

Values are mean ± S.D. ^aP<0.01, ^bP<0.001; Significantly different from control; Paired t-test. N=6.

Table 3: Effects of fruits of S. marianum ethylacetate and ethanol extracts formalin – induced pain in mice.

Material	Dose (mg/kg)	0-5 min	Inhibition (%)	15-30 min	Inhibition (%)
Control	-	89.0 ± 4.18	-	83.4 ± 3.20	-
Ethylacetate extract	100 200	84.4 ± 1.09 70.0 ± 3.80	0.04 21.35	48.2 ± 3.19^b 38.6 ± 2.60^b	42.21 53.72
Ethanol extract	100 200	69.4 ± 3.57^a 65.2±3.34^b	22.03 26.75	31.4 ± 2.60^b 24.2±2.16^b	62.36 70.99
Morphine	10	57.8 ± 2.95^b	35.06	12.2 ± 1.30^b	85.38

Values are mean ± S.D. ^aP<0.01, ^bP<0.001; Significantly different from control; Paired t-test. N=6.

The ethylacetate, ethanol extracts (100-200mg/kg, oral) and acetylsalicylic acid (100mg/kg, S.C) were administered 60 and 30min, respectively, before formalin injection Control animals received 10% v/v propylene glycol (10mg/kg).

Statistical analysis:

All data were expressed as mean ± S.D. and analyzed statistically by using student’s t-test and paired t-test. A difference was considered significant at P<0.05.

Acute toxicity studies:

The method described by Lorke et al.¹² was employed in the determination of the LD₅₀. Mice were separated into two sets of five groups of animals, each consisting of five male and female mice (N=5). They were fasted overnight and then were administered with the ethylacetate and ethanol extracts both orally and intraperitoneally at the following doses; 1, 10, 100, 1000 and 2000mg/kg. Animals were observed for 24h after treatment and the final LD₅₀ value was calculated as the square root of the product of the lowest lethal dose and highest non-lethal dose.

RESULTS AND DISCUSSION:

In the mouse writhing assay, ethylacetate and ethanol extracts caused a significant and dose-dependent inhibition of the control writhes (Table 1). The inhibition produced by the highest dose (200mg/kg) of the extracts was significantly (P<0.01) lower than that by acetylsalicylic acid (100mg/kg). The effects of ethylacetate and ethanol extracts on tail immersion tests were shown in (Table 2). These extracts showed a dose dependent inhibition of pain with the ethanol extract being more active than the ethylacetate one. There was a significant, dose dependent inhibition of both phases of the formalin induced pain response in mice, with a more potent effect on the second

than the first phase (Table 3). Several tests were employed in evaluating the analgesic effect of the ethylacetate and ethanol extracts of fruits of Silybum marianum L. Gaertn. It is necessary to apply tests which differ with respect to stimulus quality, intensity and duration, to obtain as complete a picture as possible of the analgesic properties of a substances using behavioral nociceptive tests.¹³ The results obtained indicate that the extract possess a moderate dose-dependent analgesic effect on the various pain models used. A potent inhibitory effect was exerted by both extracts on the mouse writhing assay and had a significant effect in the tail immersion tests. The extracts inhibited both the phases of formalin-induced pain with a more potent effect on the second phase than the first phase. The formalin pain test is very useful for evaluating the mechanism of pain and analgesia. Drugs which act mainly centrally such as narcotic analgesic, inhibits both phases of pain in this model while peripherally acting drugs, such as acetylsalicylic acid or indomethacin, only inhibit the late phase.¹⁴ The results obtained in this study indicate that the extracts possess analgesic effects.

REFERENCES:

1. Raizada MB. Supplement to Duthies flora of the Upper Gangetic plains and of the adjacent Siwalik and Sub Himalayan Tracts. Dehradun: Bishen Singh Mahinder Pal Singh.1976; pp.132

2. Hahn G et.al . Zur pharmacologic und toxikologie von silymarin, des anti hepatotoxischen wirkprinzipes aus Silybum marianum (L.) Gaertn. Arzneim Forsch. 1968; 18: 698-704

3. Kosina P et. al. Anti-oxidant properties of Silybin glycosides. Phytother Res. 2002; 16: S33-39

4. Khayyal MT et.al. Anti-ulcerogenic effect of some gastro intestinally acting plant extracts and their combination. Arzneim Forsch.2001; 51: 545-553.

5. Kittur S et. al. Neurotrophic and neuroprotective effects of milk thistle (Silybum marianum) on neurons in culture. J Mol Neurosci 2002; 18: 265-269.

6. Wagner H, Horhammer L, Munster R. Zur chemie des Silymarins (silybin), des Wirkprinzips der fruchte von Silybum marianum (L) Gaertn. (Carduus marianus L). Arzeim Forsch 1968; 18: 688-696.

7. Wagner H et. al. Zur struktur von Silychristin, einem Zweiten silymarin-isomeren aus Silybum marianum. Tetrahedron lett. 1971; 22: 1895-1899.

8. Wichtl M, Bisset NG. Herbal Drugs and Phytopharmaceutical stuggart: Medpharm Scientific Publisher.1994; 121-123.

9. Koster R, Anderson M, DeBeer EI. Acetic acid for analgesic screening. Fed Proc.1959; 18: 418

10. Janssen PAJ, Niemegeer CJE, Dony JGH. The inhibitory effects of fentanyl and other morphine-like analgesics on warm water induced tail withdrawal reflex in rats. Arzneim Frosch Drug Res1963; 6: 502.

11. Shibata M et. al. Modified Formalin Test: Characteristic biphasic response. Pain1989 38: 347.

12. Lorke D. Arch Toxicol. A new approach to practical acute toxicity testing. 1983 54: 275.

13. Tjolsen A et. al. The Formalin Test: an evaluation of the method. Pain 1992 51:5

14. Santos ARS et. al. Analgesic effects of callus culture from selected species of Phyllanthus in mice. J Pharm Pharmacol 1994; 46: 755.

Received on 23.06.2009 Modified on 11.08.2009

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 92-94