INTRODUCTION:

Herbal medicines derived from plant extracts are being increasingly utilized to treat a variety of clinical diseases, though relatively little knowledge about their mode of action is available. There is a growing interest in pharmacological evaluation of various plants used in Indian traditional system of medicine. The study of plant species with analgesic effects is still a fruitful research in search of new analgesic-anti-inflammatory agents. Datura metel (Syn: Datura fastusa) commonly known as thorn apple grown throughout India is a large annual plant with a zigzag stem covered all over with numerous straight sharp prickles, the flowers are purple outside and usually white inside¹. The whole plant is used to cure headache, hemiplegia, delirium, convulsions, cramps and rheumatism². The roots are used for bites from mad dogs, epilepsy, insanity and in inflammations³. In Ayurvedic preparations such as Chintamani rasa, Jayamangala rasa and Dhustura oil it is used along with mercury and sulphur for curing fever, head ache, inflammations and dandruff ⁴. The plant was reported to contain triterpene^{5, 6}, withanolide ^7-11, alkaloids ^{12, 13}.The plant accumulates more hyoscine than hyoscyamine. The roots are reported to contain 0.77% of hyoscyamine². The present study was carried out to evaluate the analgesic and anti-inflammatory potential of D. metel and validate the traditional folklore claim.

MATERIALS AND METHODS:

Plant material and extraction:

The roots of the plant Datura metel were collected during the month of June, 2009 from Tirupathi hills, Andra Pradesh, India. The collected specimen was identified by Dr. P. Jayaraman, Director, Plant Anatomy research Centre, Chennai, Tamil Nadu. A voucher specimen was deposited in the department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai.

The roots were shade dried and coarsely powdered. Weighed quantity of the powder was extracted with absolute ethanol in a soxhlet extractor. The dark colored extract was recovered by distillation in vacuum. The dried, molten ethanolic extract of D. metel (EEDM) was suspended in 0.5% carboxy methyl cellulose used for animal administration.

Animals:

Healthy Swiss albino mice (20-25g) and Wistar rats (180-200 g) of either sex were used for analgesic and anti-inflammatory activity respectively. The animals were housed in polypropylene cages, maintained under standard conditions (12h light: 12 h dark cycles; 24±2°C; 60% humidity) and fed with standard pellet diet and water ad libitum. The study was approved by the Animal Ethical Committee of Madras Medical College.

Evaluation of Analgesic activity:

The following experimental protocol was used to study the analgesic activity. The mice were divided into four groups comprising of six animals each.

Group I: Control (0-.5% CMC)

Group II: Reference standard (Pentazocine 5mg/kg. i.p. for thermal models and aspirin 100 mg/kg. i.p. for acetic acid writhing test)

Group III: EEDM (200 mg/kg. p.o.)

Group IV: EEDM (400 mg/kg. p.o.)

Writhing Test:¹⁴

Acetic acid (0.6 % v/v) was administered intraperitonially to all the groups at the dose of 1 ml/kg body weight 60 min after the administration of test compounds. Abdominal constriction and full extension of hind limbs, called a writhe was produced. Analgesia was recorded by counting the number of writhing after the injection of acetic acid for a period of 10 min. Significant reduction in the number of writhes by drug treatments as compared to vehicle control animals was considered as a positive analgesic response and the percentage inhibition of writhing was calculated

Hot-plate Test:¹⁵

In hot-plate test, mice were preselected on the hot plate at 55±0.5°C. Animals were then treated with standard (pentozocine, 5 mg / kg) and test compounds (EEDM, 200 and 400 mg / kg). The reaction time for each mouse was determined on the hot plate at 60 min after treatment. To avoid possible injury, a cut-off period of 15 sec was followed while measuring the reaction time, the time taken in sec for fore paw licking or jumping was taken as reaction time7.

Tail Immersion test:¹⁶

Basal reaction time of mice towards the heat source was recorded by immersing the lower 5 cm portion if the tail into a beaker of water maintained at 55±0.5°C. The time in sec for tail withdrawal 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 1 h after administration of EEDM, 200 and 400 mg / kg and Pentozocine (5 mg /kg).

The percentage of analgesia (pain inhibition) was determined using the formula, Pain inhibition = (1-D/C) x 100. [D represents nociception EEDM administration and C represents nociception in control animals].

Anti-inflammatory activity:

The following experimental protocol was used to study the anti-inflammatory activity. The rats were divided into four groups comprising of six animals each.

Group I: Control (0-.5% CMC)

Group II: Reference standard (Indomethacin 10 mg/kg. i.p.)

Group III: EEDM (200 mg/kg. p.o.)

Group IV: EEDM (400 mg/kg. p.o.)

Carrageenan induced rat paw edema:¹⁷

Acute inflammation was produced by the sub plantar administration of 0.1 ml of 1% carrageenan in normal saline in the right paw of rats. The different groups were treated with test and reference drugs respectively. The paw volume was measured at 1h, 3h, 5h, after carrageenan injection using plethysmometer. The animals were pretreated with the extract 1h before administration of carrageenan. The percentage of inhibition in the paw volume was determined using the formula, Percentage inhibition = (1-D/C) x 100. [D represents percentage difference in the paw volume after EEDM administration and C represents the percentage difference of paw volume in control animals]¹⁷.

RESULTS:

Analgesic activity

EEDM at both the tested concentrations (200 and 400 mg / kg) produced significant analgesia (p<0.001) which was evidenced from the reduction in the writhing movements evoked by the injection of acetic acid (Table 1). In the thermal methods a significant effect was produced (p<0.001) with both the concentrations of EEDM (Tables 2 and 3).

Table 1: Effect of EEDM on acetic acid induced writhing in mice

Treatment Group	No. of Writhing (at 20 min)	Inhibition percentage
Control	33.66 ± 0.33	---
Aspirin (100 mg/kg)	12.50 ± 0.22^*	228.57
EEDM 200 mg/kg	23.66 ± 0.21^*	29.70
EEDM 400 mg/kg	15.50 ± 0.88^*	180.28

Data represent mean ± SEM, n = 6, *p<0.001 compared to control. One way ANOVA followed by Dunnet ‘t’ test.

Table 2: Effect of EEDM on hot plate reaction in mice

Treatment Group	Reaction time at 60 min (Sec)	Inhibition percentage
Control	3.50 ± 0.22	---
Pentozocine (5 mg/kg)	11.5 ± 0.22^*	62.86
EEDM 200 mg/kg	7.12 ± 0.25^*	103.42
EEDM 400 mg/kg	9.81 ± 0.30^*	53.95

Data represent mean ± SEM, n = 6, *p<0.001 compared to control. One way ANOVA followed by Dunnet ‘t’ test.

Anti-inflammatory activity:

Pretreatment of animals with EEDM (200 and 400 mg / kg) resulted in a significant and dose related inhibition of Carrageenan induced hind paw edema (Table 4). The extracts showed mild inhibition at 1h, maximum inhibition (p<0.001) at 3h and again the edema inhibition decreased at 5h. Among the tested groups, EEDM 400 mg/kg exhibited greatest activity (69.5%) at 3h, which was comparable with the standard drug (81.45%).

DISCUSSION:

The potential analgesic effect of EEDM was estimated using both chemical and thermal methods in mice. The analgesic test used was chosen in order to test different nociceptive stimuli, namely cutaneous thermic (hot plate, tail immersion), chemical visceral (writhing) stimuli. Acetic acid-induced writhing test was used for detecting both central and peripheral analgesia, whereas hot plate and tail immersion tests are most sensitive towards centrally acting analgesics. In acetic acid-induced abdominal writhing, a visceral pain model, the release of arachidonic acid via cyclooxygenase and prostaglandin plays a role in the nociceptive mechanism¹⁸.

Table 3: Effect of EEDM on Tail Immersion method in mice

Treatment Group	Reaction time at 30 min (sec)	Inhibition percentage	Reaction time at 60 min (Sec)	Inhibition percentage
Control	4.01 ± 0.14	--	4.10 ± 0.14	--
Pentozocine (5 mg/kg)	12.50 ± 0.08^*	211.72	14.60 ± 0.06^*	256.09
EEDM 200 mg/kg	8.60 ± 0.21^*	114.46	10.63 ± 014^*	159.26
EEDM 400 mg/kg	10.11 ± 0.15^*	152.11	12.83 ± 0.14^*	212.92

Data represent mean ± SEM, n = 6, *p<0.001 compared to control. One way ANOVA followed by Dunnet ‘t’ test.

Table 4: Effect of EEDM on carrageen induced hind paw edema in rats

Treatment Group	Paw volume at 1 h (ml)	Paw volume at 3 h (ml)	Paw volume at 5 h (ml)
Control	0.353 ± 0.144	0.663 ± 0.0128	0.657 ± 0.0179
Indomethacin (10 mg/kg)	0.223±0.013^*(36.82%)	0.123±0.0095^*(81.45%)	0.277±0.0164 (57.83%)
EEDM 200 mg/kg	0.267±0.024 (24.36%)	0.240±0.0268^*(63.8%)	0.357±0.0159 (42.6%)
EEDM 400 mg/kg	0.238±0.031^*(32.57%)	0.201±0.0183^*(69.5%)	0.337±0.0212 (45.6%)

Data represent mean ± SEM, n = 6, *p<0.001 compared to control. One way ANOVA followed by Dunnet ‘t’ test.

Administration of EEDM produces a significant reduction in the number of abdominal constrictions and stretching of hind limbs induced by the injection of acetic acid in a dose-dependent manner. EEDM 200 and 400 mg/kg exhibited a writhing inhibition of 58.39% and 68.81% respectively, while the standard drug had 88.39% inhibition.

The hot plate method and tail immersion methods were found to be suitable for evaluation of centrally acting analgesic¹⁹. The centrally acting analgesic exerts their action through μ receptors indicating narcotic involvement²⁰. These analgesics generally increase the pain threshold of the animals towards thermal nociception. EEDM significantly increased the reaction time of the animals towards the thermal source in a dose-dependent manner. In hot plate method, EEDM 200 and 400 mg/kg showed a pain inhibition of 83.03% and 121.01% respectively, standard drug Pentozocine producing 262.54 % inhibition. In tail immersion method, the drug exhibited greater activity at 60 min post drug administration, in which EEDM 200 and 400 mg/kg exhibited an inhibition of 30.76 % and 84.61 % as against 130.76 % inhibition produced by Pentozocine 5 mg / kg. In thermal methods, EEDM exhibited greater percentage of inhibition and comparable with the standard drug. This clearly indicates that the analgesic activity of EEDM may be mediated by central mechanism.

Accumulation of edema fluid as a function of time after the injection of the irritant substance Carrageenan in rats is a biphasic reaction²¹. Histamine and serotonin are usually responsible for eliciting the immediate response of the inflammation in rats called first phase, whereas the kinins and prostaglandins mediate the more prolonged delayed onset responses called second phase ²². From the results, it can be inferred that the inhibitory effects of the drug EEDM in rats could be due to inhibition of prostaglandins at 60 min. The decrease in the edema fluid may be due to increased synthesis of leukotrienes at that stage because inhibition of prostaglandin synthesis diverts the reaction toward increase in leukotrienes synthesis²³.

From the results, it could be concluded that the extract exhibits analgesic activity by both central and peripheral mechanisms. Even if further studies are needed this seems to provide a rationale for the use of this plant in pain and associated inflammatory disorders.

REFERENCES:

1. Kritikar KR, Basu BD. Indian Medicinal Plants, International Book Distributors, Dehradun, Vol I, 1999.

2. Khare CP. Indian Medicinal Plants, An Illustrated Dictionary. Springer, New Delhi 1^st Ed, 2007.

3. Jain SK, Sinha BK, Gupta RC. Notable Plants in Ethno medicine of India, Deep Publications, 1991.

4. Sen Gupta. The Ayurvedic system of Medicine, Lagos Press, New Delhi, Vol I, 1906.

5. Ahmed et al. Daturolone, A triterpene from Datura fastuosa Linn. (Solanaceae). Ind J Chem Sect B. 1976; 614B (12): 1007-1008.

6. Ahmad et al. Daturadiol, A triterpene from Datura fastuosa Linn. (Solanaceae). J Bangladesh Acad Sci. 1977; 2: 71-74.

7. Siddiqui S et al. A novel withanolide Daturilin, from Datura metel. Phytochemistry. 1987; 26(9): 2642-2643.

8. Mahmood L et al. A new withanolide Datumetelin from the leaves of Datura metel Linn. Ind Chem Society. 1988; 26(3): 526-527.

9. Gupta et al. Additional withanolides from Datura metel. J Nat Prod. 1991; 54(2): 599-602.

10. Gupta et al. Withanolides of Datura metel. Phytochemistry. 1992; 31(7): 2423-2425.

11. Manickam M, Srivastava A, Ray Anil B. Withanolides from the flowers of Datura fastuosa. Phytochemistry. 1988; 47(7): 1427-1429.

12. Afsharypuor S, Mostajeran A, Mokhtary R. Variations of scopolamine and atropine in different parts of Datura metel during development. Planta Medica. 1995; 61(4): 383-384.

13. Kundu D, Sarkar AK. Identification of new alkaloid in the seeds of Datura metel Linn. Ind J Pharmacol. 1991; 23(3): 177-178.

14. Balamurugan G, Muralidharan P. Antinociceptive effect of Abutilon indicum Linn leaf extract. Research J Pharm and Tech. 2009; 2(3): 544-547.

15. Muralidharan P, Balamurugan G. Analgesic and anti-inflammatory activities of aqueous extract of Pedalium murex Linn. Biomedicine. 2008; 28(2): 84-87.

16. Ramabadran K, Bansinath M, Turndrof H, Puig MM. tail immersion test for the evaluation of nociceptive reaction in mice. J Pharmacol Methods. 1989; 21: 21-31.

17. Shanmuga Sundaram M, Sivakumar T, Balamurugan G. Anti-inflammatory effect of Cyperus rotundus Linn leaves on acute and sub acute inflammation in experimental rat models. Biomedicine. 2008; 28(4): 302-304.

18. Deraedt R, Joughney S, Delevakeo F, Falhour M. Release of Prostaglandin E and F in an algogenic reaction and its inhibition. Eur J Pharmacol 1980; 51: 17-24.

19. Woolfeg G, Mac Donald AD. The evaluation of analgesic action of Pethidine hydrochloride. J Pharmacol Exp Ther1994; 80: 300.

20. Besra SE, Sharma RM, Gomes A. Anti-inflammatory effect of petroleum ether extract of leaves of Litchi chinensis Gaertn (Sapinadaceae). J Ethnopharmacol 1996; 54: 1-6.

21. Vinegar R, Schreiber W, Hugo R. Biphasic development of carrageenan edema in rats. J Pharmacol Exp Ther 1969; 166: 96-103.

22. Vane J and Botting R. Inflammation and the mechanism of action of anti-inflammatory drugs. FASEB J. 1987; 1: 89-96.

23. Mayes PA. Metabolism of unsaturated fatty acids and eicosanoids. In: Murray eds. Harper’s Biochemistry. Englewood Cliff. NJ, Prentice Hall, 24^th Ed, 1996.

Received on 04.03.2010 Modified on 28.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 897-899