INTRODUCTION:

Currently available antipsychotics are associated with variety of autonomic, endocrine, allergic, hematopoietic and neurological side effects. As a result there is high prevalence of usage of complementary and alternative medicines for treatment of psychiatric disorders. In the search for new therapeutic products for the treatment of neurological disorders, medicinal plant research, worldwide, has progressed constantly, demonstrating the pharmacological effectiveness of different plant species in a variety of animal models¹.

Argyreia speciosa (L.f.) Sweet (convolvulaceae) commonly known as ‘Elephant creeper’ is a woody climber distributed throughout the India up to an altitude of 300 meters². It has been used as ‘Rasayan’ drug in the ayurvedic system of medicine to cure diseases of nervous system. The roots of this plant have been regarded as tonic, aphrodisiac, bitter and used in rheumatism, gonorrhea, chronic ulcer and diseases of nervous system^2,3. Previous phytochemical studies reveal the presence of lipids⁴, flavanoids⁵, triterpenes⁶, steroids⁷, phenylpropanoids⁸ and coumarins⁹ in the plant. Several investigations have proposed that this plant possess hypotensive¹⁰, anti-inflammatory¹¹, immunomodulator¹², antidiabetic¹³, Nootropic¹⁴, antibacterial and antifungal effect¹⁵.

Generally, plants possess many pharmacological actions, since they contain numerous constituents of active chemicals in it. Based on the above criteria, this study was designed to evaluate the activity of different fractions of hydroalcoholic extract on central nervous system in mice.

MATERIALS AND METHODS:

Animals:

Wistar albino mice (25-35 g) bred in Central Animal House facility of the Institute, were used. They were housed under standard conditions, maintained on a 12 h light/dark cycle and had free access to food and water up to the time of experimentation. The mice were acclimatized to the laboratory environment 1 h before the experiments. All experiments were conducted during the light period (08.00-16.00 h). During the experiments animals were free access to water only. All the protocols were approved by the Institutional Animal Ethical Committee (IAEC) and conducted according to the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).

Plant material and preparation of the extracts:

The roots of A. speciosa were collected from Balasinor (Gujarat). Their authenticity was confirmed by a Taxonomist, Department of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Gujarat. A specimen of plant is kept in the herbarium of our institute (Voucher No. ARGH8). The roots were completely dried in the sunlight and powdered. Root powder was extracted exhaustively with 50% ethanol by maceration for 2 days at room temperature with occasional shaking. Crude (hydroalcoholic) extract was filtered and dried under reduced pressure at 40˚C (Yield - 9.3% w/w of dried plant material). After preparing the aqueous solution of the crude extract, was transferred to separating funnel and fractionated with successive portion of n-hexane, chloroform and ethyl acetate. The solvent were used according to their increasing polarity. The n-hexane, chloroform, ethyl acetate and water extracts were concentrated and dried under vacuum. The yellowish green sticky mass (1.86% w/w) of n-hexane fraction (n-HF), greenish brown sticky mass (7.83% w/w) of chloroform fraction (CF), yellowish brown sticky mass (0.65% w/w) of ethyl acetate fraction (EAF) and dark brown sticky mass (78.75% w/w) of water fraction (WF) obtained were used in this study.

Fig. 1: Effect of Argyreia speciosa roots on spontaneous locomotor activity. n=10. Each bar expressed as mean ± SEM. Anova : F_{(4, 45)}= 15.18(n-HF), 21.08(CF), 12.76(EAF), 9.41(WF) followed by Dunnett’s test, ^*p<0.05 when compared with control 1 (2.5% Tween 80) group. ⁺p<0.05 when compared with control 2 (Distilled water) group. (Pretreatment-and 1 hr post treatment

Preliminary phytochemical screening:

Preliminary phytochemical screening was performed of different fractions of hydroalcoholic extracts to check the presence of various phytoconstituents¹⁶.

Drugs:

Chlorpromazine (Sun Pharmaceutical, India) was used as positive control for spontaneous motor activity and for prolongation of pentobarbital induced sleep. Sodium pentobarbital (Sigma, St. Louis, MO, USA) was used for induction of sleep. It was dissolved in 0.9% saline solution prior to administration.

Treatment:

Suspension of n-HF, CF and EAF were freshly prepared in distilled water using Tween 80 (2.5%) as suspending agent. WF solution is also prepared freshly in distilled water. All the fractions were screened for locomotor activity and pentobarbital sodium induced sleep (100, 200 and 500 mg/kg, p.o.) in mice. Mice were divided in to 15 groups, each consisting of 10 mice. Group 1-3 received n-HF, group 4-6 received CF, group 7-9 received EAF, and group 10-12 received WF. Group 13 for conrol 1, group 14 for control 2 and group 15 for positive control received chlorpromazine (2 mg/kg, i.p). All administrations were performed in a dose volume of 10 ml/kg body weight of animal. Control 1 group received Tween 80 (2.5%) in distilled water and control 2 received only distilled water in the same volume by the same route.

Neuropharmacological activity:

Spontaneous motor activity:

The Spontaneous motor activity (SMA) was measured using an actophotometer. The movement of the animal cuts off a beam of light falling on the photocell and a count was recorded and displayed digitally. Each mouse was placed individually in the actophotometer for 10 min and basal activity score was obtained. 1 h after treatment, mice was placed again in the actophotometer for recording the activity score¹⁷.

Fig. 2: Effect of Argyreia speciosa roots on duration of sleep induced by pentobarbital sodium in mice. n=10. Each bar expressed as mean ± SEM. Anova : F_{(4, 45)}= 25.95(n-HF), 27.77(CF), 32.29(EAF), 53.78(WF) followed by Dunnett’s test, ^*p<0.05 when compared with control 1 (2.5% Tween 80) group. ⁺p<0.05 when compared with control 2 (Distilled water) group.

Pentobarbitone induced hypnosis:

This test determines the effect of a test drug or extract on the duration of sleep induced by a barbiturate hypnotic such as pentobarbital sodium in mice. After 30 min of treatment, pentobarbitone sodium (35 mg/kg, i.p.) was administered to all animals. The interval between the administration of pentobarbital until the loss of the righting reflex was recorded as on set of sleep, while the time from the loss to regaining of the righting reflex as the duration of sleep.

Statistical analysis:

The data was expressed as mean ± S.E.M. Statistical analysis was done using one way analysis of variance (ANOVA) followed by Dunnett’s test. Results were considered significant at p<0.05.

RESULTS:

Result of phytochemical screening indicated presence of various phytoconstituents in different fractions of hydroalcoholic extract of Argyreia speciosa root (Table 1). The results of spontaneous motor activity study were presented in the figure 1. All the extracts produced significant and dose dependent reduction in spontaneous motor activity. Similarly, positive control chlorpromazine (2 mg/kg, i.p.) produced significant reduction in SMA. The percentage reduction obtained with respect to different extracts (at 500mg/kg dose) and standard drug chlorpromazine were shown in the following ascending rank order.

EAF(42.06)< WF(45.56)< n-HF (57.42)<cpz (65.16)<CF(67.39)

Table 1: Preliminary phytochemical screening of Argyreia speciosa roots.

Class of compounds	n-HF extract	CF extract	EAF extract	WF extract
Alkaloids	-	-	-	-
Aminoacids	-	-	-	+
Flavanoids	+	+	+	+
Saponins	+	+	+	+
Steroids	+	+	+	+
Triterpenoids	+	+	-	-
Tannins	+	+	+	+
Carbohydrates	-	+	+	+
Volatile oils	+	+	-	-
Anthraquinones	-	-	-	-
Coumarins	+	+	+	+

“-” denotes not found.

Significant, dose dependent potentiation of pentobarbital sodium induced sleep duration with n-HF extract (200, 500 mg/kg), CF extract (200, 500 mg/kg), EAF extract (200, 500 mg/kg) and WF extract (500 mg/kg) were observed (figure 2). However, reduction in onset of sleep was not observed except CF extract (500 mg/kg). (figure 3). Similarly, positive control chlorpromazine reduced onset of sleep and potentiated duration of sleep in significant manner. The value obtained (% potentiation of pentobarbitone induced sleep) with respect to different extracts (at 500 mg/kg dose) and chlorpromazine was shown in the following ascending order.

EAF(40.81)< WF(78.83)< n-HF (126.03)<cpz (187.12)<CF(255.92)

Fig. 3: Effect of Argyreia speciosa roots on onset of sleep induced by pentobarbital sodium in mice. n = 10. Each bar expressed as mean ± SEM. Anova : F_{(4, 45)}= 4.36(n-HF), 8.81(CF), 8.11(EAF), 3.18(WF) followed by Dunnett’s test,^*p<0.05 when compared with control 1 (2.5% Tween 80) group. ⁺p<0.05 when compared with control 2 (Distilled water) group.

DISCUSSION:

The results of this study indicate that the root of Argyreia speciosa may possess central nervous depressant activity. This is shown by reduction in spontaneous motor activity in mice. Spontaneous motor activity is considered as an index of alertness and a decrease in it leads to sedation¹⁸ as a result of reduced excitability of the central nervous system¹⁹. It is generally well accepted that the sedative effect of drugs can be evaluated by measurement of pentobarbital sleeping time in laboratory animals. The central nervous depressant activity of the root of Argyreia speciosa was confirmed by its ability to potentiate the pentobarbital induced sleep, which may be attributed to an action on the central mechanisms involved in the regulation of sleep²⁰ or an inhibition of pentobarbital metabolism²¹. The potentiation of pentobarbital sleep and the decrease in spontaneous locomotor activity strongly suggests central depressant effect²². The efficacy of most herbal remedies is attributed to various active principles in combination. Results of phytochemical screening showed presence of carbohydrates, proteins, steroids, saponins, tannins, flavanoids and coumarins in the root of Argyreia speciosa. Chemical studies have also reported the presence of several compounds on different parts of plant. Some compound already described in this plant could depress the central nervous system. Saponins have been shown to have sedative property and decrease spontaneous motor activity in experimental animals^23,24.

Essential oil, triterpenoids and flavanoids also display nervous system depressor activities^25-27. At present, it is not clear which of the chemical constituents of the plant may be responsible for the observed pharmacological effects of the extracts seen in the present study.

In conclusion, the neuropharmacological effects determined in the present study suggest that roots of Argyreia speciosa produce a significant central nervous depressant activity which may be due to sedative property. This study provides experimental support for the medicinal traditional use of this plant in nervous disorders.

REFERENCES:

1. Zhang ZJ. Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders. Life Science 2004;75: 1659–1699.

2. Wealth of India, Raw materials. Publication and Information Directorate, CSIR, New Delhi, 1985 pp.418.

3. Kirtikar KR., Basu BD and ICS. Indian medicinal plants, Lalit Mohan Basu publication, 1981; 2^nd edn, Vol II: pp. 1707-1708.

4. Batra A, Mehta BK. Chomatographic analysis and antibacterial activity of the seed oil of Argyreia speciosa. Fitoterapia. 1985; 56: 357-359.

5. Ahmad M, Jain N, Khan MS, Shafiullah and Ilyas M. Two new flavone glycosides from the leaves of Argyreia speciosa. J Chem Res (S). 1993; 7: 248.

6. Khan MS, Kamil SM and Ilyas M. Phytochemical investigation on the leaves of Argyreia speciosa. J Indian Chem Soc. 1992; 69: 110.

7. Chandler RF and Hooper SN. Friedelin and associated triterpenoids. Phytochemistry. 1979; 18: 711-724.

8. Shrivastava A and Shukla YN. Aryl esters and a coumarin from Argyreia speciosa. Indian J Chem. 1998; 37B: 192-194.

9. Shukla YN, Shrivastava A and Kumar SA. coumarin glycoside from Argyreia speciosa roots. Indian drugs. 2001; 38(9): 487-488.

10. Bhukani DS, Dhar ML, Dhar MM., Dhawan BN. and Mehrotra BN. Screening of Indian plants for biological activity Part II. Indian J Exp Biol. 1969; 7: 250-262.

11. Gokhle AB, Damre AS, Kulkarni SK and Saraf MN. Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera. Phytomedicine. 2002; 9(5): 433-437.

12. Gokhle AB, Damre AS and Saraf MN. Investigation in to the immunomodulatory activity of Argyreia speciosa. J Ethanopharmacol. 2003; 84(1): 109-114.

13. Akhtar MS. Hypoglycemic activities of some indigenous medicinal plants traditionally used as antidiabetic drugs. J Pak Med Ass. 1992; 42(11): 271-277.

14. Joshi H, Kaur N, Chauhan J. Evaluation of Nootropic effect of Argyreia speciosa in mice. J Health Sci. 2007; 53(4): 382-388.

15. Mishra SH and Chaturvedi SC. Antibacterial and antifungal activity of the oil and unsaponifiable matter of Argyreia speciosa Sweet. Indian Drugs. Pharmaceut. Ind. 1978; 13(5): 29-31.

16. Kokate C.K. Practical Pharmacognosy. Vallabh prakashan, Delhi. 1994; 4^th ed: pp. 107-109.

17. Kulkarni S.K., Hand book of Experimental Pharmacology, Vallabh Prakashan, New Delhi. 1999; 3^rd ed:pp.117-118.

18. Ozturk Y, Aydini S, Beis R, Baser KHC. and Berberoglu H. Effect of Hypericum perforatum L. and Hypericum calximun L. extract on the central nervous system in mice. Phytomedicine. 1996; 3: 139-146.

19. Masur J, Martz RMW and Carlini EA. Effects of acute and chronic administration of cannabis sativa and (-)9-trans tetrahydro-cannabinol on the behaviour of rats in an open-field arena. Psychopharmacology. 1971; 19: 338-397.

20. N’Gouemo P, Nguemby-bina C and Baldt-Moulinier M. Some neuropharmacological effects of an ethanolic extract of Maprounea africana in rodents. J Ethnopharmacol. 1994; 62: 57–263.

21. Kaul PN and Kulkarni SK. New drug metabolism inhibitor of marine origin. J Pharma Sci. 1978; 67: 1293–1296.

22. Perez RM, Parez JA, Garcia LM. and Sossa H. Neuropharmacological activity of Solanum nigrum fruit. J Ethanopharmacol. 1998; 62: 43-48.

23. Wagner H, Ott S, Jurcic K, Morton J and Neszmelyi A. Chemistry, 13C NMR study and pharmacology of two saponins from Colubrina asiatica. Planta Med. 1983; 48: 136-141.

24. Dubois MA, Ilyas M and Wagner H. Cussonosides A and B, two triterpene saponins from Cussoria barteri. Planta Med. 1986; 56: 80–83.

25. Hendriks H, Bos R, Allersma DP, Malingre TM. and Koster AS. Pharmacological screening of valerenal and some other components of essential oil of Valeriana officinalis. Planta Med. 1981; 42(1): 62-68.

26. Chattopadhyay D, Arunachalam G, Mandal SC, Bhadra R and Mandal AB. CNS activity of the methanol extract of Malloatus (Geist) Muell Arg. Leaf: An ethnomedicine of Onge. J Ethnopharmacol. 2003; 85: 99-105.

27. Datta BK, Datta SK, Chowdhury MM, Khan TH, Kundu JK, Rashid MA, Nahar L and Sarker SD. Analgesic, antiinflammatory and CNS depressant activities of sesquiterpenes and a flavonoid glycoside from Polygonum viscosum. Pharmazie. 2004; 59(3): 222-5.

Received on . .2008 Modified on . .2008

Research J. Pharm. and Tech.2 (2): April.-June.2009; Page 331-334