International Journal of Pharmaceutical Research Volume 2, issue 2, 2010 ISSN 0975-2366

Research Article Pharmacological Evaluation of Clerodendrum Philippinum Schauer Flowers for Antianxiety and Central Nervous System Depressant Activity
K.G.Lalitha1*, R Sathish2, R.Gayathri3, S.Karthikeyan3, P.Kalaiselvi3,G.Muthuboopathi3, T.Venkatachalam3 1 Department of Pharmaceutical Chemistry, 2Department of Pharmacology, Ultra College of Pharmacy, Madurai 625020 Tamil Nadu, India. 3 Department of Pharmaceutical Chemistry, Annai JKK Sampoorani Ammal College of Pharmacy, B. Komarapalayam - 638 183 Tamilnadu, India. *Corresponding author: Tel. (0) 91452 2534593, E-mail: kg.lalitha@gmail.com Received: 19/12/2009, Revised: 25/02/2010, Accpeted: 15/03/2010 ABSTRACT The research work deals with the ethanolic extract of Clerodendrum philippinum schauer flowers for anti anxiety and central nervous system depressant activity. The C.philippinum flowers are known to contain sterols, flavonoid, glycosides and phenyl propanoid derivatives as chemical components and are reported to have antifungal activity. It is used as folk medicine in the treatment of colic pain. In the present study the elevated plusmaze test models were used to evaluate anti-anxiety activity and locomotor activity was evaluated on actophotometer with 125 and 250 mg/kg p.o. doses of C.philippinum. The ethanolic extract of the C.philippinum flowers (125 and 250 mg/kg p.o) significantly (P > 0.01) decreased loco motor activity (45.20%, 53.47 %) and significantly (P > 0.01) increased the number of entries, time spent and rears in open arm of the elevated plus maze model. Keywords: Clerodendrum philippinum, locomotor activity, plus maze INTRODUCTION At present, anxiety and depression are the most frequent psychiatric condition commonly found and number of population suffers from the conditions at some time during their life. The efficacies of the drugs for the conditions are very limited, so the need for newer, better-tolerated and more efficacious treatment is remaining high. Therefore herbal therapy should be considered as alternative or complementary medicines. Recently the search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly [1]. This has been reflected in the large number of herbal medicines whose psychotherapeutic potential has been assessed in a variety of animal models. Clerodendrum philippinum schauer, Syn: C.fragrans Vent, (Family: Verbenaceae) is a shrub partly wild and often cultivated throughout India [2]. Leaves of C.philippinum have been used as traditional medicine for treatment of colic pain. The leaf extract exhibited anti-fungal activity. Sterol, cholesterol, 24-methyl cholesterol, sitosterol, stigmasterol, 24 methyl cholesta 5, 22 E, 25-triene 3 -ol and 24-ethyl -5 cholest 22 E-en-3 ol and phenyl propanol are isolated from this plant [3]. However there is no scientific evidence about potential effects of this plant against neuropsychological diseases, the present study is carried out to determine the effect of the plant extract against those disorders particularly against anxiety and depression, the most psychological diseases commonly found. MATERIALS AND METHODS Preparation of plant material The plant was authenticated by Dr. Danial (Botanical survey of India, Coimbatore, India) and the herbarium was kept in chemistry department for the future reference. The flowers of C.philippinum were collected from Bhavani, Tamilnadu, India. For preparation of alcoholic extract, air-dried and powdered sample of the flowers (100 g) was extracted simultaneously with petroleum ether (60-80 0C) and 80 %v/v ethyl alcohol in soxhlet apparatus for 72 h. The ethanolic extract was filtered and evaporated in a rotary evaporator under reduced pressure to give a residue (4 g, 25 %).The residue of ethanolic extract of C.philippinum (EECP) was suspended in 0.5 % tween-80 in saline for final suitable concentration. Phytochemical screening Phytochemical screening of extract for the presence of phenolic compounds, flavonoids, tannins, triterpenes, anthocyanins, anthroquinones and sterols were carried out using the standard methods [4]. Animals Albino male Swiss mice (18-22g) were used for the study. The animals were housed in colony cages and maintained under standard environmental conditions 252C temperature; 12:12 light dark cycle and 4555% relative humidity with free access to food and water ad libitum. The animals were fasted overnight and experiment carried out during the light period

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Lalitha et al / International Journal of Pharmaceutical Research 2010 2(2) 13-15 (08.00-16.00 h). The institutional animal ethical committee approved the protocol of the study. The animals were divided into five groups each containing six mice. The groups of mice were assigned to receive one of the following, Group I (vehicle (0.5 % tween 80) 1 ml/100g of body weight, p.o); Group II (EECP, 125 mg/kg, p.o) ; Group III (EECP, 250 mg /kg, p.o) and Group IV (Diazepam, 0.5 mg/kg, p.o). Drug and chemicals Diazepam (Ranbaxy Laboratories Ltd, Mumbai) was used as the standard anxiolytic agent. Petroleum ether 60-800C and ethanol were procured from Merck Ltd (India). Acute oral toxicity studies The OECD guidelines 425 were followed for the acute oral toxicity study for fixing the dose. The dose level up to 2500mg/kg of C.philippinum were not produced any mortality on oral administration. So the 125 and 250mg/kg doses were fixed for the study [5]. Behavioral parameters used to test anti-anxiety activity In Elevated plus-maze test an entry was defined as having all four paws within the arm. EPM consist of two open arms (16x5 cm) crossed with two closed arms (16x5x12cm) [6]. The arms were connected together with a central square of 5x5 cm. The apparatus was elevated to the height of 25cm in a dimly illuminated room. Mice were treated with EECP (125, 250mg/kg), diazepam (1mg/kg, p.o) and vehicle 30 min before being placed individually in the center of the EPM facing a closed arm. The time spent in both the open and closed arms were recorded for 5 min. The number of entries in to the open and closed arms was also counted during the test. Locomotor activity The Locomotor activity was studied using actophotometer [6]. The movement of the animal interrupts the beam of light falling on a photocell at which the count was recorded and displayed digitally. Each mouse was placed individually in the actophotometer for 10 min and the basal activity was obtained. Subsequently the animals were divided in to 4 groups each consists of six animals. Vehicle, EECP (125 mg/kg, 250 mg/kg), (or) diazepam (1mg/kg, p.o) was administered and after 30 min of administration, the mice were placed again in the actophotometer for recording the activity score. Statistical analysis: Results are expressed as mean + S.E.M. The statistical analysis of data was done using the one way analysis of variance (ANOVA) followed by Dunnetts test .A probability level less than 0.05 was considered statistically significant. RESULTS Phytochemical screening of EECP revealed the presence of phenolic compounds, phenyl propanoid, flavanoids and sterols. Oral administration of EECP up to 2.5 g/kg did not produce any toxic effects in mice. No mortality was observed and EECP was found to be safe at the given doses. Diazepam treated rats showed a significant increase (P > 0.01) in the number of open arm entries, percentage ratio of open arm to total arm entries, time spent in the open arms and number of rears in the open arms. EECP treated rats exhibited a significant increase (P > 0.01) in the number of open arm entries, time spent in the open arm, percentage ratio of open arm to total arm entries and number of rears in the open arms at both dose levels (125 & 250mg/kg) when compared with normal animals (Table 1). EECP in doses of 125 and 250mg/kg produced significant (P<0.01) reduction in locomotors activity as compared to the control animals. The diazepam treated group also revealed a statistically significant decrease in locomotor activity (P<0.01) (Table-2). DISCUSSION The etiology of most anxiolytic disorders is not yet fully understood but the picture has become a little clearer in the recent post. The benzodiazepines are relatively safe and are widely used anxiolytic agents. The EPM is one of most popular animal test for research on behavioral pharmacology of anxiety. It involves spontaneous or natural aversive stimuli (ie) height, unprotected opening and novelty [7]. Several plants that are used in folk medicine to diminish anxiety are reported to bring about an increase in the exploration of the open arms in EPM test [8]. In EPM native mice will normally prefer to spend much of their allotted time in the closed arms. This preference appears to reflect an aversion towards open arms that is generated by fear of open spaces. Drugs that increase open arm exploration are considered as anxiolytics and the reverse holds true are anxiogenis [9]. In our study we observed that EECP (125 & 250mg/kg) induced significant increases in the both the number of entries and time spent in the open arms. Locomotor activity is considered as an index of alertness, and a decrease indicates the sedative effect. The extract was able to induce a motor depressant effect, indicates a significant skeletal muscle relaxant and sedative effect of the plant [10]. An earlier report of chemical constituents of plants and their pharmacology suggests that plants containing flavanoids and steroids, posses activity against many CNS disorders [11]. Phytochemical tests of EECP revealed the presence of flavanoids, steroids. It is possible that the

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Lalitha et al / International Journal of Pharmaceutical Research 2010 2(2) 13-15 mechanism of anxiolytic action of EECP could be due to the binding of any of these phytochemicals to the GABA- BZD complex [9]. Table 1: Effect of Clerodendurm philippinum on behavior of rats in elevated plus maze paradigm Treatment groups Control Drug dose No. of arm entries Open Total Percentage preference to open arm Time spent in open arm (sec) No. of rears in open arm

Vehicle 2.320.21 7.501.21 30.930.07 19.140.16 2.00.1 (1ml/100gm) EECP (Dose 1) 125 mg/kg 5.420.16a 10.260.1 52.820.19 a 107.160.87a 4.050.36 a EECP (Dose 2) 250 mg/kg 6.051.12 a 9.891.12 61.671.63 a 146.20.19 a 4.262.12 a a a a STD(Diazepam) 0.5mg/kg 5.710.31 9.021.16 63.302.04 172.50.24 5.020.37 a a Values are mean SEM, n=6, -P<0.01, compared to respective vehicle treated group, one way ANOVA, followed by Dunnetts test. Table 2: Effect of Clerodendurm philippinum on locomotor acitivty in actophotometer Treatment groups Control EECP (Dose 1) EECP (Dose 2) STD (Diazepam) Values are mean SEM, n=6, followed by Dunnetts test.
a

Drug dose Vehicle (1ml/100gm) 125mg/kg 250mg/kg

Actophotometer score in 10 min 412.660.54 226.421.23 a 1921.52

a a

Percentage Decrease in activity 45.20 % 53.47 %

57.83 % 0.5mg/kg 174.161.06 -P<0.01 when compared with vehicle treated control group, one way ANOVA, Kulkarni SK. Handbook of experimental pharmacology. 3 ed. Vallabh Prakashan, New Delhi, India; 2007. 7. Adeyemi OO, Yetmitan OK, Taiwo AE, Neurosedative and muscle relaxant activities of ethyl acetate extract of Baphia nitida AFZEL. J Ethnopharmacol 2006; 106,312-316 8. Bhatacharya SK, Satyan KS, Experimental methods for evaluation of psychotropic agents in rodents. Anti- anxiety agents, Indian J Exp Biol 1997; 35: 565-575. 9. Dhonnchadha BA, Boorin M, Hascoet M, Anxiolytic-like effects of 5-HT2 ligands on three mouse models of anxiety. Behav Brain Res 2003; 140: 203- 214. 10. Thakur VD, Mengi SA, Neuropharmacological profile of Edipta alpa L. Hassk.J Ethnopharmacol 2005; 102: 23-31. 11. Hellion Ibarrola MC, Ibarrola DA, Montalbetti Y, Kennedy ML, Heinichen O, Campuzano M, The anxiolytic like effects of Aloysia polystachya [Griseb] Moldenke (Verbenaceae) in mice. J Ethnopharmacol 2006; 105: 400 408. 6.

The results obtained in this study suggest that the ethanolic extract of flowers of C.philippinum posses anti anxiety and CNS depressant properties, which is possibly mediated through the GABA- BZD mechanism. Further investication of mechanisms of action of the plant extract as well as the active substance(s) responsible for its biological action is necessary. REFERENCE 1. Zhang ZJ. Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders. Life Sci. 2004; 75: 1659-1699. 2. Willis JC, Airyshaw HK, A Dictionary of Flowering Plants and Ferns, Cambridge University Press, London, 1973, p.1214. 3. Anonymous, The Wealth of India, First supplement series, Raw Materials, CSIR, New Delhi, 2001: 2: p.69 4. Evan WC. Trease and Evans Pharmacognosy. 15 ed. W.B. Saunders, New York, 2002. 5. OECD Guidelines 425 for the testing of chemicals adopted 23.03.2006. Acute oral toxicity up and down procedure (UDP)

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