PHYTOCHEMICAL AND ANTIMALARIAL STUDIES ON SWERTIA ALATA ROYLE

Different species of genus Swertia are used in different countries for a variety of ailments including malaria. More than 85 xanthones and 17 secoiridoid bitters have been isolated from different species of Swertia. In the present study, the whole plant of Swertia alata Royle was investigated and three xanthones; 1,8-dihydroxy3,7-dimethoxyxanthone, 1,7,8-trihydroxy-3-methoxyxanthone and 1-hydroxy-3,7,8trimethoxyxanthone were isolated from different extracts. The total methanolic extract, its hexane soluble portion, chloroform soluble portion of petroleum ether extract and three isolated xanthones were tested in vitro at different dose levels for antimalarial activity. The activity was expressed as percent invasion inhibition of the tested parasite. The results indicated that all extracts and xanthones possessed some protective effect, however only 1,8-dihydroxy-3,7-dimethoxyxanthone exhibited significant activity and was therefore, further tested in vivo in Plasmodium berghei test model. 1,8-dihydroxy-3,7-dimethoxyxanthone produced the most significant reduction in percent parasitaemia at 10 mg/kg dose level. INTRODUCTION The medicinal properties of the genus Swertia are well recognized in different parts of the world, especially in Asia. The genus is held in high esteem in the Indian system of medicine and is one of the top five drugs of Kampo medicine in Japan (Fuchino, 2002). In ancient medical texts of India, S. chirata is described as the principal species of Swertia (Chunekar, 2002). The plant is known for bitter, digestive and antimalarial properties and is one of the ingredients of a popular Ayurvedic formulation Ayush 64. Unsustainable collection of this once abundantly available plant has nearly depleted its natural reserves in India resulting in diverted attention to other commonly available species like S. alata, S. angustifolia, S. paniculata and S. purpurascens. In view of this, comparative studies on different Swertia species were initiated in our laboratory to evaluate their industrial potential based on the presence of secoiridoid bitters and xanthones. It was observed in previous studies that chemically no other species is identical to S. chirata although most of them are akin in possessing derivatives of xanthones but different in bitter content of secoiridoids (Karan et al., 1997; Kumar, 2001). There is a worldwide resurgence of interest in developing antimalarial agents from plants following the discovery of artemisinin. It has been realized that plants possess unique potential to provide novel structures for drug development. The traditional use of S. alata in malaria prompted our laboratory to investigate its antimalarial potential. The phytochemical investigations led to the isolation of three 1,3,7,8-tetraoxygenated xanthones namely 1,8-dihydroxy-3,7-dimethoxyxanthone, 1,7,8-trihydroxy-3-methoxyxanthone and 1-hydroxy-3,7,8-trimethoxyxanthone. The structures of these compounds were elucidated on the basis of interpretation of their spectral details. The different extracts and pure compounds were evaluated in vitro and in vivo for their antimalarial activity. MATERIALS AND METHODS Plant Material The whole plant of S. alata was collected from Mussoorie (Uttaranchal State, Proc. WOCMAP III, Vol. 1: Bioprospecting & Ethnopharmacology Eds. J. Bernath, E. Nemeth, L.E. Craker and Z.E.Gardner Acta Hort 675, ISHS 2005 140 India). The plant was identified on the basis of taxonomic characters and by direct comparison with herbarium specimens available at the Forest Research Institute, Dehradun. The voucher specimen of the plant (No.1127) has been deposited at the Museum-cum-Herbarium of our Institute. Preparation of Extracts for Preliminary Analysis, Fractionation and Isolation Powdered plant material (1 kg), after defatting with petroleum ether was macerated with methanol at room temperature to yield 130 g of methanolic extract ('M1'). This extract 'M1' (100 g) was suspended in water and partitioned with hexane and chloroform to yield hexane soluble portion 'M2' (11 g) and chloroform soluble portion 'M3' (20 g). Both 'M2' and 'M3' were subjected to chromatographic separation by using eluants of increasing polarity (hexane, chloroform, methanol) which resulted in the isolation of 1,8-dihydroxy-3,7-dimethoxyxanthone (1) from 'M2', and 1,7,8-trihydroxy-3methoxyxanthone (2) and 1-hydroxy-3,7,8-trimethoxyxanthone (3) from 'M3' (Fig. 1). The structures of these isolated compounds were elucidated on the basis of spectral studies including UV, IR, H-NMR, and MS. The three extracts 'M1', 'M2', 'M3' and three isolates were tested for antimalarial activity. Experimental Animals, Chemicals and Reagents Adult Swiss mice (BALBc strain of either sex), organism Plasmodium berghei (NK 65 strain) and RPMI 1640 culture medium (Gibco-USA) were used in the biological studies. All other chemicals and solvents used were of analytical grade (E. Merck). In Vitro Antimalarial Studies The in vitro antimalarial activity was studied using cultures of P. berghei maintained in the laboratory following standard reported procedure (O'Neill et al., 1985; Desjardins et al., 1979; Ekong et al., 1990). The test samples were dissolved in minimal volume of chloroform and diluted with RPMI to desired strength. A suspension (0.9 ml) of red blood cells infected with P. berghei (0.1% parasitaemia, 3-5% cell hematocrit) was added to each well of a standard 24-well tissue culture plate containing different doses of test substance or control. Each test compound was assayed in duplicate. Microtiter plates were incubated for 20 h at 37°C in a sealed candle jar and the assay was terminated by aspirating the culture medium. The smears were prepared, air dried, fixed in methanol (2 min), stained with Giemsa solution (30 min), washed under running water and air dried. The number of parasites per 2000 RBCs in each smear were counted under microscope. The results are expressed as per cent invasion inhibition. No of rings in test % Invasion inhibition = 100 -------------------------x 100 No of rings in control In Vivo Antimalarial Studies The 4-day suppressive test model was used in P. berghei infected mice to determine the in vivo activity (Peters, 1987; Dutta, 1992). The doses were dispensed using 2% aqueous solution of gum acacia. Chloroquine at a dose of 4 mg/kg prepared in vehicle was used as a reference standard. The mice were randomly divided into different groups (control, test and reference) of six animals each. On day '0', animals in all the groups were inoculated with 1x10 P. berghei infected RBCs (Table 1). The animals in test group were treated with test substance while the animals of control and reference group received vehicle and chloroquine respectively on first four days. On day '4', the tail blood smears of all the animals were prepared, stained with Giemsa solution and