CHIEF CONSTITUENTS AND BIOLOGICAL ACTIVITIES DETERMINATION OF STEPHANIA VENOSA

Accessible utilization study of Stephania venosa was carried out on TLC fingerprints and various biological activity tests. Chief constituents in tuberous roots extractives of S. venosa were detected on TLC fingerprints as 3 different zones of steroids and terpenes, 8 different zones of alkaloids, 6 different zones of flavonoids and 2 different zones of phenol carboxylic acids as indicated by the hRf values and visualized colours resulting from spraying with special detection reagents. It was found that ethanol extract from tuberous roots of S. venosa displayed marked inhibitory activity on Artemia sp. with the LC50 of 260.26 μg/ml. The extract exhibited antioxidant activity with the EC50 of 4.98 μg/ml by DPPH radical scavenging assay. The microplate assay using the Dopachrome method indicated that the extract inhibited mushroom tyrosinase with the IC50 of 1.74 mg/ml. The extract given at the dose of 250 mg/kg orally, presented inconsistent antiinflammatory activity as an antioedematogenic effect in rat paw oedema induced by carrageenin only at 1 h (74.3%), and the activity was reduced thereafter; toxic/lethal dose of the extract was 750 mg/kg with anticholinesterase-like effects. The agar dilution method assay showed that the extract was active against various microbial enteropathogens and dermatopathogens, with the minimum inhibitory concentrations (MICs) between 5-30 mg/ml. INTRODUCTION Stephania venosa (Blume) Spreng (syn. S. rotunda Lour.), Menispermaceae, locally known in Thailand as Sabu Luad due to its blood-red sap, is an interesting medicinal vine with large tuberous root. The plant has simple alternate sub-orbicular leaf, with noticeable reddish brown veins on the lighter lower surface; flowers are reddishorange, in axillarys compound umbels, drupes are pisiform, becoming red on ripening. In Thai folk medicine, the tubers are used as a bitter tonic and rejuvenating medication; the red sap is used as an invulnerable ingredient in tattoo (Mahidol University Faculty of Pharmacy, 1995). In Vietnam, the plant is used as hypnotic tranquilizer (Nguyen Xuan and Do Tat Loi, 1991). Isoquinoline alkaloids from the tubers were reported as potent antimalarial on Plasmodium falciparum (Guinaudeau et al., 1982; Charles et al., 1987; Banerji et al., 1994; Likhitwitayawuid et al., 1999). The total alkaloid extract exhibited relaxant activities on isolated smooth muscle organs [Hoang, Khai Toan (Trung Tam KNNCD-QD, Vietnam), 1993]. The chief alkaloid L-tetrahydropalmatine is prescribed for neurasthenia and psychoses (Prior and Cao, 2000). The methanol extract was reported to be a potential source of new acetylcholinesterase inhibitors (Ingkaninan et al., 2001). TLC fingerprints and various biological activity tests on S. venosas tubers were reported in this paper. Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H. C. Baser, G. Franz, S. Canigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS 2005 58 MATERIALS AND METHODS Plant Materials Provided large tubers of S. venosa were purchased from Chatuchak weekend market, Bangkok, Thailand, on 13 Oct. 2001 by the Lam Ta Khong Plant Research Station, Thailand Institute of Scientific and Technological Research. The tubers were cleaned, peeled, sliced into pieces, dried in a hot air oven at 40-50°C, and then powdered. Chief Constituents Determination on TLC Fingerprints TLC fingerprints of sample solutions containing steroids-terpenes, alkaloids, and flavonoids were performed on 0.25mm thick TLC plates (Merck Silica gel 60 F254precoated), developed by suitable solvent systems, and visualized by special detection reagents (Merck, 1980; Wagner and Bladt, 1996). 1. TLC Fingerprint of Sample Solution Containing Steroids and Terpenes. 1 g of the dry powdered tubers was extracted by stirring with hexane (3 x 50 ml) for 30 min, filtered and evaporated the combined extracts to dryness under reduced pressure at 40-50°C, dissolved in 5 ml chloroform, and then applied 5 μl onto a TLC plate to performed chromatography with a suitable solvent system to a distance of 10 cm. Sprayed the developed plate with vanillin-sulfuric acid reagent, and then heated with a blower to achieved visualized zones of the existing steroids-terpenes. β-sitosterol in chloroform (1:1 w/v) was used as the reference compound. 2. TLC Fingerprint of Sample Solution Containing Alkaloids. 2.5 g of the dry powdered tubers was extracted by stirring with 60 ml 0.1 N sulphuric acid for 20 min, filtered and washed with 20 ml 0.1 N sulphuric acid, alkalinized the acid extract to pH 8-9 with 1 ml 5% ammonium hydroxide, and then extracted the resulted free alkaloids by partitioning with chloroform (3 x 10 ml). Dried the combined chloroform extracts over anhydrous sodium sulphate, filtered and evaporated the filtrate to dryness under reduced pressure at 40-50°C. Dissolved the dried extract in 0.5 ml methanol, and then applied 3 μl onto a TLC plate to performed chromatography with a suitable solvent system to a distance of 10 cm. Sprayed the developed plate with Dragendroffs reagent, and then heated with a blower to achieved visualized zones of the existing alkaloids. Quinine sulphate in methanol (1:1 w/v) was used as the reference compound. 3. TLC Fingerprint of Sample Solution Containing Flavonoids. 1 g of the dry powdered tubers was extracted by stirring with 10 ml methanol on a water bath (60°C, 5 min), allowed to cool, filtered and evaporated the filtrate to dryness under reduced pressure at 40-50°C, dissolved in 2 ml methanol, and then applied 5 μl onto a TLC plate to performed chromatography with a suitable solvent system to a distance of 10 cm. Sprayed the developed plate with natural products-polyethylene glycol (NP/PEG) reagent, dried with a blower, and then examined under UV-365 nm to detect fluorescing zones of the existing flavonoids. Rutin in methanol (1:1 w/v) was used as the reference compound. Crude Ethanol Extract Preparation The dry powdered tubers (800 g) was macerated at room temperature with 95% ethanol (6 x 2 L) for 3 days, filtered and evaporated the combined extracts to dryness under reduced pressure at 40-50°C to give 93 g of crude ethanol extract (11.62% yield), and then stored at 4°C. Brine Shrimp Micro-plate Assay Modified method of Solis et al. (1992) was used to determine inhibitory activity on Artemia sp. Five newly hatched brine shrimps in 100 μl artificial seawater was pipette into each well of a 96-well micro-plate; added 100 μl of the crude ethanol extract solution (in 0.25% Tween80-artificial seawater) which made the final concentrations of 1000, 100 and 10 μg/ml. Covered the micro-plate and incubated at room temperature for 24 h, then the dead organisms were counted under a binocular microscope (4x) compared with control wells. Thymol and kojic acid were used as positive controls. Determined %