Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo

A rapid 96-well plate assay using sulphorhodamine B (SRB) protein stain for cell number has been adopted to screen herbs used in traditional treatment s of vitiligo for substances capable of stimulating melanocyte proliferatio n. Its applicability to melan-a cells, a mouse pigmented cell line, has bee n validated. SRB assay produced good linearity up to 11 x 10(4) cells/well and interference by melanin present in the cells accounted for less than 10 % of the total optical density readings. The intra-assay variation was smal l but interassay variation was marked. For better assay precision, it is re commended that the results to be compared should be performed on the same d ay and controls should be plated in the same experiment, ideally in the sam e plate. Optimum conditions for exponential melan-a cell growth were establ ished: viz. initial plating density (3-8 x 10(3) cells/well), incubation. p eriod (4 days) and foetal bovine serum concentration (5%). Under these cond itions cells were responsive to the mitogen tetradecanoyl phorbol acetate ( TPA). Out of 28 herbal extracts screened in this assay, significant stimula tion (P < 0.05) of melanocyte proliferation was observed, in the absence of TPA, using aqueous extracts of Astragalus membranaceous root, Citrus retic ulata peel, Dictamnus dasycarpus root bark, Ophiopogon japonicus root, Pori a cocos sclerotium and Tribulus terrestris fruit. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.