The effect of isolates from Cassipourea flanaganii (Schinz) Alston, a plant used as a skin lightening agent, on melanin production and tyrosinase inhibition

Ethnopharmacological relevance: The Zulu and Xhosa people of South Africa use the stem bark of Cassipourea flanaganii as a skin-lightening cosmetic.

Aim of the study: To isolate and identify compounds responsible for the skin lightening properties from the stem bark of Cassipourea flanaganii and to evaluate their cytotoxicity towards skin cells.

Materials and methods: Extracts from the stem bark of Cassipourea flanaganii were isolated using chromatographic methods and structures were determined using NMR, IR and MS analysis. The tyrosinase inhibitory activity and the ability to inhibit the production of melanin were determined using human primary epidermal melanocyte cells. Cytoxicity was established using the same melanocytes and a neutral red assay.

Results: One previously undescribed compound, ent-atis-16-en-19-al (1) along with the known ent-atis-16-en-19-oic acid (2), ent-atis-16-en-19-ol (3), ent-kaur-16-en-19-oic acid (4), ent-kaur-16-en-19-al (5), ent-manoyl oxide (6), guinesine A (7), guinesine B (8), guinesine C (9), lichenxanthone (10), 2,4-dihydroxy-3,6-dimethyl benzoic acid methyl ester (11), lynoside (12), lupeol (13), β-amyrin (14), docosyl ferulate (15), stigmasterol, sitosterol and sitosterol-O-glucoside were isolated in this investigation. An impure fraction containing compound 3 was acetylated to obtain 19-acetoxy-ent-atis-16-ene (3a). Compounds 10 and 11 are usually isolated from lichen, hence they are possible contaminants of lichen harvested with the bark. Compounds 1, 3a, 5-14 were not significantly cytotoxic to the primary epidermal melanocyte cells (P > 0.05) when compared to the negative and positive controls (DMSO, 0.1% and hydrogen peroxide, 30 wt% in water). Inhibition of tyrosinase was significantly greater with respect to the negative control (P < 0.001) for compounds 3a, 5-8 and 9-10 at 10 μM and for compounds 5-8 and 9-10 at 100 μM. Compared to hydroquinone (the positive control) at 10μM, the level of inhibition was comparable or to that of compounds 3a, 5, 6, and 8-10 at 10μM, with 9 and 10 showing a greater level of inhibition. Inhibition of melanin was both concentration and time dependent for all compounds tested with higher melanin content at 24 hours compared to 48hrs and at 10mM compared to100 mM at both time points; melanin content was significantly lower for hydroquinone at both time points and concentrations.

Conclusions: Compounds 1, 5-14, isolated from Cassipourea flanaganii and the derivative 3a showed low cytotoxicity. All compounds had a clear time and concentration dependent effect on melanin content which did not appear to be dependent on their inhibition of tyrosinase.

Keywords: Cassipourea flanaganii; ent-atis-16-en-19-al and melanin inhibition; ent-atisane diterpenoids.