Mushroom tyrosinase activity of novel and known phenolic compounds isolated from Greyia Flanaganii (Bolus)

Abstract

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin, the pigment that is responsible for the colour of hair and skin in humans. Tyrosinase is known to be the key enzyme in melanin production. Forty plants belonging to different families were tested for their activity on tyrosinase using L-tyrosine as a substrate. Ethanolic leaf extract of Greyia flanaganii (Bolus) showed significant (p<0.05) inhibitory activity exhibiting the 50% inhibitory concentration (IC50) of 32.62 μg/ml. It was further investigated for its toxicity and effect on melanin production by melanocytes cells. G. Flanaganii showed significant (P<0.05) 20% reduction of melanin at 6.25 μg/ml while 80% of cells were found to be viable, thus indicating low levels of cytotoxicity (IC50 < 400μg/ml). The 1.2-diphenyl-2-picrylhydrazyl (DPPH) method was used to quantify the total radical scavenging capacity (RSC) of the extract. The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC50) by the ethanol extract of the leaves of G. Flanaganii was found to be 22.01 μg/ml. ‘Acne vulgaris’ is a chronic inflammatory follicular disorder of the skin that affects areas containing thelargest oil glands including face, neck, back and chest. It affects up to 80% of adolescents to some extent and less commonly occurs in infants (Shaw and Kennedy, 2007). Propionibacterium acnes is an obligate anaerobic gram-positive bacterium that is associated with or linked to ‘Acne vulgaris’. It is largely commensal and is present on most people’s skin and live on fatty acids in the sebaceous glands on sebum secreted by pores. The effect Greyia flanaganii (Bolus) against acne causing bacteria, P. acnes was investigated using microdilution assay. The minimum inhibitory concentration (MIC) of the extract of G. Flanaganiiwas found to be 250 μg/ml. A bioassay guided fractionation of the ethanolic leaf extract of G. Flanaganii let to the isolation of six known compounds and one novel phenolic compound. Isolated compounds were; 2’,6’,4-trihydroxy-4’-methoxydihydrochalcone (asebogenin/lyonogenin) (C1), 2’,4’,6’-trihydroxydihydrochalcone (C2), 2’,6’- dihydroxy-4’,4-dimethoxydihydrochalcone (C3), (2R,3R)-3,.5,7-trihydroxy-3-Oacetylflavanone (pinobanksin-3-acetate/dihydrogalangin-3-acetate) (C4), 2’,6’- dihydroxy-4’-methoxydihydrochalcone (C5), 5,7-dihydroxyflavanone [(2S)- pinocembrin] (C6) and 4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (C7).C1-C6 are well known compounds whereas C7 is a novel compound. The isolated compounds were tested for their cytotoxicity, antioxidant, tyrosinase inhibitory and antibacterial activities. C3and C4 were only tested on tyrosinase due to low yield of these compounds during isolation. C2 exhibited significant (p<0.05) antityrosinase activity exhibiting the IC50 of 69.15 μM. <b<C1, C4, C5, C6 and C7 also showed significant (p<0.05) inhibitory activity of the tyrosinase enzyme exhibiting 11.5, 35, 24, 22 and 31 % enzyme inhibition respectively at 200 μg/ml. C3 did not show any significant inhibition of the enzyme at the highest concentration tested (200μìg/ml). C6 showed low toxicity effect on melanocytes cells, IC50 of 373 μM was observed and about 10% reduction of melanin content was found at 1.5 μg/ml. All the compounds tested for antioxidant activity exhibited good radical scavenging activity. The EC50 (concentration of extract which decreases the initial DPPH absorbance by 50%) values of C2, C1, C5 and C7 was found to be ranging from 3.46 to 76.1 μM. The EC50 of the positive control used (vitamin C) was found to be 11.3 μM. The isolated compounds were also tested for their activity against P. Acnes. C2, C7 and C1 exhibited MIC values of 968, 1590 and 418 μM respectively. The positive drug control; erythromycin exhibited an MIC of 136 μM. The cytotoxicity, antityrosinase, antioxidant and antibacterial activity of G. flanaganii extract are being reported for the first time. Other flavonoid compounds such as pinistrobin, 7- methyleriodictyol, dihydrogalangin, chrysin etc have been previously isolated from this plant but all the isolated compounds in this study have been isolated for the first time from G. Flanaganii. C2 which showed the best antityrosinase activity (IC50 value of 69.1 μM) as compared to the other samples tested in the present study. This sample also showed low cytotoxicity on melanocytes cells (IC50 219 μM) and resulted in 10% reduction of melanin at 3.125 μg/ml. Therefore, this compound can be considered for its use for the treatment of skin hyper-pigmentation problems. C1 which showed low MIC (418 μM) as compared to positive drug should be considered for further studies in clinical trials for a possible antiacne agent. C2 and C1 which exhibited an EC50 value of 3.46 and 6.69 μM respectively seem to have good antioxidant activity and can be considered for their use as UV protecting agents. Based on these results, it shows that the leaf extract of Greyia flanaganii and C2 have some potential to be considered as a possible anti-tyrosinase agent though their mechanism studies need to be done in detail to support their possibilities of being used to treat hyper-pigmentation problems. Copyright