Fariba Sharifi far1, Gholamreza Dehghan-Nudeh2, Zeinab Raeiat3, Bagher Amirheidari1, Mandan Moshrefi4,5, Amin Purhemati*4,5
1Traditional and Herbal Medicines Research Center, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
2Pharmaceutics Research center, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
3Student Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
4Department of Plant Protection, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.
5American Chemical Society, Washington, DC 20036, USA.
Objective: Our previous studies have shown high tyrosinase inhibitory effect of Quercus infectoria galls, an endemic plant to Iran. Considering the potency of tyrosinase inhibitors in cosmetic as a skin depigmentation and lightening agent, we have studied the Q. infectoria galls, and its major fractions for tyrosinase-inhibitory activity. Materials and methods: A methanolic extract of Q. infectoria galls was evaporated in vacuum. The resulting residue was suspended in water and extracted successively with a combination of petroleum ether, chloroform, ethyl acetate and methanol in increasing order of polarity. As a result, fractions 1–18 were obtained. The fractions were initially screened for the O-diphenolase inhibitory activity of tyrosinase using L-tyrosine as a substrate on TLC plate by the bioautography method. All the active inhibitors from the fi rst test were dissolved in methanol to give different concentrations. 80 microliter of L-tyrosine (0.5mM) was added to wells containing a 50 μl sample, and incubated for 10 minutes at room temperature. Seventy μl of mushroom tyrosinase (1000 units/ml) was added and incubated again for 10 min at 35°C. The enzyme reaction was monitored by measuring the change in absorbance at 475 nm (at 37°C) for 10 and 20 min after incubation. The percent of inhibition of the enzyme was measured and IC50 values of each sample were calculated by probit analysis. Results: Amongst the separated fractions, fractions V and VЦ separated in ethyl acetate-methanol exhibited the highest inhibition of mushroom tyrosinase (89.2% and 93.65% inhibition respectively) in comparison to kojic acid (96.54% inhibition). The least IC 50 values were due to fractions of V and VЦ (IC50 values of 12.0 and 8.0 μg/ml respectively). The total extract induced 86.34% inhibition with IC50 value of 42.0 μg/ml (kojic acid with IC50 = 2.7 μg/ml). Conclusion: Our fi ndings indicated that the fractions with potent antityrosinase effect were extracted in the ethyl acetate-methanol fraction, so may have semi-polar nature like gallic acid and/or ellagic acid or might be from fl avonoids. The confi rmation of this needs further fractionation which is the subject of further study in our laboratory.
Keywords: Fractionation , Quercus infectoria galls , Tyrosinase inhibition , Phenolic acids.