In the underlying study, the effect of an 85% ethanol extract from dried leaves of the mulberry tree on the biosynthesis of melanin was investigated. This extract inhibited the activity of the enzyme tyrosinase, which converts DOPA to dopachrome in the process of melanin biosynthesis.
The mulberroside F (moracin M-6, 39-di-O-bb-D-gucopyranoside) obtained after extraction showed inhibitory effects on tyrosinase activity and on the melanin information of melan A cells in the study. Mulberoside F also proved to be a free radical scavenger in the study, a property that plays a role in protecting against auto-oxidation by having an anti-oxidative effect. The results of the study show that the extract from dried leaves of the mulberry tree can be used as a skin whitening agent.
Examination of fungal tyrosinase
One mM of the test substance was dissolved in MeOH : H2 O51 : 1.5 ml L-tyrosine (1.5 mM). 40 ml L-Dopa (25mM), 80 ml 67 mM phosphate-buffered saline (pH 6.8) and 40 ml of the same buffer solution or test sample were applied to a 96-well microtiter plate and then mixed with 80 ml fungal tyrosinase (60U). After a thirty-minute incubation at 37 °C, the amount of dopachrome in the reaction mixture was determined. Based on an optical density of 490 nm (OD 490), the inhibitory activity of the sample was determined as the concentration that inhibits 50 % of the enzyme activity (IC 50). Koji acid was used as the reference substance.
Examination of tyrosinase in a cultured HM3KO melanoma cell line
HM3KO cell lines were cultured in modified Eagles medium supplemented with 10% fetal calf serum. Tyrosinase activity was assayed using cultured HM3KO melanoma cell homogenate using a slightly modified Pomerantz method. Cell pellets were treated for a short period of time in an ultrasonic bath containing 0.1 mM sodium phosphate buffer (pH 6.8) with 0.1 mM phenylmethylsulfonylfluoride (PMSF) and 1% Triton X-100. After a one-hour incubation period in ice, 25 mg of solution protein was used for an enzyme assay. Each concentration of compounds were incubated for one hour at 37 °C in 0.5 ml of a reaction mixture of 0.25 mM tyrosine, 5mCi[3H]tyrosine, 0.25 mM l-3,4-dihydroxyphenylalanine (DOPA) and 0.1 mM PMSF in 0.1 M sodium phosphate buffer (pH 6.8).
Here too, the inhibitory activity of the sample was determined as the concentration that inhibits 50 % of the enzyme activity (IC 50). Koji acid was used as the reference substance. 85% of the extract from dried leaves of the mulberry tree inhibited the tyrosinase activity.
In the underlying study, it was shown that the inhibitory effects of an extract of dried mulberry leaves can be attributed to its components mulberroside F, moracin M-6 and 39-di-O-b-D-glucopyranoside. The hyperoxide scavenging properties and the inhibitory effect on the dopa-oxidase activity of the tyrosinase of each compound were evaluated to determine the depigmenting agents.
A compound at a concentration of 100mg/ml produced a 51.6% inhibition of fungal tyrosinase dopa-oxidase activity, with 50% inhibition (IC 50) detected at a concentration of 0.29 mg/ml. This compound was found to be approximately 4.5 times more potent than kojic acid in its inhibitory effect on enzyme activity.
The underlying study also showed that the inhibitory effect of naturally isolated mulberroside F on tyrosinase activity was stronger than that of kojic acid. Remarkably, the compound showed not only inhibition of tyrosinase activity, but also hyperoxide scavenging properties and inhibited melanin production in cultured melan A cells.
Conclusion
The results of the underlying study support the significant value of dried mulberry leaf extract in the treatment or prevention of hyperpigmentation of the skin.
Underlying study:
Lee, S. H.et al: Mulberroside F Isolated from the Leaves of Morus alba Inhibits Melanin Biosynthesis; in: Biol. Pharm. Bull. 25(8), 1045-1048 (2002)