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The color of human skin and hair is determined by diversiform factors, of which the degree and distribution of melanin in the epidermal layer is the most important. It is well established that tyrosinase is a pivotal enzyme in the process of melanin biosynthesis.［1,2］Furthermore, melanogenesis inhibitory compounds are useful not only as skin lightening agents added in cosmetics but also as a remedy for disturbances in hyperpigmentation such as melasma, lentigo and melanosis.［2,3］Now, it has been demonstrated that most lightening agents reduce the melanin contents of the skin through their inhibitory effects on tyrosinase.
In China, herbs such as genus ligusticum, largehead atractylodes rhizome, common bletilla tuber, agaric and others have been widely used in the treatment of hyperpigmentation of the skin or as cosmetics lightening agents, though their mechanisms still require clarification. Our previous study revealed that genus ligusticum and largehead atractylodes rhizome are potent inhibitors of tyrosinase.［4,5］Cosmetic products originating from aloes are now becoming fashionable among the public. Coarse extracts from it can positively inhibit the activity of tyrosinase in vitro.［6］But which elements of these herbs function predominantly as the inhibitor is still unknown. Natural chemicals such as aloin, cinnamic acid and sophorcardipine are key reactive elements in those plants. To evaluate their effects on tyrosinase, this study was performed.
METHODS
Reagents
Mushroom tyrosinase and L-dopa were purchased from Sigma Chemical Co., U.S.A. Aloin, cinnamic acid, amygdalin, cadabine, ferulic acid, paeonal, tanshinon, farrerol, peucidanin, ginkalide A, decholin, evodine, pinene were purchased from National Institute for the Control of Pharmaceutical and Biological Products （NICPBP), China. Other chemicals used were commercially available analytical agents. All reagents were dissolved in triple distilled water or with the help of 2% DMSO as a flux. Work solutions of natural chemicals were prepared at three different concentrations: high (2 mmol/L), medium (0.5 mmol/L) and low (0.125 mmol/L).
Enzyme assays
Tyrosinase activity was measured by determining the oxidation rate of L-dopa. The assay was performed in wells of 96-well microplates. Each well contained 200 μl reactive compounds: 40 μl L-dopa (2.5 mmol/L), 40 μl test sample (2 mmol/L), 80 μl phosphate buffer (67 mmol/L, pH 6.8) plus a final addition of 40 μl tyrosinase (25 IU/ml). Wells with test chemicals replaced the same volume phosphate buffer set as the negative control with hydroquinone (2 mmol/L) as a positive standard. The zeroing well contained 200 μl sodium chloride. Reactions were terminated after 20-minute incubation at 37℃. Absorbance was measured at a wavelength of 490 nm, following adjustment with the zeroing well. The percentage of inhibition rate to the tyrosinase was calculated as: inhibition rate to the tyrosinase (%)=(A-B)/A×100，where A represented absorbance of the negative control and B represented the test sample. The assays were repeated eight times for each natural chemical. The mean of the eight repeated readings of the final absorbance for each chemical was recorded. The final inhibitory rate data was statistically analyzed using SPSS 10.0. Afterwards, the most potent inhibitors were diluted in concentration to 1 mmol/L, 0.5 mmol/L, 0.25 mmol/L and 0.125 mmol/L. The reaction procedure was repeated with exception to the concentrations of the chemicals (the volume was still 40 μl).
Kinetic analysis
To evaluate the kinetics of each inhibitor, tyrosinase activity corresponding to the altered concentrations of L-dopa was determined as before and was diluted to six different concentrations (0.0625 mmol/L, 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1 mmol/L and 2 mmol/L). The reaction mixture was almost unchanged except for the addition of 40 μl different concentrations of L-dopa to total 200 μl in volume of reaction compounds. To each fixed concentration of inhibitor, six readings of the absorbance for every changed concentration of L-dopa was converted to the reciprocal of the value of absorbance. Afterwards, the reciprocal of V［0］ (A［490］) was plotted versus the reciprocal of concentration of the substrate (1/S) with each chemical. The Michaelis constant (Km) of each inhibitor to tyrosinase was determined by Lineweaver-Burk plots.
RESULTS
Effect of cinnamic acid, aloin, sophorcarpidine and other natural chemicals on the activity of tyrosinase
Of the 15 natural chemicals studied, cadabine, paeonal, farrerol, evodin, cinnamic acid, aloin and sophorcarpidine inhibited tyrosinase at different intensities. Cinnamic acid, aloin and sophorcarpidine were the most potent (Table 1) . A further investigation of high, medium and low concentrations of cinnamic acid, aloin and sophorcarpidine revealed that inhibition of tyrosinase at high concentrations of aloin and sophorcarpidine and low concentrations of sophorcarpidine are stronger than inhibition by hydroquinone at the medium concentration (analysis of variance, F=14.448; Fig. 1) . Inhibitory rate of tyrosinase using cinnamic acid (2 mmol/L), aloin (0.5 mmol/L) and sophorcarpidine (0.5 and 0.125 mmol/L) were nearly identical to that of hydroquinone (0.5 mmol/L). The effects appeared to be dose-dependant when tested at different concentrations.
Effects of cinnamic acid, aloin and sophorcarpidine on tyrosinase.
Effect of cinnamic acid, aloin and sophorcarpidine on the kinetics of tyrosinase The effects of cinnamic acid, aloin and sophorcarpidine on the kinetics of tyrosinase were shown in Table 2. The plot for Sophorcarpidine obtained at 1 mmol/L inhibitor concentrations intersects that of the control at the negative abscissa (Km remains unchanged, a noncompetitive inhibition pattern). Other plots intersect in the second or third quadrant (left of the ordinate, mixed inhibition pattern) (Fig. 2) . The Km value of aloin was observably larger than the others.
DISCUSSION
Human appearance is greatly influenced by skin and hair color. The phylogenetic pathway underlying this phenomenon is called melanogenesis and is the production of melanin pigments in neural crest-derived melanocytes followed by its transfer to epithelial cells. The tyrosinase gene family in humans consists of four loci coding the enzymes tyrosinase (TYR), tyrosinase related protein 1 (TRP1), tyrosinase related protein 2 (TRP2) and gp100. These four proteins have been shown to be active during melanin synthesis. In combination with the lysosome-associated membrane protein-1 (Lamp-1), they have been shown to interact as a high molecular weight, multimeric enzymatic complex that range in size from 200 to>700 kd.［4,5］Co-purification of these proteins with anti-tyrosinase antibodies suggests a stable interaction between these proteins in which the EGF motif may be important in the formation and stabilization of this complex.［6］Recent studies have shown that tyrosinase activity is stabilized in the presence of TRP1 and TRP2, providing further evidence of a melanogenic complex.［7,8］Pmel17 protein is an abundant molecule and one of its possible biological roles is cellular protection from the cytotoxic intermediates of melanin synthesis.［9］Tyrosinase is a copper-containing monophenol monooxy~genase and is well known as the rate-limiting enzyme in the biosynthesis of the skin pigment melanin. Its disruption plays an important role in local hyperpigmentation diseases, such as melasma, ephelide and lentigo. Therefore, tyrosinase may be a useful target in the areas of hyperpigmentation and cosmetics.
Traditional Chinese herbs are a very popular mode for the treatment of hyperpigmentation disorders. We have screened 219 kinds of herbs, among them 19 kinds have been shown to inhibit tyrosinase in vitro.［10-12］Cosmetic products originating from aloes are now becoming fashionable among the public, especially for women. Chinese women believe this plant can lighten skin color and youthen appearance. However, it is currently unclear which components of these herbs function as inhibitors. This study focuses on the exploration of the effects of important plant chemicals on tyrosinase. Results show that cadabine, paeonal, farrerol, evodin, cinnamic acid, aloin and sophorcarpidine do inhibit tyrosinase at different intensities at a concentration of 2 mmol/L. The latter three are most effective and can inhibit tyrosinase in a dose-dependent manner. Several tyrosinase inhibitors extracted from plants have been reported. Arbutin was reported to be a competitive inhibitor with L-tyrosine as a substrate and as an uncompetitive inhibitor with L-dopa. Azelaic acid, curcumin, and minosine were reported to be competitive inhibitors with L-tyrosine as a substrate. Furthermore, tropolone is a competitive inhibitor with L-dopa as a substrate. Another study showed oxyresveratrol was identified as a noncompetitive inhibitor and α-viniferin as a mixed-type inhibitor with L-dopa as the substrate.［12-14］
This study revealed that sophorcarpidine functions as an uncompetitive inhibitor compared to aloin and cinnamic acid, which are mixed-type. From this, we can deduce that sophorcarpidine, aloin and cinnamic acid can not only bind to the enzyme, but also to the enzyme-substrate complex as well, leading to the inactivation of tyrosinase. The Km value of aloin is larger than others. Km is generally interpreted in terms of the strength of the enzyme-substrate complex. Large values of Km for the aloin reaction signify it to be more weakly bound than the others. The effect of traditional Chinese herbs and their key elements on tyrosinase is not a rare contradiction. We previously discovered that genus ligusticum inhibits tyrosinase, but one of its main products, known as ligustilide, is not. Therefore, although this herb can inhibit tyrosinase, the element that really functions is not included in the experiment and deserves further investigation.
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