Characterization of Melanogenesis Inhibitory Constituents of Morus alba Leaves and Optimization of Extraction Conditions Using Response Surface Methodology

Melanin is a natural pigment that plays an important role in the protection of skin, however, hyperpigmentation cause by excessive levels of melatonin is associated with several problems. Therefore, melanogenesis inhibitory natural products have been developed by the cosmetic industry as skin medications. The leaves of Morus alba (Moraceae) have been reported to inhibit melanogenesis, therefore, characterization of the melanogenesis inhibitory constituents of M. alba leaves was attempted in this study. Twenty compounds including eight benzofurans, 10 flavonoids, one stilbenoid and one chalcone were isolated from M. alba leaves and these phenolic constituents were shown to significantly inhibit tyrosinase activity and melanin content in B6F10 melanoma cells. To maximize the melanogenesis inhibitory activity and active phenolic contents, optimized M. alba leave extraction conditions were predicted using response surface methodology as a methanol concentration of 85.2%; an extraction temperature of 53.2 °C and an extraction time of 2 h. The tyrosinase inhibition and total phenolic content under optimal conditions were found to be 74.8% inhibition and 24.8 μg GAE/mg extract, which were well-matched with the predicted values of 75.0% inhibition and 23.8 μg GAE/mg extract. These results shall provide useful information about melanogenesis inhibitory constituents and optimized extracts from M. alba leaves as cosmetic therapeutics to reduce skin hyperpigmentation.


Introduction
Melanin is a dark macromolecular pigment produced by melanogenesis. It determines skin colors and also plays an important role in protecting the skin from UV radiation and toxic chemicals. However, excessive accumulation of melanin in specific parts induces diverse pigmentation problems [1,2]. Melanogenesis is a complex biosynthetic process controlled by a cascade of enzymatic reactions. Tyrosinase, the enzyme that catalyzes the initial step of melanin synthesis, is the rate-limiting enzyme of melanogenesis. Melanin synthesis are also regulated by various melanogenic enzymes such as the tyrosinase-related protein 1 (TRP-1) and TRP-2, and cellular signaling [3,4]. Melanogenesis inhibitors have become important targets, especially for cosmetic products to treat hyperpigmentation [5][6][7][8].
Morus alba is a deciduous tree that belongs to the Moraceae family. This tree is widely distributed in Asia and all parts of this tree including roots, fruits, twigs and leaves are of great importance in traditional medicine. Among them, the leaves of M. alba has been used in traditional medicine for the treatment of metabolic disorders [9]. Recently, the anti-melanogenesis activity of extracts from M. alba leaves also have been reported [10,11]. In addition, stilbenoids and chalcones of M. alba leaves were reported as active constituents that inhibit tyrosinase activity and reduce melanin content [12][13][14][15]. Therefore, M. alba extracts and their constituents are suggested as promising natural sources for dietary supplements or for development as cosmetic products, especially for whitening.
For the development of products using plants, an efficient extraction procedure is indispensable. During the extraction procedure, many factors such as extraction solvent, extraction time, extraction temperature and solid-liquid ratios affect the composition of the resulting extract as well as its biological activity [16][17][18]. Therefore, optimization of extraction condition is essential for maximum efficacy. Response surface methodology that consists of mathematical and statistical techniques is an efficient tool for optimization. Response surface methodology can take into several factors simultaneously, thus it is fast and reasonable for optimization of extraction conditions, especially in the case of several variables [19][20][21].
In the present study, we attempted to characterize the melanogenesis inhibitory constituents of M. alba leaves. For optimization, response surface methodology with a three-level-three-factor Box-Behnken design (BBD) was employed to evaluate the effect of multiple factors of the extraction conditions such as methanol concentration, extraction time and extraction temperature on tyrosinase activity and total phenolic content.

Effect on Melanogenesis
The effect of the isolated compounds on melanogenesis was first evaluated in vitro using mushroom tyrosinase. Among the compounds isolated from M. alba leaves, compound 18 showed the most potent inhibition on tyrosinase activity, followed by compounds 2, 3, 4, 8, 13 and 17 ( Figure 2A). The melanogenesis inhibitory effect of isolated compounds was also evaluated by measuring the melanin content in B16F10 melanoma cells. Stimulation of B16F10 melanoma cells with α-MSH significantly increased the melanin synthesis. However, all the compounds except for compounds 3 and 20 reduced the melanin content at 10 μM without any cytotoxicity ( Figure 2B). Taken together, phenolic compounds of M. alba leaves are active constituents for anti-melanogenesis activity.

Extraction Method Development
The optimized extraction conditions for maximum extraction efficacy was investigated using response surface methodology. In our present study, phenolic compounds significantly inhibited melanogenesis, therefore, tyrosinase inhibition and phenolic content were chosen as target responses. Three extraction variables, namely extraction solvent, extraction temperature and extraction time were selected on the basis of preliminary single factor experiments. Variable ranges were set as X1 (extraction solvent): methanol concentration 0%-100%; X2 (extraction temperature): 20-60 °C and X3 (extraction time: 2-24 h. To evaluate any multiple effects of the extraction factors on tyrosinase inhibition and phenolic content, a three-level-three-factors Box-Behnken design (BBD) was employed, as shown in Table 1. The significance of each coefficient was determined using t-test and p-values. Multiple regression analysis on the experiment data yielded the second-order polynomial regression equations as follows: For the determination of significance and suitability of regression equation, ANOVA analysis was used. Greater F-value and smaller p-value were considered as significant. Lack of fit was also determined to check the quality of the model. ANOVA analysis of the models for tyrosinase inhibition and total phenolic content showed the high F-values (16.83 and 44.00, respectively), low p-values (0.003 and 0.000, respectively) and insignificant p-value (0.897 and 0.247, respectively) of lack of fit, which supported the reliability of this model (Table 2). Three dimensional response surface plots for tyrosinase inhibition and total phenolic content are shown in Figure 3. These response surface plots clearly showed linear effect of methanol concentration on tyrosinase inhibition. Tyrosinase inhibition was greatly increased with an increase in methanol concentration ( ), whereas extraction temperature ( ) and extraction time ( ) showed only slight quadratic effects on tyrosinase inhibition. Three dimensional response surface plots for total phenolic content, however, showed the quadratic effect of methanol concentration, extraction temperature and extraction time. Total phenolic content increased with increasing methanol concentration and extraction temperature but decreased with continuing increase of methanol concentration and extraction temperature.

Optimization of Extraction Parameters and Verification
Based on our results, an optimization for extraction condition for both responses was evaluated and verified by experiment. The target was to obtain maximum tyrosinase inhibition and high total phenolic content.
Optimal condition for both maximum tyrosinase inhibition and total phenolic content was determined as methanol concentration of 85.2%; temperature of 53.2 °C; and extraction time of 2.0 h, which predicted 75.0% tyrosinase inhibition and 23.8 μg GAE/mg extract. These conditions gave 74.7% tyrosinase inhibition and 24.7 μg GAE/mg extract, which showed good correlation between predicted and actual values (Table 3). Thus, this model can be used to optimize the M. alba leaves extraction process.

Discussion
In our present study, twenty compounds were isolated from M. alba leaves and their effects on melanogenesis was evaluated by direct measuring the inhibitory effect on tyrosinase activity and melanin content in B16F10 melanoma cells. The inhibitory effect of isolated compounds on tyrisonase activity was first assessed in vitro using mushroom tyrosinase. Tyrosinase catalyzes the first rate-limiting step in the melanogenesis and plays a pivotal role in melanin synthesis [34,35]. In our present study, compound 18 showed the most potent inhibition on tyrosinase activity, followed by compounds 2, 3, 4, 8, 13 and 17. Considering the structure of compounds 1-20, all the compounds isolated from M. alba leaves are phenolic compounds and can be divided into benzofurans 1-8, flavonoids 9-18, a stilbenoid 19 and a chalcone 20. Concerning the structure activity relationship, flavonoids 9-18 exerted strong inhibition at 50 μM and 100 μM. However, addition of prenyl groups (compound 13) or replacement of the 4ʹ-hydroxyl group by a methoxyl moiety (compound 18) reduced the inhibitory activity. 2-Phenylbenzofurans are also good inhibitors in our study. Moracin M (1), which is 2-(3,5-dihydroxyphenyl)-5-hydroxybenzofuran is most potent and addition of hydroxyl or prenyl groups to the benzofuran skeleton (compounds 2-4) decreased the inhibitory activity. The stilbenoid 19 and chalcone 20 also showed potent inhibition, consistent with previous reports [12][13][14]. The melanogenesis inhibitory effect of isolated compounds was also evaluated by measuring the melanin content in B16F10 melanoma cells. All the phenolic compounds except for compounds 3 and 20 reduced the melanin content at 10 μM without cytotoxicity ( Figure 2B). Taken together, we can conclude that the phenolic compounds of M. alba leaves are active constituents with anti-melanogenesis activity.
For maximum efficacy for the anti-melanogenesis effect, the extraction condition of M. alba leaves was optimized using response surface methodology. Our present study demonstrated the combinatorial effect of phenolic constituents of M. alba leaves on anti-melanogenesis activity, thus, tyrosinase inhibitory activity and phenolic content were selected as targets for optimization. Response surface analysis as well as statistical analysis showed that tyrosinase inhibition and total phenolic content were noticeably affected by the methanol concentration, followed by extraction temperature and extraction time. In addition, optimized extraction conditions were suggested for both maximum tyrosinase inhibition and total phenolic content, which was confirmed by experimental data. Taken together, our present study demonstrated that phenolic constituents are active constituents for the anti-melanogenesis activity of M. alba leaves. Our study also suggested the optimized extraction conditions of M. alba leaves as methanol concentration of 85.2%, temperature of 53.2 °C, and extraction time of 2.0 h, which gave 74.7% tyrosinase inhibition and 24.7 μg GAE/mg extract for maximum tyrosinase inhibition and total phenolic content. Conclusively, M. alba leaves are promising natural resources for development as cosmetics and food supplements and our study gives a strong support for their economic efficiency.

Assessment of Tyrosinase Activity
Tyrosinase inhibitory assays were performed using enzyme solution, which was prepared by the reconstitution of mushroom tyrosinase (Sigma, St. Louis, MO, USA) in 0.1 U/mL phosphate buffer (pH 6.5). Test sample was mixed with 50 μL enzyme buffer, and incubated for 5 min at 37 °C. Then, 50 μL tyrosine solution, which was diluted with phosphate buffer to 1 mM, was added and the enzyme reaction was allowed to proceed for 20 min at 37 °C. After incubation, the amount of dopachrome formed in the reaction mixture was determined by measuring the absorbance at 490 nm in an ELISA reader (Bio-Tek Synergy HT, Winooski, VT, USA).

Measurement of Melanin Contents
B16F10 mouse melanoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin.
Cells were maintained at 37 °C in a humidified atmosphere of 95% air-5% CO2. For the measurement of melanin content, B16F10 cells were stimulated with α-MSH and then treated with samples for 72 h. After washing with phosphate buffered saline (PBS), the cells were harvested and solubilized the melanin by vortexing in 1 N NaOH-10% DMSO at 80 °C. The melanin contents were measured by absorbance value at 490 nm with synthetic melanin as a standard. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay in an ELISA plate reader.

Conclusions
M. alba leaves exert anti-melanogenesis inhibitory activity as measured by tyrosinase inhibition and melanin content in B16F10 melanoma cells. Further fractionation of M. alba leaves resulted in the isolation of 20 phenolic compounds as active constituents. For maximum efficacy, optimized extraction conditions was derived using response surface methodology as methanol concentration of 85.2%, an extraction temperature, 53.2 °C, and an extraction time 2 h. Therefore, these results provide useful information about melanogenesis inhibitory constituents and optimized extraction conditions for M. alba leaves as potential cosmetic therapeutics to reduce skin hyperpigmentation.