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Article

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

*
Author to whom correspondence should be addressed.
College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 362-763, Korea
Molecules 2015, 20(5), 8730-8741; https://doi.org/10.3390/molecules20058730
Submission received: 17 March 2015 / Accepted: 11 May 2015 / Published: 14 May 2015
(This article belongs to the Section Natural Products Chemistry)

Abstract

:
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.

1. 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.

2. Results and Discussion

2.1. Characterization of Compounds

The leaves of M. alba were extracted twice with 80% MeOH, which yielded the methanolic extract. The methanolic extract was then fractionated into n-hexane, CH2Cl2, EtOAc and n-BuOH fractions. Further fractionation of the CH2Cl2 and EtOAc-soluble fractions resulted in the isolation of 20 compounds (Figure 1).
Figure 1. Chemical structures of compounds 120.
Figure 1. Chemical structures of compounds 120.
Molecules 20 08730 g001
The structures of the isolated compounds were determined as eight 2-phenylbenzofurans, moracin M (1), 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran (2), wittifuran E (3), moracin N (4), moracin C (5), albafuran A (6), moracin X (7), and morunigrol C (8); ten flavonoids: norartocarpetin (9), kaempferol (10), quercetin (11), isorhamnetin (12), kuwanon C (13), steppogenin (14), 7,2',4'-trihydroxyflavanone (15), astragalin (16), quercetin-3-O-β-d-glucopyranoside (17), and kaempferide 3-O-β-d-glucoside (18), one stilbenoid: oxyresveratrol (19), and one chalcone: morachalcone A (20) by spectroscopic analysis and comparison of literature values [14,22,23,24,25,26,27,28,29,30,31,32,33].

2.2. 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.
Figure 2. Effects of compounds 120 on (A) tyrosinase inhibition and (B) melanin content in B16F10 melanoma cells. Data was expressed as mean ± S.D. (n = 3). NC, normal control; PC, α-MSH stimulated positive control; KA, kojic acid. * p < 0.05 compared to NC (A), # p < 0.05 compared to PC (B).
Figure 2. Effects of compounds 120 on (A) tyrosinase inhibition and (B) melanin content in B16F10 melanoma cells. Data was expressed as mean ± S.D. (n = 3). NC, normal control; PC, α-MSH stimulated positive control; KA, kojic acid. * p < 0.05 compared to NC (A), # p < 0.05 compared to PC (B).
Molecules 20 08730 g002

2.3. Optimization of Extraction Conditionsadmas

2.3.1. 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.
Table 1. A Box-Behnken design for independent variables and their responses.
Table 1. A Box-Behnken design for independent variables and their responses.
RunActual VariablesObserved Values
MeOH Concentration (%)Extraction Temperature (°C)Extraction Time (h)Tyrosinase Inhibition (%)Total Phenolic Content (μg GAE/mg extract)
1060130.04.1
2020130.05.0
350401330.920.4
45020220.025.3
504020.013.9
6100402483.120.1
750202415.325.1
8040240.016.7
950401322.021.7
1010040281.615.7
11100601381.516.8
125060246.025.6
1350401359.820.4
14100201382.57.6
1550602436.423.9
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:
Tyrosinase inhibition = 37.86 + 60.40X1
Phenolic content = 21.34 + 3.19 X 1 + 1.25 X 2   10.41 X 1 2   2.48 X 2 2 + 5.36 X 3 2 + 2.63 X 1 X 2
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).
Table 2. ANOVA analysis for second order polynomial models for tyrosinase inhibition and total phenolic content.
Table 2. ANOVA analysis for second order polynomial models for tyrosinase inhibition and total phenolic content.
[A] Tyrosinase inhibition
VariationSum of SquareDegree of FreedomMean SquareF-Valuep-Value
Model30301.5093366.8316.830.003
Residual error1000.205200.04
Lack-of-fit219.40373.300.190.897
Pure error11.2525.62
Total31301.7014
R2 = 0.968, adjusted R2 = 0.911
[B] Total phenolic content
VariationSum of SquareDegree of FreedomMean SquareF-ValueP-Value
Model687.36976.3744.00<0.001
Residual error8.6851.74
Lack-of-fit7.1832.393.190.247
Pure error1.5020.75
Total696.0414
R2 = 0.987, adjusted R2 = 0.965
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.
Figure 3. Response surface plots show the effect of extraction variables on (A) tyrosinase inhibition and (B) total phenolic content. Three variables are methanol concentration (X1), extraction temperature (X2) and extraction time (X3).
Figure 3. Response surface plots show the effect of extraction variables on (A) tyrosinase inhibition and (B) total phenolic content. Three variables are methanol concentration (X1), extraction temperature (X2) and extraction time (X3).
Molecules 20 08730 g003
Tyrosinase inhibition was greatly increased with an increase in methanol concentration ( X 1 ), whereas extraction temperature ( X 2 ) and extraction time ( X 3 ) 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.

2.3.2. 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.
Table 3. Predicted and observed values of tyrosinase inhibition and total phenolic content under optimized condition.
Table 3. Predicted and observed values of tyrosinase inhibition and total phenolic content under optimized condition.
Extraction ConditionTyrosinase Inhibition aTotal phenolic Content b
MeOH Concentration (%)Extraction Temperature (°C)Extraction Time (h)PredictedObserved Predicted Observed
85.253.22.075.074.723.824.7
a Tyrosinase inhibition (%) was measured at 100 μg/mL; b Total phenolic content was expressed as μg GAE/mg extract.

2.4. 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 120, all the compounds isolated from M. alba leaves are phenolic compounds and can be divided into benzofurans 18, flavonoids 918, a stilbenoid 19 and a chalcone 20. Concerning the structure activity relationship, flavonoids 918 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 24) 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.

3. Experimental Section

3.1. General Information

NMR spectra were recorded on a DRX 500 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). EI-mass spectra were obtained on a VG Autospec Ultima mass spectrometer (Waters, Milford, MA, USA). Semipreparative HPLC was performed using a Waters HPLC system (Waters) equipped with Waters 600 Q-pumps, a 996 photodiode array detector, and Waters Empower software using a Gemini-NX ODS-column (5 μm, 10 × 150 mm). Silica gel (70–230 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (25–100 μm, Amersham Biosciences, Uppsala, Sweden) were used for open column chromatography (CC). Thin-layer chromatography (TLC) was performed on a precoated silica gel 60 F254 (0.25 mm, Merck). All other chemicals and reagents were analytical grade.

3.2. Isolation of Compounds 120

The leaves of M. alba (10.0 kg) were extracted twice with 80% MeOH (50 L, 24 h, room temperature) which yielded the methanolic extract (1.65 kg). The methanolic extract was then suspended in H2O and partitioned successively with n-hexane, CH2Cl2, EtOAc and n-BuOH (2 L each, twice, room temperatures). The CH2Cl2 fraction (109.2 g) was subjected to silica gel column chromatography with the mixture of CH2Cl2/MeOH to give seven fractions (M1–M7). M5 was subjected to column chromatography over Sephadex LH-20 eluting with MeOH to give five fractions (M5D1-M5D5). Compound 20 (2.5 mg) was obtained from M5D4 by semipreparative HPLC eluting with CH3CN/H2O. M4 was subjected to silica column chromatography with the mixture of n-hexane-EtOAc to give seven fractions (M4A-M4G). M4C was subjected to column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH to give five fractions (M4C1-M4C5). Semipreparative HPLC of M4C4 eluting with CH3CN/H2O yielded compounds 13 (0.9 mg), 2 (0.2 mg), 3 (0.4 mg), 6 (0.6 mg), 7 (0.4 mg) and 8 (0.6 mg). M4B was subjected to column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH to give seven fractions (M4B1-M4B7). M4D was subjected to column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH to give four fractions (M4D1-M4D4). Compounds 4 (2.5 mg) and 5 (1.5 mg) was purified from M4D4 and M4B7, respectively, by semipreparative HPLC eluting with CH3CN/H2O.
The EtOAc fraction (17.7 g) was subjected to silica column chromatography with the mixture of CH2Cl2/MeOH to give nine fractions (E1-E9). Compounds 9 (14.1 mg) was obtained from E3 by column chromatography over Sephadex LH-20 eluting with MeOH. Compound 19 (19.9 mg) was obtained from E7 by column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH (1:1). E5 was subjected to RP-silica column chromatography with MeOH/H2O to give 6 fractions (E5A-E5F). E5B was subjected to column chromatography over Sephadex LH-20 eluting with MeOH to give 11 fractions (E5B1-E5B11). Compound 10 (13.0 mg) was obtained from E5B11 by recrystallization. Compounds 11 (2.6 mg), 14 (1.5 mg), and 15 (1.5 mg) were obtained from M5B6 by semipreparative HPLC eluting with CH3CN/H2O. E6 was subjected to column chromatography over Sephadex LH-20 eluting with MeOH to give eight fractions (E6A-E6H). E6F was subjected to column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH to give six fractions (E6F1-E6F6). Compound 12 (5.0 mg) was obtained from E6F5 by recrystallization. Compound 1 (10.0 mg) was obtained from E6F6 by semipreparative HPLC eluting with MeOH/H2O. E8 was subjected to column chromatography over Sephadex LH-20 eluting with CH2Cl2/MeOH to give seven fractions (E8A-E8G). Column chromatography of E8D over Sephadex LH-20 eluting with MeOH yielded five fractions (E8D1-E8D5). Compounds 16 (0.5 mg) and 17 (0.5 mg) were obtained from E8D2 by semipreparative HPLC eluting with MeOH/H2O. Recrystallization of E9 yielded compound 18 (341.0 mg).

3.3. Evaluation of Anti-Melanogenesis Activity

3.3.1. 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).

3.3.2. 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.

4. 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.

Acknowledgments

This work was supported by Basic Science Research Program (2010-0025054) and Medical Research Center program (2008-0062275) through the National Research Foundation of Korea.

Author Contributions

J.Y.J. and E.J.M. designed the experiments and executed the isolation and optimization. Q.L., Y.H.J., S.B.K., H.H.Y. and D.H.S. performed the biological assay. B.Y.H. and M.K.L. analyzed the data and wrote the paper. All authors discussed the results and commented the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Prota, G. Progress in the chemistry of melanins and related metabolites. Med. Res. Rev. 1988, 8, 525–556. [Google Scholar] [CrossRef] [PubMed]
  2. Rees, J.L. Genetics of hair and skin color. Annu. Rev. Genet. 2003, 37, 67–90. [Google Scholar] [CrossRef] [PubMed]
  3. Hah, Y.S.; Cho, H.Y.; Lim, T.Y.; Park, D.H.; Kim, H.M.; Yoon, J.; Kim, J.G.; Kim, C.Y.; Yoon, T.J. Induction of melanogenesis by rapamycin in human MNT-1 melanoma cells. Ann. Dermatol. 2012, 24, 151–157. [Google Scholar] [CrossRef] [PubMed]
  4. Liu, W.S.; Kuan, Y.D.; Chiu, K.H.; Wang, W.K.; Chang, F.H.; Liu, C.H.; Lee, C.H. The extract of Rhodobacter sphaeroides inhibits melanogenesis through the MEK/ERK signaling pathway. Mar. Drugs 2013, 11, 1899–1908. [Google Scholar] [CrossRef] [PubMed]
  5. Solano, F.; Briganti, S.; Picardo, M.; Ghanem, G. Hypopigmenting agents: An updated review on biological, chemical and clinical aspects. Pigment Cell Res. 2006, 19, 550–571. [Google Scholar] [CrossRef] [PubMed]
  6. Chang, T.S. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 2009, 10, 2440–2475. [Google Scholar] [CrossRef] [PubMed]
  7. Kim, Y.-J.; Uyana, H. Tyrosinase inhibitors from natural and synthetic sources: Structure, inhibition mechanism and perspective for the future. Cell. Mol. Life Sci. 2005, 62, 1707–1723. [Google Scholar] [CrossRef] [PubMed]
  8. Parvez, S.; Kang, M.; Chung, H.W.; Bae, H. Naturally occurring tyrosinase inhibitors: Mechanism and applications in skin health, cosmetics and agriculture industries. Phytother. Res. 2007, 21, 805–816. [Google Scholar] [CrossRef] [PubMed]
  9. Tang, W.; Eisenbrand, G. Morus alba. In Handbook of Chinese Medicinal Plants, 1st ed.; Wiley-VCH Verlag GmbH & Co. KGaA.: Weinheim, Germany, 2011; Volume 2, pp. 777–781. [Google Scholar]
  10. Wang, K.H.; Lin, R.D.; Hsu, F.L.; Huang, Y.H.; Chang, H.C.; Huang, C.Y.; Lee, M.H. Cosmetic applications of selected traditional Chinese herbal medicines. J. Ethnopharmacol. 2006, 106, 353–359. [Google Scholar] [CrossRef] [PubMed]
  11. Lee, K.T.; Lee, K.S.; Jeong, J.H.; Jo, B.K.; Heo, M.Y.; Kim, H.P. Inhibitory effects of Ramulus mori extracts on melanogenesis. J. Cosmet. Sci. 2003, 54, 133–142. [Google Scholar] [PubMed]
  12. Lee, S.H.; Choi, S.Y.; Kim, H.; Hwang, J.S.; Lee, B.G.; Gao, J.J.; Kim, S.Y. Mulberroside F isolated from the leaves of Morus alba inhibits melanin biosynthesis. Biol. Pharm. Bull. 2002, 25, 1045–1048. [Google Scholar] [CrossRef] [PubMed]
  13. Park, K.T.; Kim, J.K.; Hwang, D.; Yoo, Y.; Lim, Y.H. Inhibitory effect of mulberroside A and its derivatives on melanogenesis induced by untraviolet B irradiation. Food Chem. Toxicol. 2011, 49, 3038–3045. [Google Scholar] [CrossRef] [PubMed]
  14. Takahashi, M.; Takara, K.; Toyozato, T.; Wada, K. A novel bioactive chalcone of Morus australis inhibits tyrosinase activity and melanin biosynthesis in B16 melanoma cells. J. Oleo Sci. 2012, 61, 585–592. [Google Scholar] [CrossRef] [PubMed]
  15. Shin, N.H.; Ryu, S.Y.; Choi, E.J.; Kang, S.H.; Chang, I.M.; Min, K.R.; Kim, Y. Oxyresveratrol as the potent inhibitor on dopa oxidase activity of mushroom tyrosinase. Biochem. Biophys. Res. Commun. 1998, 243, 801–803. [Google Scholar] [CrossRef] [PubMed]
  16. Zhang, W.M.; Huang, W.Y.; Chen, W.X.; Han, L.; Zhang, H.D. Optimization of extraction condition of areca seed polyphenols and evaluation of their antioxidant activities. Molecules 2014, 19, 16416–16427. [Google Scholar] [CrossRef] [PubMed]
  17. Lu, C.-L.; Zhu, Y.-F.; Hu, M.-M.; Wang, D.-M.; Zu, X.-J.; Lu, C.-J.; Zhu, W. Optimization of astilbin extraction from the rhizome of Smilax glabra, and evaluation of its anti-inflammatory effect and probable underlying mechanism in lipopolysaccharide-induced RAW264.7 macrophages. Molecules 2015, 20, 625–644. [Google Scholar] [CrossRef] [PubMed]
  18. Jeong, J.Y.; Jo, Y.H.; Lee, K.Y.; Do, S.-G.; Hwang, B.Y.; Lee, M.K. Optimization of pancreatic lipase inhibition by Cudrania tricuspidata fruits using response surface methodology. Bioorg. Med. Chem. Lett. 2014, 24, 2329–2333. [Google Scholar] [CrossRef] [PubMed]
  19. Bezerra, M.A.; Santelli, R.E.; Oliveira, E.P.; Villar, L.S.; Escaleira, L.A. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008, 76, 965–977. [Google Scholar] [CrossRef] [PubMed]
  20. Xu, Q.; Chen, Y.; Wang, H.; Zhang, N.; Xu, S.; Zhang, L. Application of response surface methodology to optimize extraction of flavonoids from Fructus sophorae. Food Chem. 2013, 138, 2122–2129. [Google Scholar] [CrossRef] [PubMed]
  21. Ferreira, S.L.C.; Bruns, R.E.; Ferreira, H.S.; Matos, G.D.; David, J.M.; Brandao, G.C.; da Silva, E.G.P.; Portugal, L.A.; Reis, P.S.; Souza, A.S.; et al. Box-Behnken design An alternative for the optimization of analytical methods. Anal. Chim. Acta 2007, 597, 179–186. [Google Scholar] [CrossRef] [PubMed]
  22. Eumkeb, G.; Siriwong, S.; Phitaktim, S.; Rojtinnakirn, N.; Sakdarat, S. Synergistic activity and mode of action of flavonoids isolated from smaller galangal and amoxicillin combinations against amoxicillin-resistant Escherichia coli. J. Appl. Microbiol. 2011, 112, 55–64. [Google Scholar] [CrossRef] [PubMed]
  23. Chang, Y.C.; Chang, F.R.; Wu, Y.C. The constituents of Lindera glauca. J. Chin. Chem. Soc. 2000, 47, 373–380. [Google Scholar] [CrossRef]
  24. Choi, S.W.; Jang, Y.J.; Lee, Y.J.; Leem, H.H.; Kim, E.O. Analysis of functional constituents in mulberry (Morus alba L.) twigs by different cultivars, producing areas, and heat processing. Prev. Nutr. Food Sci. 2013, 18, 256–262. [Google Scholar] [CrossRef] [PubMed]
  25. Miyazawa, M.; Hisama, M. Antimutagenic activity of flavonoids from Chrysanthemum morifolium. Biosci. Biotechnol. Biochem. 2003, 67, 2091–2099. [Google Scholar] [CrossRef] [PubMed]
  26. Zheng, Z.P.; Cheng, K.W.; To, J.T.; Li, H.; Wang, M. Isolation of tyrosinase inhibitors from Artocarpus heterophyllus and use of its extract as antibrowning agent. Mol. Nutr. Food Res. 2008, 52, 1530–1538. [Google Scholar] [CrossRef] [PubMed]
  27. Ryu, Y.B.; Ha, T.J.; Curtis-Long, M.J.; Ryu, H.W.; Gal, S.W.; Park, K.H. Inhibitory effects on mushroom tyrosinase by flavones from the stem barks of Morus lhou (S.) Koidz. J. Enzym. Inhib. Med. Chem. 2008, 23, 922–930. [Google Scholar] [CrossRef]
  28. Tan, Y.; Yang, Y.; Zang, T.; Chen, R.; Yu, D. Bioactive 2-arylbenzofuran derivatives from Morus wittiorum. Fitoterapia 2010, 81, 742–746. [Google Scholar] [CrossRef] [PubMed]
  29. Han, J.T.; Bang, M.H.; Chun, O.K.; Kim, D.O.; Lee, C.Y.; Baek, N.I. Flavonol glycosides from the aerial parts of Aceriphyllum rossii and their antioxidant activities. Arch. Pharm. Res. 2004, 27, 390–395. [Google Scholar] [CrossRef] [PubMed]
  30. Wei, Y.; Xie, Q.; Fisher, D.; Sutherland, L. Separation of patuletin-3-O-glucoside, astragalin, quercetin, kaempferol and isorhamnetin from Flaveria bidentis (L.) Kuntze by elution-pump-out high-performance counter-current chromatography. J. Chromatogr. A 2011, 1218, 6206–6211. [Google Scholar] [CrossRef] [PubMed]
  31. Takasugi, M.; Ishikawa, S.; Masamune, T. Albafurans a and b, geranyl 2-phenylbenzofurans from mulberry. Chem. Lett. 1982, 8, 1221–1222. [Google Scholar] [CrossRef]
  32. Yang, Y.; Gong, T.; Liu, C.; Chen, R.Y. Four new 2-arylbenzofuran derivatives from leaves of Morus alba. Chem. Pharm. Bull. 2010, 58, 257–260. [Google Scholar] [CrossRef] [PubMed]
  33. Wang, L.; Cui, X.Q.; Gong, T.; Yan, R.Y.; Tan, Y.X. Three new compounds from the barks of Morus nigra. J. Asian Nat. Prod. Res. 2008, 10, 897–902. [Google Scholar] [CrossRef] [PubMed]
  34. Mayer, A.M. Polyphenol oxidases in plant: Recent progress. Phytochemistry 1987, 26, 11–20. [Google Scholar] [CrossRef]
  35. Seo, S.Y.; Sharma, V.K.; Sharma, N. Mushroom tyrosinase: Recent prospects. J. Agric. Food Chem. 2003, 51, 2837–2853. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the compounds are not available from the authors.

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MDPI and ACS Style

Jeong, J.Y.; Liu, Q.; Kim, S.B.; Jo, Y.H.; Mo, E.J.; Yang, H.H.; Song, D.H.; Hwang, B.Y.; Lee, M.K. Characterization of Melanogenesis Inhibitory Constituents of Morus alba Leaves and Optimization of Extraction Conditions Using Response Surface Methodology. Molecules 2015, 20, 8730-8741. https://doi.org/10.3390/molecules20058730

AMA Style

Jeong JY, Liu Q, Kim SB, Jo YH, Mo EJ, Yang HH, Song DH, Hwang BY, Lee MK. Characterization of Melanogenesis Inhibitory Constituents of Morus alba Leaves and Optimization of Extraction Conditions Using Response Surface Methodology. Molecules. 2015; 20(5):8730-8741. https://doi.org/10.3390/molecules20058730

Chicago/Turabian Style

Jeong, Ji Yeon, Qing Liu, Seon Beom Kim, Yang Hee Jo, Eun Jin Mo, Hyo Hee Yang, Dae Hye Song, Bang Yeon Hwang, and Mi Kyeong Lee. 2015. "Characterization of Melanogenesis Inhibitory Constituents of Morus alba Leaves and Optimization of Extraction Conditions Using Response Surface Methodology" Molecules 20, no. 5: 8730-8741. https://doi.org/10.3390/molecules20058730

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