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Flavonoids, apigenin and icariin exert potent melanogenic activities in murine B16 melanoma cells.

ABSTRACT

We aimed to screen for melanogenic agents among 35 botanical compounds. The compounds were first assessed with regard to their effects on tyrosinase activity in B16 cells. At 100 [mu]M, 13 compounds showed tyrosinase activity-enhancing effects, ranging from 2.6 to 372.8% activation. Five of them showed more than 50% enhancement and were further tested for their [EC.sub.50] values. Compared with 8-Methoxypsoralen, an effective tyrosinase activator with an [EC.sub.50] of 7.26 [mu]M, 3 compounds exhibited smaller [EC.sub.50] values (apigenin, 0.45 [mu]M; hyperosid, 0.92 [mu]M; and icariin, 1.01 [mu]M for enhancing tyrosinase activity). The 3 compounds significantly increased cellular melanin contents without affecting cell proliferation. Compared with 8-Methoxypsoralen ([EC.sub.50], 35.94 [mu]M for stimulating pigmentation), apigenin ([EC.sub.50], 17.46 [mu]M) and icariin ([EC.sub.50], 32.77 [mu]M) showed better melanogenic activity, while hyperosid ([EC.sub.50], 70.4 [mu]M) was less potent. Western blot analysis demonstrated that the 3 compounds could differentially increase the expression levels of tyrosinase, and tyrosinase-related proteins 1 and 2. Together these data suggest that apigenin and icariin exert potent melanogenic activities through, at least in part, upregulating the protein expression levels of melanogenic enzymes in B16 cells. Thus, further investigations are merited to ascertain their potential application in treating hypopigmentation disorders.

ARTICLE INFO

Keywords: Apigenin B16 cells Icariin Melanogenesis Tyrosinase

Article history:

Received 24 February 2010

Received in revised form 3 May 2010

Accepted 2 June 2010

[C] 2010 Elsevier GmbH. All rights reserved.

Introduction

Insufficient skin pigmentation will leave the underlying tissue improperly protected from ultraviolet (UV) radiation (Costin and Hearing 2007) and result in the development of hypopigmentation disorders (Rose 2009). The maintenance of skin pigmentation relies on the normal biosynthesis of melanin in melanocytes, which is controlled by various factors (Hearing 1999). Among them the enzymatic melanosomal proteins tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2 [L-3,4-dihydroxyphenylalanine (DOPA) chrome tautomerase (DCT)] play an important role (Kobayashi et al. 1994; Yasumoto et al. 1997). Of the three enzymes, tyrosinase is the most critical because in the de novo synthesis pathway of melanin it catalyses the two rate-limiting steps; namely, the hydroxylation of tyrosine to DOPA and the oxidation of DOPA to dopaquinone. Thus, melanin production is mainly dependent on tyrosinase expression and activation (Chakraborty et al. 1996; Costin and Hearing 2007; Eves et al. 2006), and tyrosinase activity-enhancing agents are valuable for skin care.

In the present study we first screened for tyrosinase activity enhancers among 35 phytocompounds in B16 mouse melanoma cells. Potent tyrosinase activators were further assessed for their potential use as novel melanogenic agents by comparing their melanogenesis-stimulating activities with that of a positive control, 8-Methoxypsoralen (Lei et al. 2002). The melanogenic mechanisms of the identified compounds were preliminarily investigated by checking their influence on the protein expression levels of melanin biosynthesis enzymes.

Materials and methods

Reagents and phytocompounds

Triton X-100, L-3,4-dihydroxyphenylalanine, sodium hydroxide (NaOH), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), and 8-Methoxypsoralen (purity [greater than or equal to] 98%) were purchased from Sigma (St. Louis, MO, USA) purchased from Sigma (purity [greater than or equal to] 98%). Other compounds tested were obtained from Shanghai R&D Center for Standardization of Chinese Medicines (Shanghai, China, purity > 99%).

Cell culture

Murine melanoma B16 cells (Shanghai Institutes for Biological Science, Chinese Academy of Sciences, China) grown in DMEM medium (GIBICO, USA) supplemented with 10% fetal bovine serum (GIBICO, USA), 1% penicillin/streptomycin (P/S, GIBCO, USA) were cultured at 37 [degrees]C in a humidified atmosphere of 5% [CO.sub.2].

Tyrosinase activity assay

Cellular murine tyrosinase activity was measured as previously described (Hunt et al. 1994) with slight modification. Briefly, B16 cells were seeded in 96-well plates (4 x [10.sup.3] cells/well) and allowed to adhere at 37 [degrees]C for 12 h. Test compounds (dissolved in DMSO) were then added to individual wells. Control cells were treated with 0.1% DMSO in this experiment and the following experiments. After a 72-h incubation, cells were washed with PBS and lysed in PBS (pH 6.8) containing 0.1% Triton X-100 by freezing and thawing. Then, 100 [mu]l of freshly prepared substrate solution (0.1% L-DOPA) was added to each lysate. Following 1-h incubation, optical densities were determined at 475 nm with a Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA). Each treatment was performed in triplicate and each experiment was repeated three times. [EC.sub.50] values were determined using GraphPad Prism 5.0 software.

Measurement of cellular melanin contents

Cells were seeded in 60 mm dishes (30 x [10.sup.4] cells/well) and allowed to adhere at 37 [degrees]C for 12 h. After adding samples, cells were incubated for 72 h and then washed with PBS and lysed in 150 [mu]l of 1 M NaOH followed by a 1-h heating at 80 [degrees]C to solubilize the melanin. Each lysate (100 [mu]l) was put in a well on a 96-well microplate, and the absorbance was measured at 475 nm with a microplate spectrophotometer (BD Bioscience, USA). Protein concentration of each sample was determined by Bio-Rad Protein Assay (BIO-RAD, USA). Each experiment was repeated three times. Intracellular melanin amount/protein amount was shown as percentage values. Each percentage value in the compound treated cells was calculated with respect to that in the solvent DMSO treated control cells. [EC.sub.50] values were determined using GraphPad Prism 5.0 software.

Measurement of cell viability

Cell viability was determined using the MTT assay. Briefly, B16 cells were cultured as in tyrosinase activity assay section. After a 72-h incubation, 10 [mu]l of MTT solution (5 mg/ml in PBS) was added to each well and cells were incubated at 37 [degrees]C for 4h. Then, 50 [mu]l of stop solution (10% SDS, 5% n-butanol, 0.01 M HCl) was added to dissolve the crystals in each well. Following a 12-h incubation, optical densities were determined at 570 nm with a microplate spectrophotometer (BD Bioscience, USA). Each treatment was performed in triplicate and each experiment was repeated three times.

Western blot analysis

Cells were collected and lysed with RIPA lysis buffer [50 mM Tris-Cl, 1%, v/v, NP-40, 0.35%, w/v, sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 1 mM [Na.sub.3][VO.sub.4] containing a protease inhibitor cocktail (Roche, Germany). Protein concentration was determined by Bio-Rad Protein Assay (BIO-RAD, USA). Equal amounts of individual protein samples were separated by SDS-PAGE and then electro-transferred onto the nitrocellulose membrane (Amersham Biosciences, USA). Membranes were blocked for 30 min with 5% skim milk in TBST buffer composed of 50 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20 and incubated with the primary antibody overnight at 4 [degrees]C. Antibodies against tyrosinase and TRP-2 were purchased from Bioworld Technology. Anti-TRP-1 was from Abeam Technology. GADPH was used as loading control and was detected using an anti-GADPH polyclonal antibody (Santa Cruze Biotechnology). After incubation with secondary antibodies, ECL detection reagents (Amersham Biosciences, USA) were used to detect signals.

Statistics

Results were expressed as the mean[+ or -] S.D. Statistical analyses were performed using Student's t-test. P < 0.05 was considered to be statistically significant.

Results and discussion

Firstly we tested the effects of 35 botanical compounds on tyrosinase activity using L-DOPA as the substrate in B16 cells. They were assessed at 100 [mu]M. Thirteen compounds showed stimulatory activity, ranging from 2.6 to 372.8% activation. Five of the 13 compounds showed more than 50% activation and were further tested for their [EC.sub.50] values. Compared with 8-Methoxypsoralen, an effective tyrosinase activator (Lei et al. 2002), 3 compounds (apigenin, hyperosid and icariin) showed smaller [EC.sub.50] values (Table 1).
Table 1

Effects of tested compounds on tyrosinase activity. Data are
represented as the mean [+ or -] S.D. of three independent assays.

No.  Chemical   Compound           Tyrosinase activity
     family

                                   Enhancement (%)     [EC.sub.50]
                                                       ([mu]M)

1    Flavonoid  Alpinetin           82.8 [+ or -] 0.9  10.18
2    Flavonoid  Apigenin           372.8 [+ or -] 1.1   0.45
3    Flavonoid  Calycosin          -19.9 [+ or -] 2.8
4    Flavonoid  Chrysin            -37.4 [+ or -] 4.3
5    Flavonoid  Galangin           -97.1 [+ or -] 1.2
6    Flavonoid  Hesperidin          48.8 [+ or -] 1.2
7    Flavonoid  Hyperosid          238.6 [+ or -] 3.2   0.92
8    Flavonoid  Icariin            177.6 [+ or -] 1.6   1.01
9    Flavonoid  Isorhamnetin       -21.5 [+ or -] 0.9
10   Flavonoid  Luteolin           100.0 [+ or -] 1.7   22.6
11   Flavonoid  Morusin             -2.1 [+ or -] 0.4
12   Flavonoid  Naringenin         -13.7 [+ or -] 2.1
13   Flavonoid  Puerarin           -10.7 [+ or -] 2.1
14   Flavonoid  Baicalin           -21.7 [+ or -] 4.3
15   Lignan     Anwuligan          -86.1 [+ or -] 1.3
16   Lignan     Gaultherin          29.7 [+ or -] 1.9
17   Lignan     Magnolol           -96.6 [+ or -] 1.3
18   Lignan     Podophyllotoxin    -20.0 [+ or -] 1.8
19   Lignan     Schizandrin B       40.4 [+ or -] 2.1
20   Lignan     Sesamin             36.3 [+ or -] 1.1
21   Phenol     Curcumin           -76.3 [+ or -] 5.3
22   Phenol     Ferulic acid       -23.9 [+ or -] 2.5
23   Phenol     Menisdaurin        -10.5 [+ or -] 2.1
24   Phenol     Rhodioloside        34.9 [+ or -] 1.1
25   Phenol     Rosavin             -7.2 [+ or -] 0.6
26   Phenol     Salicin             -8.5 [+ or -] 0.4
27   Phenol     Sodium Danshensu   -13.4 [+ or -] 2.1
28   Phenol     Syringin            25.0 [+ or -] 0.3
29   Terpenoid  Bilobalide          20.9 [+ or -] 2.6
30   Terpenoid  Geniposide           2.6 [+ or -] 0.3
31   Terpenoid  Gentiopicroside     -8.8 [+ or -] 1.2
32   Terpenoid  Ginkgolide A        -2.8 [+ or -] 0.8
33   Terpenoid  Ginkgolide B        -1.8 [+ or -] 0.9
34   Terpenoid  Ophiopogonin D      -4.5 [+ or -] 1.0
35   Terpenoid  Ursolic acid       -47.5 [+ or -] 1.1
36   Coumarin   8-Methoxypsoralen   57.7 [+ or -] 2.7   7.26
                (a)

(-): Inhibits the activity.
(a) Positive control.


We next investigated the effects of the 3 identified compounds on melanogenesis and cell proliferation in B16 cells. Melanogenesis was assessed by determining intracellular melanin amount/protein amount which is shown in percentage value calculated with respect to that in the control cells. Apigenin, hyperosid and icariin increased cellular melanin contents in a concentration-dependent manner after a 72-h treatment (Fig. 1). Compared with 8-Methoxypsoralen ([EC.sub.50], 35.94 [mu]M), 2 compounds exhibited smaller [EC.sub.50] values (apigenin, 17.46 [mu]M and icariin, 32.77 [mu]M), while hyperosid had a greater [EC.sub.50] value of 70.4 [mu]M for stimulating pigmentation. All 3 compounds did not significantly affect cell proliferation determined by MTT assay under our experimental conditions (data not shown). In vivo potential tolerability of the 3 compounds remains to be evaluated when they are used as pigmentation stimulators.

[FIGURE 1 OMITTED]

Melanin synthesis is mainly regulated by melanogenic enzymes such as tyrosinase, TRP-1, and TRP-2 (Hearing 1999). Tyrosinase catalyzes the hydroxylation of tyrosine to DOPA and the oxidation of DOPA to dopaquinone as mentioned in the introduction. TRP-2, which functions as dopachrome tautomerase, catalyzes the rearrangement of dopachrome to 5,6-Dihydroxyindole-2-Carboxylic acid (DHICA), and TRP-1 oxidizes DHICA to a carboxylated indole-quinone; both work at downstream points in the melanin biosynthetic pathway (Yokoyama et al. 1994). To determine whether the melanogenic properties of the 3 compounds can be attributed to their capacity to increase the expression levels of the 3 melanogenic enzymes, Western blot analysts was conducted. As shown in Fig. 2, apigenin elevated the expression of all the 3 enzymes; hyperosid increased the expression of TRP-1 and TRP-2 but not tyrasinase (TYR); while icariin did not significantly influence the expression level of any enzyme, except for a slight elevation of TYR expression. These data suggest that apigenin and hyperosid may stimulate melanogenesis by, at least in part, increasing the expression levels of the main melanogenic enzymes. Of course, we are aware that the melanin biosynthetic pathway is complicated, and whether other mechanisms are responsible for the melanogenic activities of apigenin and hyperosid remains to be elucidated. Although icariin did not affect the expression of TRP-1 and TRP-2 and only slightly increased TYR expression, it exhibited more potent melanogenic activity than hyperosid. Icariin may promote melanogenesis mainly by increasing melanogenic enzymes activities, through enhancing catalytic capacity, but not expression levels, of the enzymes.

[FIGURE 2 OMITTED]

Since apigenin and icariin exerted better melanogenic activity than the positive control 8-Methoxypsoralen, further investigations are merited for developing them as novel melanogenic agents. Both compounds are naturally occurring flavonoids. Apigenin is abundantly present in a variety of fruits and leafy vegetables (Czeczot et al. 1990) and has been shown to possess various bioactivities such as anti-oxidant, anti-mutagenic, anti-carcinogenic, anti-inflammatory, anti-proliferative, and anti-progression properties (Patel et al. 2007). Icariin is the main bioactive constituent of plants in the Epimedium family and shows anti-thrombosis, anti-tumor, anti-aging, anti-osteoporosis, and immune-enhancing activities (Liu and Wu 2009). We are the first to report their melanogenic activity.

In summary, we identified 2 flavonoid compounds, apigenin and icariin, that demonstrated potent melanogenic activity in B16 cells without affecting proliferation. Further investigations are warranted to explore their potential application in the management of hypopigmentation disorders.

Acknowledgements

This study was supported by grants from Shanghai key disciplines-funded construction projects (Y0301), Modernization of Traditional Chinese Medicine, Shanghai Science and Technology Commission (05DZ19742), and Hong Kong Baptist University (FRG/08-09/II-30).

References

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Czeczot, H., Tudek, B., Kusztelak, J., Szymczyk, T., Dobrowolska, B., Glinkowska, G., Malinowski, J., Strzelecka, H., 1990. Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs. Mutat. Res. 240, 209-216.

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Yasumoto, K., Yokoyama, K., Takahashi, K., Tomita, Y., Shibahara, S., 1997. Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes. J. Biol. Chem. 272, 503-509.

Yokoyama, K., Yasumoto, K., Suzuki, H., Shibahara, S., 1994. Cloning of the human DOPAchrome tautomerase/tyrosinase-related protein 2 gene and identification of two regulatory regions required for its pigment cell-specific expression. J. Biol. Chem. 269, 27080-27087.

Yan Ye (a), (b), Gui-Xin Chou (b), (c), **, Hui Wang (a), Jian-Hong Chu (a), Zhi-Ling Yu (a), *

(a) Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

(b) Key Laboratory of Standardization of Chinese Medicines, Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Zhungjiang, Shanghai, China

(c) Shanghai R & D Center for Standardization of Chinese Medicines, Shanghai, China

* Corresponding author. Tel.: +852 3411 2465; fax: +852 3411 2461.

** Corresponding author at: Tel: +86 21 5027 1706; fax: +86 21 50271708.

E-mail addresses: chouguixin@yahoo.com.cn (G.-X. Chou), zlyu@hkbu.edu.hk (Z.-L. Yu).
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Title Annotation:Short communication
Author:Ye, Yan; Chou, Gui-Xin; Wang, Hui; Chu, Jian-Hong; Yu, Zhi-Ling
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Dec 15, 2010
Words:2732
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