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Ha, S., Jung, J., Kang, H., Kim, T., and Yang, J. (2020). "Tyrosinase activity and melanogenic effects of Lespedeza bicolor extract in vitro and in vivo," BioRes. 15(3), 6244-6261.

Abstract

Lespedeza bicolor (L. bicolor) is used for medicinal purposes because of its various biological and pharmacological activities. In this study, the effects of L. bicolor ethanol extract on the treatment of vitiligo were investigated. The determination of melanin content in melanocytes was measured using B16 melanoma cells and C57BL/6J Ler-vit/vit mice. Finally, the quercetin content in L. bicolor were qualitatively analyzed using HPLC. The results obviously indicated that the L. bicolor extract enhanced melanogenesis and increased tyrosinase activity in cultured melanoma cells and C57BL/6J Ler-vit/vit mice. Treatment with L. bicolor extract led to a higher content of melanin and eumelanin in C57BL/6J Ler-vit/vit mice hair than in control (untreated) mice, which demonstrated the therapeutic effect of hair-graying associated with vitiligo. There was a notable increase in melanocytes in the skin of C57BL/6J Ler-vit/vit mice treated with L. bicolor extract compared with the control. L. bicolor extract was a potent tyrosinase and melanin synthesis activator in B16 melanoma cells. C57BL/6J Ler-vit/vit mice treated with L. bicolor extract had significantly higher melanin content in hair than the untreated control. The results suggest that L. bicolor extract is a potential alternative treatment for improvement of vitiligo.


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Tyrosinase Activity and Melanogenic Effects of Lespedeza bicolor Extract in vitro and in vivo

Si Young Ha,a Ji Young Jung,a Hee Young Kang,b Tae-Heung Kim,c and Jae-Kyung Yang a*

Lespedeza bicolor (L. bicolor) is used for medicinal purposes because of its various biological and pharmacological activities. In this study, the effects of L. bicolor ethanol extract on the treatment of vitiligo were investigated. The determination of melanin content in melanocytes was measured using B16 melanoma cells and C57BL/6J Ler-vit/vit mice. Finally, the quercetin content in L. bicolor were qualitatively analyzed using HPLC. The results obviously indicated that the L. bicolor extract enhanced melanogenesis and increased tyrosinase activity in cultured melanoma cells and C57BL/6J Ler-vit/vit mice. Treatment with L. bicolor extract led to a higher content of melanin and eumelanin in C57BL/6J Ler-vit/vit mice hair than in control (untreated) mice, which demonstrated the therapeutic effect of hair-graying associated with vitiligo. There was a notable increase in melanocytes in the skin of C57BL/6J Ler-vit/vit mice treated with L. bicolor extract compared with the control. L. bicolor extract was a potent tyrosinase and melanin synthesis activator in B16 melanoma cells. C57BL/6J Ler-vit/vit mice treated with L. bicolor extract had significantly higher melanin content in hair than the untreated control. The results suggest that L. bicolor extract is a potential alternative treatment for improvement of vitiligo.

Keywords: Lespedeza bicolor; Melanogenesis; Tyrosinase activity; Vitiligo

Contact information: a: Division of Environmental Forest Science/Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Republic of Korea; b: Department of Dermatology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea; c: White-Line Skin Clinic, Changwon, 51496, Republic of Korea; *Corresponding author: jkyang68@gmail.com

INTRODUCTION

Vitiligo is usually an acquired pigment disorder, a skin disease caused by the loss of the melanin pigment in the epithelium. Vitiligo occurs in 1% of the world’s population and affects all races. Different studies suggest that some genetic mechanisms are involved in the etiology of vitiligo; in some cases, at least 30% of people have a family history. The exact cause of this disease is not yet known, and only the period of remission and exacerbation is known. Depigmentation might be an option, but such radical transformations may have a serious impact on a patient’s cultural identity.

In vitiliginous areas, melanocytes are present in the skin but are in an inhibited state of functional activity. When melanocyte activity is affected, melanin is not synthesized in the melanosomes of melanocytes. In addition, the development of vitiligo is related to the rates of synthesis and decay of tyrosinase. Many studies have investigated the importance of tyrosinase in the regulation of racial pigmentation. Song et al. (1994) showed that tyrosinase was an enzyme important in melanin formation.

The current treatment options for vitiligo include medication, surgery, and adjunctive therapies (those used along with surgical or medical treatments). Whitton et al. (2016) reported that there are several ways to improve the appearance of vitiligo, but the effect is limited. Narrowband ultraviolet B (NB-UVB) phototherapy is problematic because the resulting repigmentation is transient. Skin grafting techniques are the most effective interventions, but they can only be used with stabilized or segmental types of vitiligo, which are less common. In summary, there is currently no satisfactory solution to vitiligo, and the patients are impacted for life.

The study of local knowledge of natural resources has become increasingly important in the investigation of the development of medicines without side effects. Many studies are underway to find potential pigment for the treatment of vitiligo. Piper nigrum L. fruit extract has growth-stimulatory activity in melanocytes. In addition, the extracts from Cucumis melo and Ammi visnaga fruits have been studied. Wood extracts have been tested also as a treatment for vitiligo. In studies by Szczurko et al. (2011), the potency of oral Ginkgo biloba extract to halt progression of active vitiligo was evaluated. The wood extract treatments studied were Angelica sinensis root extract and Ammi majus L. extract. Lin et al. (1999) found that out of 28 herbal extracts screened, significant stimulation (p<0.05) of melanocyte proliferation was observed using aqueous extracts of herbs. Tahir et al. (2010) reported that Polylpodium leucotomos extract has been used for the treatment of vitiligo for more than 10 years in Europe. Madhogaria and Ahmed (2010) reported a patient who developed depigmented patches after using a cream containing kojic dipalmitate, licorice root extract, and Mitracarpus scaber extract.

As a medicinal plant, Lespedeza bicolor (L. bicolor) is used in folk medicine for white patches on the body, such as leucoderma and vitiligo. Despite its established popular use for the treatment of vitiligo, there are few reports in the literature on this property. Similar previous studies have investigated the effects of flavonoids, contained in many L. bicolor extracts, on tyrosinase and the therapeutic effects on inflammation (Samiullah et al. 2012; Rafiq et al. 2013). In addition, quercetin, a component of L. bicolor extract, was reported to induce the upregulation of melanogenesis and enhance tyrosinase activity in dose- and time-dependent manners (Nagata et al. 2004).

Therefore, L. bicolor extract could be used as a potential resource for plant-based pharmaceutical products for melanogenesis. In vitro and in vivo experiments were conducted to determine the effect of L. bicolor extract on vitiligo treatment. The cytotoxicity, tyrosinase activity, and melanin content were analyzed in B16 melanoma cells in vitro. The melanin content, eumelanin content, and histologic analysis were examined in C57BL/6J Ler-vit/vit. A mice model for vitiligo, an acquired cutaneous depigmentary disorder, has been established and given the provisional genetic designation C57BL/6J Ler-vit/vit on Boissy et al. (1987). Through this study, the potential of L. bicolor for the treatment of vitiligo was examined.

EXPERIMENTAL

Materials

Preparation of extract

The plant materials were obtained from the Gyeongsang National University Research Forest, Jinju, South Korea and identified by comparison with the voucher specimen (HCCN: 24239 GBH0000003470) deposited at the Herbarium of the Rural Development Administration-Genebank Center, Jeonju, South Korea. The plants were identified by Dr. Hak-gon Kim in the GyeongNam Forest Enviromental Research Institute, Jinju, South Korea. The plants were authenticated by Hee-gon Kang in Gyeongsang National University Research Forest, Jinju, South Korea. The fresh stem of L. bicolor (Fig. 1) was cut into small pieces (length 3 cm) and dried over-night at room temperature. The dried, L. bicolor stem (500 g) was soaked in 60% ethanol 10 L for 1 week at room temperature. After vacuum filtration (Whatman No. 2 filter paper), the residue was extracted twice more in the same way. The filtrates were evaporated at 45 °C under reduced pressure using a rotary evaporator to remove the solvent and lyophilized to obtain the crude extract at a yield of 21.8% (109 g). The extract was stored at 4 °C.

Fig. 1. Lespedeza bicolor

Cell culture

Mouse melanoma cell lines were provided by Ajou University Medical Center. The B16 melanomas growth medium was composed of Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA), 10% fetal bovin serum (FBS), and 1% penicillin/streptomycin (PS). The culture condition was 37 °C in a humidified atmosphere of 0.5% CO2.

Cell Cytotoxicity Assay

Subcultures of B16 cells were seeded in 96 well plates at a density of 7 × 103 B16 cells and cultured for 24 h. The medium was then replaced with 500 μL fresh DMEM medium containing 10% FBS and 1% PS. Next, 1 μL (5, 10, 20 μg L. bicolor ethanol crude extract/1 mL 60% ethanol) of the L. bicolor ethanol extract was added to each well and cultured for 3 day. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was dissolved in phosphate-buffered saline (PBS). After 3 days, 50 μL of MTT (at a final concentration of 5 mg/mL) were added, and the cells were incubated in 0.5% CO2 at 37 °C for 1 h. The plates were shaken with dimethylsulphoxide (DMSO) for 15 min to dissolve the blue/purple formazan crystals. The percentage of viable cells was quantified by identifying the ability to reduce MTT. The optical density was measured in a ELISA reader (Tetertek Multiskan MCC/340, Labsystem, Helsinki, Finland) at 540 nm.

Determination of Melanin Content in Melanocytes

The assay followed Kubo’s method (Kubo et al. 2004) with slight modification. Briefly, subcultures of B16 cells were seeded in 60 ø plates at a density of 8 × 104 B16 melanoma cell and cultured for 24 h. The medium was replaced with 3 mL fresh DMEM medium containing 10% FBS and 1% PS. The 1 μL of the L. bicolor ethanol extract was added to each well and cultured for 3 day. After 3 days, the cells were harvested, suspended in 0.1 mL 1N NaOH-10% DMSO solution (v/v), and kept at 60 °C for 6 h in a water bath. The 90 μL test solution was transferred into a 96 well plate and measured in a ELISA reader (Tetertek Multiskan MCC/340, Labsystem, Helsinki, Finland) at 490 nm. The melanin content was determined by calculation from a synthetic melanin standard curve.

Mushroom Tyrosinase Assay

Measurement of tyrosinase in L-DOPA oxidation of mushroom extracts was carried out as described by Masamoto et al. (2003). The experiment was performed by partially modifying the method. First, 100 μL of 0.1 M phosphate buffer was mixed with 20 μL of different concentrations from L. bicolor ethanol extract. Then, 20 μL of mushroom tyrosinase (2,000 U/mL in phosphate buffer) were added to initiate the reaction. The mixture was incubated at 37 °C for 5 days and then incubated at 37 °C for 10 min with the addition of 40 microliters of L-DOPA (4 mM in 0.1 M phosphate buffer). The mixture was measured in a ELISA reader at 475 nm. The percentage activity of tyrosinase was calculated as follows,

% activity = 100 – (B/A x 100) (1)

where A is absorbance in 10 min without sample, and B is absorbance in 10 min with tested sample.

Determination of Cellular Tyrosinase Activity

Subcultures of B16 cells were seeded in 60 ø plates at a density of 8 × 104 B16 melanoma cell and cultured for 24 h. The medium was replaced with 3 mL fresh DMEM medium containing 10% FBS and 1% PS. The 1 μL of the L. bicolor ethanol extract was added to each well and cultured for 3 days. After 3 days, the cells were harvested and lysed by incubation at -4 °C for 1 h in 100 μL lysis buffer (PBS pH 6.8, trytone×100 1%). The lysates were centrifuged at 10,000 × g for 30 min (4 °C) to obtain the supernatant as a source of tyrosinase. Tyrosinase activity was assayed as described previously (Nagata et al. 2004). The reaction mixture contained 20 μg of lysate, 180 μL of 2 mM L-DOPA/pH 6.8 PBS, and pH 6.8 PBS. After incubation at 37 °C for 1 h, Absorbance was measured at a wavelength of 490 nm to observe the dopachrome formation.

Determination of Melanogenesis in C57BL/6J Ler-vit/vit Mice

Cell culture

All the experimental procedures were performed according to the guidelines of the Committee for Ethical Usage of Experimental Animals at Gyeongsang National University, South Korea: 1.) Act on Experimental Animals Act No. 9932 2010.1.18, 2010.3.19 Other laws amended by the Ministry of Health and Welfare; 2.) Enforcement Decree of the Experimental Animal Act Presidential Decree No. 22075 2010.3.15, 2010.3.19 Other laws amended by the Ministry of Health and Welfare; 3.) Enforcement Rules of the Act on Experimental Animals Ministry of Health and Welfare No. 1 2010.3.19, 2010.3.19 Other laws amended by the Ministry of Health and Welfare. The C57BL/6J Ler-vit/vit mice with melanocyte disappearance (Medrano and Nordlund 1990; Slominski and Paus 1993) were used as in vivo animal models. Female C57BL/6J Ler-vit/vit mice were purchased from SAMTAKO (Gyeonggi-do, South Korea). These mice were stored under the conditions of temperature (20 to 26 °C), humidity (30 to 70%), and illumination (lit from 08:00 to 20:00) and used for the experiment. The type of food for mice was standard diets, and crude nutrients were 20% protein, 4.5% fat, 6% fiber, 7% ash, 0.5% calcium, and 1% phosphorus. The bedding material was GLP bedding (SAMTAKO, Gyeonggi-do, South Korea), and number of cage companions was one. The C57BL/6J Ler-vit/vit mice used in this experiment were 10 to 15 weeks of age. Food and tap water were provided ad libitumL. bicolor ethanol extract was dissolved in 60% ethanol and used in the experiment. The mice were randomly divided into two groups of five mice as normal control group with mice treated 60% ethanol and mice treated 0.2 mL/cm2 L. bicolor ethanol extract for 5 months without intermission. The control was treated with 60% ethanol on the opposite side of the same mouse. Repeated experiments were conducted with three sets and changes in hair color was monitored on once a day. When the experiment finished, euthanization was performed by 10% isoflurane with prolonged exposure at 1, 2, and 4 h after administration, and death was confirmed by exsanguination.

Melanin and eumelanin content measurement in mice hair

Samples of hair were incubated overnight in 1M NaOH, as previously described (Green and Wilson 1996). Standards were prepared by dissolving synthetic melanin (Sigma Chemical Co., Poole, Dorset, UK) over the concentration range 0.05 to 0.4 mg/mL in 1 M NaOH. The absorbance at 500 nm (total melanin) and 650 nm (eumelanin) of both standards and sample digests was measured using a Pye Unicam SP8-100 ultraviolet-visible spectrophotometer. Because synthetic and endogenous melanin differ in structure, the values presented are comparative rather than absolute.

Histologic examination of C57BL/6J Ler-vit/vit mice

Throughout this investigation, the standard procedure of Laidlaw and Blackberg (1932) was used, and for carrying out the reaction both freshly prepared sheets of pure epidermis and frozen sections were employed. Where paraffin sections were found to be necessary, as when studying the distribution of melanocytes in hair follicles, specimens comprising the full thickness of the skin were subjected to Dopa treatment after a brief preliminary period of formol fixation. They were then given an additional period in the fixative and sectioned by ordinary methods. The dermo-epidermal was incubated with EDTA solution (pH 7.4) at 37 °C for 2 h. Dermo-epidermal was separated with microforcep and washing with saline for 1 min. The dermo-epidermal was incubated with the L-dopa solution at 37 °C for 1 h. The dopa solution after the reaction was replaced with a new dopa solution, and dermo-epidermal was once more incubated at 37 ° C for 8 h.

Finally, dermo-epidermal was washed with saline for 1 min. Dyed dermo-epidermal was fixed with 10% formalin for 20 min and washed with distilled water for 3 min. Finally, the dermo-epidermal was dehydrated with 95% alcohol and 100% alcohol for 20 min each and cleared with the xylene for 20 min (three times). The dermo-epidermal was mounted on slide glass with Canada balsam.

HPLC Analysis

Chromatographic analysis was carried out by DAD following RP-HPLC separation installed with HIQ SIL C18V reversed-phase column (ø 4.6 mm × 250 mm, Young-In Chromass, Anyang-si, South Korea) packed with 5 μm diameter particles. The mobile phase was methanol-acetonitrile-water (40:15:45, v/v/v) containing 1.0 % acetic acid. The flow rate and injection volume were 1.0 mL/min and 10 μL, respectively. The standard was quercetin, and both sample and standard were filtered through a 0.45 μm membrane filter. HPLC analysis was performed at ambient temperature, and the peak analysis of the chromatography was confirmed by comparison with the retention time of the standard.

Statistical Analysis

All experiments were run at least in triplicate. SPSS 11.5 (Chicago, IL, USA) and PROC GLM in SAS 9.1 software (Cary, NC, USA) were use for the statistical analysis. A descriptive statistical analysis was made by calculating the mean and standard deviation, and comparison between groups was complemented by a comparison between means (pairwise t-test). P <0.05 and p <0.001 were interpreted as significant.

RESULTS AND DISCUSSION

Toxicity to B16 Melanoma Cells

The effects of the extract on cell proliferation were investigated. The cells were exposed to various doses of extract for 72 h, and cytotoxicity was determined by the MTT assay. As shown in Fig. 2, cell viability was maintained for 72 h after exposure to L. bicolor extract. The cells were treated with various concentrations of L. bicolor extract (5, 10, and 20 µg/mL), and the cell viability was calculated relative to the control. Cell viability was maintained as L. bicolor extract concentration increased (5, 10, and 20 µg/mL). These results indicated that L. bicolor extract effectively induced the survival of B16 cells. In addition, L. bicolor extract did not exert cytotoxic effects on B16 melanoma cell proliferation. The percentage viabilities of control cells and those treated with ethanol and various concentration of were not significantly different. Mosmann (1983) reported that the amount of formazan produced in the MTT assay is exactly proportional to the viability of the cells. Therefore, the effects of L. bicolor extract on melanin synthesis and cellular tyrosinase activity were examined subsequently.

Fig. 2. Effect of L. bicolor ethanol extract on cytotoxicity in B16 melanoma cells. Control: untreated; Ethanol: 60 % ethanol; Concentration: L. bicolor ethanol extract

Effect of L. bicolor Extract on Melanin Content in B16 Melanoma Cells

B16 melanoma cells offer quantifiable markers of cytodifferentiation, such as melanin production, as well as a morphological marker (dendrite formation) (Pomerantz 1964). There is considerable experimental evidence to indicate that growth and melanization are intimately related in melanoma cells (Huberman and Callaham 1979). Lan et al. (2005) reported that melanin content was significantly higher in vitiligo lesions. Itoh and Furuichi (2005) used melanin content as an indicator for the evaluation of anti-graying effects in hair and improvements in vitiligo vulgaris. Niu et al. (2016) reported that the main cause of vitiligo was anti-melanogenic activity and that the analysis of melanin content was essential to improve vitiligo. To examine the melanogenic activity of the L. bicolor extract, the stimulatory effect of L. bicolor extract on melanin was evaluated in B16 melanoma cells. The B16 melanoma cells were treated with the L. bicolor extract at 5, 10, and 20 μg/mL for 72 h. The melanin content was presented as a percentage of the control (vehicle). The following effects of L. bicolor extract on melanogenesis of the B16 melanoma cells were noted. L. bicolor ethanol extract exerted a marked stimulatory effect on melanogenesis, without affecting cell proliferation, at concentrations of 5 to 20 µg/mL (Fig. 3).

Fig. 3. Effect of L. bicolor ethanol extract on melanin content in B16 melanoma cells. Control: untreated; Ethanol: 60 % ethanol; Concentration: L. bicolor ethanol extract.* p<0.05 compared to the untreated control; ** p<0.01 compared to the untreated control.

The L. bicolor extract exhibited a significant dose-dependent increase on melanin content. The melanin content was 142.11% ± 0.07%, 180.00% ± 0.10%, and 181.95% ± 0.38% after treatment with 5, 10, and 20 µg/mL L. bicolor extract, respectively. Kang et al. (2018) reported that the melanin content was 146% and 110% after treatment with 8 µg/mL and 40 µg/mL of Euphorbia supina extract in B16F10 cells, respectively. After the addition of 0.5 mM glycyrrhizin, the cellular melanin content reaches approximately 160% of control cells (Jung et al. 2001). The Tunisian Capparis spinosa extract stimulates melanogenesis in B16 cells and has been found to increase melanin content by 12% and 60% at 0.005% and 0.05% extract concentrations, respectively (Matsuyama et al. 2009). The L. bicolor extracts induced similar levels of melanin as previous studies, with a maximum melanin content of 181.95% after treatment with 20 µg/mL extract. Therefore, the L. bicolor extract has the potential as a new natural resource to increase melanin content.

Fig. 4. L. bicolor extract increases melanogenesis in B16 melanoma cells. The dark-black color of the B16 melanoma cell pellets demonstrate that control (untreated), ethanol and the L. bicolor extract. Control: untreated; Ethanol: 60 % ethanol

In confirmation of the results of Fig. 4, the increase of melanin biosynthesis and secretion shows a distinct black color of the cell pellet. Hamid et al. (2012) reported that the dark-black color of the B16F1 melanoma cell pellets demonstrated that α-MSH, forskolin, and mangosteen leaf extract stimulated melanogenesis activity. Nair et al. (2001) evaluated repigmentation, as determined by cell pellet color and melanin assays. The colors of cell pellets are evaluated visually for melanin content assay (Usuki et al. 2003). These observations suggest that the L. bicolor extract increased the melanogenic activity of B16 melanoma cells.

Effect of L. bicolor Extract on Tyrosinase Activity

Effect of L. bicolor extract on mushroom tyrosinase activity

To determine whether L. bicolor ethanol extract had a direct effect on the major enzyme in the melanogenesis, an in vitro cell-free mushroom tyrosinase assay was conducted. The effects of L. bicolor ethanol extract on mushroom tyrosinase activity are shown in Fig. 5. The effects of the extract on the oxidation of L-DOPA by mushroom tyrosinase occurred in a dose-dependent manner. At 10 µg/mL and 20 µg/mL, L. bicolor ethanol extract exerted activity on the oxidation of L-DOPA by mushroom tyrosinase; the 10 µg/mL L. bicolor ethanol extract showed lower activity than the 20 µg/mL L. bicolor ethanol extract. However, there was no significant difference between the two concentrations. Tyrosinase catalyzes 3,4-dihydroxyphenylalanine (DOPA) quinone formation from DOPA, and melanin formation from DOPA quinone via autoxidation and enzymatic reaction (Jimenez et al. 1993). Therefore, melanin production is related to tyrosinase expression; the results presented here were similar. More specifically, both the melanin content and tyrosinase activity were increased by 10 µg/mL and 20 µg/mL L. bicolor extract (Figs. 3 and 5). It is anticipated that continued research will increase knowledge concerning the activity of tyrosinase in L. bicolor ethanol extract, and continue to shed light on therapeutic strategies that can be used to reduce or eliminate vitiligo. Most studies have been performed on the effect of the mushroom tyrosinase from various plant extracts (Kim et al. 2003; Hsu et al. 2007). Thus, L. bicolor extract may be noted as an effective material with mushroom tyrosinase activity.

Fig. 5. Effects of L. bicolor ethanol extract on mushroom tyrosinase activity. Purified tyrosinase was mixed with L. bicolor ethanol extract and incubated with L-DOPA. Control: untreated; Ethanol: 60 % ethanol; Concentration: L. bicolor ethanol extract. * p<0.05 compared to the untreated control.

Effect of L. bicolor extract on tyrosinase activity in B16 melanoma cells

Tyrosinase catalyzes three steps in the biosynthesis process of melanin; hence, the measurement of tyrosinase activity is very important. The intracellular tyrosinase activity was measured after the culture of B16 melanoma cells with L. bicolor ethanol extract. Different concentrations of L. bicolor extract, not mushroom tyrosinase, were used to treat cell lysats extracted from B 16 melanoma cells. Equal masses of cell lysate were prepared with respect to the protein concentration. At 5 µg/mL L. bicolor extract, a weak effect on the direct activation of intracellular tyrosinase was observed, but tyrosinase-inducing activity was increased significantly by 10 µg/mL and 20 µg/mL L. bicolor extract (Fig 6). Chen et al. (2012) showed that tyrosinase was regarded as the rate-limiting enzyme of melanogenesis, which modulates this process through the catalysis of the hydroxylation of tyrosine into DOPA and the further oxidation of DOPA into dopaquinone. Matsuda et al. (2005) confirmed the potential stimulation of melanogenesis from tyrosinase activity by using cultured B16 melanoma cells. The activity of tyrosinase in melanocytes may be expressed in tyrosinase cells due to direct activity or an increase in the total amount of protein in the cell (Oh et al. 2011). In the previous results, L. bicolor extract exerted a significant influence on tyrosinase activity. This result shows that the L. bicolor extract can be used directly as a tyrosinase activator. In a previous study (Jung et al. 2001), the cellular tyrosinase activity was also increased dose-dependently by glycyrrhizin, reaching 220% of the value in control cells at a treatment concentration of 1 mM. Adzuki bean extract is known to have a weak effect on the direct activation of tyrosinase in cells (Itoh and Furuichi 2005). Tuerxuntayi et al. (2014) reported that, compared with untreated conditions, treatment with Kaliziri extract at 5 to 40 µg/mL resulted in a dose-dependent increase in tyrosinase activity in B16 cells (to a maximum of 138% tyrosinase activity). Treatment with forskolin significantly increased the intracellular tyrosinase activity by more than three-fold, whereas treatment with 32 µg/mL extract demonstrated a four-fold increase in intracellular tyrosinase activity (Hamid et al. 2012). These results suggested that L. bicolor extract is a stimulant of tyrosinase, similar to other natural extracts; moreover, melanin synthesis increases as tyrosinase activity increased.

Fig. 6. Effect of L. bicolor ethanol extract on intracellular tyrosinase activity in B16 melanoma cells (Control: untreated; Ethanol: 60% ethanol treated).

* p<0.05 compared to the untreated control.

Effect of L. bicolor Extract on Melanin Content in C57BL/6J Ler-vit/vit Mice Hair

Before the experiment, the health status of the mice were normal. The experiment was conducted using a total of 15 mice, which were used for all experiments. Melanin contents in the hair samples taken from the back of C57BL/6J Ler-vit/vit mice were significantly higher in those treated with L. bicolor ethanol extract than in the control group (p < 0.01) (Fig. 7). Therefore, the L. bicolor extract has the potential to assist repigmentation in vitiligo because it increased melanin content by approximately 40% compared with the control. In vertebrates and higher mammals, melanin plays an important role in thermoregulation, gastrointestinal tract, sexual attraction, and photoprotection. These melanin pigments can be distinguished chemically by the red-yellow pheomelanin and the brown-black eumelanin. Both types of melanin are found in human hair, the epidermis, and cultured melanocytes. The main synthesis process for these two pigments is similar and is controlled by tyrosinase. However, the production of eumelanin is important for repigmentation in the treatment of vitiligo.

Fig. 7. Effect of L. bicolor ethanol extract on melanin content of hair in C57BL/6J Ler-vit/vit mice. The mice hair was lysed with 1N NaOH and the absorbance of the solution at 500nm was measured. C57BL/6J Ler-vit/vit mice: vitiligo mice hair; C57BL/6J: normal mice hair.

** p<0.01 compared to the control