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Journal of Ethnopharmacology 148 (2013) 199–204

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Journal of Ethnopharmacology

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Effect of a gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells

Patrícia Mazureki Campos a, Cíntia Delai da Silva Horinouchi b, Arthur da Silveira Prudente b, Valdir Cechinel-Filho c, Daniela de Almeida Cabrini b, Michel Fleith Otuki b,d,n

a Department of Pharmaceutical Sciences, Universidade Federal do Paraná, CEP 80210-170, Curitiba, PR, Brazil b Department of Pharmacology, Universidade Federal do Paraná, PO Box 19031, CEP 81530-900, Curitiba, PR, Brazil c Núcleo de Investigacões Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí, PO Box: 360, CEP 88302-202, Itajai, SC, Brazil d Department of Pharmaceutical Sciences, Universidade Estadual de Ponta Grossa, CEP 84030-900, Ponta Grossa, PR, Brazil

article info abstract

Article history: Ethnopharmacology relevance: Garcinia gardneriana (Planchon and Triana) Zappi () is popularly Received 27 July 2012 called “bacopari” in southern Brazil. The leaves of this are traditionally used to treat skin disorders. Received in revised form Aim of study: This study evaluated the effects of a hydroalcoholic extract of Garcinia gardneriana leaves 26 March 2013 (HEGG) on B16F10 murine melanoma cells in order to search for new depigmenting agents. Accepted 31 March 2013 Materials and methods: The effects of HEGG were assessed in melanin content assays in B16F10 Available online 17 April 2013 melanoma cells compared with the reference drug kojic acid (500 mM). Melanin content was measured Keywords: after spontaneous melanogenesis, UVB-induced melanogenesis and melanogenesis induced by α-MSH. Garcinia gardneriana At the same time, cell viability assays were conducted. Intracellular and mushroom tyrosinase activity Clusiaceae assays were employed to evaluate the effect of HEGG on tyrosinase activity. Melanin Results: HEGG decreased the level of melanin under all three experimental conditions of melanin Tyrosinase ' Hyperpigmentation content evaluation without reducing cell viability. In intracellular tyrosinase assays, the enzyme s activity was reduced about 19% with extract concentrations ranging 0.1–10 mg/mL. In the mushroom tyrosinase activity assay a maximal inhibition of 35% (1000 mg/mL) was observed. Conclusion: These results suggest that HEGG inhibition relates to its tyrosinase activity. Therefore, the hydroalcoholic extract of Garcinia gardneriana shows great potential for use as a depigmenting agent in hyperpigmentation disorders. & 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction followed by oxidation of L-DOPA to form dopaquinone. The second and third enzymes are tyrosinase-related protein-1 (TRP-1) and Melanin is the most widespread natural pigment. Pigmentation dopacromo tautomerase (DCT), which are more concerned with of hair, skin and eyes is mainly due to melanin. This pigment is the generation of different types of melanin (Hearing and produced by melanocytes, which are cells derived from the neural Yamaguchi, 2006; Slominski et al., 2004). Since tyrosinase is the crest (melanoblasts) during embryogenesis that migrate to the limiting step enzyme in melanogenesis, tyrosinase activity is often skin in the basal layer of the epidermis. They are dendritic cells evaluated in investigations into the effects of depigmenting that synthesize melanin within vesicles called melanosomes, agents. which are later transferred to neighbouring keratinocytes Melanin is a polymer that protects the skin against the (Hearing and Tsukamoto, 1991; Park et al., 2008). deleterious effects of ultraviolet radiation. The melanosomes are Melanosomes contain three enzymes that are absolutely neces- scattered inside the adjacent keratinocytes and remain in the sary for the synthesis of both types of melanin (pheomelanin and perinuclear area to form supranuclear melanin caps that protect eumelanin). The first enzyme is tyrosinase, which is responsible DNA from incident UV rays (Choi et al., 2010; Slominski et al., for the critical and limiting step in melanogenesis, the hydroxyla- 2004). However, in some situations an abnormal accumulation of tion of tyrosine to form 3,4-dihydroxyphenylalanine [L-DOPA] pigment can occur, resulting in hyperpigmentation disorders. For example, skin insults such as acne lesions and scratches, which induce acute inflammation, may promote postinflammatory n Correspondence to: Departamento de Farmacologia, Setor de Ciências Biológicas, hyperpigmentation. In addition, chronic exposure of skin to UV, Universidade Federal do Paraná, Av. Francisco Hoffman dos Santos, s/n, PO Box: 19031, 81530-900 Curitiba, PR, Brazil. Tel.: + 55 (41) 3361-1539; fax: + 55 (41) 3266-2042. specially the hands, arms and face, leads to sun-induced dark E-mail address: [email protected] (M.F. Otuki).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.03.079 200 P.M. Campos et al. / Journal of Ethnopharmacology 148 (2013) 199–204 lesions known as solar lentigos (Choi et al., 2010; Ortonne and from Biotec (Porto Alegre, RS, Brazil) and kojic acid from Galena Bissett, 2008). (Campinas, SP, Brazil). Dulbecco's modified Eagle's medium Skin hyperpigmentation is the most common complaint from (DMEM) and foetal bovine serum were obtained from Cultilab patients who consult a dermatologist, normally seeking to restore (Campinas, SP, Brazil). a homogeneous skin colour. The most frequent types of hyper- chromic patches include: melasma, ephelides, solar melanosis, senile lentigos and postinflammatory hyperpigmentation. Aging, 2.2. Plant material pregnancy, endocrine disorders, treatment with sex hormones and sun exposure are the main factors that trigger skin hyperpigmen- The leaves of Garcinia gardneriana were collected next to FURB, tation (Ortonne and Bissett, 2008; Pandya and Guevara, 2000). in Blumenau, Brazil. Prof. Marcos Sobral identified the plant and Treatment of the skin with depigmenting agents, from the the vouchers were deposited at Dr. Roberto Miguel Klein Herbar- pharmacological and cosmetological views, should provide safety ium (FURB, Blumenau) under accession numbers 534–540. and efficacy without lead to side effects. Hydroquinone, one of the most used depigmenting agents, is efficient in reducing the amount of melanin; however, it causes many harmful and cyto- 2.3. Preparation of hydroalcoholic extract of Garcinia gardneriana toxic effects. This undesirable effect is due to its strong oxidizing activity, which generates toxic by-products inside melanocytes Air-dried leaves (150 g) were cut into small pieces and macer- and causes permanent depigmentation (Draelos, 2007). The activ- ated with 50% ethanol at room temperature for approximately ity of kojic acid, also used in skin lightening, is based on blocking 2 weeks. After solvent removal, a hydroalcoholic extract was the activity of tyrosinase. However, there are reports showing skin obtained. A part of this extract was used in the cell culture studies sensitization, including dermatitis and cytotoxicity, which have and the other part was used for phytochemical analysis. resulted in some countries discontinuing its use (Serra-Baldrich et al., 1998). In view of the lack of safe depigmenting agents, studies have been conducted to find natural or synthetic com- 2.4. Cell culture pounds that block exacerbated melanogenesis without promoting side effects (Chang, 2009). Murine B16F10 melanoma cells (from American Type Culture In folk medicine, the leaves of Garcinia gardneriana are nor- Collection, Rockville, MD, USA) were cultured in DMEM supple- mally used to treat inflammation, particularly in skin disorders, as mented with 10% heat-inactivated foetal bovine serum (FBS) and well as to treat pain, urinary tract and other infections (Cechinel 1% penicillin/streptomycin (10,000 U/100 mg/mL) at 37 1Cina

Filho et al., 2000). In order to verify its possible anti-inflammatory humidified atmosphere containing 5% CO2. activity, a hydroalcoholic extract of Garcinia gardneriana was evaluated in a rat paw edema model, where the extract showed anti-inflammatory activity after application of carrageenan 2.5. Cell viability (Castardo et al., 2008). Phytochemical studies with Garcinia garderiana revealed the Cell viability was determined after treatment with the tested presence of several xanthones, the triterpenes lupeol and betulin, compounds using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- and the steroid β-sitosterol (Braz Filho et al., 1970). Flavonoids zolium bromide (MTT; Sigma Chemical Co., St. Louis, USA), which were also identified, such as the biflavonoids volkensiflavone, I3- measures mitochondrial activity in living cells as described pre- naringenin-II8-eriodictyol (GB-2a), GB-1a, fukugetin (Botta et al., viously (Tada et al., 1986). Briefly, B16F10 cells were cultured at 1984), fukugiside (Luzzi et al., 1997), and I3-naringenin-II8-4′- 7 Â 103 cells/well in a 96-well plate. After 24 h, cells were exposed OMe-eriodictyol (GB-2a-II-4′-OMe) (Verdia et al., 2004). It was to HEGG (1–100 mg/mL) or 500 mM kojic acid under the same also observed that flavonoids isolated from the leaves of Garcinia conditions used in the melanin content assays. At the end of the gardneriana, such as fukugetin, fukugiside and volkensiflavone, incubation, the medium with treatment was removed, the cells possess bactericidal activity against many Gram-positive bacteria were washed twice with phosphate buffer and then incubated (Guimarães et al., 2004). with MTT solution (0.5 mg/mL) for 3 h at 37 1C. The medium was Plant extracts with antimelanogenic activity typically possess discarded and 200 μL of ethanol was added. The absorbance of polyphenols such as flavonoids, which are usually the factors each well was measured at 570 nm using a plate reader (EL 808B, responsible for the activities in plant extracts (Chang, 2009). The BioTech Instruments, Inc., Winooski, VT, USA). aim of this study was to evaluate the effects of a hydroalcoholic extract from Garcinia gardneriana (HEGG) rich in flavonoids on murine B16F10 melanoma cells. 2.6. Determination of melanin biosynthesis in B16F10 cells

The amount of melanin was used as an index of melanogenesis. 2. Materials and methods Determination of melanin content was performed using the published protocol with minor modifications (Chen et al., 2009). 2.1. Chemicals and reagents In brief, B16F10 cells (4 Â 104/well) were seeded in 24-well plates and incubated for 24 h. At the end of each desired treatment, the α-MSH, L-tyrosine, L-DOPA, mushroom tyrosinase, 3-(4,5-dime- cells were washed twice with phosphate buffer and then dissolved tylyhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in 500 mL of 1 N NaOH containing 10% DMSO. Samples were dimethyl sulfoxide were purchased from Sigma-Aldrich Aldrich incubated at 80 1C for 1 h and mixed to solubilize the melanin. (St. Louis, MO, USA). Penicillin/streptomycin and trypsin were The optical density of the mixed homogenate was then measured purchased from Invitrogen (Carlsbad, CA, USA). Potassium dihy- at 490 nm. To measure the amount of melanin in each experiment, drogen phosphate and Triton-X were purchased from Merck the total amount of melanin (100%) produced during the experi- (Frankfurt, Germany). Ultrapure water was obtained using a mental period was considered as the control group and the rate of Milli-Q purification system from Millipore (Bedford, MA, USA). inhibition in the treatment groups was calculated in proportion to Sodium hydroxide and ethanol (analytical grade) were purchased this standard. P.M. Campos et al. / Journal of Ethnopharmacology 148 (2013) 199–204 201

2.6.1. Spontaneous melanogenesis assay (Fig.1A), after treatment with several concentrations (3– After seeding, the cells were washed with phosphate buffer and 100 mg/mL) of HEGG for 48 h, a decrease in melanin content was then treated with various concentrations of HEGG for 48 h before observed, with a maximal inhibition of 34.13710.99% (100 mg/ melanin content evaluation. mL). Whereas the tested concentrations did not decrease cell viability as evidenced by MTT assay (Fig. 1B). 2.6.2. UVB-induced melanogenesis Ultraviolet radiation-induced melanogenesis was performed to After seeding, the cells were washed with phosphate buffer and mimic the physiological stimulation from sun exposure. In this then exposed in this solution to UVB light (290–320 nm, TL01 assay, treatment with HEGG (10 mg/mL) for 24 h after UVB expo- lamp, Philips, Eindhoven, Netherlands) at a dose of 50 mJ/cm2. The sure inhibited melanogenesis by 35.276% while kojic acid inhib- phosphate buffer was removed just before the exposure. The cells ited melanogenesis by 8775.7% (Fig. 2A). Withal, the MTT assay were incubated with various concentrations of HEGG dissolved in was performed to verify the treatment's effect on cell viability, medium for 24 h before melanin content evaluation. which was not reduced during this period (Fig. 2B). A third evaluation of melanin content was performed by first α 2.6.3. α-MSH-induced melanogenesis inducing melanogenesis with -MSH followed by treatment with After 48 h from seeding, the cells were washed with phosphate HEGG at several concentrations (Fig. 3A). In this assay, total m buffer and then treated with 100 nM α-MSH plus HEGG at various inhibition was observed at a concentration of 3 g/mL HEGG, concentrations for 48 h before melanin content evaluation. overcoming the inhibition of kojic acid. The MTT assay was applied to verify cell viability, and revealed no damage from HEGG when 2.7. Intracellular tyrosinase activity assay compared to the control group (Fig. 3B).

Tyrosinase activity was determined according to the method described by Li et al. (2010) with slight modification. B16F10 3.2. Inhibitory effect of HEGG on tyrosinase activity murine melanoma cells were seeded at a density of 2.5 Â 103 cells/well in 96-well plates. Afterward, the cells were incubated Tyrosinase is the key enzyme in melanin biosynthesis; there- with HEGG and 500 mM kojic acid for 48 h, washed with ice-cold fore, the effects of HEGG on intracellular tyrosinase activity were phosphate buffer and lysed with phosphate buffer (pH 6.8) evaluated in B16F10 cells (Fig. 4). The intracellular tyrosinase containing 1% Triton-X/PBS (90 μL/well), then frozen at −80 1C activity was decreased by about 19% at the tested concentrations (10–0.1 mg/mL). for 30 min. After thawing and mixing, 10 μLof1%L-DOPA was fi added to each well. Following incubation at 37 1C for 2 h, the In order to con rm the inhibition of enzyme activity, the effect absorbance was measured at 490 nm. of HEGG was evaluated in an in vitro assay using mushroom

2.8. Mushroom tyrosinase activity assay

The influence of HEGG on tyrosinase activity was determined as previously described with minor modifications (Hyun et al., 2008). To the wells in a 96-well microplate were added: HEGG in various concentrations, 20 mL of mushroom tyrosinase (500 U/mL in phosphate buffer, pH 6.5), 170 mL of a mixture containing 1 mM L-tyrosine solution, 50 mM potassium phosphate buffer, pH 6.5, and distilled water (10:10:9, v/v/v), respectively. The microplate was then incubated at 37 1C for 40 min. The amount of dopaqui- none resulting from the reaction was determined based on the optical density at 490 nm using a microplate reader (EL808B, BioTech Instruments, Inc., Winooski, VT, USA). One unit (U) of enzymatic activity was defined as the amount of enzyme needed to increase the absorbance at 280 nm by 0.001 per min in a 3 mL reaction mixture containing L-tyrosine at pH 6.5 and 25 1C. Kojic acid (500 mM) was used as the positive control and ultrapure water as the negative control.

2.9. Statistical analysis

Each experiment was performed at least in triplicate. All data were presented as mean7S.E.M. Statistical analysis was per- formed by one-way analysis of variance (ANOVA) complemented by post hoc Newman–Keuls test. Differences were considered significant when the P-value was less than 0.05. All tests were carried out using GraphPad Software (San Diego, CA, USA).

3. Results

3.1. Effect of HEGG on melanin biosynthesis and cell viability Fig. 1. Effects of HEGG on melanin content (A) and cell viability (B) during spontaneous melanogenesis. B16F10 cells were incubated for 48 h in the presence of HEGG at different concentrations. Kojic acid was used as a reference drug. Data fi ** Intracellular melanin released in B16F10 lysates was quanti ed are the means7S.E.M from three separate experiments. Po0.01 : statistically in melanin content assays. In the spontaneous melanogenesis significant vs. control group (DMEM). 202 P.M. Campos et al. / Journal of Ethnopharmacology 148 (2013) 199–204

Fig. 2. Effects of HEGG on melanin content (A) and cell viability (B) after UVB exposure. B16F10 cells were incubated for 24 h in the presence of HEGG at different Fig. 3. Effects of HEGG on melanin content (A) and cell viability (B) after α-MSH concentrations after exposure to UVB. Kojic acid was used as a reference drug. Data exposure. B16F10 cells were incubated for 48 h in the presence of HEGG at different are the means7S.E.M from three separate experiments. **Po0.01 and ***Po0.001: concentrations together with α-MSH. Kojic acid was used as a reference drug. Data statistically significant vs. control group (DMEM). The naïve group represents the are the means7S.E.M from three separate experiments. **Po0.01 and ***Po0.001: basal melanin content in unexposed cells. statistically significant vs. control group (DMEM). The naïve group represents the basal melanin content in unexposed cells. tyrosinase with L-tyrosine as substrate, plus HEGG at several concentrations (Fig. 5). It was observed that HEGG promoted a maximal inhibition of 34.3472.51% (1000 mg/mL).

4. Discussion

In order to identify new depigmenting agents, the effect of a hydroalcoholic extract from the leaves of Garcinia gardneriana on melanogenesis was investigated in the B16F10 cell line. In the evaluations of melanin content, the influence of HEGG on sponta- neous melanogenesis was verified; it was able to inhibit this process, Fig. 4. Effects of HEGG on intracellular tyrosinase activity. Inhibition of intracellular decreasing the amount of melanin produced by melanoma cells. tyrosinase activity by HEGG at different concentrations. Kojic acid was used as a One of the most relevant physiological stimuli of melanogen- reference drug. Tyrosinase activity was examined in the presence of L-DOPA as a esis is the ultraviolet radiation from solar light. UV rays directly substrate and is represented as percent inhibition. Each value is given as the 7 fi affect melanocytes and keratinocytes. Once stimulated, keratino- mean S.E.M. from independent triplicate experiments. Signi cantly different from the control group (**Po0.01) and ***Po0.001). cytes release keratinocyte-derived factors such as α-MSH, a hor- mone that stimulates melanocytes to produce melanin (Hunt et al., 1994). Since stimulation of melanogenesis by ultraviolet radiation regulating pigmentation via GPCR (G protein-coupled receptor)– is one of the main causes of hyperpigmentation and further cAMP–MITF (microphthalmia-associated transcription factor), promotes inflammation, B16F10 cells received UVB radiation to increasing the expression of MITF, which in turn induces target simulate this stimulus (Abdel-Malek et al., 2010). Treatment with genes for key melanogenic enzymes such as TRP-1 and DCT (Park HEGG promoted a reduction in melanin synthesis after UVB et al., 2009). In the midst of this, HEGG was able to decrease stimulus, suggesting a possible protective effect against hyperpig- melanin synthesis induced by α-MSH. This result supports the mentation induced by sun exposure. potential of Garcinia gardneriana to interfere with the melanin The hormone α-MSH is an important component of the production cascade in melanocytes. melanogenesis stimulatory cascade, and both human skin mela- HEGG impaired melanin synthesis during normal melanin nocytes and murine melanoma cells respond to α-MSH stimulation production and under two different stimuli in B16F10 cells, (Schwahn et al., 2001). This hormone binds to the melanocortin proving the melanogenesis inhibitory effect of this extract. In 1 receptor (MC1R) on melanocytes, which plays a crucial role in order to evaluate the possible mechanisms by which HEGG acts, P.M. Campos et al. / Journal of Ethnopharmacology 148 (2013) 199–204 203

2006). Considering this, HEGG seems to be a potential source of an interesting depigmenting agent, since it was effective in inhibiting melanogenesis and also includes biflavonoids in its phytochemical composition, combining two beneficial mechanisms for the treat- ment of hyperpigmenting skin. According to Solano et al. (2006), dermatologists face great difficulty in treating disorders of hyperpigmentation and skin disorders related to suppression of melanogenesis. Efficient agents should modulate melanogenic biochemical pathways to achieve a good outcome without promoting side effects. Thus, the ideal clinical strategy should be a combination of depigmenting agents fi Fig. 5. Effects of HEGG on mushroom tyrosinase activity. Inhibition of mushroom in order to increase ef ciency while reducing treatment time and tyrosinase activity by HEGG at different concentrations. Kojic acid was used as a the risk of adverse effects. Our results suggest that HEGG could be reference drug. Tyrosinase activity was examined in the presence of L-tyrosine as a a relevant candidate for the development of a safe depigmenting substrate and is represented as percent inhibition. Each value is given as the agent for skin lightening. However, additional studies are neces- mean7S.E.M. from independent triplicate experiments. Significantly different sary to elucidate the mechanism of action and safety of Garcinia from the control group (**Po0.01) and ***Po0.001). gardneriana. its influence on tyrosinase activity was investigated. First of all, the involvement of intracellular tyrosinase was evaluated through Acknowledgements quantification of dopaquinone formation in melanoma cells. Treat- ment with HEGG promoted an inhibition in the enzyme's activity, This study was supported by a grant from Conselho Nacional de as evidenced by a decrease in the quantity of dopaquinone. Desenvolvimento Científico e Tecnológico (CNPq). P.M. Campos, C. Tyrosinase is a multifunctional enzyme responsible for the first D.S. Horinouchi and A.S. Prudente are PhD students and thank two steps of melanogenesis. Being a rate-limiting enzyme, block- CAPES and REUNI for fellowship support. ing its activity impairs melanin synthesis. Thus, agents capable of ' inhibiting tyrosinase activity or reducing the enzyme s expression References are considered potential targets in research for new depigmenting agents (Chang, 2009). Abdel-Malek, Z., Kadekaro, A.L., Swope, V.B., 2010. Stepping up melanocytes to the The intracellular tyrosinase activity assay clearly demonstrated challenge of UV exposure. Pigment Cell Melanoma Research 23, 171–186. the influence of HEGG on enzyme activity. However, with this test Botta, B., Mac-Quhae, M.M., Delle Monache, G., Delle Monache, F., Demello, J.F., it was not possible to determine if the effect is really due to direct 1984. Chemical investigation of the Rheedia.V:Biflavonoids and xanthochymol. Journal of Natural Products. 47, 1053. inhibition of the enzyme, since the extract could be interfering Braz Filho, R., Magalhães, Cavalcante De, G, Gottlieb, O.R., 1970. Xanthones of with the enzyme synthesis pathway. In order to confirm the direct Rheedia gardneriana. Phytochemistry 9, 673. interaction of the extract with the enzyme, the effect of HEGG on Castardo, J.A., Prudente, A.S., Ferreira, J., Guimarães, C.L., Delle Monache, F., Cechinel Filho, V., Otuki, M.F., Cabrini, D.A., 2008. Anti-inflammatory effects of hydro- mushroom tyrosinase activity was evaluated. Mushroom tyrosi- alcoholic extract and two biflavonoids from Garcinia gardneriana leaves in nase is a commercially available enzyme that is often used as a mouse paw oedema. Journal of Ethnopharmacology 118, 405–411. substitute for human tyrosinase in order to perform screenings for Cechinel Filho, V., Da Silva, K.L., De Souza, M.M., Oliveira, A.E., Yunes, R.A., Guimarães, C.L., Verdi, L.G., Simionatto, E.L., Delle Monache, F., 2000. I3- tyrosinase inhibitors (Parvez et al., 2007). Once again, HEGG naringenina-II8-4_-OMe-eriodictyol: a new potential analgesic agent isolated decreased dopaquinone formation via the mushroom tyrosinase, from Rheedia gardneriana leaves. Zeitschrift für Naturforschung 55, 820–823. supporting the evidence that HEGG directly blocks the enzyme. Chang, T.S., 2009. An updated review of tyrosinase inhibitors. International Journal – The results obtained in the two tyrosinase activity assays suggest of Molecular Science 10, 2440 2475. Chen, L.G., Chang, W.L., Chia, J.L., Lee, T.L., Chwen, M.S., Wang, C.C., 2009. that a possible mechanism by which HEGG is reducing the amount Melanogenesis inhibition by gallotannins from chinese galls in B16 mouse of melanin is via inhibition of tyrosinase activity. This finding melanoma cells. Biological Pharmaceutical Bulletin 32, 1447–1452. corroborates other studies conducted in of the genus Choi, W., Miyamura, Y., Wolber, R., Smuda, C., Reinhold, H.L., Kolbe, L., Hearing, V.J., 2010. Regulation of human skin pigmentation in situ by repetitive UV exposure: Garcinia, such as the study by Masuda et al. (2005), where the molecular characterization of responses to UVA and/or UVB. Journal of authors screened 39 from the coast of Japan and found Investigative Dermatology 130, 1685–1696. Garcinia subelliptica was among those that exhibited tyrosinase Draelos, Z.D., 2007. Skin lightening preparations and the hydroquinone contro- versy. 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