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Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells
Journal of Ethnopharmacology 137 (2011) 1183–1188
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Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells Y.Y. Chan, K.H. Kim, S.H. Cheah ∗ Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
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Article history: Received 23 December 2010 Received in revised form 27 May 2011 Accepted 18 July 2011 Available online 26 July 2011 Keywords: Sargassum polycystum Melanin Melanogenesis Tyrosinase B16F10 melanoma cells
a b s t r a c t Ethnopharmacological relevance: Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine. Aim of the study: The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. Materials and methods: Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in ␣-MSH-stimulated and unstimulated B16F10 cells. Results: Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and ␣-MSHstimulated melanin production in B16F10 cells. Conclusions: Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Melanin is the main component determining the color of skin and up to 10% of cells in the innermost layer of the epidermis produce melanin pigments (Vamos-Vigyazo, 1981; Hearing, 2005). The major role of melanin is to protect the skin from damaging effects of ultraviolet radiation (Tsatmali et al., 2002). Melanin biosynthesis, or melanogenesis, is a well-known physiological response of human skin upon exposure to ultraviolet light and other stimuli. Melanogenesis is regulated by enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinaserelated protein-2 (TRP-2) (Kameyama et al., 1995). The inhibition of tyrosinase is the most common approach to achieve skin whiteness as it is the key enzyme that catalyzes the rate-limiting step of melanin biosynthesis (Solano et al., 2006). Tyrosinase catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenylalanine (l-
∗ Corresponding author. Tel.: +60 3 79674925; fax: +603 79674775. E-mail address: [email protected] (S.H. Cheah). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.050
DOPA), followed by the oxidation of l-DOPA to dopaquinone. Oxidative polymerization of several dopaquinone derivatives gives rise to melanin (Parvez et al., 2006). Thus substances that inhibit tyrosinase may be useful ingredients to be incorporated into cosmetic preparations. Marine algae (seaweeds) have long been used as food and medicine in Asian countries such as Japan, China and Korea. For centuries, Greeks and Romans have used them as medicinal remedies and in cosmetics. In folk medicine, seaweeds have been utilised for a wide range of remedial purposes such as treatment of gallstone, vermifuges, stomach ailments, eczema, cancer and renal disorders (Hoppe and Lerving, 1982; Srivastava and Kulshreshtha, 1989). Presently, a variety of seaweeds have been used extensively in cosmetic applications. Research on seaweeds with the purpose of developing novel skin-whitening agents from marine sources is of great interest in recent years. Sargassum polycystum, belonging to family Sargassaceae, is a kind of edible brown alga (Phaeophyta) that has been used as food and medicine. It is commonly found in tropical and sub-tropical countries such as Indonesia, Malaysia, Thailand and Vietnam
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(Linsey and Ohno, 1999). It has been used traditionally in folk medicine for treatment of skin-related disorders (i.e. eczema, scabies and psoriasis), renal dysfunction, heart ailments, lung diseases, ulcer, and also to promote secretion of bile (Raghavendran et al., 2006). Previous studies have shown that organic solvent and water extracts of Sargassum polycystum possess hepatoprotective, antilipidemic, antioxidant and membrane stabilizing properties (Raghavendran et al., 2004, 2005). It has been reported that antioxidants may reduce hyperpigmentation and support skin health (Ma et al., 2001). Thus, in view of the traditional use of Sargassum polycystum in skin-related disorders and its antioxidant property which may reduce hyperpigmentation, a study was undertaken to investigate if Sargassum polycystum possesses any antimelanogenesis effects with a view of its possible use as a treatment for hyperpigmentation and its use as a skin-whitening agent in cosmetics. In this report we describe the differential extraction of dried, powdered Sargassum polycystum with solvents of different polarity. The ability of the different extracts to act as a skin-whitening agent was tested by its ability to inhibit tyrosinase, the rate limiting enzyme in melanogenesis, initially using a cell-free mushroom tyrosinase system, which has commonly been employed for the testing and screening of potential skin-whitening agents (Song et al., 2009). The ability to inhibit cellular tyrosinase derived from melanin producing B16F10 melanoma cells as well as its ability to inhibit melanogenesis was also tested. Kojic acid, a fungal metabolite produced by some species of Aspergillus and Penicillium, which is well known to be an inhibitor of tyrosinase and melanogenesis (Garcia and Fulton, 1996), was used as a positive control. 2. Materials and methods 2.1. Seaweed material Seaweed Sargassum polycystum was collected from the low tide area of Pulau Seri Buat (Pulau Sri Buat) and Pulau Sembilan, Endau, Johor, Malaysia. The species identification was done by Prof. Phang Siew Moi of the Algae Research Laboratory, University of Malaya (UMalgae). The collected seaweed was thoroughly washed in running tap water to remove the salt, epiphytes and sand, air-dried in the shade and coarsely powdered. 2.2. Preparation of seaweed extract The powdered seaweed (2.0 kg) was first soaked in 6 L of 95% ethanol for 3 days at room temperature. The solvent was then decanted and filtered. The procedure was repeated 2 times. The filtrate from each extraction was combined and the solvent was evaporated at 40 ◦ C under reduced pressure using a rotary evaporator to give an ethanolic crude extract (134.77 g). This ethanolic crude extract was further extracted with 400 ml of hexane to give a hexane-soluble fraction (30.42 g) and a hexane insoluble residue. The hexane-insoluble residue was partitioned between ethyl acetate–water (100 ml:100 ml) to give an ethyl acetatesoluble fraction and a water soluble fraction. The ethyl acetate fraction was dried in a rotary evaporator at 40 ◦ C to yield a dry extract of 1.35 g. The water layer was lyophilized to give a water fraction (60.66 g). The ethanolic crude extract and its fractionated extracts were dissolved in dimethyl sulphoxide (DMSO, Sigma, St. Louis, MO, USA), while the water fraction was dissolved in water, to create stock solutions of 250 mg/ml. The stock solutions were diluted appropriately with buffer or media at the time of testing. The final concentration of DMSO in test wells was 1% for cell-free assay and 0.1% for cell-based assay.
2.3. Mushroom tyrosinase assay The effect of Sargassum polycystum extracts on cell free mushroom tyrosinase activity was determined spectrophotometrically as described previously (Rahman et al., 2001) with minor modifications. The tyrosinase activity was determined using l-DOPA (Sigma, St. Louis, MO, USA) as a substrate. In brief, 50 l of 700 units/ml of mushroom tyrosinase (EC 1.14.18.1) (Sigma, St. Louis, MO, USA) in 0.1 M phosphate buffer, pH 6.8, 50 l of the different concentrations (100, 250 and 500 g/ml) of the different extracts and 100 l of 0.1 M phosphate buffer (pH 6.8) were added to each well of a 96-well plate (TPP, Switzerland) and mixed. The assay mixture was pre-incubated at room temperature for 10 min; 100 l of 2.5 mM lDOPA in 0.1 M phosphate buffer (pH 6.8) was then added into each well and incubation was continued for 20 min at room temperature. The quantity amount of dopachrome formed in the reaction mixture was determined against blank (solution without enzyme) at 475 nm in a microplate reader (Tecan Sunrise, Switzerland). Kojic acid (Sigma, St Louis, MO, USA) at a concentration of 100 g/ml was used as a standard tyrosinase inhibitor in order to confirm that the assay was working (Arung et al., 2009). The percentage of tyrosinase activity was calculated as follows: tyrosinase activity (%) = [(A − B)/(C − D)] × 100, where A is the absorbance of reaction mixture containing test sample and mushroom tyrosinase, B is the absorbance of blank sample containing test sample but without mushroom tyrosinase, C is the absorbance of reaction mixture without test sample and with mushroom tyrosinase and D is the absorbance of the well without both test sample and mushroom tyrosinase (l-DOPA alone).
2.4. Cell culture and treatment The B16F10 murine melanoma cells (CRL-6475) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich, USA) containing 2 mM lglutamine (Applichem, Denmark), supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100 units/ml of penicillin (PAA, Austria), and 100 g/ml of streptomycin (PAA, Austria) in culture flasks in a CO2 incubator with a humidified atmosphere containing 5% CO2 in air at 37 ◦ C. The culture medium was changed every 2 days. The cells were harvested by trypsinization when they were about 70% confluent, counted with a haemocytometer (Weber, England) and seeded at the appropriate numbers into wells of cell culture plates for further experiments.
2.5. Cell viability assay (MTT assay) To determine the safety of the various extracts the viability of cells following treatment with extracts was determined by the MTT assay. This method is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial enzymes in viable cells (Mosmann, 1983). The quantity of formazan formed is proportional to the number of viable cells present and can be measured spectrophotometrically. Briefly, cells seeded at a density of 3 × 103 cells/well in a 96-well plate were left to adhere overnight. A test sample was then added to each well and incubated for another 72 h. Next, the treated cells were labelled with MTT dye reagent (Applichem, Denmark) in PBS (2 mg/ml) for 3 h. The formazan precipitates were dissolved by DMSO and the concentrations were measured at 554 nm in a microplate reader with a reference wavelength of 690 nm. Cell viability was calculated using the following formula: cell viability (%) = (Asample /Acontrol ) × 100, where Asample and Acontrol are the
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absorbances from the mixture with, or without the addition of test sample, respectively.
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3. Results
2.6. Cellular tyrosinase assay
3.1. Effects of Sargassum polycystum on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability
Cellular tyrosinase activity was measured using a previously described method (Lee et al., 2006) with small modifications. Wells of 6 well plates (TPP, Switzerland) were seeded with B16F10 melanoma cells at a density of 2 × 105 cells/well contained in 3 ml of media. Plates were incubated overnight in a humidified CO2 incubator at 37 ◦ C and 5% CO2 to allow cells to adhere. Cells were then exposed to increasing doses of test extracts or kojic acid for 72 h in the presence or absence of 100 nM alpha-melanocyte stimulating hormone (␣-MSH, Sigma, USA). The cells were then washed with sodium phosphate buffered saline (pH 6.8) and lyzed with M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA). The lysates were then clarified by centrifugation at 13,000 rpm for 15 min at 4 ◦ C. The protein concentration was determined by the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA) using bovine serum albumin (BSA, Sigma, USA) as the standard. The reaction mixture consisting of 40 g protein (adjusted to 100 l with 0.1 M PBS, pH 6.8) and 100 l of 5 mM l-DOPA was added to each well of a 96-well plate. After incubation at 37 ◦ C for 1 h, the absorbance was measured at 475 nm using a microplate reader. Tyrosinase activity in the protein was calculated by the following formula:
The effects of Sargassum polycystum extracts on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability are shown in Table 1. The results show that ethanolic crude extract and its hexane and ethyl acetate fractions did not have any effect on mushroom tyrosinase activity while the water fraction had only a small inhibitory effect at the highest dose tested. On the other hand, ethanolic extract, hexane and ethyl acetate fractions reduced cellular tyrosinase activity in a dose-dependent manner. Water fraction did not have any effect on cellular tyrosinase except a slight inhibition at the highest dose tested. In the cell viability (MTT) assay, hexane fraction did not have any appreciable cytotoxic activity at a dose of 100 g/ml, but reduced viable cells slightly at the higher doses. Ethanolic extract also had some minor cytotoxic effect, while ethyl acetate fraction had the greatest cytotoxic effect. Water fraction did not show any cytotoxic effect at all. The ethyl acetate fraction was not investigated further due to its higher cytotoxicity. Kojic acid, used as a positive control, reduced mushroom tyrosinase drastically even at the lowest dose used. However, when tested on cellular tyrosinase the inhibitory effect was less severe, and showed a dose–response effect similar to the seaweed extracts and fractions.
tyrosinase activity (%) =
OD475 OD475
of sample × 100 of control
3.2. Effect of Sargassum polycystum hexane fraction (SPHF) on melanin synthesis in B16F10 melanoma cells
2.7. Melanin content assay Melanin content was measured as described previously (Hosoi et al., 1985) with slight modifications. The B16F10 melanoma cells were seeded with 2 × 105 cells/well in 3 ml of medium in 6-well culture plates and incubated overnight to allow cells to adhere. The cells were exposed to various concentrations (100, 250 and 500 g/ml) of the seaweed extracts or kojic acid for 72 h in the presence or absence of 100 nM ␣-MSH. At the end of the treatment, the cells were washed with PBS and lyzed with 800 l of 1 N NaOH (Merck, Germany) containing 10% DMSO for 1 h at 80 ◦ C. The absorbance at 400 nm was measured using a microplate reader. The melanin content was determined from a standard curve prepared from an authentic standard of synthetic melanin (Sigma, USA).
To gain further evidence of SPHF’s involvement in melanogenesis, the effect of SPHF on melanin production in B16F10 cells was tested. Fig. 1 demonstrates that SPHF reduced cellular melanin content in B16F10 cells in the absence of ␣-MSH stimulation. The inhibition was dose-dependent: SPHF at 100, 250 and 500 g/ml induced significant inhibition on melanin production by 15.13, 28.71 and 39.93%, respectively. Kojic acid at the same doses also reduced cellular melanin production in a dose-dependent manner.
2.8. Phytochemical screening The major secondary metabolites were identified by employing the methodology outlined by Harborne (1998). Briefly, qualitative phytochemical screening of the active fraction of Sargassum polycystum ethanolic crude extract was carried out for alkaloids (Meyer and Dragendoff’s tests), tannins (FeCl3 test), saponins (frothing test), lipids (Wattman paper test), flavonoids (Schinoda’s test and anisaldehyde staining), sugars (resorcinol–H2 SO4 and aniline hydrogen phthalate test), phenols (Folin-Ciocalteu test), terpenoids (vanillin–H2 SO4 test, Liberman Burchard’s test), amino acid and amines (Ninhydrin test). 2.9. Statistical analysis All data are presented as the mean ± SEM of 3–4 independent experiments. Comparisons among groups were made using ANOVA followed by Dunnett’s post hoc test. Student’s t-test was used when single comparisons were made between means of two groups. A value of P < 0.05 was regarded as statistically significant in all experiments.
Fig. 1. Effects of SPHF and kojic acid on cellular melanin content in B16F10 melanoma cells. The control readings (unfilled bar) were set as 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control.
82.35 ± 4.40** 58.63 ± 1.26*** 86.17 ± 2.54* 68.77 ± 2.04*** b b
Fig. 2. Effect of SPHF on cellular tyrosinase activity in ␣-MSH-stimulated B16F10 cells compared with kojic acid. The cells were incubated with 100 nM ␣-MSH alone or together with increasing doses of SPHF or kojic acid for 72 h following which cellular tyrosinase activity was measured. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SEM of four independent experiments. # P < 0.001 versus control group (without ␣-MSH). **P < 0.01, ***P < 0.001 versus ␣-MSH-treated group.
Data are presented as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control. a Relative activity (%) = (% of sample versus control). b 100% mushroom tyrosinase inhibitory activity was observed in the optimisation of the assay. c Not tested because the ethyl acetate fraction expressed high cytotoxicity.
c c
100 73.98 ± 2.56** 75.99 ± 4.95*** 42.99 ± 1.73*** 107.23 ± 6.63 93.55 ± 2.25
100 70.69 ± 6.75** 65.65 ± 7.35*** 40.52 ± 3.11*** 113.45 ± 4.80 86.93 ± 2.23
250 g/ml
100 86.99 ± 3.48 96.38 ± 4.77 77.32 ± 0.98* 110.22 ± 3.49 92.70 ± 1.63 100 65.04 ± 3.78*** 50.43 ± 2.63*** 100 82.03 ± 2.35** 67.94 ± 2.99***
100 g/ml 500 g/ml 250 g/ml
100 86.86 ± 2.35** 79.25 ± 2.99*** 89.08 ± 3.07* 92.05 ± 2.47 84.56 ± 1.62***
100 g/ml
100 102.53 ± 1.32 102.43 ± 1.51 95.91 ± 1.73 87.07 ± 4.88*
500 g/ml 250 g/ml
100 96.61 ± 0.80 97.91 ± 0.55 98.48 ± 3.21 99.66 ± 1.75
100 97.78 ± 1.34 97.10 ± 0.55 100.58 ± 1.81 99.49 ± 0.73 11.73 ± 1.51***
100 g/ml
Sargassum polycystum
Control Ethanolic crude extract Hexane fraction Ethyl acetate fraction Water fraction Kojic acid (positive control)
Cellular tyrosinase activity (%)a Mushroom tyrosinase activity (%)a
Cell viability (%)a
500 g/ml
Y.Y. Chan et al. / Journal of Ethnopharmacology 137 (2011) 1183–1188 Table 1 Effects of kojic acid, Sargassum polycystum ethanolic crude extract and its solvent soluble fractions on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability in B16F10 melanoma cells. Control readings (from wells containing no test material or kojic acid) were set as 100% and reading of experimental wells were expressed as a percentage of controls.
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3.3. Effects of SPHF on cellular tyrosinase activity and melanin synthesis in B16F10 cells with ˛-MSH stimulation The effects of SPHF on cellular tyrosinase activity and melanogenesis in ␣-MSH-stimulated B16F10 melanoma cells were next investigated. Upon exposure to 100 nM ␣-MSH alone, the tyrosinase activity of B16F10 cells was significantly increased compared to the controls (Fig. 2). SPHF significantly reduced the tyrosinase activity of ␣-MSH-stimulated B16F10 cells in a dose-dependent manner, with 31.85% inhibition at 100 g/ml, 39.57% at 250 g/ml and 54.41% at 500 g/ml. Kojic acid at the same doses similarly reduced tyrosinase activity in a dose-dependent manner.
Fig. 3. Effect of SPHF on cellular melanin content in ␣-MSH-stimulated B16F10 cells compared with kojic acid. The cells were incubated with 100 nM alone or together with increasing doses of SPHF or kojic acid for 72 h following which total cellular melanin activity was measured. Baseline melanin content in control wells not exposed to ␣-MSH and any test material or kojic acid was set at 100%. Data from experimental wells were expressed as a percentage of control. Each column represents the mean ± SEM of four independent experiments. # P < 0.001 versus control group (without ␣-MSH). **P < 0.01, ***P < 0.001 versus ␣-MSH-treated group.
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Fig. 3 shows the effect of similar levels of SPHF and kojic acid on melanin production in ␣-MSH-stimulated B16F10 cells. In parallel to the cellular tyrosinase activity, ␣-MSH increased the melanin concentration of B16F10 melanoma cells. The SPHF and the positive control, kojic acid, also significantly inhibited melanin production in ␣-MSH-stimulated B16F10 melanoma cells in a dose-dependent manner. After co-incubation for 72 h with 100 nM ␣-MSH and SPHF at concentrations of 100, 250 and 500 g/ml, the melanin production of ␣-MSH-treated B16F10 cells was suppressed to 62.19, 42.77 and 31.80%, respectively, compared to ␣-MSH-treated group without SPHF. Kojic acid similarly reduced melanin production to 96.81, 87.43 and 68.72% at concentrations of 100, 250 and 500 g/ml respectively. 3.4. Phytochemical screening of SPHF The phytochemical screening of the active fraction, SPHF, revealed the presence of the following classes of chemical compounds: saponins, flavonoids, tannins, terpenoids, phenols, sugars, amino acid and amines; alkaloids and lipids were absent. 4. Discussion In this study, several fractions of powdered dried Sargassum polycystum have been extracted with solvents of different polarity and tested for their possible antimelanogenesis and skin-whitening properties using inhibition of mushroom tyrosinase activity in a cell-free system and inhibition of cellular tyrosinase activity in B16F10 murine melanoma cells as screening assays. Our major findings are: (i) the cellular tyrosinase assay is a more reliable assay than the cell-free mushroom tyrosinase assay for the screening of potential skin-whitening agents and (ii) Sargassum polycystum may contain potential skin-whitening agents that inhibit melanogenesis in B16F10 melanoma cells by inhibition of cellular tyrosinase. Ethanol extraction was used in the initial experiments as ethanol can extract a wide range of agents of different polarity (Harborne, 1998) and is thus useful as a first screening step. The fact that the ethanolic extract and its hexane fraction failed to inhibit mushroom tyrosinase while being able to inhibit cellular tyrosinase activity, points to the weakness of the former protocol to screen for the presence of skin-whitening or antimelanogenesis agents in natural sources and man-made compounds. The finding that hexane fraction inhibits cellular tyrosinase activity as well as melanin production in B16F10 melanoma cells strengthens this conclusion. It must be remembered that mushroom tyrosinase is found in the cytosol while tyrosinase in melanocytes is membrane bound, and thus it can be expected that the effect of antimelanogenesis agents on these tyrosinases may not be the same. Since target cells of the agents to be screened are melanocytes, it would be more reliable to use tyrosinase derived from the melanin-producing cells instead of mushrooms. The murine B16F10 cell line was used because they produce melanin, are known to contain tyrosinase which is associated with melanogenesis, respond to ␣-MSH activation and are easy to culture in vitro (Busca and Ballotti, 2000; An et al., 2008). Kojic acid was used as a positive control in all these screenings as it has a known inhibitory effect on tyrosinase as well as melanin production (Garcia and Fulton, 1996). The water fraction did show some mild inhibitory activity both towards mushroom and cellular tyrosinase activity, and this could be due to a polar agent present in water fraction that is different from the non-polar agent seen in hexane fraction. However the inhibitory effect is small and it is not economically feasible to be developed further.
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The ethanolic extract appeared to have some cytotoxic as well as tyrosinase inhibitory effect on cellular tyrosinase activity (Table 1). However, a more highly cytotoxic fraction (ethyl acetate) and a less cytotoxic hexane fraction could be further separated. Thus, a large part of the cytotoxic effect of the ethanolic extract was due to the agents that separated out into the ethyl acetate fraction. Dooley (1997) previously speculated that a desirable skin-whitening agent should inhibit melanin synthesis in melanosomes by acting specifically to reduce the synthesis or activity of tyrosinase and with little or no cytotoxicity. Hence, the ethyl acetate fraction at higher concentration level (250 and 500 g/ml) was not used further due to its greater cytotoxicity on the melanoma cells. The hexane fraction was however less cytotoxic, having no significant direct killing effect on the B16F10 cells at the lowest dose used (100 g/ml). At this dose, however, cellular tyrosinase was already significantly inhibited. At the higher doses (250 and 500 g/ml), cellular tyrosinase inhibitory activity was increased, but there was also greater cytotoxic activity. The decrease in tyrosinase activity at the higher doses of hexane fraction cannot be attributed to the smaller number of viable cells present because assays were normalised to use the same quantity of protein (40 g) from each well. Thus the inhibition of tyrosinase activity was bona fide. The fact that the hexane fraction was also able to inhibit the increase in cellular tyrosinase in ␣-MSH-stimulated B16F10 melanoma cells provides further evidence of the direct action of hexane fraction on inhibition of cellular tyrosinase and melanogenesis. However, due to the slightly cytotoxic effects of the hexane fraction at the higher concentrations, the dose that will be used for any formulation or treatment regime will have to be carefully calibrated. In conclusion, based on the results obtained from cellular tyrosinase and melanogenesis inhibitory assays using B16F10 cells, we report here for the first time that hexane fraction of the ethanolic extract of Sargassum polycystum (SPHF) contains agent(s) that can be further developed and formulated into a skin-whitening or antimelanogenesis preparation for cosmetics and therapeutic use. The identification of active constituents that are responsible for the antimelanogenesis effect of SPHF and the phytochemical profiling are currently underway. Further studies are warranted to investigate the active components and the underlying molecular mechanisms of action involved in the melanogenesis inhibition. Acknowledgements This study was supported by the postgraduate research grant from the University of Malaya (PS280/2008A) and Fundamental Research Grant Scheme (FP021/2008C) from the Ministry of Higher Education of Malaysia. References An, S.M., Kim, H.J., Kim, J.E., Boo, Y.C., 2008. Flavonoids, taxifolin and luteolin attenuate cellular melanogenesis despite increasing tyrosinase protein levels. Phytotherapy Research 22, 1200–1207. Arung, E.T., Kusuma, I.W., Christy, E.O., Shimizu, K., Kondo, R., 2009. Evaluation of medicinal plants from Central Kalimantan for antimelanogenesis. Journal of Natural Medicines 63, 473–480. Busca, R., Ballotti, R., 2000. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Research 13, 60–69. Dooley, T.P., 1997. Topical skin depigmenting agents: current products and discovery of novel inhibitors of melanogenesis. Journal of Dermatological Treatment 7, 188–200. Garcia, A., Fulton Jr., J.E., 1996. The combination of glycolic acid and hydroquinone or kojic acid for the treatment of melasma and related conditions. Dermatological Surgery 22, 443–447. Harborne, J.B., 1998. Methods of Plant Analysis, third ed. Chapman and Hall Publishers, London. Hearing, V.J., 2005. Biogenesis of pigment granules: a sensitive way to regulate melanocyte function. Journal of Dermatological Science 37, 3–14.
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Raghavendran, H.R., Sathivel, A., Devaki, T., 2005. Effect of Sargassum polycystum (Phaeophyceae)-sulphated polysaccharide extract against acetaminopheninduced hyperlipidemia during toxic hepatitis in experimental rats. Molecular and Cellular Biochemistry 276, 89–96. Raghavendran, H.R., Sathivel, A., Devaki, T., 2006. Defensive nature of Sargassum polycystum (Brown alga) against acetaminophen-induced toxic hepatitis in rats: role of drug metabolizing microsomal enzyme system, tumor necrosis factoralpha and fate of liver cell structural integrity. World Journal of Gastroenterology 12, 3829–3834. Rahman, A.U., Choudhary, M.I., Thomsen, W.J., 2001. Bioassay Techniques for Drug Development. Harwood Academic, Australia. Solano, F., Briganti, S., Picardo, M., Ghanem, G., 2006. Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigment Cell Research 19, 550–571. Song, T.Y., Chen, C.H., Yang, N.C., Fu, C.S., Chang, Y.T., Chen, C.L., 2009. The correlation of in vitro mushroom tyrosinase activity with cellular tyrosinase activity and melanin formation in melanoma cells A2058. Journal of Food and Drug Analysis 17, 156–162. Srivastava, R., Kulshreshtha, D.K., 1989. Bioactive polysaccharide from plants. Phytochemistry 28, 2877–2883. Tsatmali, M., Ancans, J., Thody, A.J., 2002. Melanocyte function and its control by melanocortin peptides. The Journal of Histochemistry and Cytochemistry 50, 125–133. Vamos-Vigyazo, L., 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Critical Reviews in Food Science and Nutrition 15, 49–127.
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Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells Y.Y. Chan, K.H. Kim, S.H. Cheah ∗ Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
a r t i c l e
i n f o
Article history: Received 23 December 2010 Received in revised form 27 May 2011 Accepted 18 July 2011 Available online 26 July 2011 Keywords: Sargassum polycystum Melanin Melanogenesis Tyrosinase B16F10 melanoma cells
a b s t r a c t Ethnopharmacological relevance: Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine. Aim of the study: The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. Materials and methods: Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in ␣-MSH-stimulated and unstimulated B16F10 cells. Results: Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and ␣-MSHstimulated melanin production in B16F10 cells. Conclusions: Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Melanin is the main component determining the color of skin and up to 10% of cells in the innermost layer of the epidermis produce melanin pigments (Vamos-Vigyazo, 1981; Hearing, 2005). The major role of melanin is to protect the skin from damaging effects of ultraviolet radiation (Tsatmali et al., 2002). Melanin biosynthesis, or melanogenesis, is a well-known physiological response of human skin upon exposure to ultraviolet light and other stimuli. Melanogenesis is regulated by enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinaserelated protein-2 (TRP-2) (Kameyama et al., 1995). The inhibition of tyrosinase is the most common approach to achieve skin whiteness as it is the key enzyme that catalyzes the rate-limiting step of melanin biosynthesis (Solano et al., 2006). Tyrosinase catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxyphenylalanine (l-
∗ Corresponding author. Tel.: +60 3 79674925; fax: +603 79674775. E-mail address: [email protected] (S.H. Cheah). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.07.050
DOPA), followed by the oxidation of l-DOPA to dopaquinone. Oxidative polymerization of several dopaquinone derivatives gives rise to melanin (Parvez et al., 2006). Thus substances that inhibit tyrosinase may be useful ingredients to be incorporated into cosmetic preparations. Marine algae (seaweeds) have long been used as food and medicine in Asian countries such as Japan, China and Korea. For centuries, Greeks and Romans have used them as medicinal remedies and in cosmetics. In folk medicine, seaweeds have been utilised for a wide range of remedial purposes such as treatment of gallstone, vermifuges, stomach ailments, eczema, cancer and renal disorders (Hoppe and Lerving, 1982; Srivastava and Kulshreshtha, 1989). Presently, a variety of seaweeds have been used extensively in cosmetic applications. Research on seaweeds with the purpose of developing novel skin-whitening agents from marine sources is of great interest in recent years. Sargassum polycystum, belonging to family Sargassaceae, is a kind of edible brown alga (Phaeophyta) that has been used as food and medicine. It is commonly found in tropical and sub-tropical countries such as Indonesia, Malaysia, Thailand and Vietnam
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(Linsey and Ohno, 1999). It has been used traditionally in folk medicine for treatment of skin-related disorders (i.e. eczema, scabies and psoriasis), renal dysfunction, heart ailments, lung diseases, ulcer, and also to promote secretion of bile (Raghavendran et al., 2006). Previous studies have shown that organic solvent and water extracts of Sargassum polycystum possess hepatoprotective, antilipidemic, antioxidant and membrane stabilizing properties (Raghavendran et al., 2004, 2005). It has been reported that antioxidants may reduce hyperpigmentation and support skin health (Ma et al., 2001). Thus, in view of the traditional use of Sargassum polycystum in skin-related disorders and its antioxidant property which may reduce hyperpigmentation, a study was undertaken to investigate if Sargassum polycystum possesses any antimelanogenesis effects with a view of its possible use as a treatment for hyperpigmentation and its use as a skin-whitening agent in cosmetics. In this report we describe the differential extraction of dried, powdered Sargassum polycystum with solvents of different polarity. The ability of the different extracts to act as a skin-whitening agent was tested by its ability to inhibit tyrosinase, the rate limiting enzyme in melanogenesis, initially using a cell-free mushroom tyrosinase system, which has commonly been employed for the testing and screening of potential skin-whitening agents (Song et al., 2009). The ability to inhibit cellular tyrosinase derived from melanin producing B16F10 melanoma cells as well as its ability to inhibit melanogenesis was also tested. Kojic acid, a fungal metabolite produced by some species of Aspergillus and Penicillium, which is well known to be an inhibitor of tyrosinase and melanogenesis (Garcia and Fulton, 1996), was used as a positive control. 2. Materials and methods 2.1. Seaweed material Seaweed Sargassum polycystum was collected from the low tide area of Pulau Seri Buat (Pulau Sri Buat) and Pulau Sembilan, Endau, Johor, Malaysia. The species identification was done by Prof. Phang Siew Moi of the Algae Research Laboratory, University of Malaya (UMalgae). The collected seaweed was thoroughly washed in running tap water to remove the salt, epiphytes and sand, air-dried in the shade and coarsely powdered. 2.2. Preparation of seaweed extract The powdered seaweed (2.0 kg) was first soaked in 6 L of 95% ethanol for 3 days at room temperature. The solvent was then decanted and filtered. The procedure was repeated 2 times. The filtrate from each extraction was combined and the solvent was evaporated at 40 ◦ C under reduced pressure using a rotary evaporator to give an ethanolic crude extract (134.77 g). This ethanolic crude extract was further extracted with 400 ml of hexane to give a hexane-soluble fraction (30.42 g) and a hexane insoluble residue. The hexane-insoluble residue was partitioned between ethyl acetate–water (100 ml:100 ml) to give an ethyl acetatesoluble fraction and a water soluble fraction. The ethyl acetate fraction was dried in a rotary evaporator at 40 ◦ C to yield a dry extract of 1.35 g. The water layer was lyophilized to give a water fraction (60.66 g). The ethanolic crude extract and its fractionated extracts were dissolved in dimethyl sulphoxide (DMSO, Sigma, St. Louis, MO, USA), while the water fraction was dissolved in water, to create stock solutions of 250 mg/ml. The stock solutions were diluted appropriately with buffer or media at the time of testing. The final concentration of DMSO in test wells was 1% for cell-free assay and 0.1% for cell-based assay.
2.3. Mushroom tyrosinase assay The effect of Sargassum polycystum extracts on cell free mushroom tyrosinase activity was determined spectrophotometrically as described previously (Rahman et al., 2001) with minor modifications. The tyrosinase activity was determined using l-DOPA (Sigma, St. Louis, MO, USA) as a substrate. In brief, 50 l of 700 units/ml of mushroom tyrosinase (EC 1.14.18.1) (Sigma, St. Louis, MO, USA) in 0.1 M phosphate buffer, pH 6.8, 50 l of the different concentrations (100, 250 and 500 g/ml) of the different extracts and 100 l of 0.1 M phosphate buffer (pH 6.8) were added to each well of a 96-well plate (TPP, Switzerland) and mixed. The assay mixture was pre-incubated at room temperature for 10 min; 100 l of 2.5 mM lDOPA in 0.1 M phosphate buffer (pH 6.8) was then added into each well and incubation was continued for 20 min at room temperature. The quantity amount of dopachrome formed in the reaction mixture was determined against blank (solution without enzyme) at 475 nm in a microplate reader (Tecan Sunrise, Switzerland). Kojic acid (Sigma, St Louis, MO, USA) at a concentration of 100 g/ml was used as a standard tyrosinase inhibitor in order to confirm that the assay was working (Arung et al., 2009). The percentage of tyrosinase activity was calculated as follows: tyrosinase activity (%) = [(A − B)/(C − D)] × 100, where A is the absorbance of reaction mixture containing test sample and mushroom tyrosinase, B is the absorbance of blank sample containing test sample but without mushroom tyrosinase, C is the absorbance of reaction mixture without test sample and with mushroom tyrosinase and D is the absorbance of the well without both test sample and mushroom tyrosinase (l-DOPA alone).
2.4. Cell culture and treatment The B16F10 murine melanoma cells (CRL-6475) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma–Aldrich, USA) containing 2 mM lglutamine (Applichem, Denmark), supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100 units/ml of penicillin (PAA, Austria), and 100 g/ml of streptomycin (PAA, Austria) in culture flasks in a CO2 incubator with a humidified atmosphere containing 5% CO2 in air at 37 ◦ C. The culture medium was changed every 2 days. The cells were harvested by trypsinization when they were about 70% confluent, counted with a haemocytometer (Weber, England) and seeded at the appropriate numbers into wells of cell culture plates for further experiments.
2.5. Cell viability assay (MTT assay) To determine the safety of the various extracts the viability of cells following treatment with extracts was determined by the MTT assay. This method is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial enzymes in viable cells (Mosmann, 1983). The quantity of formazan formed is proportional to the number of viable cells present and can be measured spectrophotometrically. Briefly, cells seeded at a density of 3 × 103 cells/well in a 96-well plate were left to adhere overnight. A test sample was then added to each well and incubated for another 72 h. Next, the treated cells were labelled with MTT dye reagent (Applichem, Denmark) in PBS (2 mg/ml) for 3 h. The formazan precipitates were dissolved by DMSO and the concentrations were measured at 554 nm in a microplate reader with a reference wavelength of 690 nm. Cell viability was calculated using the following formula: cell viability (%) = (Asample /Acontrol ) × 100, where Asample and Acontrol are the
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absorbances from the mixture with, or without the addition of test sample, respectively.
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3. Results
2.6. Cellular tyrosinase assay
3.1. Effects of Sargassum polycystum on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability
Cellular tyrosinase activity was measured using a previously described method (Lee et al., 2006) with small modifications. Wells of 6 well plates (TPP, Switzerland) were seeded with B16F10 melanoma cells at a density of 2 × 105 cells/well contained in 3 ml of media. Plates were incubated overnight in a humidified CO2 incubator at 37 ◦ C and 5% CO2 to allow cells to adhere. Cells were then exposed to increasing doses of test extracts or kojic acid for 72 h in the presence or absence of 100 nM alpha-melanocyte stimulating hormone (␣-MSH, Sigma, USA). The cells were then washed with sodium phosphate buffered saline (pH 6.8) and lyzed with M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA). The lysates were then clarified by centrifugation at 13,000 rpm for 15 min at 4 ◦ C. The protein concentration was determined by the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA) using bovine serum albumin (BSA, Sigma, USA) as the standard. The reaction mixture consisting of 40 g protein (adjusted to 100 l with 0.1 M PBS, pH 6.8) and 100 l of 5 mM l-DOPA was added to each well of a 96-well plate. After incubation at 37 ◦ C for 1 h, the absorbance was measured at 475 nm using a microplate reader. Tyrosinase activity in the protein was calculated by the following formula:
The effects of Sargassum polycystum extracts on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability are shown in Table 1. The results show that ethanolic crude extract and its hexane and ethyl acetate fractions did not have any effect on mushroom tyrosinase activity while the water fraction had only a small inhibitory effect at the highest dose tested. On the other hand, ethanolic extract, hexane and ethyl acetate fractions reduced cellular tyrosinase activity in a dose-dependent manner. Water fraction did not have any effect on cellular tyrosinase except a slight inhibition at the highest dose tested. In the cell viability (MTT) assay, hexane fraction did not have any appreciable cytotoxic activity at a dose of 100 g/ml, but reduced viable cells slightly at the higher doses. Ethanolic extract also had some minor cytotoxic effect, while ethyl acetate fraction had the greatest cytotoxic effect. Water fraction did not show any cytotoxic effect at all. The ethyl acetate fraction was not investigated further due to its higher cytotoxicity. Kojic acid, used as a positive control, reduced mushroom tyrosinase drastically even at the lowest dose used. However, when tested on cellular tyrosinase the inhibitory effect was less severe, and showed a dose–response effect similar to the seaweed extracts and fractions.
tyrosinase activity (%) =
OD475 OD475
of sample × 100 of control
3.2. Effect of Sargassum polycystum hexane fraction (SPHF) on melanin synthesis in B16F10 melanoma cells
2.7. Melanin content assay Melanin content was measured as described previously (Hosoi et al., 1985) with slight modifications. The B16F10 melanoma cells were seeded with 2 × 105 cells/well in 3 ml of medium in 6-well culture plates and incubated overnight to allow cells to adhere. The cells were exposed to various concentrations (100, 250 and 500 g/ml) of the seaweed extracts or kojic acid for 72 h in the presence or absence of 100 nM ␣-MSH. At the end of the treatment, the cells were washed with PBS and lyzed with 800 l of 1 N NaOH (Merck, Germany) containing 10% DMSO for 1 h at 80 ◦ C. The absorbance at 400 nm was measured using a microplate reader. The melanin content was determined from a standard curve prepared from an authentic standard of synthetic melanin (Sigma, USA).
To gain further evidence of SPHF’s involvement in melanogenesis, the effect of SPHF on melanin production in B16F10 cells was tested. Fig. 1 demonstrates that SPHF reduced cellular melanin content in B16F10 cells in the absence of ␣-MSH stimulation. The inhibition was dose-dependent: SPHF at 100, 250 and 500 g/ml induced significant inhibition on melanin production by 15.13, 28.71 and 39.93%, respectively. Kojic acid at the same doses also reduced cellular melanin production in a dose-dependent manner.
2.8. Phytochemical screening The major secondary metabolites were identified by employing the methodology outlined by Harborne (1998). Briefly, qualitative phytochemical screening of the active fraction of Sargassum polycystum ethanolic crude extract was carried out for alkaloids (Meyer and Dragendoff’s tests), tannins (FeCl3 test), saponins (frothing test), lipids (Wattman paper test), flavonoids (Schinoda’s test and anisaldehyde staining), sugars (resorcinol–H2 SO4 and aniline hydrogen phthalate test), phenols (Folin-Ciocalteu test), terpenoids (vanillin–H2 SO4 test, Liberman Burchard’s test), amino acid and amines (Ninhydrin test). 2.9. Statistical analysis All data are presented as the mean ± SEM of 3–4 independent experiments. Comparisons among groups were made using ANOVA followed by Dunnett’s post hoc test. Student’s t-test was used when single comparisons were made between means of two groups. A value of P < 0.05 was regarded as statistically significant in all experiments.
Fig. 1. Effects of SPHF and kojic acid on cellular melanin content in B16F10 melanoma cells. The control readings (unfilled bar) were set as 100%. Data from experimental wells were expressed as percentage of control. Each column represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control.
82.35 ± 4.40** 58.63 ± 1.26*** 86.17 ± 2.54* 68.77 ± 2.04*** b b
Fig. 2. Effect of SPHF on cellular tyrosinase activity in ␣-MSH-stimulated B16F10 cells compared with kojic acid. The cells were incubated with 100 nM ␣-MSH alone or together with increasing doses of SPHF or kojic acid for 72 h following which cellular tyrosinase activity was measured. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SEM of four independent experiments. # P < 0.001 versus control group (without ␣-MSH). **P < 0.01, ***P < 0.001 versus ␣-MSH-treated group.
Data are presented as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control. a Relative activity (%) = (% of sample versus control). b 100% mushroom tyrosinase inhibitory activity was observed in the optimisation of the assay. c Not tested because the ethyl acetate fraction expressed high cytotoxicity.
c c
100 73.98 ± 2.56** 75.99 ± 4.95*** 42.99 ± 1.73*** 107.23 ± 6.63 93.55 ± 2.25
100 70.69 ± 6.75** 65.65 ± 7.35*** 40.52 ± 3.11*** 113.45 ± 4.80 86.93 ± 2.23
250 g/ml
100 86.99 ± 3.48 96.38 ± 4.77 77.32 ± 0.98* 110.22 ± 3.49 92.70 ± 1.63 100 65.04 ± 3.78*** 50.43 ± 2.63*** 100 82.03 ± 2.35** 67.94 ± 2.99***
100 g/ml 500 g/ml 250 g/ml
100 86.86 ± 2.35** 79.25 ± 2.99*** 89.08 ± 3.07* 92.05 ± 2.47 84.56 ± 1.62***
100 g/ml
100 102.53 ± 1.32 102.43 ± 1.51 95.91 ± 1.73 87.07 ± 4.88*
500 g/ml 250 g/ml
100 96.61 ± 0.80 97.91 ± 0.55 98.48 ± 3.21 99.66 ± 1.75
100 97.78 ± 1.34 97.10 ± 0.55 100.58 ± 1.81 99.49 ± 0.73 11.73 ± 1.51***
100 g/ml
Sargassum polycystum
Control Ethanolic crude extract Hexane fraction Ethyl acetate fraction Water fraction Kojic acid (positive control)
Cellular tyrosinase activity (%)a Mushroom tyrosinase activity (%)a
Cell viability (%)a
500 g/ml
Y.Y. Chan et al. / Journal of Ethnopharmacology 137 (2011) 1183–1188 Table 1 Effects of kojic acid, Sargassum polycystum ethanolic crude extract and its solvent soluble fractions on mushroom tyrosinase activity, cellular tyrosinase activity and cell viability in B16F10 melanoma cells. Control readings (from wells containing no test material or kojic acid) were set as 100% and reading of experimental wells were expressed as a percentage of controls.
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3.3. Effects of SPHF on cellular tyrosinase activity and melanin synthesis in B16F10 cells with ˛-MSH stimulation The effects of SPHF on cellular tyrosinase activity and melanogenesis in ␣-MSH-stimulated B16F10 melanoma cells were next investigated. Upon exposure to 100 nM ␣-MSH alone, the tyrosinase activity of B16F10 cells was significantly increased compared to the controls (Fig. 2). SPHF significantly reduced the tyrosinase activity of ␣-MSH-stimulated B16F10 cells in a dose-dependent manner, with 31.85% inhibition at 100 g/ml, 39.57% at 250 g/ml and 54.41% at 500 g/ml. Kojic acid at the same doses similarly reduced tyrosinase activity in a dose-dependent manner.
Fig. 3. Effect of SPHF on cellular melanin content in ␣-MSH-stimulated B16F10 cells compared with kojic acid. The cells were incubated with 100 nM alone or together with increasing doses of SPHF or kojic acid for 72 h following which total cellular melanin activity was measured. Baseline melanin content in control wells not exposed to ␣-MSH and any test material or kojic acid was set at 100%. Data from experimental wells were expressed as a percentage of control. Each column represents the mean ± SEM of four independent experiments. # P < 0.001 versus control group (without ␣-MSH). **P < 0.01, ***P < 0.001 versus ␣-MSH-treated group.
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Fig. 3 shows the effect of similar levels of SPHF and kojic acid on melanin production in ␣-MSH-stimulated B16F10 cells. In parallel to the cellular tyrosinase activity, ␣-MSH increased the melanin concentration of B16F10 melanoma cells. The SPHF and the positive control, kojic acid, also significantly inhibited melanin production in ␣-MSH-stimulated B16F10 melanoma cells in a dose-dependent manner. After co-incubation for 72 h with 100 nM ␣-MSH and SPHF at concentrations of 100, 250 and 500 g/ml, the melanin production of ␣-MSH-treated B16F10 cells was suppressed to 62.19, 42.77 and 31.80%, respectively, compared to ␣-MSH-treated group without SPHF. Kojic acid similarly reduced melanin production to 96.81, 87.43 and 68.72% at concentrations of 100, 250 and 500 g/ml respectively. 3.4. Phytochemical screening of SPHF The phytochemical screening of the active fraction, SPHF, revealed the presence of the following classes of chemical compounds: saponins, flavonoids, tannins, terpenoids, phenols, sugars, amino acid and amines; alkaloids and lipids were absent. 4. Discussion In this study, several fractions of powdered dried Sargassum polycystum have been extracted with solvents of different polarity and tested for their possible antimelanogenesis and skin-whitening properties using inhibition of mushroom tyrosinase activity in a cell-free system and inhibition of cellular tyrosinase activity in B16F10 murine melanoma cells as screening assays. Our major findings are: (i) the cellular tyrosinase assay is a more reliable assay than the cell-free mushroom tyrosinase assay for the screening of potential skin-whitening agents and (ii) Sargassum polycystum may contain potential skin-whitening agents that inhibit melanogenesis in B16F10 melanoma cells by inhibition of cellular tyrosinase. Ethanol extraction was used in the initial experiments as ethanol can extract a wide range of agents of different polarity (Harborne, 1998) and is thus useful as a first screening step. The fact that the ethanolic extract and its hexane fraction failed to inhibit mushroom tyrosinase while being able to inhibit cellular tyrosinase activity, points to the weakness of the former protocol to screen for the presence of skin-whitening or antimelanogenesis agents in natural sources and man-made compounds. The finding that hexane fraction inhibits cellular tyrosinase activity as well as melanin production in B16F10 melanoma cells strengthens this conclusion. It must be remembered that mushroom tyrosinase is found in the cytosol while tyrosinase in melanocytes is membrane bound, and thus it can be expected that the effect of antimelanogenesis agents on these tyrosinases may not be the same. Since target cells of the agents to be screened are melanocytes, it would be more reliable to use tyrosinase derived from the melanin-producing cells instead of mushrooms. The murine B16F10 cell line was used because they produce melanin, are known to contain tyrosinase which is associated with melanogenesis, respond to ␣-MSH activation and are easy to culture in vitro (Busca and Ballotti, 2000; An et al., 2008). Kojic acid was used as a positive control in all these screenings as it has a known inhibitory effect on tyrosinase as well as melanin production (Garcia and Fulton, 1996). The water fraction did show some mild inhibitory activity both towards mushroom and cellular tyrosinase activity, and this could be due to a polar agent present in water fraction that is different from the non-polar agent seen in hexane fraction. However the inhibitory effect is small and it is not economically feasible to be developed further.
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The ethanolic extract appeared to have some cytotoxic as well as tyrosinase inhibitory effect on cellular tyrosinase activity (Table 1). However, a more highly cytotoxic fraction (ethyl acetate) and a less cytotoxic hexane fraction could be further separated. Thus, a large part of the cytotoxic effect of the ethanolic extract was due to the agents that separated out into the ethyl acetate fraction. Dooley (1997) previously speculated that a desirable skin-whitening agent should inhibit melanin synthesis in melanosomes by acting specifically to reduce the synthesis or activity of tyrosinase and with little or no cytotoxicity. Hence, the ethyl acetate fraction at higher concentration level (250 and 500 g/ml) was not used further due to its greater cytotoxicity on the melanoma cells. The hexane fraction was however less cytotoxic, having no significant direct killing effect on the B16F10 cells at the lowest dose used (100 g/ml). At this dose, however, cellular tyrosinase was already significantly inhibited. At the higher doses (250 and 500 g/ml), cellular tyrosinase inhibitory activity was increased, but there was also greater cytotoxic activity. The decrease in tyrosinase activity at the higher doses of hexane fraction cannot be attributed to the smaller number of viable cells present because assays were normalised to use the same quantity of protein (40 g) from each well. Thus the inhibition of tyrosinase activity was bona fide. The fact that the hexane fraction was also able to inhibit the increase in cellular tyrosinase in ␣-MSH-stimulated B16F10 melanoma cells provides further evidence of the direct action of hexane fraction on inhibition of cellular tyrosinase and melanogenesis. However, due to the slightly cytotoxic effects of the hexane fraction at the higher concentrations, the dose that will be used for any formulation or treatment regime will have to be carefully calibrated. In conclusion, based on the results obtained from cellular tyrosinase and melanogenesis inhibitory assays using B16F10 cells, we report here for the first time that hexane fraction of the ethanolic extract of Sargassum polycystum (SPHF) contains agent(s) that can be further developed and formulated into a skin-whitening or antimelanogenesis preparation for cosmetics and therapeutic use. The identification of active constituents that are responsible for the antimelanogenesis effect of SPHF and the phytochemical profiling are currently underway. Further studies are warranted to investigate the active components and the underlying molecular mechanisms of action involved in the melanogenesis inhibition. Acknowledgements This study was supported by the postgraduate research grant from the University of Malaya (PS280/2008A) and Fundamental Research Grant Scheme (FP021/2008C) from the Ministry of Higher Education of Malaysia. References An, S.M., Kim, H.J., Kim, J.E., Boo, Y.C., 2008. Flavonoids, taxifolin and luteolin attenuate cellular melanogenesis despite increasing tyrosinase protein levels. Phytotherapy Research 22, 1200–1207. Arung, E.T., Kusuma, I.W., Christy, E.O., Shimizu, K., Kondo, R., 2009. Evaluation of medicinal plants from Central Kalimantan for antimelanogenesis. Journal of Natural Medicines 63, 473–480. Busca, R., Ballotti, R., 2000. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Research 13, 60–69. Dooley, T.P., 1997. Topical skin depigmenting agents: current products and discovery of novel inhibitors of melanogenesis. Journal of Dermatological Treatment 7, 188–200. Garcia, A., Fulton Jr., J.E., 1996. The combination of glycolic acid and hydroquinone or kojic acid for the treatment of melasma and related conditions. Dermatological Surgery 22, 443–447. Harborne, J.B., 1998. Methods of Plant Analysis, third ed. Chapman and Hall Publishers, London. Hearing, V.J., 2005. Biogenesis of pigment granules: a sensitive way to regulate melanocyte function. Journal of Dermatological Science 37, 3–14.
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