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Kinetic study on the tyrosinase and melanin formation inhibitory activities of carthamus yellow isolated from Carthamus tinctorius L.
Journal of Bioscience and Bioengineering VOL. 115 No. 3, 242e245, 2013 www.elsevier.com/locate/jbiosc
Kinetic study on the tyrosinase and melanin formation inhibitory activities of carthamus yellow isolated from Carthamus tinctorius L. Yi-Shyan Chen,1, * Shu-Mei Lee,2 Chih-Chien Lin,1 Chia-Yi Liu,1 Meng-Chen Wu,1 and Wun-Ling Shi1 Department of Cosmetic Science, Providence University, No. 200, Sec. 7, Taiwan Boulevard, Shalu, Taichung 43301, Taiwan, ROC1 and Department of Cosmetic Science and Management, Mackay Medicine, Nursing and Management College, 92 Shengjing Road, Beitou, Taipei 11260, Taiwan, ROC2 Received 17 July 2012; accepted 18 September 2012 Available online 11 October 2012
Carthamus yellow (CY) is the major component of the yellow pigments of Carthamus tinctorius L. CY has been extensively used as a natural color additive for food and cosmetics. Here, our results demonstrate that carthamus yellow reduced the activity of mushroom tyrosinase in a dose-dependent manner with a half maximal inhibitory concentration (IC50) value of approximately 1.01 ± 0.03 mg/mL. A kinetic study of carthamus yellow on tyrosinase exhibited a mode of competitive inhibition with a Ki of 0.607 mg/mL. Moreover, cell viability analysis indicated that carthamus yellow used at concentrations of 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells. Melanin content analysis showed that melanin production in B16F10 melanoma cells treated with 4 mg/mL carthamus yellow can decrease to 82.3 ± 0.4% of the levels of melanin production of untreated cells. Thus, carthamus yellow has the potential to become a useful skinwhitening agent in the future. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Carthamus tinctorius L.; Carthamus yellow; Enzyme kinetics; Melanin; Tyrosinase]
The control of melanogenesis is a key approach for the treatment of abnormal skin pigmentation disease or for the purpose of appearance beautification (1,2). Epidermal and dermal hyperpigmentation can be caused by an increased activity of melanogenic enzymes, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) (3,4). Tyrosinase (EC 1.14.18.1) is the key enzyme in the browning of fruits and vegetables and the darkening of skin, hair and eyes in animals (5,6). Tyrosinase plays a role in the initial step of melanin synthesis by catalyzing the oxidation of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and of DOPA to DOPAquinone, which can be processed to DOPAchrome (7). Thus, this enzyme is a good target for inhibition in the search for different types of depigmenting agents. Numerous tyrosinase inhibitors have been developed from both natural (8,9) and synthetic sources (10,11). In practical applications, however, just a small number of tyrosinase inhibitors are used as skin-whitening agents, due mainly to safety concerns (12). Therefore, the discovery of new and safe tyrosinase inhibitors is an important and ongoing focus for the medicinal, nutritional and cosmetic industries. More commonly known in Asia as safflower or red flower, Carthamus tinctorius L. is an annual plant belonging to the Compositae family and has been used as a natural pigment additive for food and cosmetics (13). C. tinctorius is also a famous traditional Chinese medicine that promotes blood circulation by treating blood stasis * Corresponding author. Tel.: þ886 4 26328001x15407; fax: þ886 4 26311167. E-mail address: [email protected] (Y.-S. Chen).
(14). The petals of C. tinctorius are commonly used for the treatment of trauma, chronic gastritis and cardiovascular and gynecological diseases (13,15). Moreover, several studies have demonstrated that C. tinctorius extract can have many effects, such as an anticoagulant, vasodilator, antioxidant, immunosuppressant and neuroprotector (16e18). Carthamus yellow (CY; CI Natural Yellow 5) is the major component of the yellow pigments of C. tinctorius. Two chalcones, safflomin A (Fig. 1A) and safflomin B (Fig. 1B), are the main compounds of carthamus yellow (19). Previous studies also demonstrated that carthamus yellow, including safflomin A and safflomin B, provides the main biological functions of C. tinctorius, such as vasodilative activity to reduce blood pressure and heart rate and anti-inflammatory, neuroprotective, anti-oxidative and antihepatic fibrosis activities (14,19e21). Although the biological activities of carthamus yellow have been reported broadly and some compounds from safflower seeds have been found to have anti-melanogenesis activity (22), no studies have directly revealed the inhibitory effect of carthamus yellow on tyrosinase and melanin production. Furthermore, the utilization of botanical tyrosinase inhibitors in medicine, food and cosmetics would increase the additive value of agricultural products. Thus, this present study is the first report to investigate carthamus yellow’s inhibition kinetics on tyrosinase and its effects on melanin production in melanoma cells. The results of this study indicate that carthamus yellow has the potential to become a depigmenting agent for medical, food and cosmetic applications.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.013
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RESULTS AND DISCUSSION
FIG. 1. Chemical structures of the two major compounds of carthamus yellow: (A) safflomin A, (B) safflomin B. MATERIALS AND METHODS Materials Carthamus yellow (no. 300-31782, safflomin A and safflomin B) was purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Arbutin, vitamin C (ascorbic acid), mushroom tyrosinase, L-tyrosine, Dimethyl sulfoxide (DMSO) and other chemicals were purchased from SigmaeAldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA were purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Affymetrix/USB (Cleveland, OH, USA). Deionized distilled water (ddH2O) for solutions and buffers was obtained from the Milli-Q system (Millipore, Bedford, MA, USA). Tyrosinase activity assay and kinetic study For the tyrosinase activity assay, each 60 mL sample (containing 0.4e4.0 mg/mL carthamus yellow) was mixed with 100 mL of 1 mM L-tyrosine in phosphate buffer solution (pH 6.8). Then, 40 mL of mushroom tyrosinase solution (100 units/mL) was added to the mixture, which was then incubated for 25 min at 37 C. The spectrophotometric analysis was performed at 475 nm, and the inhibition of DOPAchrome formation was calculated as the inhibition percentage (23). The unit definition of tyrosinase activity is as follows: one unit will cause an increase in A280 of 0.001 per min at 25 C in a 3 mL reaction mixture containing L-tyrosine. For the kinetic study, different concentrations of Ltyrosine solution (0.3, 0.5 and 0.8 mM) were used. The velocities of the enzymatic reaction in 1.0 and 1.5 mg/mL carthamus yellow were calculated and compared with that of the non-inhibitory enzymatic reaction. The Michaelis constant (Km) and the maximal velocity (Vmax) of the tyrosinase activity were determined by the LineweavereBurk plot at various concentrations of L-tyrosine as a substrate. The Ki values were calculated from shared regression fits of substrateevelocity curves taken at different concentrations of the inhibitors (24). Cell line and cell culture The B16F10 melanoma cells (BCRC 60031) were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan, R.O.C.). The cells were maintained in DMEM supplemented with 10% FBS containing 100 units/mL penicillin and 100 mg/mL streptomycin at 37 C in a 5% CO2 incubator. Cells grown in various culture dishes at 80% confluence were used in all of the experiments. MTT assay for cell viability The B16F10 cells were seeded in 96-well plates (6 103 cells/well) using DMEM medium supplemented with 10% FBS for 24 h, after which the medium was replaced with FBS-free DMEM medium for another 24 h. The prepared cells were subsequently treated with different concentrations of carthamus yellow (1.0e4.0 mg/mL) for 48 h. Next, 100 mL (0.5 mg/mL) of MTT solution was added to cells, which were then incubated at 37 C for 30 min and washed twice with PBS. Finally, the PBS cleaned cells were lysed with 100 mL DMSO, and the absorbance was measured spectrophotometrically at 540 nm using an ELISA reader (25). Melanin content analysis For melanin content analysis, either 1.0e4.0 mg/ mL of carthamus yellow or 0.1 mg/mL of control inhibitors (vitamin C or arbutin) was used to treat B16F10 cells. B16F10 cells were seeded in 6-well plates (2 105 cells/well) using DMEM medium supplemented with 10% FBS for 24 h. The prepared cells were subsequently treated with CY or control inhibitors for 24 h. Cells were then detached using 0.05% trypsin-EDTA. Finally, the cell pellets were dissolved in 120 mL of 1 N NaOH for 1 h at 65 C, and the melanin content of melanoma cells was measured spectrophotometrically at 405 nm by an ELISA reader (26). Statistical analysis Quantitative data were analyzed using Student’s t-tests and are presented as mean standard error (S.E.) of three independent experiments. The P-values less than 0.05 were considered to be significant.
Inhibition of tyrosinase activity by carthamus yellow In the present study, we investigated the effect of carthamus yellow on tyrosinase inhibition to find a novel and effective substance for skin whitening. We found that carthamus yellow clearly reduced mushroom tyrosinase activity in a dose-dependent manner (Fig. 2). Our results showed that 0.4e4.0 mg/mL carthamus yellow reduced the activity of tyrosinase from 27.9% to 85.1%, respectively (Fig. 2), and that carthamus yellow suppressed tyrosinase with a half maximal inhibitory concentration (IC50) value of approximately 1.01 0.03 mg/mL. Comparatively, the IC50 value of vitamin C to mushroom tyrosinase in our experiments was approximately 0.12 0.01 mg/mL (data not shown). Thus, while the IC50 value of carthamus yellow was higher than vitamin C, we also found that carthamus yellow had a strong inhibitory activity toward mushroom tyrosinase. Kinetic analysis of carthamus yellow on tyrosinase The mode of tyrosinase inhibition by carthamus yellow was determined by LineweavereBurk plot analysis as shown in Fig. 3. The kinetic parameters of tyrosinase inhibition by carthamus yellow revealed that the Ki was 0.607 mg/mL (Table 1). Carthamus yellow at 1.0 and 1.5 mg/mL exhibited Km values of 1.678 and 2.709 mM, respectively (Table 1). However, carthamus yellow at 0, 1.0 and 1.5 mg/mL showed almost the same Vmax values of either 0.023 or 0.024 mmol/min (Table 1). In Fig. 3, the regressions of various carthamus yellow concentrations were fitted to intersect with the Y-axis at the same point. As the substrate (L-tyrosine) concentration varies, the Km value of tyrosinase increased in a dose-dependent manner without changing the Vmax, indicating that carthamus yellow is a competitive tyrosinase inhibitor (Fig. 3 and Table 1). Tyrosinase inhibitors with established efficacy include competitive inhibitors such as arbutin, azelaic acid, deoxyArbutin, hydroquinone and kojic acid (10,12), as well as the non-competitive inhibitors ellagic acid, glabridin and oxyresveratrol (27,28). Additionally, there are mixed-type tyrosinase inhibitors such as phthalic acid, heptadecahydroquinone and p-coumaric acid (29e31). Moreover, the major component of the yellow pigments of C. tinctorius is carthamus yellow, which in turn is mainly composed of the chalcone compounds safflomin A and safflomin B (Fig. 1). Several studies have demonstrated that chalcones are potent tyrosinase inhibitors with competitive inhibition abilities (32,33). In this study, carthamus yellow also exhibited potent inhibitory activity on tyrosinase through competitive inhibition, a feature similar to other chalcone compounds (Fig. 3 and Table 1).
FIG. 2. Inhibition of tyrosinase activity by carthamus yellow (CY) at various concentrations. Each value is expressed as mean S.E. (n ¼ 3).
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FIG. 4. Cell viability of B16F10 melanoma cells treated with various concentrations of carthamus yellow. Each value is expressed as mean S.E. (n ¼ 3). FIG. 3. LineweavereBurk plot of tyrosinase activity inhibited by carthamus yellow.
In an earlier study, Khatib et al. (33) demonstrated that the tyrosinase inhibitory activity of chalcones is due to its phenol group(s). The most important factor of the inhibitory activity of chalcones on tyrosinase is the position of the phenolic hydroxyl group(s) rather than their number (33). Consequently, we hypothesize that the tyrosinase inhibitory activity of carthamus yellow might also be provided by the phenol groups on the aromatic rings of the safflomin A and B molecules (Fig. 1). Effects of carthamus yellow on B16F10 melanoma cells To evaluate the effects of carthamus yellow on melanogenesis, we used B16F10 melanoma cells as a model. First, the effects of concentrations of 1.0e4.0 mg/mL carthamus yellow on the cell viability of B16F10 melanoma cells were assessed by a standard MTT method (Fig. 4). The result of this cell viability assay indicated that carthamus yellow at 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells (Fig. 4). Moreover, 4.0 mg/ mL carthamus yellow is almost the highest concentration that can be obtained in aqueous solution using a normal dissolving procedure (stir at room temperature). Thus, we used the concentrations of 1.0e4.0 mg/mL carthamus yellow for our later melanin content analysis. Although there were no statistically significant differences in cell viability between the control and carthamus yellow-treated B16F10 melanoma cells, cell viability slightly increased nonetheless in carthamus yellow-treated B16F10 melanoma cells (Fig. 4). This phenomenon may result from the components of carthamus yellow possibly having an ability to slightly enhance the growth of B16F10 melanoma cells. Melanin content analysis clearly demonstrated that carthamus yellow at concentrations higher than 2 mg/mL repressed the production of melanin in B16F10 melanoma cells (Fig. 5). The percentage of melanin content in 4 mg/mL carthamus yellowtreated B16F10 melanoma cells was decreased to 82.3 0.4% of the levels of melanin production of untreated cells. The percentages of melanin content in B16F10 melanoma cells treated with 0.1 mg/mL
of the control inhibitors vitamin C or arbutin were 87.9 1.6% and 56.8 1.7%, respectively (Fig. 5). These results show that the inhibitory activity of carthamus yellow on melanogenesis is not stronger than that of vitamin C or arbutin, but instead is within a comparable range. Thus, these results demonstrate that carthamus yellow is an effective and safe melanogenesis inhibitor. Finding safe and effective skin-whitening agents that can be used to prevent hyperpigmentation is important for many applications. As melanogenesis inhibitors are being developed, safety should be the most important consideration. In this study, carthamus yellow exhibited no cytotoxicity on B16F10 melanoma cells but had an efficient inhibitory activity on melanogenesis. Furthermore, carthamus yellow is already an edible and extensively used natural color additive for food and cosmetics. Therefore, we believe that carthamus yellow could be used as a safe and effective depigmenting agent for many applications. Moreover, several studies have demonstrated that chalcones are potent tyrosinase inhibitors, and thus, it would be interesting to compare the anti-melanogenesis activities of carthamus yellow with those of other chalcones. In summary, carthamus yellow decreased the activity of mushroom tyrosinase in a dose-dependent manner with an IC50 value of approximately 1.01 0.03 mg/mL. Carthamus yellow exhibited a mode of competitive inhibition with a Ki of 0.607 mg/mL. Moreover, the MTT assay indicated that carthamus yellow concentrations
TABLE 1. Kinetic parameters of tyrosinase inhibited by carthamus yellow. Inhibitor None CY
Conc. (mg/mL)
Km (mM)a
Vmax (mmol/min)a
1.0 1.5
0.923 1.678 2.709
0.023 0.024 0.023
Ki (mg/mL)a
0.607 a The kinetic parameters Km, Vmax and Ki were calculated from the LineweavereBurk plot.
FIG. 5. Melanin content of B16F10 melanoma cells treated with various concentrations of carthamus yellow. The vitamin C (VitC) and arbutin (Arb) treatments are positive controls. Each value is expressed as mean S.E. (n ¼ 3). *P < 0.05, compared with the control.
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of 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells. Melanin content analysis revealed that the percentage of melanin production in B16F10 melanoma cells could be decreased to 82.3 0.4% if treated with 4 mg/mL carthamus yellow. Altogether, the results of this study indicate that carthamus yellow has the potential to become a useful depigmenting agent for medical, food and cosmetic products in the future.
16.
17.
ACKNOWLEDGMENTS This work was supported by grants from the National Science Council, Taiwan, R.O.C. (NSC99-2313-B-126-002-MY3).
18. 19.
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Kinetic study on the tyrosinase and melanin formation inhibitory activities of carthamus yellow isolated from Carthamus tinctorius L. Yi-Shyan Chen,1, * Shu-Mei Lee,2 Chih-Chien Lin,1 Chia-Yi Liu,1 Meng-Chen Wu,1 and Wun-Ling Shi1 Department of Cosmetic Science, Providence University, No. 200, Sec. 7, Taiwan Boulevard, Shalu, Taichung 43301, Taiwan, ROC1 and Department of Cosmetic Science and Management, Mackay Medicine, Nursing and Management College, 92 Shengjing Road, Beitou, Taipei 11260, Taiwan, ROC2 Received 17 July 2012; accepted 18 September 2012 Available online 11 October 2012
Carthamus yellow (CY) is the major component of the yellow pigments of Carthamus tinctorius L. CY has been extensively used as a natural color additive for food and cosmetics. Here, our results demonstrate that carthamus yellow reduced the activity of mushroom tyrosinase in a dose-dependent manner with a half maximal inhibitory concentration (IC50) value of approximately 1.01 ± 0.03 mg/mL. A kinetic study of carthamus yellow on tyrosinase exhibited a mode of competitive inhibition with a Ki of 0.607 mg/mL. Moreover, cell viability analysis indicated that carthamus yellow used at concentrations of 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells. Melanin content analysis showed that melanin production in B16F10 melanoma cells treated with 4 mg/mL carthamus yellow can decrease to 82.3 ± 0.4% of the levels of melanin production of untreated cells. Thus, carthamus yellow has the potential to become a useful skinwhitening agent in the future. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Carthamus tinctorius L.; Carthamus yellow; Enzyme kinetics; Melanin; Tyrosinase]
The control of melanogenesis is a key approach for the treatment of abnormal skin pigmentation disease or for the purpose of appearance beautification (1,2). Epidermal and dermal hyperpigmentation can be caused by an increased activity of melanogenic enzymes, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) (3,4). Tyrosinase (EC 1.14.18.1) is the key enzyme in the browning of fruits and vegetables and the darkening of skin, hair and eyes in animals (5,6). Tyrosinase plays a role in the initial step of melanin synthesis by catalyzing the oxidation of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and of DOPA to DOPAquinone, which can be processed to DOPAchrome (7). Thus, this enzyme is a good target for inhibition in the search for different types of depigmenting agents. Numerous tyrosinase inhibitors have been developed from both natural (8,9) and synthetic sources (10,11). In practical applications, however, just a small number of tyrosinase inhibitors are used as skin-whitening agents, due mainly to safety concerns (12). Therefore, the discovery of new and safe tyrosinase inhibitors is an important and ongoing focus for the medicinal, nutritional and cosmetic industries. More commonly known in Asia as safflower or red flower, Carthamus tinctorius L. is an annual plant belonging to the Compositae family and has been used as a natural pigment additive for food and cosmetics (13). C. tinctorius is also a famous traditional Chinese medicine that promotes blood circulation by treating blood stasis * Corresponding author. Tel.: þ886 4 26328001x15407; fax: þ886 4 26311167. E-mail address: [email protected] (Y.-S. Chen).
(14). The petals of C. tinctorius are commonly used for the treatment of trauma, chronic gastritis and cardiovascular and gynecological diseases (13,15). Moreover, several studies have demonstrated that C. tinctorius extract can have many effects, such as an anticoagulant, vasodilator, antioxidant, immunosuppressant and neuroprotector (16e18). Carthamus yellow (CY; CI Natural Yellow 5) is the major component of the yellow pigments of C. tinctorius. Two chalcones, safflomin A (Fig. 1A) and safflomin B (Fig. 1B), are the main compounds of carthamus yellow (19). Previous studies also demonstrated that carthamus yellow, including safflomin A and safflomin B, provides the main biological functions of C. tinctorius, such as vasodilative activity to reduce blood pressure and heart rate and anti-inflammatory, neuroprotective, anti-oxidative and antihepatic fibrosis activities (14,19e21). Although the biological activities of carthamus yellow have been reported broadly and some compounds from safflower seeds have been found to have anti-melanogenesis activity (22), no studies have directly revealed the inhibitory effect of carthamus yellow on tyrosinase and melanin production. Furthermore, the utilization of botanical tyrosinase inhibitors in medicine, food and cosmetics would increase the additive value of agricultural products. Thus, this present study is the first report to investigate carthamus yellow’s inhibition kinetics on tyrosinase and its effects on melanin production in melanoma cells. The results of this study indicate that carthamus yellow has the potential to become a depigmenting agent for medical, food and cosmetic applications.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.013
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RESULTS AND DISCUSSION
FIG. 1. Chemical structures of the two major compounds of carthamus yellow: (A) safflomin A, (B) safflomin B. MATERIALS AND METHODS Materials Carthamus yellow (no. 300-31782, safflomin A and safflomin B) was purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Arbutin, vitamin C (ascorbic acid), mushroom tyrosinase, L-tyrosine, Dimethyl sulfoxide (DMSO) and other chemicals were purchased from SigmaeAldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and trypsin-EDTA were purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Affymetrix/USB (Cleveland, OH, USA). Deionized distilled water (ddH2O) for solutions and buffers was obtained from the Milli-Q system (Millipore, Bedford, MA, USA). Tyrosinase activity assay and kinetic study For the tyrosinase activity assay, each 60 mL sample (containing 0.4e4.0 mg/mL carthamus yellow) was mixed with 100 mL of 1 mM L-tyrosine in phosphate buffer solution (pH 6.8). Then, 40 mL of mushroom tyrosinase solution (100 units/mL) was added to the mixture, which was then incubated for 25 min at 37 C. The spectrophotometric analysis was performed at 475 nm, and the inhibition of DOPAchrome formation was calculated as the inhibition percentage (23). The unit definition of tyrosinase activity is as follows: one unit will cause an increase in A280 of 0.001 per min at 25 C in a 3 mL reaction mixture containing L-tyrosine. For the kinetic study, different concentrations of Ltyrosine solution (0.3, 0.5 and 0.8 mM) were used. The velocities of the enzymatic reaction in 1.0 and 1.5 mg/mL carthamus yellow were calculated and compared with that of the non-inhibitory enzymatic reaction. The Michaelis constant (Km) and the maximal velocity (Vmax) of the tyrosinase activity were determined by the LineweavereBurk plot at various concentrations of L-tyrosine as a substrate. The Ki values were calculated from shared regression fits of substrateevelocity curves taken at different concentrations of the inhibitors (24). Cell line and cell culture The B16F10 melanoma cells (BCRC 60031) were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan, R.O.C.). The cells were maintained in DMEM supplemented with 10% FBS containing 100 units/mL penicillin and 100 mg/mL streptomycin at 37 C in a 5% CO2 incubator. Cells grown in various culture dishes at 80% confluence were used in all of the experiments. MTT assay for cell viability The B16F10 cells were seeded in 96-well plates (6 103 cells/well) using DMEM medium supplemented with 10% FBS for 24 h, after which the medium was replaced with FBS-free DMEM medium for another 24 h. The prepared cells were subsequently treated with different concentrations of carthamus yellow (1.0e4.0 mg/mL) for 48 h. Next, 100 mL (0.5 mg/mL) of MTT solution was added to cells, which were then incubated at 37 C for 30 min and washed twice with PBS. Finally, the PBS cleaned cells were lysed with 100 mL DMSO, and the absorbance was measured spectrophotometrically at 540 nm using an ELISA reader (25). Melanin content analysis For melanin content analysis, either 1.0e4.0 mg/ mL of carthamus yellow or 0.1 mg/mL of control inhibitors (vitamin C or arbutin) was used to treat B16F10 cells. B16F10 cells were seeded in 6-well plates (2 105 cells/well) using DMEM medium supplemented with 10% FBS for 24 h. The prepared cells were subsequently treated with CY or control inhibitors for 24 h. Cells were then detached using 0.05% trypsin-EDTA. Finally, the cell pellets were dissolved in 120 mL of 1 N NaOH for 1 h at 65 C, and the melanin content of melanoma cells was measured spectrophotometrically at 405 nm by an ELISA reader (26). Statistical analysis Quantitative data were analyzed using Student’s t-tests and are presented as mean standard error (S.E.) of three independent experiments. The P-values less than 0.05 were considered to be significant.
Inhibition of tyrosinase activity by carthamus yellow In the present study, we investigated the effect of carthamus yellow on tyrosinase inhibition to find a novel and effective substance for skin whitening. We found that carthamus yellow clearly reduced mushroom tyrosinase activity in a dose-dependent manner (Fig. 2). Our results showed that 0.4e4.0 mg/mL carthamus yellow reduced the activity of tyrosinase from 27.9% to 85.1%, respectively (Fig. 2), and that carthamus yellow suppressed tyrosinase with a half maximal inhibitory concentration (IC50) value of approximately 1.01 0.03 mg/mL. Comparatively, the IC50 value of vitamin C to mushroom tyrosinase in our experiments was approximately 0.12 0.01 mg/mL (data not shown). Thus, while the IC50 value of carthamus yellow was higher than vitamin C, we also found that carthamus yellow had a strong inhibitory activity toward mushroom tyrosinase. Kinetic analysis of carthamus yellow on tyrosinase The mode of tyrosinase inhibition by carthamus yellow was determined by LineweavereBurk plot analysis as shown in Fig. 3. The kinetic parameters of tyrosinase inhibition by carthamus yellow revealed that the Ki was 0.607 mg/mL (Table 1). Carthamus yellow at 1.0 and 1.5 mg/mL exhibited Km values of 1.678 and 2.709 mM, respectively (Table 1). However, carthamus yellow at 0, 1.0 and 1.5 mg/mL showed almost the same Vmax values of either 0.023 or 0.024 mmol/min (Table 1). In Fig. 3, the regressions of various carthamus yellow concentrations were fitted to intersect with the Y-axis at the same point. As the substrate (L-tyrosine) concentration varies, the Km value of tyrosinase increased in a dose-dependent manner without changing the Vmax, indicating that carthamus yellow is a competitive tyrosinase inhibitor (Fig. 3 and Table 1). Tyrosinase inhibitors with established efficacy include competitive inhibitors such as arbutin, azelaic acid, deoxyArbutin, hydroquinone and kojic acid (10,12), as well as the non-competitive inhibitors ellagic acid, glabridin and oxyresveratrol (27,28). Additionally, there are mixed-type tyrosinase inhibitors such as phthalic acid, heptadecahydroquinone and p-coumaric acid (29e31). Moreover, the major component of the yellow pigments of C. tinctorius is carthamus yellow, which in turn is mainly composed of the chalcone compounds safflomin A and safflomin B (Fig. 1). Several studies have demonstrated that chalcones are potent tyrosinase inhibitors with competitive inhibition abilities (32,33). In this study, carthamus yellow also exhibited potent inhibitory activity on tyrosinase through competitive inhibition, a feature similar to other chalcone compounds (Fig. 3 and Table 1).
FIG. 2. Inhibition of tyrosinase activity by carthamus yellow (CY) at various concentrations. Each value is expressed as mean S.E. (n ¼ 3).
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FIG. 4. Cell viability of B16F10 melanoma cells treated with various concentrations of carthamus yellow. Each value is expressed as mean S.E. (n ¼ 3). FIG. 3. LineweavereBurk plot of tyrosinase activity inhibited by carthamus yellow.
In an earlier study, Khatib et al. (33) demonstrated that the tyrosinase inhibitory activity of chalcones is due to its phenol group(s). The most important factor of the inhibitory activity of chalcones on tyrosinase is the position of the phenolic hydroxyl group(s) rather than their number (33). Consequently, we hypothesize that the tyrosinase inhibitory activity of carthamus yellow might also be provided by the phenol groups on the aromatic rings of the safflomin A and B molecules (Fig. 1). Effects of carthamus yellow on B16F10 melanoma cells To evaluate the effects of carthamus yellow on melanogenesis, we used B16F10 melanoma cells as a model. First, the effects of concentrations of 1.0e4.0 mg/mL carthamus yellow on the cell viability of B16F10 melanoma cells were assessed by a standard MTT method (Fig. 4). The result of this cell viability assay indicated that carthamus yellow at 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells (Fig. 4). Moreover, 4.0 mg/ mL carthamus yellow is almost the highest concentration that can be obtained in aqueous solution using a normal dissolving procedure (stir at room temperature). Thus, we used the concentrations of 1.0e4.0 mg/mL carthamus yellow for our later melanin content analysis. Although there were no statistically significant differences in cell viability between the control and carthamus yellow-treated B16F10 melanoma cells, cell viability slightly increased nonetheless in carthamus yellow-treated B16F10 melanoma cells (Fig. 4). This phenomenon may result from the components of carthamus yellow possibly having an ability to slightly enhance the growth of B16F10 melanoma cells. Melanin content analysis clearly demonstrated that carthamus yellow at concentrations higher than 2 mg/mL repressed the production of melanin in B16F10 melanoma cells (Fig. 5). The percentage of melanin content in 4 mg/mL carthamus yellowtreated B16F10 melanoma cells was decreased to 82.3 0.4% of the levels of melanin production of untreated cells. The percentages of melanin content in B16F10 melanoma cells treated with 0.1 mg/mL
of the control inhibitors vitamin C or arbutin were 87.9 1.6% and 56.8 1.7%, respectively (Fig. 5). These results show that the inhibitory activity of carthamus yellow on melanogenesis is not stronger than that of vitamin C or arbutin, but instead is within a comparable range. Thus, these results demonstrate that carthamus yellow is an effective and safe melanogenesis inhibitor. Finding safe and effective skin-whitening agents that can be used to prevent hyperpigmentation is important for many applications. As melanogenesis inhibitors are being developed, safety should be the most important consideration. In this study, carthamus yellow exhibited no cytotoxicity on B16F10 melanoma cells but had an efficient inhibitory activity on melanogenesis. Furthermore, carthamus yellow is already an edible and extensively used natural color additive for food and cosmetics. Therefore, we believe that carthamus yellow could be used as a safe and effective depigmenting agent for many applications. Moreover, several studies have demonstrated that chalcones are potent tyrosinase inhibitors, and thus, it would be interesting to compare the anti-melanogenesis activities of carthamus yellow with those of other chalcones. In summary, carthamus yellow decreased the activity of mushroom tyrosinase in a dose-dependent manner with an IC50 value of approximately 1.01 0.03 mg/mL. Carthamus yellow exhibited a mode of competitive inhibition with a Ki of 0.607 mg/mL. Moreover, the MTT assay indicated that carthamus yellow concentrations
TABLE 1. Kinetic parameters of tyrosinase inhibited by carthamus yellow. Inhibitor None CY
Conc. (mg/mL)
Km (mM)a
Vmax (mmol/min)a
1.0 1.5
0.923 1.678 2.709
0.023 0.024 0.023
Ki (mg/mL)a
0.607 a The kinetic parameters Km, Vmax and Ki were calculated from the LineweavereBurk plot.
FIG. 5. Melanin content of B16F10 melanoma cells treated with various concentrations of carthamus yellow. The vitamin C (VitC) and arbutin (Arb) treatments are positive controls. Each value is expressed as mean S.E. (n ¼ 3). *P < 0.05, compared with the control.
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of 1.0e4.0 mg/mL had no cytotoxicity in B16F10 melanoma cells. Melanin content analysis revealed that the percentage of melanin production in B16F10 melanoma cells could be decreased to 82.3 0.4% if treated with 4 mg/mL carthamus yellow. Altogether, the results of this study indicate that carthamus yellow has the potential to become a useful depigmenting agent for medical, food and cosmetic products in the future.
16.
17.
ACKNOWLEDGMENTS This work was supported by grants from the National Science Council, Taiwan, R.O.C. (NSC99-2313-B-126-002-MY3).
18. 19.
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