Antioxidant and Anti-Melanogenesis Effects of Colloidal Gold Camellia sinensis L. Extracts

Green tea extract derived from the leaves of Camellia sinensis L. (CS), is a representative beverage with antioxidant, anti-cancer, and anti-viral properties. CS extract is also used in cosmetics. Colloidal gold is generally a sol or colloidal suspension of gold nanoparticles in water. Colloidal gold green tea (CGCS), cultivated as a fertilizer using this colloidal gold solution, contains gold minerals and possesses anti-inflammatory, analgesic, and anti-tumor properties. However, the skin bioactivity of CGCS has not yet been investigated. In this study, we investigated the effect of the CGCS extract on skin whitening. CGCS extract contained high levels of phenols and flavonoids and displayed 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in a concentration-dependent manner. CGCS extract inhibited melanin synthesis and tyrosinase activity in B16F10 cells more effectively than the CS extract. Moreover, the CGCS extract decreased the expression levels of the melanogenesis-related proteins, tyrosinase, tyrosinase-related proteins (TRPs), and microphthalmia-associated transcription factor (MITF). In conclusion, our study showed that the CGCS extract inhibits the expression of tyrosinase, TRP-1, and TRP-2 via the downregulation of MITF, thereby inhibiting melanin synthesis. Therefore, CGCS can potentially be used as a skin-whitening ingredient in the cosmetic industry.


Introduction
Recently, the desire for life extension and a better quality of life have increased owing to the improved standards of living, which has also increased the interest in skincare. The destruction of the ozone layer due to environmental pollution increases human exposure to ultraviolet (UV) rays, ultimately increasing the incidence of skin diseases, such as pigmentation [1].
UV rays are directly involved in melanin pigment formation. Melanin is a natural pigment that is commonly found in nature and determines the color of the skin, hair, and eyes. Melanin protects the skin cells from damage by external stimulation, such as UV rays, and removes toxic substances. However, the overproduction of melanin induces the formation of freckles and melasma and causes skin cancer by promoting skin aging [2,3].
Currently, various substances, such as arbutin, kojic acid, ascorbic acid, linoleic acid, and hydroquinone, are used as whitening materials. These substances exhibit a whitening effect by inhibiting the activity of tyrosinase and are widely used commercially in the cosmetic, pharmaceutical, and food industries. However, their use is limited by their side effects, such as skin irritation, vitiligo, and cytotoxicity [11][12][13][14]. Therefore, there is a need for effective whitening materials from natural sources that exhibit few side effects are required [15][16][17].
Gold is reported to have calming, nourishing, and detoxification effects in the traditional medical book, Donguibogam. Recently, studies on the various effects of gold, such as skin activation, blood circulation promotion, arthritis pain relief, immunity, and brain activity enhancement have been published in Europe and the United States. Gold nanoparticles play an effective role as antioxidants by inhibiting the formation of reactive oxygen species (ROS) and scavenging free radicals [27]. In addition, colloidal gold green tea (colloidal gold Camellia sinensis L., CGCS) processed into nanoparticles aids in hepatocyte protection and exerts anti-tumor activity in tumor-causing cells [28].
Although the biological activities of gold and green tea are well known, the effect of CGCS on anti-melanogenesis in skin cells has not yet been reported. Therefore, in this study, we investigated the anti-melanogenic effect of the CGCS extract to determine its potential application as a skin-whitening agent.

Total Polyphenol and Flavonoid Contents
Polyphenols, which include catechins, resveratrol, and flavonoids, etc., are one of the antioxidant substances that convert free radicals in the human body into non-harmful substances. Polyphenols are known to have anti-aging, DNA and cellular protein protection, and anti-cancer effects [29]. Herein, the content of polyphenols and flavonoids in the CGCS extract was measured, with gallic acid and catechin as the respective standards. Based on the results, the content of polyphenols and flavonoids in the CGCS extract was 41 ± 0.05 mg GAE/g and 11 mg CE/g, respectively.

DPPH Radical Scavenging Activity of the CGCS Extract
DPPH radical scavenging activity is an indicator of the free radical scavenging ability of a sample and is widely used to evaluate antioxidant activity [30]. To measure the antioxidant effect of the CGCS extract, the DPPH radical scavenging activity was measured at treatment concentrations of 10-200 µg/mL. The CGCS extract increased the DPPH radical scavenging activity in a concentration-dependent manner and showed an EC 50 of 24.3 µg/mL. In particular, 100-200 µg/mL of the CGCS extract led to more than 95% radical scavenging activity, with values higher than those obtained with L-ascorbic acid (positive control) ( Figure 1).

Figure 1.
DPPH radical scavenging activity of CGCS extracts. The results are expressed as the mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001 compared with the control.

Cell Viability of the CGCS Extract
Cell viability was measured by a method using the ability of mitochondria to infiltrate the yellow water solubility substrate MTT tetrazolium into cells by the dehydrogenase action and reduce it from mitochondria to nonreceptivity MTT formazan [31]. When the viability of the B16F10 melanoma cells treated with 10-200 µg/mL of the CGCS extract was measured, no significant cytotoxicity was found. Therefore, subsequent experiments were performed with a concentration of 200 µg/mL or less ( Figure 2).

Cell Viability of the CGCS Extract
Cell viability was measured by a method using the ability of mitochondria to infiltrate the yellow water solubility substrate MTT tetrazolium into cells by the dehydrogenase action and reduce it from mitochondria to nonreceptivity MTT formazan [31]. When the viability of the B16F10 melanoma cells treated with 10-200 µg/mL of the CGCS extract was measured, no significant cytotoxicity was found. Therefore, subsequent experiments were performed with a concentration of 200 µg/mL or less ( Figure 2).

Inhibitory Effect of the CGCS Extract on Melanogenesis
Melanin is a dark brown or black pigment produced in an organelle called melanosomes in melanocytes. Skin color is determined by the amount of melanin and darkens as the amount of melanin increases [32]. To confirm the effect of the CGCS extract on melanogenesis, the melanin content in B16F10 melanoma cells was measured following treatment with α-MSH, CGCS extract, CS extract, and arbutin (the positive control). Compared with the α-MSH treatment group alone, the group treated with the CGCS extract had a decrease in melanin content in a concentration-dependent manner. Further, 200 µg/mL of

Inhibitory Effect of the CGCS Extract on Melanogenesis
Melanin is a dark brown or black pigment produced in an organelle called melanosomes in melanocytes. Skin color is determined by the amount of melanin and darkens as the amount of melanin increases [32]. To confirm the effect of the CGCS extract on melanogene-sis, the melanin content in B16F10 melanoma cells was measured following treatment with α-MSH, CGCS extract, CS extract, and arbutin (the positive control). Compared with the α-MSH treatment group alone, the group treated with the CGCS extract had a decrease in melanin content in a concentration-dependent manner. Further, 200 µg/mL of the CGCS extract reduced the melanin content by 7.6% relative to the CS extract and had a higher melanin inhibitory activity than arbutin ( Figure 3). . Cells were exposed to α-MSH (100 nM) alone or with the different samples or arbutin (100 µg/mL) for 72 h. The results are expressed as the mean ± SD from three independent experiments. ### p < 0.001 compared with the control, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the α-MSH.

Inhibitory Effect of the CGCS Extract on Intracellular Tyrosinase Activity
Tyrosinase is an enzyme that acts in the early stages of the melanin biosynthetic pathway and inhibits the activity of tyrosinase to prevent melanin synthesis [33]. Intracellular tyrosinase activity induced by α-MSH was measured after treatment with the CGCS extract, CS extract, and arbutin for 48 h. The CGCS extract decreased intracellular tyrosinase activity in a concentration-dependent manner. In addition, 200 µg/mL of the CGCS extract reduced tyrosinase activity by 23.6% compared to the CS extract and induced a higher tyrosinase inhibitory activity than the positive control, arbutin ( Figure 4). . Cells were exposed to α-MSH (100 nM) alone or with the different samples or arbutin (100 µg/mL) for 72 h. The results are expressed as the mean ± SD from three independent experiments. ### p < 0.001 compared with the control, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the α-MSH.

Inhibitory Effect of the CGCS Extract on Intracellular Tyrosinase Activity
Tyrosinase is an enzyme that acts in the early stages of the melanin biosynthetic pathway and inhibits the activity of tyrosinase to prevent melanin synthesis [33]. Intracellular tyrosinase activity induced by α-MSH was measured after treatment with the CGCS extract, CS extract, and arbutin for 48 h. The CGCS extract decreased intracellular tyrosinase activity in a concentration-dependent manner. In addition, 200 µg/mL of the CGCS extract reduced tyrosinase activity by 23.6% compared to the CS extract and induced a higher tyrosinase inhibitory activity than the positive control, arbutin ( Figure 4).

Inhibitory Effect of the CGCS Extract on MITF, Tyrosinase, TRP-1 and TRP-2 Protein Expression
MITF is an important transcriptional regulator of melanin synthesis. MITF regulates tyrosinase and several factors, such as TRP-1 and TRP-2. The effect of the CGCS extract on the expression of transcription factors involved in melanin synthesis was determined by western blot. The CGCS extract was found to decrease the expression of MITF, tyrosinase, TRP-1, and TRP-2 induced by α-MSH at 12-48 h ( Figure 5).

Inhibitory Effect of the CGCS Extract on MITF, Tyrosinase, TRP-1 and TRP-2 Protein Expression
MITF is an important transcriptional regulator of melanin synthesis. MITF regulates tyrosinase and several factors, such as TRP-1 and TRP-2. The effect of the CGCS extract on the expression of transcription factors involved in melanin synthesis was determined by western blot. The CGCS extract was found to decrease the expression of MITF, tyrosinase, TRP-1, and TRP-2 induced by α-MSH at 12-48 h ( Figure 5). (a)

Discussion
Melanin is an important pigment that determines the color of the skin, hair, and eyes [34]. Melanin also protects the skin from harmful effects, such as UV radiation, and DNA damage [35,36]. However, excessive production of melanin due to excessive UV exposure causes abnormal hyperpigmentation, which may result in freckles, age spots, and melasma [37].
In this study, we evaluated the antioxidant and anti-melanogenic effects of the CGCS extracts grown using a colloidal gold solution as a fertilizer. The antioxidant effect of the CGCS extract was evaluated by measuring the polyphenol and flavonoid contents, and DPPH radical scavenging activity. The total polyphenol and flavonoid contents of the extract were 41 ± 0.05 mg GAE/g and 11 mg CE/g, respectively. In addition, the CGCS extract increased the DPPH radical scavenging activity in a concentration-dependent manner.

Discussion
Melanin is an important pigment that determines the color of the skin, hair, and eyes [34]. Melanin also protects the skin from harmful effects, such as UV radiation, and DNA damage [35,36]. However, excessive production of melanin due to excessive UV exposure causes abnormal hyperpigmentation, which may result in freckles, age spots, and melasma [37].
In this study, we evaluated the antioxidant and anti-melanogenic effects of the CGCS extracts grown using a colloidal gold solution as a fertilizer. The antioxidant effect of the CGCS extract was evaluated by measuring the polyphenol and flavonoid contents, and DPPH radical scavenging activity. The total polyphenol and flavonoid contents of the extract were 41 ± 0.05 mg GAE/g and 11 mg CE/g, respectively. In addition, the CGCS extract increased the DPPH radical scavenging activity in a concentration-dependent manner. According to previous studies, water and 40% ethanol green tea extracts showed the highest polyphenol and flavonoid contents. Notably, 100 µg/mL green tea extract shows activity similar to that of L-ascorbic acid [38,39]. In our experiment, the CGCS extract showed superior scavenging activity compared to L-ascorbic acid.
CGCS extract inhibited α-MSH-induced melanin production and tyrosinase activity compared to the CS extract. In a previous study, green tea extract has been reported to inhibit tyrosinase activity and melanin production via its active ingredients, such as ECG, GCG, EGCG, and EGC [40]. In addition, gold nanoparticles act as antioxidants by inhibiting the formation of ROS and scavenging free radicals [27]. Therefore, CGCS extract shows excellent anti-melanogenic activity owing to the synergistic effect of gold and the active compounds of green tea.
These results suggest that the CGCS extract inhibits melanogenesis via MITF downregulation and the inhibition of tyrosinase and TRPs signaling pathways in B16F10 mouse melanoma cells.

Preparation of the CGCS Extract
The CGCS and CS extracts were purchased from Bohyang Dawon (Boseong, Korea), and authentication and identification of samples were conducted for the study by Mr. Youngki Choi at Bohyang Dawon, who has expertise in classifying green tea. The dried samples were milled and added to distilled water, which was extracted at 100 • C for 6 h. The extracts were filtered using No.2 filter paper (Advantec, Tokyo, Japan), evaporated to dryness with a rotary evaporator, and then lyophilized to eliminate residual water. The yields of the CGCS and CS extracts were approximately 27.0% and 15.7%. All extracts were stored at −20 • C until used.

Total Polyphenol Contents
The total polyphenol content was measured using the Folin-Denis method with some modifications [46]. Briefly, 500 µL of sample diluted to an appropriate concentration and 500 µL of Folin reagent were mixed and reacted for 3 min at room temperature. Thereafter, 500 µL of 10% Na 2 CO 3 was added to the mixture, which was allowed to react for 1 h in the dark at room temperature. The absorbance was measured at 760 nm using a UV/Vis Spectrophotometer (Optizen 2120 UV, Mecasys, Daejeon, Korea). The calibration curve was prepared using gallic acid as standard.

Total Flavonoid Content
The total flavonoid content was measured using the method of Woisky and Salatino, with some modifications [47]. Briefly, 200 µL of sample diluted to an appropriate concentration and 800 µL of 80% ethanol were added to 60 µL of 5% NaNo 2 , mixed, and allowed to react for 5 min. Thereafter, 10% AlCl 3 was added to the mixture, which was allowed to react for 5 min at room temperature.
Then, in 1 M NaOH solution, the absorbance was measured at 510 nm using a UV/vis Spectrophotometer (Optizen 2120 UV, Mecasys, Daejeon, Korea). The calibration curve was prepared using catechin as standard.

DPPH Radical Scavenging Activity
DPPH radical scavenging activity was measured using the Blois method [48], with modifications. Different concentrations of the extract and ascorbic acid (positive control) were added to 0.2 mM DPPH solution. Thereafter, the solution was mixed and incubated for 30 min at room temperature in the dark. The absorbance was measured at 515 nm using a Microplate Spectrophotometer (Multiskan Sky; Thermo Fisher Scientific, Waltham, MA, USA). The DPPH radical scavenging rate was calculated using the following formula (Equation (1)

Cell Culture
B16F10 melanoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA). B16F10 cells were cultured in DMEM containing 10% FBS and 1% P/S in a humidified atmosphere with 5% CO 2 at 37 • C.

Cell Viability Assay
The cell viability was determined using the MTT assay method [49]. B16F10 melanoma cells were seeded at a density of 1 × 10 4 cells/well in a 96-well plate and incubated with various concentrations of the extracts (10-200 µg/mL) for 48 h at 37 • C. After incubation, 0.5 mg/mL MTT solution was added to the wells, and the plate was further incubated for 3 h at 37 • C. The medium was subsequently removed and 200 µL DMSO was added.
The plate was then gently shaken for 15 min. The absorbance was measured at 570 nm using a Microplate Spectrophotometer (Multiskan Sky; Thermo Fisher Scientific, Waltham, MA, USA).

Measurement of Melanin Content
B16F10 melanoma cells were seeded in a 60-mm dish at a density of 2 × 10 5 cells/well and incubated for 24 h at 37 • C. The medium was replaced with fresh medium containing various concentrations of extracts (10-200 µg/mL), α-MSH (100 nM), arbutin (100 µg/mL), and incubated for 48 h. The cells were harvested after washing with PBS. The pellet obtained via centrifugation (12,000 rpm, 15 min) was dissolved in 1N NaOH containing 10% DMSO at 80 • C for 1 h. The absorbance was measured at 475 nm using a Microplate Spectrophotometer (Multiskan Sky; Thermo Fisher Scientific, Waltham, MA, USA).

Intracellular Tyrosinase Activity Assay
B16F10 melanoma cells were seeded in a 60-mm dish at a density of 2 × 10 5 cells/well and incubated for 24 h at 37 • C. The medium was replaced with fresh medium containing various concentrations of extracts (10-200 µg/mL) and α-MSH (100 nM). Arbutin (100 µg/mL) was administered to cells as a positive control and the cells were incubated for 48 h. The cells were then washed with cold PBS and lysed with Pro-Prep lysis solution (iNtRON Biotechnology, Seongnam, Korea). The cell lysates were clarified by centrifugation at 13,000 rpm for 5 min at 4 • C. The lysates were dissolved in 0.1 M sodium phosphate buffer (pH 6.8) and treated with L-dopa (1 mg/mL) in a 96-well plate at 37 • C for 1 h. The absorbance was measured at 475 nm using a Microplate Spectrophotometer (Multiskan Sky; Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot Analysis
B16F10 cells were seeded at densities of 2 × 10 5 cells/well in a 60-mm dish and incubated with extracts and α-MSH (100 nM). Thereafter, the cells were washed with cold PBS and lysed with Pro-Prep lysis solution for 15 min on ice. The cell lysates were centrifuged at 13,000 rpm for 5 min at 4 • C and the lysed supernatant protein was measured using the Bradford assay. A total of 20 µL of protein was digested via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was incubated with a blocking buffer (5% skim milk and 0.1% Tween 20 in TBS) for 1 h and then a primary antibody for 1 h. GAPDH was used as an internal control. The membrane was washed four times with TBS containing 0.1% Tween 20, and then incubated with horseradish peroxidase (HRP) conjugated anti-mouse and anti-rabbit secondary antibody for 1 h at room temperature. Protein band detection on the PVDF membrane was performed using Western Bright TM ECL reagent and Davinch-Western TM Imaging System (Davinch-K, Seoul, Korea).

Statistical Analysis
The results are presented as mean ± standard deviation (SD) of three independent experiments, and statistical significance was obtained using Student's t-test and ANOVA. All statistical analyses were conducted using SPSS statistical software 22.0. * p < 0.05, ** p < 0.01, *** p < 0.001 values were considered to indicate significant difference.

Conclusions
In this study, we demonstrated that the CGCS extract has excellent antioxidant activity and inhibitory effect on melanin synthesis in cells as it reduced the protein expression of MITF and related enzymes (tyrosinase, TRP-1, TRP-2). Altogether, these findings suggest that the CGCS extract has high potential as a skin-whitening agent, additional studies, such as extraction conditions, molecular mechanism analysis, and clinical tests are needed for the application as a functional cosmetics ingredient.