Food Sci. Biotechnol. 24(3): 1087-1096 (2015) DOI 10.1007/s10068-015-0139-3

RESEARCH ARTICLE

Skin Whitening Activity of Supercritical Fluid Extract from Spent Coffee in B16F10 Melanoma Cell Hea Mi Sung, Hyun Jung Jung, Ji Sun Sin, Ki Myong Kim, and Ji-Hyang Wee

Received July 18, 2014; revised January 23, 2015; accepted January 25, 2015; published online June 30, 2015 © KoSFoST and Springer 2015

Abstract In the present study, we evaluated the skin whitening effects of supercritical fluid extract (SFE) of the spent coffee (SC) on tyrosinase inhibition activity, melanin synthesis inhibition activity and DOPA staining. Hot water extracts from coffee bean (CB) and SC and drip extracts from CB were employed for control. Inhibitions of melanin activities of SFE (SC) at short processing time were significantly higher than other values under the same extract processing. In comparison, inhibition of melanin activities appeared at high pressure in CB. Tyrosinase inhibition activity of SFE (SC) extracted for 1 h at 150 bar was significantly higher than those of SFE (CB and SC) even of controls. According to the above results, SFE (SC) extracted for 2 h or less at 450 bar showed significantly high activities in the melanin synthesis inhibition and tyrosinase inhibition. Keywords: coffee canephora var., melanoma cell, melanin synthesis, tyrosinase, fatty acid

Introduction Coffee is currently produced in over 70 countries, and 11,700 tons were imported into South Korea in 2010 alone. The consumption of coffee has been increasing at an exponential rate (1). Spent coffee are a byproduct of coffee consumption, produced when the beverage is extracted Hea Mi Sung, Hyun Jung Jung, Ji Sun Sin, Ji-Hyang Wee (
) Food Research Institute, Jeonnam Bioindustry Foundation, Naju, Jeonnam 520-330, Korea Tel: +82-61-339-1210; Fax: +82-61-336-9627 E-mail: [email protected] Ki Myong Kim Department of Food and Nutrition, Honam University, Gwangju 506-714, Korea

from the coffee beans at home or at coffee shops. Approximately 0.91 kg of spent coffee is generated as waste per kilogram of coffee beans. However, a significant quantity of coffee oil, which is composed of fatty acids and sterol groups, has been reported to remain in spent coffee (2). Thus, further research on the potential utility of the active components in spent coffee is needed. Coffee can be divided into two primary varieties: Arabica and Robusta. Depending on the variety, the coffee exhibits differences in sugar, protein, and fat content. In particular, amino acid, chlorogenic acid, caffeine, and trigonelline content has been reported to be higher in the Robusta variety (3). The characteristic fatty acid components of coffee include palmitic acid, stearic acid, linoleic acid, and oleic acid, and these components have been shown to be effective in the treatment of diabetes, heart disease, and hypercholesterolemia. They have also been shown to be effective for wrinkle reduction and skin whitening (4). The sterol components of coffee include β-stigmasterol, campesterol, and stigmasterol. These sterols have been reported to reduce serum cholesterol (5). Therefore, the extraction and utilization of these fatty acids and sterols from spent coffee by appropriate methods may allow the use of spent coffee as a functional raw material rather than a waste product. A supercritical fluid is a substance in a liquid or gaseous state that surpasses a given high-pressure, high-temperature point, called the supercritical point, to reach a critical state. As the substance exceeds the critical state, the thermal motion of the molecules becomes violent. However, the violent molecular movements are not accompanied by phase changes; therefore, the substance can undergo continuous changes between a low-density state, similar to that of an ideal gas, and a high-density state, similar to that of a standard liquid. Extraction at the supercritical state may induce greater intra-substance permeability, permitting

1088

a level of extraction difficult to achieve with conventional techniques. The supercritical fluid extraction (SFE) of isomers and natural ingredients facilitates the purification of polymeric materials and protects certain useful components from chemical modification or destruction. Currently, carbon dioxide is the most widely used solvent for supercritical extraction, because its critical temperature is near room temperature. Carbon dioxide is non-flammable, non-polar, and dissolves organic substances by behaving like an organic solvent in its supercritical state (6). Due to such characteristics, supercritical extraction with carbon dioxide is suitable for extracting natural compounds with low polarity and is commonly used for the extraction of fatty acids. Melanin, one of the major pigments present in the skin, is produced by melanin vesicles (melanosomes) in melanocytes residing in the basal layer of the epidermis. After it is produced, melanin is transported through dendrites to the keratinocytes of the epidermis. Through a continuous keratinization process, melanocytes are produced and cause pigmentation of the skin. Melanin protects the skin against ultraviolet radiation. However, excess production or abnormal distribution of melanin produces spots, freckles, and speckles (7-9). The most important factor in the synthesis of melanin is tyrosinase, a polymerase that catalyzes the rate-determining step. α-Melanocyte stimulating hormone (α-MSH) is a neuropeptide produced from proopiomelanocortin (POMC) and secreted by melanocytes; it has been shown to stimulate tyrosinase activation. Constant secretion of αMSH increases tyrosinase activation in melanosomes, as well as tyrosinase mRNA expression. In addition, it increases melanin synthesis and deposition, resulting in skin pigmentation (10,11). Consequently, the identification of inhibitors of tyrosinase activation has been the focus of much research on skin whitening (12,13). The food, cosmetic, and pharmaceutical industries have been actively conducting research to produce functional food products, functional cosmetics, and medications that inhibit tyrosinase activity (14-16). In the present study, we investigated whether spent coffee, usually discarded as waste after the brewing process, could be used as a functional raw material for the production of skin whitening agents. We varied the conditions of supercritical fluid extraction with canbon dioxide and evaluated the potential skin-whitening effects of the various extracts to identify the optimal conditions for supercritical fluid extraction. To objectively evaluate the skin whitening effects of the coffee ground extracts produced using the supercritical method, we compared their potential skin whitening effects with those of supercritical fluid extracts of coffee bean, hot-water extracts of coffee bean, and hot-water extracts (HWE) of

Sung et al.

spent coffee. Finally, we comprehensively evaluated the above results to investigate the recyclable value of spent coffee.

Materials and Methods Materials The coffee used in the present study was a Robusta variety of Vietnamese origin. We purchased coffee that had already been roasted and ground (1kgcoffee Co., Gwangju, Korea), and refrigerated the coffee prior to use. The B16F10 melanoma cells used for skin-whitening evaluation were purchased from Korean Cell Line Bank (Seoul, Korea). 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide inner salt (XTT), phenazine methosulfate (PMS), α-melanocyte stimulating hormone (α-MSH), arbutin, theophylline, L-3,4-dihydroxyphenyl alanine (L-DOPA), tyrosinase, Bradford Reagent, and dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich (St. Louis, MO, USA). The materials used for cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibioticantimycotics, were purchased from Gibco BRL (Grand Island, NY, USA). Hot water extract and drip extract Coffee (90 g) was placed in a coffee maker (DW-101 TS; Seoul, Korea), and 1,800 mL of tap water was poured into the coffee maker. The resulting drip coffee produced at a temperature of approximately 80-85oC was used as the drip extract (DE). The coffee residue left in the filter after dripping was subjected to drying in a 40oC oven to produce spent coffee. In order to ensure that the spent coffee had the same water content as coffee bean, the spent coffee were dried to a water content below 4%. The HWE of coffee bean were prepared by pouring 600 mL of distilled water at 80oC into 30 g of coffee bean, and letting the mixture stand for 1 h. The HWE of spent coffee were produced using the process described above. DE and HWE were freeze-dried and pulverized into powder for use in the following experiments (Table 1). The HWE from coffee bean (CB) and spent coffee (SC) were used as control groups to compare the effects of the supercritical extracts. Supercritical fluid extract SFE was performed using a SFE system (SFE 5L; Natex Prozesstechnologie GmbH, Ternitz, Austria) supplied by UMax Ltd. One kilogram of coffee bean and 1 kg of spent coffee were inserted into the closed extraction system. The ground coffee and spent coffee were subject to extraction at a temperature of 40oC under varying pressure conditions of 150, 250, 350, and 450 bar (Table 1). Carbon dioxide was used as the supercritical fluid, and it was supplied using a diaphragm

1089

Whitening Effects of Spent Coffee Table 1. Coffee bean and spent coffee of extract conditions Item

Extract method1)

Pressure (bar)

Temperature (oC)

Time (h)

HWE

-

80

1

DE

-

80-90

0.016

SFE

150 250 350 450

40

1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3

150

HWE

-

80

1

250

SFE

150 250 350 450

40

1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3

Coffee bean

Spent coffee

Table 2. Yield in coffee extracts Pressure (bar)

Time (h)

DE

-

0.016

27.1

-

HWE

-

1

24.9

24.9

1 2 3 1 2 3 1 2 3 1 2 3

2.1 2.5 1.7 3.0 1.1 1.8 3.9 1.6 2.0 3.9 1.5 3.9

3.3 3.1 2.7 9.1 0.8 0.7 9.8 0.4 0.4 9.9 0.3 0.5

SFE 350

1)

HWE, hot water extract; DE, Drip hot water extract; SFE, supercritical fluid extract.

metering pump LDE1 type mass flow meter (LEWA GmbH, Leonberg, Germany) at a rate of 30 kg/h. The SFE samples were acquired at predetermined time intervals of 1, 2, and 3 h after initiating extraction. In other words, 1 h after initiating extraction, a sample was acquired and the extraction recommenced. The above process was repeated at the 2 and 3 h marks to acquire samples at predetermined time intervals. Measurement of total phenolic compounds The total polyphenol content were determined by Folin-Denis colorimetric method (17). After pouring 900 µL of distilled water and extracts of coffee bean or spent coffee (1 mg/100 µL) into a test tube, 100 µL of Folin-Ciocalteu reagent was added and mixed thoroughly. The mixture was incubated for 5 min at room temperature; then, 1 mL of 7% sodium carbonate solution and 400 µL of distilled water were added and mixed thoroughly. The resulting mixture was incubated at room temperature for 90 min to allow development of the color reaction. The absorbance of the mixture was measured at 750 nm. The standard calibration curve was constructed using of gallic acid. This curve was used to calculate the total phenolic compounds present in test samples. Measurement of fatty acids Fatty acid analysis was performed using the method provided in the Food and Drugs Safety Office’s Food Standards Codex (18). First, 25 mg of test sample was measured into a screw-cap tube. Then, we added 1.5 mL of a solution consisting of 0.5 N sodium hydroxide and methanol and heated the resulting mixture at 100oC for 5 min. The mixture was then cooled to room temperature. Next, we added 2 mL of a solution consisting of methanol and boron trifluoride (Fluka Co., Buchs, Germany). The resulting mixture was subjected to

Yield (%)

Extraction1)

450

Coffee bean Spent coffee

1)

DE, Drip hot water extract; HWE, hot water extract; SFE, supercritical fluid extract

100oC for 30 min and cooled to room temperature. Finally, 1 mL of isooctane was added to the solution, mixed, and left to stand to allow phase separation. The top layer (isooctane) was then removed and subjected to gas chromatographic analysis (Shimadzu GC 2010; Shimadzu, Kyoto, Japan). The conditions for analysis are shown in Table 2. Cell culture The B16F10 melanoma cells were maintained in DMEM containing 10% FBS, penicillin, and streptomycin (100 g/mL). Cells were placed in an incubator at 37oC under 5% CO2 and 95% humidity. The cultured cells were fed 2-3 times per week and rinsed with phosphate buffer saline (PBS) within 6-7 days of cultivation. The cells were detached from culture dishes using 0.05% trypsin-0.02% EDTA in PBS. Cell viability assay Cell survival was quantified by a colorimetric XTT assay that measures mitochondrial activity in viable cells (19). First, the B16F10 cells were seeded in 6-well plates at 1×105 cells/well. The cells were cultured for 24 h; then, DMEM was removed and the cells were treated with extracts of CB and SC at concentrations of 0-1,000 µg/mL. On the eighth day, the medium was removed, the plate was rinsed with PBS, and 250 µL of XTT-phenazine methosulfate (PMS) solution (1 mg XTT10 µg PMS/mL) was added to the cells, and incubated at 37oC for 2 h. After incubation, the absorbance was measured at a wavelength of 450 nm by a spectrophotometer. Measurement of tyrosinase inhibition activity Intracellular tyrosinase activity was measured using the

1090

method described previously by Martínez-Esparza et al. (20). First, the B16F10 cells were placed in 6-well plates at 1×105 cells/well. The cells were cultured for 24 h and then coffee extracts (100 µg/mL) and α-MSH (2 µM) were added to the wells. The cells were subjected to seven additional days of culture. After seven days, the plates were rinsed with PBS. 200 µL of lysis buffer [5 mM EDTA, 0.1 M SPB (pH 6.8), 1% Triton X-100, 0.1% 0.1 M PMSF] was added to the wells and the cells were collected. After disrupting the cells on ice for 30 min, the disrupted cells were centrifuged for 30 min at 4oC at 16,000×g to separate the supernatant from the pellet. The supernatant was used to measure tyrosinase activity. Protein quantification was performed to using the Bradford reagent and measured at an absorbance of 595 nm. The calculated protein quantity was equally divided and 0.1 M SPB was added to yield a volume of 50 µL. The mixtures were subsequently treated with 50 µL of 10 mM L-DOPA and 10 µL of tyrosinase (20 units) at 37oC for 30 min. The changes in absorbance were measured at 10 min intervals at 475 nm. Measurement of melanin content Total melanin content was measured using a modification of the method published by Hosoi et al. (21). B16F10 cells were seeded in 6-well plates at 1×105 cells/well. The cells were incubated for 24 h, and then coffee extract (100 µg/mL) and α-MSH (2 µM) were added to each well. After the 7-day culture period, the plates were rinsed with PBS. Then, a lysis buffer [5 mM EDTA, 0.1 M sodium phosphate buffer (SPB pH 6.8), 0.1% Triton X-100] was used to disrupt the cells. The disrupted cells were placed in tubes and centrifuged for 20 min at 16,000×g. After the supernatant was removed from the pellet, 300 µL of 1 N sodium hydroxide and 10% DMSO was added and incubated at 90oC for 1 h. After the pellet was dissolved, the absorbance of the solution was measured at 475 nm. DOPA staining DOPA staining was performed to visualize the capacity of the extracts to inhibit melanin synthesis, using a modification of the method of Kim et al. (22). B16F10 cells were seeded in 6-well plates at 1×105 cells/ well. The cells were cultured for 24 h and then coffee extracts (100 µg/mL) and α-MSH (2 µM) were added to each well. The cells were then cultured for seven additional days. At the end of the 7-day culture period, the cells were fixed for 30 min with 4% formalin, rinsed with PBS, and reacted with 0.1% L-DOPA for 4 h at room temperature. After incubation with L-DOPA, the cells were subject to a second PBS rinse; then, the cells were fixed for 30 min with 10% formalin, rinsed with PBS and dehydrated using ethanol. The fixed cells were observed under an optical microscope (BX41-PH; Olympus Co., Tokyo, Japan).

Sung et al.

Statistical analysis The results are shown as mean± standard deviation (SD). Significance tests among the experimental groups were performed using Student’s t-test and Duncan’s multiple range test. The significance level of the statistical analysis was p<0.05.

Results and Discussion Extraction yields The yield of HWE (CB) was 24.9%, whereas DE was 27.1%; thus, DE produced a slightly higher yield than HWE. The yield of HWE (SC) was 24.9%, identical to the yield from HWE (SC) (Table 2). The yield of SFE (CB) was confirmed from 1.1 to 3.9%. And, the yield of SFE (SC) ranged from 0.3 to 9.9%. Under identical pressure conditions, the yield of SFE were highest at 1 h, both CB and SC. At 1 h extraction, the yield SFE (SC) was higher than SFE (CB). In particular, the yields from SFE (SC) were high at pressures of 250, 350, and 450 bar (9.1, 9.8, and 9.9%) respectively. However, SFE (CB) produced yields of 3.0, 3.9, and 3.9% under the same pressures. The above results show that SFE (SC) produced a higher yield than SFE (CB) at short-time extracts. Under supercritical conditions, carbon dioxide exhibits characteristics similar to organic solvents, and the use of SC, which had already lost many water-soluble substances through drip extraction, allowed for more efficient extraction than CB. At 250 bar, however, yields of SFE (SC) were 9.1, 0.8, and 0.7% at 1, 2, and 3 h, respectively, showing a dramatic decrease in yield at 2 h. Similar results were observed at pressures of 350 and 450 bar. Whereas SFE (CB) exhibited slow and consistent extraction over time, SFE (SC) were almost completely extracted within the first time, demonstrating the superiority of SFE (SC) for extraction. Furthermore, both SFE (CB) and SFE (SC) exhibited high yields under pressures above 250 bar. These results assure the findings of a previous study by Couto et al. (23), which showed that an increase in pressure during the extraction process increases the carbon dioxide density, thereby increasing the permeability of the coffee microstructure to the supercritical fluid (6). Total polyphenolic compound extraction Phenolic compounds serves as anti-oxidants by eliminating reactive oxygen species (ROS) within the human body. Such antioxidant activity has been shown to inhibit melanin production (24). To evaluate the influence of experimental sample, we analyzed the total polyphenolic content of experimental extracts. We found that DE was confirmed to contain 21.5 g/100 g. HWE (CB) contained 11.2 g/100 g, HWE (SC) contained 9.9 g/100 g, and SFE (CB) and SFE (SC)

Whitening Effects of Spent Coffee

1091

Fig. 1. Total phenol contents of coffee extracts. Pressure (bar), super critical fluid extract; HWE (CB), hot water extract of coffee bean; DE (CB), drip hot water extract of coffee bean; HWE (SC), hot water extract of spent coffee. Each value was expressed as the mean±SD; *Significantly different from that of the DE treatment at p<0.05; #Significantly different from that of the HWE treatment at p<0.05.

contained under 1 g/100 g of polyphenolic compounds. Thus, HWE were contained significantly higher total polyphenolic compound content than SFE (Fig. 1). In the present study, HWE was shown to solubilize hydrophilic substances more effectively than SFE (6), as demonstrated by the finding that the total polyphenolic compound content of DE and HWE were higher than SFE. Fatty acid extraction The results of fatty acid analysis of the extracts are shown in Table 3. The fatty acid content of HWE (both of CB, SC) was minimal or non-existent. However, the fatty acid content of SFE was significantly higher than HWE. In SFE, the carbon dioxide exhibits characteristics similar to those of organic solvents and more effectively extracts hydrophobic fatty acids. SFE (both of CB, SC) exhibited higher total fatty acid and individual fatty acid content following at pressures >250 bar for 1 h than for 2 or 3 h. The total fatty acid and individual fatty acid content decreased when extracting for 3 h. SFE (SC) exhibited a higher total fatty acid content than SFE (CB). When CB was extracted to SFE at pressures of 150, 250, 350, and 450 bar for 1 h, the total fatty acid content was 21.7, 32.4, 41.7, and 42.0 g/kg, respectively. However, the total fatty acid content of SC was 32.0, 93.3, 129.5, and 121.2 g/kg, respectively. Due to the high diffusion rate and low viscosity of supercritical carbon dioxide, the permeability of non-polar substances is higher than that of polar substances (6). Thus, because the SC exhibited lower polarity due to the elimination of polar substances through the drip extraction,

the microstructure of SC was more permeable to the SFE solvent, effectively increasing the fatty acid content. Cell toxicity In order to investigate the toxicity of extracts of CB and SC on B16F10 cells, an XTT assay was performed. As shown in Fig. 2, no extracts exhibited cell toxicity at a concentration of 100 µg/mL. However, at a concentration of 300 µg/mL, HWE of CB, SC and DE all exhibited cell toxicity. SFE (CB or SC) did not exhibit cell toxicity at the same concentration (300 µg/mL). Based on these results, we performed assays to determine inhibition of melanin synthesis and tyrosinase activity using a concentration of 100 µg/mL, at which no extracts exhibited cell toxicity. Inhibition of tyrosinase activity Among the HWE, the tyrosinase inhibition by the SC was highest, at 33.5% inhibition, followed by CB (29.3% inhibition) and DE (27.5% inhibition). Among the SFE, SC processed at 150 bar for 1 h showed the highest capacity to inhibit tyrosinase (37.3% inhibition), which was significantly higher (p<0.05) than the capacity of the other SFE (both of CB, SC) (Fig. 3). However, SFE (CB) processed at pressures above 350 bar exhibited high tyrosinase inhibition. The SFE (CB) did not inhibit tyrosinase activity when produced at pressure conditions below 250 bar, i.e., inhibition of tyrosinase activity was only exhibited at pressures above 350 bar. Based on these results, the activity of SFE (SC), which exhibits high tyrosinase inhibition activity, was expected to be high even at low extraction pressure and short extraction duration.

1092

Sung et al.

Table 3. Compositions of fatty acids in coffee extracts (g/kg equivalent of coffee powder) Coffee bean1)

Pressure (bar)

Time (h)

Palmitic acid

Stearic acid

Oleic acid

Linoleic acid

Arachidic acid

Linolenic acid

Total Fatty acid

HWE

-

1

00.2

0.0

0.0

0.2

0.0

0.0

0.4

DE

-

0.016

00.0

0.0

0.0

0.0

0.0

0.0

0.0

1 2 3 1 2 3 1 2 3 1 2 3

07.4 08.2 04.4 10.1 04.0 05.6 12.8 05.4 06.1 13.0 04.9 11.5

1.3 1.9 1.1 2.1 0.9 1.3 2.7 1.2 1.4 2.8 1.1 2.6

2.2 2.7 1.6 3.3 1.3 1.9 4.3 1.9 2.1 4.3 1.7 3.9

10.4 13.0 07.4 15.8 06.3 08.9 20.4 08.8 09.9 20.5 07.7 18.2

0.4 0.7 0.4 0.8 0.3 0.5 1.1 0.4 0.6 1.0 0.4 1.1

0.0 0.2 0.0 0.3 0.1 0.0 0.4 0.2 0.2 0.4 0.0 0.0

21.7 26.7 14.9 32.4 12.9 18.3 41.7 17.9 20.2 42.0 15.7 37.3

150

250 SFE 350

450 Spent coffee

Pressure (bar)

Time (h)

Palmitic acid

Stearic acid

Oleic acid

Linoleic acid

Arachidic acid

Linolenic acid

Total Fatty acid

HWE

-

1

00.0

0.0

0.0

00.0

0.0

0.0

00.0

1 2 3 1 2 3 1 2 3 1 2 3

10.9 11.7 05.0 28.4 02.0 01.5 39.8 01.2 01.1 37.4 00.9 01.3

1.8 2.0 1.9 6.0 0.6 0.4 8.5 0.3 0.3 8.1 0.2 0.3

3.2 3.5 2.4 9.5 0.8 0.6 13.40 0.4 0.4 12.60 0.3 0.4

15.5 17.7 10.4 45.4 03.6 02.5 63.3 02.0 01.7 59.1 01.4 02.0

0.6 0.6 1.0 2.3 0.4 0.2 3.1 0.1 0.1 3.1 0.1 0.1

0.0 0.3 0.0 0.9 0.0 0.0 1.3 0.0 0.0 0.9 0.0 0.0

32.0 35.9 20.8 93.3 07.3 05.2 129.50 04.0 03.5 121.20 02.9 04.2

150

250 SFE 350

450 1)

HWE, hot water extract; DE, Drip hot water extract; SFE, supercritical fluid extract.

The inhibition of tyroisnase activity can be accomplished by several mechanism, as follows: First, several polyphenols are accepted as substrates by tyrosinase. It is serves by the same slow-binding and competitive inhibition mode against mushroom tyrosinase (25,26). Second, a long-chain fatty acid inhibited monophenolase and diphenolase of mushroom tyrosinase (27,28). Third, the inhibitory mechanism was proposed by binding of the compound to some site of the tyrosinase. A compounds of various plants reversibly bind to tyrosinase and reduce its catalytic capacity (29). In this study, although skin whitening efficacy does not increase proportionally depending on the fatty acid and phenolics, fatty acid was co-act with various compounds of SFE on tyrosinase inhibitory. It is determined that to counteract or enhance the efficacy. However, it is necessary to additional study how individual fatty acid and another compounds of SFE affect on tyrosinase inhibitory. Inhibition of melanin synthesis in melanocytes The

present study evaluated the capacity of extracts of CB and SC in inhibiting melanin synthesis using the B16F10 mouse cell line. The results are shown in Fig. 4. Comparison with the findings for the control group showed that HWE, DE, and SFE all inhibited melanin synthesis significantly. Among the HWE, CB (29% inhibition) exhibited significantly greater capacity to inhibit melanin synthesis than SC (21% inhibition) or DE (17% inhibition). Among the SFE, CB produced under 450 bar and 2 h duration (35% inhibition), SC produced under 250 bar and 2 h duration (34% inhibition), and SC produced under 350 bar and 1 h duration (34% inhibition) exhibited a strong inhibitory effect on melanin synthesis, similar to that observed with the positive control, arbutin. This effect was significantly greater than that of HWE (CB) (29% inhibition), demonstrating that SFE (SC) exhibited a greater inhibitory effect on melanin synthesis than HWE (CB). Alhough SFE (SP) contained less than 1 g/100 g of polyphenolic compounds, SFE exhibited significantly greater

Whitening Effects of Spent Coffee

1093

Fig. 2. Cell viability of B16F10 cells after treatment with coffee extracts. Pressure (bar), super critical fluid extract; HWE (CB), hot water extract of coffee bean; DE (CB), drip hot water extract of coffee bean; HWE (SC), hot water extract of spent coffee. Each value was expressed as the mean±SD; *Significantly different from that of the control treatment at p<0.05.

inhibition of melanin synthesis than HWE. In previous studies, all plants which had abundant polyphenol content was not exhibited in the tyrosinase inhibition (30). In other study, also an unsaturated fatty acid suppressed melanin production and a saturated fatty acid enhanced the melanin production by Aida et al. (4). In this study, all experiment extracts were contained unsaturated fatty acids than saturated fatty acids, seems to inhibition of melanin synthesis. SFE (SC) which contained much of fatty acid seems to have effective in inhibition of melanin synthesis. SFE (CB) only at 450 bar for 2 h exhibited inhibition of melanin synthesis similar to that seen for the positive control. Whereas, SFE (SC) exhibited significant (p<0.05) inhibition of melanin synthesis under various extraction conditions, including

150 bar for 2 h, 250 bar for 2 h, and 350 bar for 1 h. SFE (SC) exhibited a greater capacity to inhibit melanin synthesis than the majority of SFE (CB). These indicates that higher polarity decreases solubility, while lower polarity induces complete solubility in SFE using carbon dioxide (14). SFE (SC) showed a higher capacity to inhibit melanin synthesis than HWE (both of CB, SC). Furthermore, SFE (SC) exhibited greater inhibition of melanin synthesis than SFE (CB), even when extracted at lower pressures and for a shorter duration. These results slightly differed from the results for tysoinase inhibition activity. SFE (SC) obtained under a pressure of 450 bar and 2 h duration lacked the capacity to inhibit tyrosinase activity, showing no difference from the control group. However, the capacity to inhibit

1094

Sung et al.

Fig. 3. Tyrosinase inhibition activity of B16F10 melanocyte treated with coffee extracts (100 µg/mL). Pressure (bar), super critical fluid extract; HWE (CB), hot water extract of coffee bean; DE (CB), drip hot water extract of coffee bean; HWE (SC), hot water extract of spent coffee; A, arbutin 100 µg/mL. Each value was expressed as the mean±SD; Different letters show a significantly difference at p<0.05 as determined by Duncan's multiple range test.

Fig. 4. Melanin inhibition activity of B16F10 melanocyte treated with coffee extracts (100 µg/mL). Pressure (bar), super critical fluid extract; HWE (CB), hot water extract of coffee bean; DE (CB), drip hot water extract of coffee bean; HWE (SC), hot water extract of spent coffee; A, arbutin 100 µg/mL. Each value was expressed as the mean±SD; Different letters show a significantly difference at p<0.05 as determined by Duncan's multiple range test.

melanin synthesis of the SFE of SC produced under identical conditions was significantly higher (28%) than that observed in the control group. Although the extracts were obtained under identical SFE conditions, their inhibition of tyrosinase activity and melanin synthesis differed, indicating that factors other than tyrosinase interfere with the melanin synthesis process to prevent melanin formation. Melanin synthesis may have been inhibited not by action

on tyrosinase, but other proteins, such as TRP1 and TRP2 (11,12). Consequently, the capacity to inhibit melanin synthesis by extracts of CB and SC should be investigated further in association with tyrosinase-related proteins, including TRP1 and TRP2, and their mRNA expression levels, to clarify how extracts of CB and SC affect melanin synthesis.

Whitening Effects of Spent Coffee

1095

Fig. 5. Observation of melanin inhibition activity by DOPA stain after treated with coffee extract (100 µg/mL) in B16F10 cells. Control, water; Arbutin, arbutin 100 µg/mL; SFE Pressure (bar), super critical fluid extract; HWE (CB), hot water extract of coffee bean; DE (CB), drip hot water extract of coffee bean; HWE (SC), hot water extract of spent coffee

DOPA staining The formation of melanocytes occurs simultaneously with the development of dendrites, such that they connect with the neighboring keratinocytes to form epidermal-melanocyte units, ultimately resulting in excessive pigment deposition in the keratinocytes. DOPA reacts with melanin cells to form a dark brown precipitate (7). Using DOPA staining, formation of melanin and development of dendrites can be observed. We performed DOPA staining in B16F10 cells to observe the effect of extracts of CB and SC on melanin production and dendrite formation. Morphological changes in the cells revealed by DOPA staining are shown in Fig. 5. Control cells, which

were treated with α-MSH only, exhibited dense deposition of melanin, in conjunction with increased dendrite development. However, the group treated with the positive control, arbutin, a tyrosinase inhibitor, rarely showed melanin deposition or dendrite formation. Also, the experimental group treated with extracts from CB or SC had lesser melanin synthesis and dendrite formation than the control group. SFE (CB extracted for 2 h at 450 bars) and SFE (SC extracted 2 h at 250 bar and for 1 h at 350 bar) reduced dendrite formation and melanin synthesis more effectively than HWE. In particular, DOPA staining showed that SFE (SC) at 150 bar for 2 h produced effects similar to those of

1096

arbutin treatment, suggesting the efficacy of SC extracts for skin whitening may be similar. This result is concordant with the melanin synthesis. In this study, we showed that when SFE (SC) were produced at 150 bar pressure for 2 h, at 250 bar pressure for 2 h, and at 350 bar pressure for 1 h, inhibition of melanin synthesis was greater than that noted for HWE (CB). Furthermore, inhibition of tyrosinase activity was also found to be significantly higher in the SFE (SC) produced under a pressure of 150 bar for 1 h than with the other extracts. Thus, if SC was subjected to SFE with carbon dioxide at a pressure of 150 bar for less than 2 h, an extract was obtained that exhibited a high capacity to inhibit both melanin synthesis and tyrosinase activity. In addition, the SFE (SC) exhibited a higher capacity to inhibit both melanin synthesis and tyrosinase activity than SFE (CB) extracts obtained using either HWE or SFE. These results indicated that SC might be utilized as a functional raw material to produce substances with skinwhitening effects, rather than simply being a waste byproduct of coffee brewing. However, we observed that high fatty acid content, traditionally associated with skinwhitening effects, did not correspond with the capacity to inhibit melanin synthesis or tyrosinase activity. We observed that the extracts exhibited a large difference in their capacity to inhibit melanin synthesis and their capacity to inhibit tyrosinase activity indicating that further research is necessary to isolate the active substances associated with these mechanisms for skin whitening. Acknowledgments This study was supported by the 3GBio-Based Eco-Biomaterial R&D Project (No. R0000480) of the Ministry Of Trade, Industry & Energy. Disclosure The authors declare no conflict of interest.

References 1. Borrelli RC, Visconti A, Mennella C, Anese M, Fogliano V. Chemical characterization and antioxidant properties of coffee melanoidins. J. Agr. Food Chem. 50: 6527-6533 (2002) 2. Silva MA, Nebra SA, Machado Silva MJ, Sanchez CG. The use of biomass residues in the brazilian soluble coffee industry. Biomass Bioenerg. 14: 457-467 (1998) 3. Kim KJ, Park SK. Changes in major chemical constituents of green coffee beans during the roasting. Korean J. Food Sci. Technol. 38: 153-158 (2008) 4. Aida K, Shinmoto H, Kobori M, Tsushida T. Up and down regulation of melanin production of mouse melanoma B16C7 cells with fatty acid. J Jpn. Soc. Food Sci. 47: 793-796 (2000) 5. Itoh T, Tamura T, Matsumoto T. Sterol composition of 19 vegetable oils. J. Am. Oil Chem. Soc. 50: 122-125 (1973) 6. Kim KJ, Lee YW. Supercritical fluid technology for green food processing. Food Sci. Ind. 43: 35-52 (2010) 7. Bell AA, Weeler MH. Biosynthesis and function of fungal melanin. Annu. Rev. Phtophathol. 24: 411-451 (1986)

Sung et al. 8. Chen JS, Wei CI, Marshall MR. Inhibition mechamism of kojic acid on polyphenol oxidase. J. Agr. Food Chem. 39: 1897-1901 (1991) 9. Urabe K, Aroca P, Tsukamoto K, Mascagna D, Palumbo A, Prota G, Hearing VJ. The inherent cytotoxicity of melanin precursors: A revision. Biochim. Biophys. Acta 1221: 272-278 (1994) 10. Buscà R, Ballotti R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigm. Cell Res. 13: 60-69 (2000) 11. Saha B, Singh SK, Sarkar C, Bera R, Ratha J, Tobin DJ, Bhadra R. Activation of the Mitf promoter by lipid-stimulated activation of p38-stress signalling to CREB. Pigm. Cell Res. 19: 595-605 (2006) 12. Cabanes J, Chazarra S, Garcia-Carmona F. Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J. Pharm. Pharmacol. 46: 982-985 (1994) 13. Jung SW, Lee NK, Kim SJ, Han D. Screening of tyrosinase inhibitior from plants. Korean J. Food Sci. Technol. 27: 891-896 (1995) 14. Kim HB. Studies on enhancement functional substances in coffee by supercritical fluid extraction. MS thesis, Ajou University, Suwon, Korea (2007) 15. Suk KD, Lee SJ, Bae JM. Inhibitory effects of Cuscuta japonica extract and C. australis extract on mushroom tyrosinase activity. Korean J. Pharmacogn. 35: 380-383 (2004) 16. Lee HB, Bai S, Chin JE. Inhibitory effect of lithospermum erythrorhizon extracts on melanin biosynthesis. J. Korean Soc. Food. Sci. Nutr. 34: 1325-1329 (2005) 17. Roura E, Andrés-Lacueva C, Estruch R, Lamuela-Raventós RM. Total polyphenol intake estimated by a modified Folin-Ciocalteu assay of urine. Clin. Chem. 52: 749-752 (2006) 18. MFDS. Food Standards Codex. Method 9. Ministry of Food and Drug Safety, Cheongwon, Korea. pp. 52-66 (2013) 19. Lim YM, Kim BR, Hong KY. Antioxidant effect of Crataegi Fructus extract on the oxidative stress of reactive oxygen species in cultured human skin fibroblasts. Koroean J. Orient. Physiol. Pathol. 22: 115-119 (2008) 20. Martínez-Esparza M, Jiménez-Cervantes C, Solano F, Lozano JA, García-Borrón JC. Mechanisms of melanogenesis inhibition by tumor necrosis factor-alpha in B16/F10 mouse melanoma cells. Eur. J. Biochem. 255: 139-146 (1998) 21. Hosoi J, Abe E, Suda T, Kuroki T. Regulation of melanin synthesis of B16 mouse melanoma cells by 1α, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 45: 1474-1478 (1985) 22. Kim SK, Kim DS, Won HW, Mun YJ. Effect of Saururus chinensis BAILL extract for pharmacopuncture on the melanogenesis in B16F10 cells. Korean J. Acupunct. 29: 117-130 (2012) 23. Couto RM, Fernandes J, Gomes da Silva MDR, Simoes PC. Supercritical fluid extraction of lipids from spent coffee grounds. J. Supercrit. Fluid. 51: 159-166 (2009) 24. Jo YN, Jeong HR, Jeong JH, Heo HJ. The skin protecting effects of ethanolic extracts of eggplant peels. Korean J. Food Sci. Technol. 44: 94-99 (2012) 25. Arung ET, Shimizu K, Kondo R. Inhibitory effect of artocarpanone from Artocarpus heterophyllus on melanin biosynthesis. Biol. Pharm. Bull. 29: 1966-1969 (2006) 26. Zheng ZP, Cheng KW, To JT, Li H, Wang M. Isolation of tyrosinase inhibitors from Artocarpus heterophyllus and use of its extract as antibrowning agent. Mol. Nutr. Food Res. 52: 1530-1538 (2008) 27. Jeon HJ, Noda M, Maruyama M, Matoba Y, Kumagai T, Suqivama M. Identification and kinetic study of tyrosinase inhibitors found in sake lees. J. Agr. Food Chem. 54: 9827-9833 (2006) 28. Maqid AA, Voutquenne-Nazabadioko L, Bontemps G, Litaudon M, Lavaud C. Tyrosinase inhibitos and sesquiterpene diglycosides from Guioa villosa. Planta Med. 74: 55-60 (2008) 29. Cooksey CJ, Garratt PJ, Land EJ, Pavel S, Ramsden CA, Riley PA, Smit NPM. Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase. J. Biol. Chem. 272: 26226-26235 (1997) 30. Cho EJ, Ju HM, Jeong CH, Eom SH, Heo HJ, Kim DO. Effect of phenolic extract of dry leaves of Lespedeza cuneata G. Don on antioxidant capacity and tyrosinase inhibition. Korean J. Hort. Sci. Technol. 29: 358-365 (2011)