정금나무 열매(Fruit of Vaccinum oldhami)의 분리 정제물(클로로겐산, 퀘르세틴 및 퀘르시트린)에 관한 항산화 및 미백활성 검증

Antioxidant and Whitening Activities of Chlorogenic Acid, Quercetin, and Quercitrin from the Fruit of Vaccinum oldhami

Abstract

정금나무 열매(Fruit of Vaccinum oldhami)를 분리 정제하여 클로로겐산(CA), 퀘르세틴(QT) 및 퀘르시트린(QR)을 얻었다. 전자공여능 측정 결과, CA, QT, QR은 각각 1,000 ㎍/ml의 농도에서 91.9%, 89.9%, 77.4%의 효과를 나타내었다. ABTS⁺ 라디칼 소거능 측정 결과, QT 및 QR은 1,000 ㎍/ml의 농도에서 각각 99.5%, 91.4%를 나타내었으며, CA는 100 ㎍/ml의 농도에서 90% 이상의 소거능을 나타내었다. Tyrosinase 억제 활성 측정 결과, CA, QT, QR은 각각 1,000 ㎍/ml이 농도에서 29.5%, 34.7%, 23.7%의 저해율을 나타내었다. MTT assay를 통해 B16F10의 세포 생존율을 측정한 결과, CA, QT, QR 모두 100 ㎍/ml의 농도에서 80% 이상의 생존율을 나타내었다. 단백질 발현을 측정한 결과, 농도가 증가함에 따라 CA에서 TRP-1, TRP-2의 단백질 발현이 억제되는 것을 확인할 수 있었으며 QT에서는 TRP-1, TRP-2 및 tyrosinase에서 우수한 단백질 발현 억제율을 나타내었다. CA의 mRNA 발현 억제율을 측정한 결과, MITF의 mRNA 발현 억제 효과가 우수함을 확인하였으며, TRP-1, TRP-2 및 tyrosinase의 mRNA 발현량은 농도가 증가할수록 감소하였다. QT는 농도가 증가할수록 MITF, TRP-2, tyrosinase의 mRNA 발현이 감소하였다. QR의 mRNA 발현 억제율을 측정한 결과, 농도가 증가할수록 mRNA 발현량이 감소하는 것을 확인할 수 있었다. 이하의 결과에 따라 CA, QT, QR의 항산화 및 미백 효과를 확인할 수 있었다.

The fruit of Vaccinum oldhami was separated and purified to obtain the compounds chlorogenic acid (CA), quercetin (QT), and quercitrin (QR). The electron-donating abilities of CA, QT, and QR at 1,000 ㎍/ml were 91.9%, 89.9%, and 77.4%, respectively QT and QR showed 99.5% and 91.4% ABTS⁺ radical scavenging ability at a 1,000 ㎍/ml concentration, respectively, and CA showed a 95% ability or higher at 100 ㎍/ml. Regarding tyrosinase inhibitory activity, CA, QT, and QR exhibited 29.5%, 34.7%, and 23.7% efficacy, respectively, at 1,000 ㎍/ml. Regarding the cell viability for melanoma cells (B16F10) assessed through MTT assay, CA, QT, and QR showed cell a viability of 80% or more at 100 ㎍/ml. To measure the deterrent of protein expression, CA affected TRP-1 and TRP-2 in accordance with increases in concentration. The protein expression inhibition rate of QT was excellent for TRP-1, TRP-2, and tyrosinase. CA was confirmed to have an excellent mRNA expression inhibitory effect against MITF, and the amount of mRNA expression of TRP-1, TRP-2, and tyrosinase decreased with an increase in the CA concentration. As the concentration of QT increased, the mRNA expression of MITF, TRP-2, and tyrosinase decreased. QR decreased the amount of mRNA as the QR concentration increased. The excellent antioxidant and whitening effects of CA, QT, and QR were thus confirmed.

Introduction

Melanin is a blackish-brown pigment that absorbs ultraviolet rays and blocks the penetration of ultraviolet rays through the skin. Melanin includes both the blackish-brown eumelanin and the reddish-yellow pheomelanin. These pigments determine the color of human skin, hair, and pupils and inhibit damage to skin cells by absorbing ultraviolet rays. However, when melanin is excessively synthesized and accumulated, it can cause spots, freckles, blemishes, and, in severe cases, even skin cancer [10]. Melanin synthesis is regulated by the three enzymes, namely tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2, whereas melanin synthesis through melanocytes by ultraviolet rays involves cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB) [11,12]. In melanin synthesis, tyrosine oxidizes 3, 4-dihydroxyphenylalanine (DOPA) by tyrosinase and then converts DOPA to DOPA quinone to produce melanin [5,8]. Although arbutin is mainly used for improving whitening in functional cosmetics, researchers have recently focused on developing functional cosmetics for whitening using natural materials.

The Vaccinum oldhami F. is a deciduous, broad-leaved shrub that belongs to the azalea family. The species grows in the southern regions of Korea and in the middle of the mountains of Jeju-do. It ranges from 1 to 4 m in height, has dark brown branches with alternate elliptical or ovate leaves and from May to July, produces 4 to 5 mm bell-shaped flowers at the ends of its vertical branches. The species’ fruits range from 6 to 8 mm in diameter, ripen to black in September, and are covered with a white powder [18-20]. Due to the efficacy of Fruit of Vaccinum oldhami preservation, convergence and as a diuretic, it has been used to treat cystitis, vomiting and rashes. Moreover, researchers have studied the antioxidant and anticancer effects of Fruit of Vaccinum oldhami grown in Japan [6,13].

Even though research is currently being conducted on Fruit of Vaccinum oldhami, there is still little research on Fruit of Vaccinum oldhami. Therefore, the aim of the present study was to investigate the antioxidant effects and whitening activities of Fruit of Vaccinum oldhami by examining the effects of purified isolates of chlorogenic acid (CA), quercetin (QT), and quercitrin (QR) on B16F10 cells.

Materials and Methods

Sample extraction

The Fruit of Vaccinum oldhami were collected from the Gyeonggi-do Forest Environment Research Institute and ethanol extracted by immersing dried plant material in 70% ethanol (10 times the sample) at room temperature for 24 hr according to the procedure in the Fig. 1. The supernatant was separated and extracted, the sample extracts were filtered (Whatman No. 2) and concentrated (EYELA evaporator), after which the solvent was completely removed, lyophilized and stored at -20℃ for further analysis.

Fig. 1. The procedure for extraction from Fruit of Vaccinum oldhami.

Purification and identification of active compounds

The Fruit of Vaccinum oldhami ethanol extract (30 g) was suspended in water, filtered and fractionated by adding different solvents with different polarities. A 1:1 mixture of water-suspended material and n-hexane were added to a separating funnel, in order to fractionate the water and hexane layers. The n-hexane layer was then subjected to vacuum concentration to obtain the n-hexane fraction (1.52 g). Through the same procedure, the ethyl acetate (EtOAc, 16.8 g) and H₂O (10.48 g) layers were sequentially added to obtain the fractions, which were then subject to vacuum concentration as well. The EtOAc fraction was suspended in water and filtered, after which the H₂O-MeOH concentration was increased from 0 to 100% using middle-pressure liquid chromatography (MPLC) in a column filled with Sephadex LH-20 (10-25 μm, GE Healthcare Bio-Science AB, Uppsa la, Sweden) and then separated. Finally, thin-layer chromatography (TLC) was performed to separate the material into seven fractions (Fr. 1 - Fr. 7; Fig. 2). The seven fractions were repeatedly subject to Sephadex LH-20, MCI CHP 20, ODS-H-AQ column chromatography to separate the final three phenolic compounds. According to the color development and 1D/2D NMR measurement results by 10% H₂SO₄ and FeCl₃ solutions on the TLC plate of the three compounds, compounds 1-3were identified as chlorogenic acid (CA, 491 mg), quercetin (QT, 48 mg) and quercitrin (QR, 68 mg; Fig. 3). For UPLC analysis, a CORTECS C18 (4.6×150 mm, 2.7 μm) column was used, and various solvent compositions and wavelengths were analyzed. As a result, in the UV 280 nm detection wavelength, by subjecting the acetonitrile and water gradient profile to elution at a flow rate of 0.5 ml/min, we were able to satisfactorily confirm the peaks of compounds 1-3. Compounds 1-3 were diluted by concentration and the results indicated linearity. The detection concentrations of compounds 1-3 ranged from 15.625 to 250 ug/ml. The analysis results indicated linearity by concentration, with slopes of y = 781.38 – 1613.3 (R2 = 0.9916), y = 333.79 – 148.76 (R2 = 0.9987) and y = 101.01 – 2748.7 (R2 = 0.9936) respectively (Fig. 4). Fig. 5 shows a chromatogram of the ethyl acetate fractions of Vaccinum oldhami analyzed in the same amount of 10 μl under the established UPLC conditions. Peak area was substituted by compound for the y value of the regression equation, in order to calculate the concentrations of compounds 1–3 in the Fruit of Vaccinum oldhami. The concentrations of compounds 1-3 were 280.14, 0.63, and 0.47 mg/g, respectively. Accordingly, compounds 1-3 (CA, QR, QT), which were obtained by separating and purifying Fruit of Vaccinum oldhami were used as the experimental samples.

Fig. 2. Separation flow diagram of Fruit of Vaccinum oldhami Extracts.

Fig. 3. Chemical structure of compound 1-3. Compound 1 - ¹H-NMR (600 MHz, MeOD) δ : 7.07 (1H, brs, H-2'), 6.96 (1H, brd, J = 6.6 Hz, H-5'), 6.80 (1H, dd, J = 2.4, 7.2 Hz, H-6'), 7.59 (1H, d, J = 15.6 Hz, H-7'), 6.31 (1H, d, J = 15.6 Hz, H-8'), 2.10 – 2.21 (4H, m, H-2, 6), 3.73 (1H, brs, H-3), 4.19(1H, brs, H-4), 5.39(1H, s, H-5). ¹³C-NMR (150MHz, MeOD) δ : 167.7 (COO), 148.1 (C-4'), 145.4 (C-3'), 145.3 (C-7') 126.4 (C-1'), 121.5 (C-6'), 115.1 (C-2') 114.1 (C-5'), 113.7 (C-8'), 73.5 (C-1), 71.5 (C-3), 71.2 (C-4), 39.0 (C-2), 37.5 (C-6). Compound 2 - ¹H-NMR (600 MHz, MeOD) δ : 7.33 (1H, brs, H-2'), 7.32 (1H, dd, J = 2.4, 8.4, H-6'), 6.96 (1H, d, J = 8.4, H-5'), 6.40 (1H, d, J = 1.8, H-8), 6.23 (1H, d, J = 1.8, H-6), 5.36 (1H, s, anomeric), 4.20 (1H, s, H-2''), 3.87 (1H, m, H-4''), 3.65 (1H, m, H-3''), 3.07 (1H, t, J = 11.4, H-5'', 1.05, 3H, s, rha-CH3), ¹³C-NMR (150 MHz, MeOD) δ : 178.3 (C=O), 164.5 (C-7), 161.8 (C-5), 157.8 (C-2), 157.1 (C-9), 148.4 (C-4'), 145.1 (C-3'), 135.3 (C-3), 121.4 (C-1'), 121.0 (C-6'), 115.4 (C-2'), 115.1 (C-5'), 104.4 (C-10), 101.8 (C-1''), 98.5 (C-6), 93.4 (C-8), 72.3 (C-4''), 70.5 (C-2''), 70.4 (C-3''), 69.5 (C-4''), 16.2 (C-6''). Compound 3 - ¹H-NMR (600 MHz, MeOD) δ : 7.75 (1H, brs, H-2'), 7.66 (1H, dd, J = 2.4, 8.4, H-6'), 6.91 (1H, d, J = 8.4, H-5'), 6.40 (1H, d, J = 1.8, H-8), 6.20 (1H, d, J = 1.8, H-6) ¹³C-NMR (150 MHz, MeOD) δ : 175.9 (C=O), 164.1 (C-7), 161.1 (C-5), 156.8 (C-9), 147.3 (C-4'), 146.6 (C-2), 144.8 (C-3'), 135.8 (C-3), 122.7 (C-1'), 120.2 (C-6'), 114.8 (C-5'), 114.5 (C-2'), 103.1 (C-10), 97.8 (C-6), 93.0 (C-8).

Fig. 4. Linearity review results of compound 1-3.

Fig. 5. Chromatogram of ethyl acetate fractions from Fruit of Vaccinum oldhami.

Reagents and devices

2,2-diphenyl-1-picrylhydrazyl (DPPH) and potassium persulfate were purchased from Sigma-Aldrich Coporation (St. Louis, MO, USA). 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) were purchased from Wako Pure Chemical Industries. Ltd. (Japan). Tyrosinase mushroom, L-3,4-dihydroxy-phenyl-alanine (L-DOPA) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A). B16F10 were purchased from ATCC (U.S.A). Fetal bovine serum (FBS), dulbecco`s modified eagle medium (DMEM), penisillin, phosphate bufferd saline (PBS) and trypsin were purchased from thermo scientific hyclone (USA). 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A). Tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), Tyrosinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, primary anti body and mouse-anti-goat and rabbit-anti-mouse were purchased from Santacruz (CA, U.S.A), microphthalmia-associated transcription factor (MITF) were purchased from Abcam (U.S.A). Sephadex LH-20 (10-25 μm, GE Healthcare Bio-Science AB, Uppsa la, Sweden), MCI-gel CHP 20 (75-150 μm, Mitsubishi Chemical, Tokyo, Japan), YMC GEL ODS-A-HG (50 μm, YMC group, Japan), TLC silica gel 60 (Merck, Darmstadt, Germany), Medium pressure liquid chromatography (MPLC) used Buchi (Sepacore 50, Swiss), and a glass column (100×920) was used for columns. ¹H and ¹³C-NMR spectra used AVANCEIII HD 600 (Bruker Biospin GmbH, Rheinstetten, Germany). Freeze drier (ILShin Bio Base Co. Korea), vortex (Scientific Industries, INC, USA), ELISA reader (Tecan, Austria), CO₂ incubator (vision scientific, Korea), pH meter (Mettler-Toledo AG, Swltzerland), centrifuge (Hanil Science Industrial Co. Korea), autoclave (JS ResearchInc, Korea), microscope (Olympus, Japan), digital shaker (Deihan Scientific, Korea), Davinch-Chemi™ imager CAS-400SM System (Davinch-K Co, Korea), PCR (C-100, Bio-Rad. U.S.A).

Electron-donating ability

Electron donating ability (EDA) was measured using a modified version of the method of Blois [1]. More specifically, 60 μl 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the extracts by concentration were mixed 120 μl at a time and incubated for 15 minutes, after which the absorbance (517 nm) each solution was measured using a microplate reader. The EDA was then calculated as the absorbance reduction rate of the additive and non-additive groups of the sample solution.

ABTS⁺ radical scavenging activity assay

Antioxidant activity was measured using previously described methods [23]. Briefly, ABTS⁺ radicals, 7 mM 2,2-azino-bis (3-ethyl- benthiazoline-6-sulfonic acid) and 2.45 mM potassium persulfate were mixed and incubated at room temperature for 24 hr. After forming ABTS⁺ , it was diluted with ethanol, 100 μl of the sample was added to 100 μl ABTS⁺ and absorbance was measured at 700 nm.

Tyrosinase-inhibition activity

Tyrosinase-inhibition activity was measured as described previously [24]. For the reaction zone, 40 μl 200 U/ml mushroom tyrosinase was combined with a mixture of the substrate (40 μl), sample solution (40 μl) and L-DOPA (10 mM, dissolved in 80 μl 67 mM sodium phosphate buffer, pH 6.8). The resulting solutions were then incubated at 37℃ for 10 min and the DOPA chrome produced in the reaction solutions was determined by measuring absorbance (492 nm). Finally, tyrosinase-inhibition activity was calculated as the absorbance reduction rate of the additive and non-additive groups of the sample solution.

Cell line and culture

B16F10 melanoma cells were incubated in DMEM with 10% FBS and 1% penicillin/streptomycin (100 U/ml) at 37℃and 5% CO₂ incubator.

Cell viability

The MTT assay of the melanoma cells (B16F10) was conducted as described previously [2]. The melanoma cells were seeded into 96-well plates at a density of 1×10 ⁵ cells/well (180 μl). The samples were prepared by concentration and added by 20 μl, after which the cells were incubated at 37℃and 5% CO₂ incubator for 24 hr. After adding 40 μl MTT solution (2.5 μg/ml), the cells were cultured for an additional 3 hr. Next, the culture medium was removed, 100 μl DMSO was added to each well, the cells were incubated at room temperature for 10 min, and then the absorbance (540 nm) of the wells was measured using an ELISA reader. Cell viability was calculated as the absorbance reduction rate of the additive and non-additive groups of the sample solution.

Protein expression measurement through Western Blot

To investigate the activity of the whitening factors MITF, TRP-1, TRP-2, and tyrosinase, B16F10 melanoma cells were seeded into a 100-mm tissue culture dish, at 1×10 ⁶ cells/well and cultured for 24 hr. After removing the medium, α-MSH was treated for 2 hr and the extract was cultured for 24 hr in the medium treated by concentration. The medium was removed again and the cells were washed twice using 1×PBS. One Complete mini tablet was dissolved in 10 ml of RIPA buffer at 100 μl and the solution was centrifuged at 13,200 rpm for 20 min at 4℃. A BCA protein assay kit was used to quantify the supernatant obtained by centrifugation, and 20 μl protein was separated by electrophoresis using a 10% acrylamide gel. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer device and incubated in a blocking buffer (5% skim milk in tween 20) for 1 hr at room temperature. The primary antibody was diluted and left overnight at 4℃, after which it was washed three times at 10 min intervals using Tris-buffered saline and tween 20. The secondary antibody was diluted at a ratio of 1:1,000, incubated at room temperature for 2 hr, and washed three times using tween 20, after which the band was confirmed and quantified using the Davinch-Chemi Imager CAS-400SM System.

Measurement of mRNA expression through Reverse transcription-polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) was used to investigate the mRNA expression of the whitening factors MITF, TRP-1, TRP-2, and tyrosinase. Table 1 shows the primer sequences used in the experiment. The PCR reactions included 5×Green GoTaq Flexi buffer, MgCl₂, nucleotides (10 mM), primers, GoTaq DNA Polymerase, nuclease-free water, and synthesized cDNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using 40 cycles of 10 s at 96℃, 30 s at 64℃, and 60 s at 72℃. Tyrosinase was amplified using 40 cycles of 10 s at 96℃, 30 s at 64℃, and 60 s at 72℃. MITF, TRP-1, and TRP-2 were amplified using 40 cycles of 10 s at 96℃, 30 s at 56℃, and 60 s at 72℃. The quality of the PCR-amplified fragments was confirmed using 1.5% agarose gel with 0.002% ethidium bromide, electrophoresis for 30 min at 100 V, and visualization using a UV transilluminator.

Table 1. Sequence of the primers used for RT-PCR

Results

Electron-donating ability

The DPPH used in the EDA measurement experiment is a stable free radical that appears purple in itsnon-reduced and is decolorized upon reduction by antioxidants, such as ascorbic acid, butylated hydroxyanisole (BHA), and sulfur-containing amino acids. As a result, DPPH is widely used to verify the antioxidant ability of a variety of natural materials [25]. The present study investigated the effects of three materials (CA, QT, QR) purified isolates from Fruit of Vaccinum oldhami EDA (Fig. 6-8). Analysis revealed that EDA increased with increasing concentrations of the three compounds. Indeed, QT and QR exhibited EDA values of 89.9% and 77.4%, respectively, at the highest concentration (1,000 μ g/ml), whereas CA exhibited an EDA value of 86.2% at 500 μg/ml and 91.9% 1,000 μg/ml.

Fig. 6. Electron donating ability of Fruit of Vaccinum oldhami separation and purification chlorogenic acid.

Fig. 7. Electron donating ability of Fruit of Vaccinum oldhami separation and purification quercetin.

Fig. 8. Electron donating ability of Fruit of Vaccinum oldhami separation and purification quercitrin.

ABTS⁺ radical scavenging activity assay

To measure ABTS⁺ radical scavenging ability, ABTS⁺ radicals that are generated by the reaction of ABTS diammonium salt and potassium persulfate are removed from reaction solutions by antioxidants, thereby changing the color of the solutions from radical specific cyan to light green [14]. Fig. 9-11 show the ABTS⁺ radical scavenging ability of CA, QT, and QR purified isolates from Fruit of Vaccinum oldhami. The purified QT and QR xhibited scavenging abilities of 99.5% and 91.4%, respectively, at their highest concentration (1,000 μg/ml), whereas CA exhibited a scavenging ability of 95.4% at only 100 μg/ml, thereby demonstrating excellent ABTS⁺ radical scavenging ability.

Fig. 9. ABTS⁺ radical scavenging ability of Fruit of Vaccinum oldhami separation and purification chlorogenic acid.

Fig. 10. ABTS⁺ radical scavenging ability of Fruit of Vaccinum oldhami separation and purification quercetin.

Fig. 11. ABTS⁺ radical scavenging ability of Fruit of Vaccinum oldhami separation and purification quercitrin.

Tyrosinase-inhibition activity

Melanin synthesis is regulated by the enzymatic action of tyrosinase, which promotes the oxidation of tyrosine and functions as the main enzyme for melanin biosynthesis in the melanosomes of melanocytes of the epidermis. Therefore, tyrosinase inhibitors can effectively inhibit the synthesis of melanin polymers in the skin and have been recognized as a useful evaluation method in the cosmetics industry [3,9].

Fig. 12 - Fig. 14 depicts the tyrosinase-inhibition activity of CA, QT, and QR purified isolates from Fruit of Vaccinum oldhami. Analysis revealed that tyrosinase-inhibition activity increased with increasing concentrations of all three compounds. Indeed, CA and QR exhibited tyrosinase-inhibition activities of 29.5% and 23.7%, respectively, at the highest concentration (1,000 μg/ml), whereas QT exhibited greater tyrosinase-inhibition activity (34.7% at 1,000 μg/ml) than either CA or QR.

Fig. 12. Inhibition rate of Fruit of Vaccinum oldhami separation and purification chlorogenic acid on tyrosinase.

Fig. 13. Inhibition rate of Fruit of Vaccinum oldhami separation and purification quercetin on tyrosinase.

Fig. 14. Inhibition rate of Fruit of Vaccinum oldhami separation and purification quercitrin on tyrosinase.

Cell viability

In MTT assays, which measure cell viability, the yellow water-soluble substance (MTT tetrazolium) is reduced to a purple water-insoluble substance (MTT formazan), owing to the dehydrogenase action of the mitochondria during metabolism, which reflects the concentration of living cells producing famazan [22]. Fig. 15-17 depict the effects of CA, QT and QR on the viability of B16F10 melanoma cells, as indicated by the MTT assay. At 100 μg/ml, the viabilities of the CA, QT and QR-treated cell cultures were 88%, 92.57% and 94.7%, respectively, with QR yielding the greatest viability measurement. This experiment was conducted at a concentration of 100 μg/ml or below, which exhibited cell viability of more than 85%.

Fig. 15. Cell viability of extracts from Fruit of Vaccinum oldhami separation and purification chlorogenic acid on melanoma cell (B16f10).

Fig. 16. Cell viability of extracts from Fruit of Vaccinum oldhami separation and purification quercetin on melanoma cell (B16f10).

Fig. 17. Cell viability of extracts from Fruit of Vaccinum oldhami separation and purification quercitrin on melanoma cell (B16f10).

Protein expression measurement through Western Blot

Western blotting was used to measure the inhibitory effects of CA, QT and QR (25, 50 and 100 μg/ml) on the expression of whitening factors (MITF, TRP-1, TRP-2 and tyrosinase) by B16F10 melanoma cells. The control sections were set to those with over expressed melanin in which α-MSH was treated, whereas the normal sections were set to those with untreated α-MSH. Here, β-actin, which exhibits relatively constant expression, regardless of cell conditions, was used as the positive control. First, the expression of both TRP-1 and TRP-2 in was significantly higher in the cells treated with CA (100 μg/ml) than in the control group, which was treated with kojic acid (Fig. 18). Meanwhile, the inhibition of MITF and tyrosinase expression was greater (30% and 82%, respectively) in the cells treated with CA (100 μg/ml) than in the control group. The expression inhibition rates of MITF, TRP-1, TRP-2, and tyrosinase in the QT-treated cells were 40.4%, 61.2%, 55% and 44.4%, respectively at 100 μ g/ml (Fig. 19), all of which were greater than the rate observed in the control group. The expression of MITF in QR-treated cells was significantly greater, at 100 μg/ml, than that of the control group (Fig. 20), and the expression inhibition rates of TRP-1, TRP-2, and tyrosinase were 85%, 79.9% and 31%, respectively, at 100 μg/ml, all of which were greater than the rate observed in the control group.

Fig. 18. MITF, TRP-1, TRP-2 and tyrosinase protein expression rate of Fruit of Vaccinum oldhami separation and purification chlorogenic acid on melanoma cell (B16f10).

Fig. 19. MITF, TRP-1, TRP-2 and tyrosinase protein expression rate of Fruit of Vaccinum oldhami separation and purification quercetin on melanoma cell (B16f10).

Fig. 20. MITF, TRP-1, TRP-2 and tyrosinase protein expression rate of Fruit of Vaccinum oldhami separation and purification quercitrin on melanoma cell (B16f10).

Measurement of mRNA expression through Reverse transcription-polymerase chain reaction (PCR)

To investigate the inhibitory effects of CA, QT and QR on the expression of MITF, TRP-1, TRP-2, and tyrosinase mRNA, B16F10 melanoma cells were treated purified CA, QT and QR (25, 50 and 100 μg/ml). The control sections were set to those with over expressed melanin in which α -MSH was treated, whereas the normal sections were set to those with untreated α-MSH. After 24 hr, mRNA expression was measured using RT-PCR and GAPDH, a housekeeping gene that exhibits relatively consistent expression, as a positive control. The mRNA was over expressed in the control group that was treated with 25, 50 and 100 μg/ml concentrations of purified CA, QT and QR (Figs. 21-23). The expression of MITF, TRP-1, TRP-2, and tyrosinase mRNA was inhibited as the concentrations of the treatment compounds increased. The expression of TRP-1, TRP-2, and tyrosinase mRNA was significantly inhibited in the CA-treated cells (100 μg/ml), when compared to the control group, which was treated with kojic acid, and the expression of MITF mRNA was 44.5% in the CA-treated cells (100 μg/ml), thereby indicating the excellent inhibitory effects of CA. As the concentration of QT increased, the mRNA expression of MITF, TRP-2, and tyrosinase decreased. Therefore, QR inhibited the mRNA expression of all four factors (MITF, TRP-1, TRP-2 and tyrosinase), more so as its concentration increased, and at 100 μg/ml, the mRNA expression was significantly lower than that of the control group.

Fig. 21. MITF, TRP-1, TRP-2 and tyrosinase mRNA expression rate of Fruit of Vaccinum oldhami separation and purification chlorogenic acid on melanoma cell (B16f10).

Fig. 22. MITF, TRP-1, TRP-2 and tyrosinase mRNA expression rate of Fruit of Vaccinum oldhami separation and purification quercetin on melanoma cell (B16f10).

Fig. 23. MITF, TRP-1, TRP-2 and tyrosinase mRNA expression rate of Fruit of Vaccinum oldhami separation and purification quercitrin on melanoma cell (B16f10).

Discussion

Melanin determines the color of human skin, hair, and pupils, and inhibits damage to skin cells by absorbing ultraviolet rays. However, when excessively synthesized and accumulated, melanin can cause spots, freckles, blemishes, and, in severe cases, even skin cancer [10]. In melanin synthesis, tyrosine oxidizes DOPA by tyrosinase and then converts DOPA to DOPA quinone to produce melanin [5,8].

Although arbutin is mainly used for improving whitening in functional cosmetics, researchers have recently focused on developing functional cosmetics for whitening using natural materials.

Research results have been confirmed that the fruits of Vaccinum oldhami are excellent in antioxidant and anti-inflammatory properties, but studies related to whitening are still incomplete [16]. In addition, this study studied CA, QT, and QR, which are known to be good for antioxidants [7]. CA, a phenolic antioxidant [27], has anti-diabetic effects through post-meal blood sugar drop by reducing the absorption of carbohydrates and glucose [4,17,21]. QT and QR are known to be effective in preventing degenerative diseases such as skin aging, obesity, inflammation, arteriosclerosis, diabetes, and high blood pressure caused by oxidative damage to biomaterials by silver active oxygen [15,26]. The goal of the present study was to investigate the antioxidant effects and whitening activities of three compounds (CA, QR and QT) that were purified isolates from Fruit of Vaccinum oldhami. In regard to antioxidant effects, the EDA of the three compounds increased with increasing concentration and at 1,000 μg/ml, the three compounds (CA, QT and QR) exhibited electron-donating abilities of 91.9%, 89.9% and 77.4%, respectively. Similarly, ABTS⁺ radical-scavenging activity of the three compounds increased with increasing concentration, and at 1,000 μg/ml, QT and QR exhibited scavenging abilities of 99.5% and 91.4%, respectively, whereas CA exhibited a scavenging ability of ≥ 95% at only 100 μg/ml. The tyrosinase-inhibition activity of the three compounds also increased with increasing concentration, and at 1,000 μg/ml, the three compounds (CA, QT and QR) exhibited inhibition activities of 29.5%, 34.7% and 23.7%, respectively. The present study also examined the effects of CA, QT and QR on the viability of B16F10 melanoma cells, using the MTT assay and found that the three compounds yielded cell viability values of 88%, 92.5%, and 94.7%, respectively, at 100 μg/ml. Additional experiments showed cell viability close to 100%. The inhibitory effects of CA, QT and QR on the expression of melanin biosynthesis-related genes were also investigated. The expression of TRP-1 and TRP-2 proteins decreased with increasing CA concentration, and the expression of MITF and tyrosinase were inhibited by 30 and 82%, respectively, when compared to the control treatment. Meanwhile, QT exhibited excellent inhibition of MITF, TRP-1, TRP-2 and tyrosinase expression and QR inhibited the expression levels of MITF, TRP-1, TRP-2, and tyrosinase, as well. Purified isolates CA also exhibited excellent inhibition of MITF mRNA expression, and the inhibition of TRP-1, TRP-2 and tyrosinase mRNA expression increased with increasing CA concentration. Purified isolates QT also significantly inhibited the expression of TRP-1 mRNA and the inhibition of MITF, TRP-2, and tyrosinase mRNA expression increased with increasing CA concentration. Meanwhile, the inhibition of MITF, TRP-1, TRP-2 and tyrosinase mRNA expression increased with increasing QR concentration. The results of the present study demonstrate that CA, QT and QR exhibit antioxidant effects and whitening activities and that melanin production was inhibited by interfering with the protein and mRNA expression of factors related to melanin biosynthesis. Accordingly, the results of the present study confirm the excellent antioxidant effects and whitening activities of Fruit of Vaccinum oldhami CA, QT and QR, as well as the potential utility of Fruit of Vaccinum oldhami as a functional material in cosmetics.

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.