abstract

Background: Panax ginseng is a marvelous herbal remedy for all ailments of the body. That may be why it is called Panax, which means “cure for all”. Melanin is a pigment that gives color to our skin; however, increased melanin production can lead to tumor formation. Human exposure to ultraviolet B radiation has increased extensively owing to the increased sunlight due to global warming. Consequently, a phenomenon called photoaging has been observed for all skin colors and types. As a result of this phenomenon, a set of enzymes called matrix metalloproteinases, which serve as degradation enzymes for extracellular matrix proteins, mainly collagen, is increased, causing depletion of collagen and resulting in early wrinkle formation. 

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Methods: Therefore, in our study, we used the murine melanoma cell line B16/F10 to study the inhibition of melanogenesis by Korean Red Ginseng (KRG) extract in vitro and HRM-2 hairless mice exposed to artificial ultraviolet B to examine the efficacy of KRG in vivo. We prepared a 3% red ginseng extract cream and evaluated its effects on human skin.

Conclusion: Therefore, we conclude that KRG is an excellent skin whitening and anti-aging product.

·2019 The Korean Society of Ginseng. Publishing services by Elsevier B.V. This is an open-access article

1. Introduction

Melanin is a compound responsible for skin pigmentation, and the variety of skin colors in the human race is attributable to the amount of melanin-producing cells present in the skin [1]. The process responsible for the formation of melanin is called melanogenesis. In this process, a-melanocyte-stimulating hormone (a-MSH) binds to its receptor (i.e., melanocortin 1 receptor) causing elevated levels of cAMP, which in turn activates microphthalmia-associated factor (MITF) through various pathways, such as cyclic Adenosine Monophosphate (cAMP) response element binding protein, extracellular-regulated kinase, and protein kinase B (AKT), causing its degradation. Owing to this degradation, the rate-limiting step in the process of melanogenesis (i.e., the action of the enzyme tyrosinase, TYR) is affected and becomes activated. TYR is responsible for the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which forms melanin. Tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) are further downstream factors of TYR and MITF that are activated and are involved in the formation of melanin. Consequently, throughout the whole process, it is TYR that is the most important component of the regulation of melanin production in skin cells [2-4]. 

Panax ginseng is a wonder herb that has been consumed widely in eastern Asia for over 1,000 years as it has many beneficial health effects. It is available in a variety of forms, including drinks, capsules, and tablets [5,6]. Past studies have revealed the noteworthy effects of ginseng when used to reduce the incidence of various types of tumors, many mental anomalies, diabetes, hypertension, hyperlipidemia, and inflammation [7-13]. In particular, in the Korean peninsula, ginseng supplements form part of a normal diet. Commercially, ginseng is available in the form of whole ginseng root extract, single ginsenoside extracts, or capsules. Ginsenosides are the constituent active compounds present in the whole ginseng root that are responsible for the efficacious health-enhancing properties of ginseng [14].

There have been many studies on the effects of single ginsenoside on melanin production and melasma [15]. However, at present, no study has reported the anti-melanogenic effects of Korean Red Ginseng (KRG) extract on melanin production and examined its skin whitening and antiaging effects, particularly in humans. Therefore, we investigated the TYR inhibition and melanin production inhibition by KRG in vitro in the B16/F10 melanoma cell line via a mechanistic study of the pathways involved in this process. Our results indicated that KRG markedly inhibited TYR activity and decreased melanin content via the MITF degradation pathway. Moreover, our in vivo study using an ultraviolet B (UVB) irradiated hairless mouse (HRM-2) model of photoaging and hyperpigmentation revealed that the production of melanin in HRM-2 mice was substantially and markedly reduced by the application of KRG (150 and 300mg/kg). Furthermore, the 3% red ginseng extract cream showed excellent antiwrinkle and skin-whitening qualities in humans. Thus, we concluded that KRG and KRG formulations as a cream should be useful in the cosmetic industry as skin-whitening and anti-aging agents.

2. Materials and methods

2.1. Chemicals and reagents

Dulbecco’s modified Eagle’s medium (WelGene Co, Korea); fetal bovine serum (WelGene Co., Korea); streptomycin and penicillin (Lonza, MD, USA); TRIzol reagent (Invitrogen, Carlsbad, CA, USA); oligodT, MITF, TYR, TRP-1, TRP-2, and b-actin primers were obtained from (Bioneer, Daejeon, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide was purchased from Sigma-Aldrich. Antibodies for MITF, TYR, TRP-1, and TRP-2 were obtained (Santa Cruz Biotechnology, Santa Cruz, Inc., TX, USA). Mushroom TYR and L-DOPA were purchased from Sigma (St. Louis, MO, USA). All other reagents were of local analytical grade.

2.2. Sample preparation

2.3. Evaluation of the stability of red ginseng cream

(1) pH test 

The pH of the red ginseng cream was measured on Days 1, 7, 15, and 30 of the 30-day experiment. The pH of the red ginseng cream was slightly acidic and showed almost no change over the 30 days (Table 2).

(2) Discoloration test 

The degree of discoloration of red ginseng cream was measured on Days 1, 7, 15, and 30. No change in color was observed over the 30-day experimental period (Supplemental Fig. 1).

(3) Patch test 

To check for an allergic response to ginseng cream, 1 mL of red ginseng cream was applied to the dorsal central back area (1 cm×1 cm) of the volunteers, and the skin reaction was evaluated after 24 h. Various types of skin reactions were evaluated, as shown in Supplemental Table. 3A-B, and no erythema, edema, or untoward reaction was observed. Therefore, this cream was further tested for other parameters on the skin. Furthermore, the degree of skin irritation was evaluated, as shown in Supplemental Table. 4, to yield the primary skin irritation index.

2.4. Red ginseng cream treatment regimen and evaluation of effects on oil and moisture content, elasticity, and whitening of the skin

To assess the effects of red ginseng cream on the oil content, moisture content, elasticity, and whitening of the skin, 10 women (approximately 40 years of age, nonsmokers, with no history of skin disease or facial treatment) were divided into two groups each containing five individuals: the control group, in which cream without red ginseng extract was applied (non-KRG), and the experimental group, in which cream with red ginseng extract was applied (KRG-treated group). The number of individuals was calculated by using the statistical software, G*Power 3.1 omicX; the coefficient of significance was 0.05, the power was 80, the number of groups was 2, and the number of repeated measurements was 6.

The cream was applied on the T-zone area (1) of the face, which is located 1 cm above the upper part of the middle of the T on the forehead between the eyebrows and on the right and the left sides for 3e5 seconds. The U-zone (2) was drawn horizontally from the nose and vertically down from the tail of the eye, as shown in Supplemental Fig. 2. To standardize the measurement conditions, the research was performed at 24 C ±1 C and 55% ± 10% relative humidity.

2.5. Cell culture

2.6. Cell-free TYR inhibition assay 

The assay was performed using a slightly modified protocol as previously described [3]. Briefly, 10 mL of KRG was placed in triplicate in a 96-well plate and mixed with 60 mL of 50 mmol/L of phosphate buffer on ice (pH 6.8). Subsequently, 20 mL of 0.9mg/mL L-DOPA was added to each well. Finally, 10 mL mushroom TYR was added to each well, and the plate was incubated at 27° C for 10 min. After incubation, dopachrome production was measured spectrophotometrically at 450nm by using a microplate reader (Versamax; Molecular Devices, LLC, CA, USA); in this experiment, kojic acid was used as the positive control [16].

2.7. Melanin inhibition assay

2.8. Animal experiment and grouping

2.9. UVB irradiation and induction of photoaging

2.10. Effect on melanin production in HRM-2 mice

2.11. Skin wrinkle measurement 

The degree of skin aging induced by UVB was measured through observation of the wrinkle formation. To evaluate the formation of wrinkles, the HRM-2 mice were anesthetized by the intraperitoneal injection of chloral hydrate (0.1 mL of 7% chloral hydrate/25 g mouse) in Week 5. Exposure to UVB and sample treatment were described in the previous section. Skin wrinkles were evaluated in Weeks 3, 4, and 5 by using DETAX System II (MIXPAC) and Double-Stick Disc (3M Healthcare, Germany) after UVB irradiation. Double-Stick Disc (sprayed with DETAX System II) was attached to the mouse skin and removed after 2e3 minutes. Disc wrinkles were evaluated by the scoring system of Bissett [17]: grade 0, absence of wrinkles; grade 1, several shallow wrinkles; grade 2, some wrinkles; and grade 3, some deep wrinkles. After the disc was removed and the skin was cleaned with 70% ethanol, the skin was photographed by using a USB Digital Microscope ( × 400; CE FOROHS, China), and a visual analysis of skin wrinkles was performed.

2.12. Enzyme-linked immunosorbent assay

2.13. Histological observation of skin 

The skin tissues extracted from each experimental group were fixed in 10% formalin solution for 48 h and then stained with hematoxylin and eosin by the method of Cardiff [18] to determine the epidermal thickness. For collagen visualization, Masson’s trichome staining was performed by established protocols [19].

2.14. RNA extraction and real-time polymerase chain reaction 

Total RNA was extracted from the B16/F10 cells and UVB-ira-dilated mouse skin after they were treated with KRG and stimulated with a-MSH by using TRIzol by the manufacturer’s instructions. RNA (2 mg) was annealed with oligodT (Bioneer Co, Daejeon) for 10 min at 70° C and cooled for 5 min on ice, reverse transcribed using reverse transcriptase premix (Bioneer Co., Daejeon) in 20 mL of the reaction mixture, and run for 90 min at 42.5°C in a thermal cycler. The reaction was terminated at 95°C for 5 min to inactivate the reverse transcriptase. The reverse transcription polymerase chain reaction (PCR) was performed on aliquots of cDNA obtained from Reverse Transcriptase (RT) reaction in a PCR premix (Bioneer Co, Daejeon). The PCR products were then electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized by using ImageQuant LAS 500 (GE Healthcare Life Sciences, Seoul, South Korea). The intensity of band densities were normalized to that of Glyceraldehyde 3- phosphate dehydrogenase (GAPDH), and the primer sequences are provided in Supplemental Table. 1. The expression of MMP-2, MMP-9, and interleukin (IL)-1b was analyzed by using real-time quantitative PCR using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, USA).

2.15. Western blot analysis

2.16. Statistical analysis

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