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Evelyn Saba,1,☆ Seung-Hyung Kim,2,☆ Yuan Yee Lee,1 Chae-Kyu Park,3 Jae-Wook Oh,4,☆ Tae-Hwan Kim,1 Hyun-Kyoung Kim,5 Seong-Soo Roh,6,∗∗ and Man Hee Rhee1,∗
Abstract
Background
Panax ginseng is a marvelous herbal remedy for all ailments of 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.
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.
Results
Our results demonstrated that KRG induced potent suppression of tyrosinase activity and melanin production in B16/F10 cells; moreover, it reduced the transcription and translation of components involved in the melanin production pathway. In the in vivo experiments, KRG potently suppressed the expression of matrix metalloproteinases, reduced wrinkle formation, and inhibited collagen degradation. On human skin, ginseng cream increased skin resilience and skin moisture and enhanced skin tone.
Conclusion
Therefore, we conclude that KRG is an excellent skin whitening and antiaging product.
Keywords: Antiaging, Human trials, Korean Red Ginseng extract, Melanogenesis, Wrinkles
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 . The process responsible for the formation of melanin is called melanogenesis. In this process, α-melanocyte–stimulating hormone [α-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 , , .
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 , . 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 , , , , , , . 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 .
There have been many studies on the effects of single ginsenoside on melanin production and melasma . However, at present, no study has reported the antimelanogenic 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 a skin-whitening and antiaging agent.
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 β-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 Biotechonology, 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
KRG was kindly provided by the Korea Ginseng Cooperation that consisted of the following 11 ginsenosides composition [mg/g]: Rb1 6.67, Rb2 2.79, Rc 1.01, Rd 1.01, Rg3s 2.22, Rg3r 0.79, Re 1.97, Rf 1.40, Rg1 1.67, Rg2s 1.23, and Rh1 0.77 as analyzed by High Performance Liquid Chromatography [HPLC] analysis. While 3% red ginseng cream [the composition of ginsenosides was same as described previously] was prepared by Kyungnam University, Changwon, Republic of Korea. Briefly, a mixture of 80 mL of purified water and 30 mL of either sweet almond oil or sunflower seed oil was heated at 80°C. Thereafter, purified water was added again and emulsified by using a blender. Subsequently, tocopherol was added as an antioxidant, and 3% red ginseng extract was added as the active ingredient; the mixture was termed red ginseng cream.
2.3. Evaluation of the stability of red ginseng cream
The following tests were performed to evaluate of the stability of 3% 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
Table 2
Measure of oil content in the experimental group and the control group
GroupsOil contentForeheadRight cheek ①Right cheek ②Left cheek ①Left cheek ②Average0 weeks36.8 ± 25.6737.0 ± 33.5040.4 ± 24.6022.2 ± 12.9526.6 ± 20.9632.6 ± 7.78Experimental group2 weeks85.0 ± 44.5494.0 ± 68.19123.2 ± 10.5782.0 ± 69.65106.2 ± 13.8698.08 ± 16.914 weeks104.20 ± 44.64112.20 ± 104.48114.40 ± 83.7394.00 ± 67.9277.20 ± 55.36100.4 ± 15.24Variation+67.4+75.2+74+71.8+50.667.8 ± 10.060 weeks98.0 ± 44.1280.8 ± 45.19116.4 ± 74.9391.4 ± 41.4266.8 ± 24.0490.68 ± 18.60Control group2 weeks173.4 ± 112.74199.8 ± 171.67115.2 ± 56.96124 ± 51.78114.6 ± 70.72145.4 ± 38.934 weeks144.6 ± 48.41*143.0 ± 64.25177.6 ± 57.86116.8 ± 77.1699.4 ± 91.78136.28 ± 29.84Variation+46.6+62.2+61.2+25.4+32.645.6 ± 16.56
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 [].
- [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 , and no erythema, edema, or untoward reaction was observed. Therefore, this cream was further tested for other parameters on skin. Furthermore, the degree of skin irritation was evaluated, as shown in , 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 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 past 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 were 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.
Ten individuals [five in the experimental group and five in the control group] were selected to account for a dropout rate of 20%. The human trial experiments were permitted by the Institutional Review Board of Kyungnam University [IRB # 1040460-A-2017-040].
Every day for 1 month, the cream was applied to the skin 1 h after cleansing. Thereafter, the aforementioned parameters were measured in the skin on Weeks 0, 2, and 4.
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 3–5 seconds. The U-zone [2] was drawn horizontally from the nose and vertically down from the tail of the eye, as shown in . To standardize the measurement conditions, the research was performed at 24 ° C ± 1 ° C and 55% ± 10% relative humidity.
The oil content, water content, elasticity, and melanin content were measured by using a sebumeter SM815 [CK electronics, Germany], corneometer CM825 [CK electronics, Germany], mexameter MX18 [CK electronics, Germany], and a cutometer [CK electronics, Germany], respectively, by touching these instruments to the skin for 3–5 seconds.
The moisture and oil content and skin tones of the skin were analyzed by T-test. An empirical analysis of the study results was considered to indicate statistically significant changes for p values of