Anti-aging Effect Of Mixed Extract From Medicinal Herbs Ⅱ

Results

1. Anti-aging effect of the MHE in vitro

1) Effects of the MHE on cell viability

To ensure the cell viability of the MHE on normal human dermal fibroblasts, the cells were treated with various concentrations (0.01%, 0.025%, 0.05%. 0.10%, 0.25%, 0.50%, 0.75%, and 1.00%) of the MHE. The results suggest that MHE did not significantly reduce the viability of human fibroblast cells (Figure 1) and was safe to use at all concentrations.

2) Effects of the MHE on pro-collagen expression

The effects of the MHE on pro-collagen type I expression were studied in cultured normal human dermal fibroblasts. Compared with normal cells, the MHE treatment with concentrations of 0.05%, 0.10%, 0.25%, 0.50%, and 0.75% can strongly increase the procollagen type I content. The effect of the MHE on pro-collagen type I expression was found to be dose-dependent (Figure 2).

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3) Effects of the MHE on MMP1 and TIMP1 gene expressions

The gene expressions of MMP1 and TIMP1 were examined by real-time PCR. MHE significantly downregulated the MMP1 mRNA level in a dose-dependent manner at 48 hours (Figure

3A). Additionally, MHE downregulated MMP1 gene expression level to half at concentrations of 0.05% and 0.25% at 24 hours. In contrast, TIMP1 was increased in the cells treated with MHE at concentrations of 0.05%, 0.1%, and 0.25% (Figure 3B), which was probably related to the decrease in MMP1 expression. Thus, MHE downregulated MMP1 and upregulated TIMP1 and hence has a good anti-aging effect.

4) Effects of the MHE on MMP1 protein expression

To investigate whether MHE attenuates skin aging, HDFn cells were treated with different concentrations of the MHE, and the protein expression of MMP1 was detected by Western

Blotting. The results indicate that the MHE treatment significantly decreased the expression level of MMP1 (Figure 4A, 4B) in a dose-dependent manner.

2. Skin improvement effect of the MHE in vivo

In comparison to the baseline, skin hydration significantly increased at 2 and 4 weeks after the treatment. The increments were 24.60% and 33.04%, respectively (Table 4 and Figure 5A). Skin elasticity significantly increased at 2 and 4 weeks after the treatment. The increments were 9.51% and 11.40%, respectively (Table 4 and Figure 5B). In the case of skin texture, all parameters (Sa and Stm) were significantly improved at four weeks after the treatment. The improvement rate of the Sa parameter was 10.45% and that of the Stm parameter was 11.61% (Tables 4 and 6 and Figure 5C, 5D). In the case of the crow's feet, all parameters (Ra, Rz, area and volume) were significantly improved at 4 weeks after the treatment. The improvement rates of the Ra, Rz, Area, and Volume parameters were 7.35%, 6.18%, 13.41% and 15.29%, respectively, (Table 5 and 6, and Figure 5E, 5F, 5G, 5H). No adverse reactions were observed in any of the participants during the test period. No itching, prickling, tickling, burning, stinging, stiffness, or tightening were experienced by the participants. In addition, no erythema, edema, scale, or papule were observed by the examiners.

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Discussion

Skin aging is a complex biological process that is affected by a combination of endogenous and exogenous factors. The endogenous factors are genetics, cellular metabolism, hormones, and metabolic processes. The exogenous factors are chronic light exposure, contamination, ionizing radiation, chemicals, and toxins. Various aging factors combine leading to accumulated structural and physiological changes and gradual changes in each skin layer and to changes in the appearance of the skin, especially in areas where the skin was exposed to the sun (Cevenini et al., 2008; Ganceviciene et al., 2012). The skin is an organ that shows the most marks from aging. Therefore, many researchers are making efforts to develop new skin anti-aging materials. Traditional herbal medicines provide an interesting, largely unexplored source for the development of potential new drugs. The potential use of traditional herbal medicines for the development of new skin-care cosmetics has been emphasized recently (Kiken & Chhen, 2012).

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The purpose of this study was to develop a cosmetic ingredient with anti-aging and anti-wrinkle functions based on herbal medicines. For this, six medicinal herbs were mixed and

extracted based on the "Yin-Yang" and "Five Elements" theory. To test the anti-aging effect of the MHE, we evaluated skin collagen production, MMP1 inhibition, and TIMP1-promoting

effects. To elucidate whether the MHE results in wrinkle improvement, the inhibitory activities of MMP1 and the promoting activities of collagen type I synthesis and TIMP1 were measured using HDFn cells. As a result of the test, it was revealed that the MHE has the effect of improving skin wrinkles by increasing the amount of collagen production in the HDFn

cells and inhibiting the gene expression and protein production of MMP1. In addition, there is an effect of promoting the gene expression of TIMP1, which inhibits MMP1. It was further

confirmed that hydration, elasticity, texture, and wrinkles all improved within 4 weeks compared to before use on the skin of the lotion, essence, eye cream, and cream applied with 1% MHE. Therefore, it was confirmed that MHE has an anti-aging effect by improving skin wrinkles.

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In this study, ultrasonic extraction used for medicinal herbal extraction is an extraction method widely used in plant extraction along with Soxhlet extraction, immersion extraction,

pressure extraction, reflux extraction, and accelerated solvent extraction. The ultrasonic extraction method has the advantage of low solvent usage and easy extraction; thus, it is a popular extraction method when considering aspects such as economic efficiency and time of the extraction process (Wan et al., 2006). There are various methods for extracting plant components,and different types of solvents may be used such as water and ethanol, or different extraction methods may be used including pressure extraction and reflux extraction such as ultrasonic extraction. In addition, the extraction conditions can be changed such as the extraction time, temperature, and pH. It is important to find the optimal extraction method for each plant because the type and degree of the physiological activity of a plant extract may vary depending on the extraction method. In this study, six medicinal herbs were extracted using ultrasonic extraction; however, when other extraction methods are used, whether the physiologically active ingredients and skin effects change compared to the MHE used in this study should be clarified by further study.

The six herbal medicines have various medical and skin effects including an anti-aging effect. Therefore, it is predictable that by using them, the MHE has an anti-aging effect. Additionally, the MHE has the potential to be effective in skin-related whitening, as an antioxidant and anti-inflammatory agent, and in atopy improvement. Among the components of the MHE, the Forsythiae fructus extract has been reported to have a whitening effect, and the antioxidant functions of Siberian ginseng, Solomon's seal, Ponciri focus and ginseng have been reported. Each extract contains various flavonoids, which also have antioxidant properties (Choi, 2008; Dong et al., 2017; Fathiet al., 2014; Huang et al., 2011; Kong & Kwon, 2021; Yang & Choe, 2011). Antioxidants inhibit tyrosinase and interfere with melanin biosynthesis, which can have a skin-whitening effect (Briganti et al., 2003; Solano et al., 2006). Therefore, itis possible that MHE, which is supposed to contain flavonoids or other antioxidants, may have a skin-whitening effect. In addition, the MHE, which contains herbs with anti- inflammatory and atopy-improving effects (Bao et al., 2016; Dong et al., 2017), has the potential to improve atopic dermatitis and to have anti-inflammatory and wound healing effects. All these possibilities should be confirmed by further study.

anti-aging cistanche treatment

In this study, the anti-aging effect of the MHE extracted from six medicinal herbs with various medical and skin effects was confirmed, and the skin improvement effect was confirmed

on the facial skin by applying it to cosmetics. We developed a new cosmetic ingredient, MHE, and confirmed its value as a skin anti-aging ingredient. Therefore, it is expected that cosmetics that include the MHE can significantly contribute to the improvement of skin aging.

Conclusion

In this study, MHE containing Forsythiae fructus, Tribuli fructus, Solomon's seal, Siberian ginseng, Ponciri fructus, and Ginseng showed an anti-wrinkle activity in dermal fibroblast cells. In addition, the moisturizing, elasticity-improving, and skin texture activities of the MHE were elucidated by clinical tests. It seems that the MHE can be used as a skin anti-aging ingredient in functional cosmetics.

Author's contribution

MLSY designed this investigation, performed the experiment, and wrote the manuscript with assistance from AHY. AHY, and KWS performed experiments and data analysis. HJC performed the preparation of the MHE samples. LXX and JHH performed the preparation of the cosmetics. PHW, CJH oversaw the project.

Author Details

Soyoun Lee (Senior Researcher), Skin Science Research Center, New Life Cosmetics R&D Center, New Life Group Corporation, Seoul 08594, Korea; Hongyan An (Associate Researcher), Skin Science Research Center, New Life Cosmetics R&D Center, New Life Group Corporation, Seoul 08594, Korea; Woosoo Kim (Assistant Researcher), Skin Science Research Center, New Life Cosmetics R&D Center, New Life Group Corporation, Seoul 08594, Korea; Xinxin Lu (Senior Researcher), New Life Cosmetics R&D Center Co., Ltd, Shanghai 200233, China; Hyanghwa Jeon (Principal Researcher), New Life Cosmetics R&D Center, New Life Group Corporation, Seoul08594, Korea; Han Woong Park (Director), Skin Science Research Center, New Life Cosmetics R&D Center, New Life Group Corporation, Seoul 08594, Korea; Jeongcheol Ha (Managing Director), New Life Cosmetics R&D Center Co., Ltd, Shanghai 200233, China; Jinhun Cho (Vice President), New Life Cosmetics R&D Center Co., Ltd, Shanghai 200233, China.

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Anti-aging Effect Of Mixed Extract From Medicinal Herbs