Abstract
Purpose: This study was conducted to confirm the applicability of a mixed herbal extract (MHE) as an anti-aging cosmetic ingredient by investigating its skin anti-aging activities in vitro and in vivo. Methods: In this study, we prepared MHE using an ultrasonic extraction containing Forsythiae fructus, Tribuli fructus, Solomon’s seal, Siberian ginseng, Ponciri Fructus and Ginseng. We investigated the anti-aging effect of the MHE for skin in dermal fibroblasts. The anti-aging activity was determined by the type I collagen synthesis levels. Matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinases 1 (TIMP1) mRNA levels were measured by qRT-PCR. MMP1 protein levels were evaluated by blotting analysis. Clinical tests of skin moisture, elasticity, texture, and wrinkles were performed using cosmetics containing 1% MHE. Results: The MHE induced the upregulation of pro-collagen type I synthesis and TIMP1 mRNA expression. The MHE led to the downregulation of MMP1 mRNA levels and protein levels. Furthermore, after skin application of cosmetics containing 1% MHE, skin hydration, elasticity, texture, and crow’s feet were improved 4 weeks after the treatment. Conclusion: MHE has an anti-aging effect by promoting collagen synthesis and suppressing MMP1 gene expression in vitro, and it has a skin improvement effect in vivo. Therefore, the MHE was shown to have value as a functional cosmetic ingredient.
Keywords: Skincare, Anti-aging, Anti-wrinkle, Natural cosmetics, Herbal medicine
The Herbal Supplements Cistanche For ANTI-AGING
Introduction
The skin is an organ that comes into contact with the external environment and is vital for our survival, acting as the primary barrier against external attacks such as infectious agents and physical injury; additionally, it fulfills several important physiological roles (Slominski et al., 2004; Vollmer et al., 2018). As we age, skin function gradually deteriorates with atrophic changes due to ultraviolet (U.V.) rays, pollution, and stress and by skin aging that occurs as the number of skin cells and skin thickness decrease (Farage et al., 2013).
Skin aging can be divided into endogenous aging and exogenous aging. Endogenous skin aging occurs naturally over time regardless of external influences. The Dermal-Epidermal junction (DEJ) cohesion weakens, and with the differentiation of keratinocytes, lipid formation decreases (Makrantonaki et al., 2007). On the other hand, exogenous aging is caused by external factors such as U.V. rays, infrared rays (I.R.), smoking, fine dust, and lifestyle. Among these, photoaging is caused by U.V. rays penetrating the skin and damaging collagen and elastin in the dermis. In this process, the elastic fibers and collagen fibers of the skin are deformed, resulting in decreased elasticity and sagging. Collagen is a major component of the extracellular matrix and has an important role in determining tissue function.
Among the various collagen types, type 1 collagen accounts for about 75-80% of the total collagen (Landau, 2007; Lovell et al., 1987; Mays et al., 1988). When the skin is exposed to U.V. rays for a long time, free radicals are produced. These free radicals stimulate the expression of matrix metalloproteinase-1 (MMP1) that promotes collagen degradation and inhibits collagen synthesis which reduces skin elasticity and results in wrinkles. Therefore, to prevent and improve skin wrinkles caused by aging, it is necessary to develop an ingredient that promotes collagen production and inhibits the expression and activity of MMP1 (Wlaschek et al., 2001).
Skin aging is closely related to quality of life. Because the skin is the most visible organ, it makes us aware of the aging process (Binic et al., 2013). Various efforts are ongoing in the fields of medicine, pharmaceuticals, and cosmetics to prevent and improve skin aging. The development of anti-aging ingredients is one of the most important anti-aging strategies.
We have prepared herbal extracts using ultrasonic extraction to develop new anti-aging ingredients containing Forsythiae fructus, Tribuli fructus, Solomon's seal, Siberian ginseng, Ponciri fructus, and Ginseng.
Six herbal medicines were selected from plants with various medical and skin effects. Forsythiae fructus, the dried fruit of Forsythia suspense (family Oleaceae), known as Lian Qiao in China, was first recorded in Shennong Bencao Jing, a prestigious monograph on traditional Chinese medicine (TCM) (Dong et al., 2017). It has been used as a heat-clearing and detoxifying TCM for the treatment of infectious diseases, such as acute nephritis, erysipelas, and ulcers (Wang et al., 2018). It is widely used in clinics as a single drug or compound prescription. Modern pharmacology has shown it to have a variety of bioactivities, including anti-inflammatory, antibacterial, antiviral, antioxidant, antitumor, anti-diabetic, antihyperlipidemic, antiandrogenic alopecia, anti-vomiting, anti-aging, and anti-obesity activities and neuroprotective, hepato-protective, and vasorelaxant effects (Dong et al., 2017). In addition, it has been reported that Forsythiae fructus has skin whitening, anti-atopic dermatitis, and anti-aging effects. According to Taiwan's nationwide prescription database, Forsythia Fructus has been included in the top 10 herbs most commonly used to treat atopic dermatitis (15.9%), urticaria (11.49-13.4%), and acne (22.3%) (Dong et al., 2017). Forsythiae fructus contains saponins,flavonoids, and alkaloids and oleanolic acid which has a useful pharmacological action. Additionally, it contains arctigenin and matairesinol. As physiologically active skin ingredients, they are tyrosinase inhibitors that act on the melanin metabolism pathway induced by α-melanocyte stimulating hormone (α-MSH)and are attracting attention as cosmetic whitening materials (Yang & Choe, 2011). Tribuli fructus, which is the dried fruit of Tribulus terrestris L, has been reported to have pharmacological activities of improving sexual function, preventing and treating cardiovascular disease, and improving neuroprotection and memory. It also has anti-diabetic, anti-inflammatory, and antioxidant effects (Chhatre et al., 2014). Tribuli fructus extract contains large amounts of diosgenin, tigogein, various saponins, etc. These saponins are the most important bioactive components responsible for various biological effects such as the aphrodisiac effect, antihypertensive effect and protective effect against ischemia-reperfusion injury and the anticancer, antibacterial and antifungal activities (Chhatre et al., 2014).
Other ingredients include furostanol and spirostanol saponins, polysaccharides, flavonoid glycosides, alkaloids, and amides. In addition, it is effective against atopic dermatitis in rats and has anti-inflammatory effects (Chhatre et al., 2014). It has been reported that the anti-aging effect of Tribuli fructus extract increases the production of Type I pro-collagen and Elastin in HS68 cells, promotes the lipid accumulation of adipocytes, and ultimately improves skin wrinkles and elasticity (Kim et al., 2016). Siberian ginseng (Acanthopanax senticosus) is a deciduous shrub belonging to the family Oga along with ginseng and wild ginseng. Siberian ginseng has many similarities with ginseng and is used as an herbal medicine for stroke, high blood pressure, and diabetes. Siberian ginseng extract has been used in oriental medicine for a long time, and pharmacological and physiological results have been reported in various studies until recently. Siberian ginseng components that have been identified so far include eleuthero sides A, B, C, D, E, I, K, L,and M, and sesamin, chiisanoside, caffeic acid, chlorogenic acid, campesterol, vitamins, and minerals (Park et al., 2010).
It contains triterpenoid saponins, lignans, coumarins, and flavonoids, among which the phenolic compounds are considered to be the most active components (Huang et al., 2011). There are many reports on the effectiveness of Siberian ginseng extract on the skin. The anti-wrinkle effect, antioxidant effect, collagen synthesis, and collagenase inhibitory activity against photoaging have been reported (Park et al., 2010). It has been reported that Siberian ginseng extract regulates the synthesis of Type IV collagen, affecting the proliferative capacity of epidermal stem cells and thickening the epidermis (Choi et al., 2016). Solomon's seal is a rhizome of Polygonatum odoratum (synonym: P.officinale) that has a tonic effect and is used to treat heart failure and diabetes in oriental medicine. Solomon's seal is anti-cycle and antitussive; it relieves heart tension, and is a diuretic, energizer, hypoglycemic, sedative, and tonic; it is used to treat lung diseases, including tuberculosis and gastrointestinal disorders (Haroon et al., 2012). In addition, it is widely used in the treatment of blood disorders; it has beneficial effects on the kidney ducts and prevents gray hair, vision problems, vertigo, and ringworm. It is a nervine tonic and is used in diabetes treatment (Haroon et al., 2012). Solomon's seal has a high phenol content and antioxidant activity. Homoisoflavanones from Solomon's seal inhibit the formation of advanced glycosylation end products. Solomon's seal has shown a strong intestinal immune system regulatory activity and the ability to induce osteoclast differentiation and lectin-induced apoptosis. In the case of P. odoratum leaf extract, the wound healing function is reported as an example (Fathi et al., 2014). Ponciri fructus immatures is the unripe fruit of Poncirus trifoliata Rafinesque, belonging to the Rutaceae family (Jung et al., 2016). Recently, anti-inflammatory and anti-hypersensitivity effects have been reported, and the effect of the extract of fat silt has been reported to induce the apoptosis of cancer cells (Lee et al., 2008). Ponciri fructus is known to be effective in treating atopic dermatitis. Through the skin application of Ponciri fructus extract, its effects against atopic dermatitides, such as epidermal hyperkeratosis, dermal edema, and cytokines, have been reported. In particular, significant decreases have been reported in itching, the number of mast cells, IgE production, and nerve growth factor expression. Ponciri fructus extracts have shown an effective anti-inflammatory effect against atopic dermatitis and antipruritic efficacy by regulating immune mechanisms. In addition, oral administration of Ponciri fructus extract is known to be helpful for the treatment and prevention of allergic diseases (Hwang et al., 1997). Ginseng is a widely used traditional herbal medicine with multi-functional activities. Ginseng has been used for its anti-inflammatory, antioxidant, antitumor, and anti-aging activities (Choi, 2008; Kim & Ko, 2020). Many studies have been conducted on the efficacy of ginseng on the skin (He et al., 2018). Among them, there are many reports on saponins and ginsenosides, which are representative ingredients of ginseng. Saponins are compounds found extensively in most plants and exist in various forms. Saponins can accelerate various biological activities such as hemolysis, and antibacterial, antiviral, and antioxidant functions. In addition, saponins have anti-inflammatory properties that can reduce swelling and skin inflammation. Ginsenosides increase interleukin (I.L.)-1β, one of the inflammatory cytokines known to promote angiogenesis and induce vascular endothelial growth factor and accumulationat skin burn sites in mice. Their effect of removing macrophages from skin wounds and promoting wound healing has also been reported (Kim et al., 2011).
The objective of this study was to elucidate the skin anti-aging effect of a mixed herbal extract (MHE) in vitro and in vivo.
Methods
1. In vitro efficacy evaluation
1) Preparation of the MHE
Dried fruit of Forsythia suspense, dried fruit of Tribulus Terrestris L., Siberian ginseng (Acanthopanax senticosus (Eleuthero) Root), Solomon's seal (Rhizome and root of Polygonatum Officinale), the fruit of the Poncirus Trifoliata Rafinesque, and ginseng (Panax) were purchased from a local market. Based on the "Yin-Yang" and "Five Elements" theory of Traditional Chinese Medicine (TCM), Forsythia suspensa, Tribulus terrestris L., and Poncirus trifoliata Rafinesque corresponding to "Yang" and Siberian ginseng, ginseng, and Solomon's seal corresponding to "Yin" was selected. The six medicinal herbs were again divided into the characteristics corresponding to the five elements (wood, fire, water, Metal or Gold, and Earth or soil). These medicinal herbs were mixed at the same ratio for the balance of each element, and Poncirus trifoliata and ginseng corresponding to Earth were combined with the other elements to match the proportions (Table 1). The six medicinal plants were extracted in 30% ethanol by ultra-sonication (20 kHz, 2 h). After concentrating with a rotary evaporator, 30% (v/v) butylene glycol was added, and the solution was diluted to prepare an extract containing 1% solid content.
Table 1. Composition of mixed herbs
2) Cell cultures and reagents
Human primary fibroblast cells (HDFn) were obtained from PromoCell (Normal human dermal fibroblasts juvenile foreskin, C-12300; PromoCell, Germany). Cell cultures with Dulbecco's modified Eagle's medium high glucose (DMEM high glucose, SH30243.01, Hyclone, Cytiva) containing 10% (v/v) fetal bovine serum (FBS, SH30084.03, Hyclone) and 1% antibiotic antimycotic agents (Anti-anti, 15240-062, Gibco, USA) were cultured at 37℃ in a 5% CO2 incubator.
3) Cell viability assay (MTT assay)
The MTT assay measured the cell viability and determined the cytotoxicity of the MHE. Cells were seeded at 5×103 cells/well on a 96-well plate and cultured for 24 h under cell culture conditions. Then, we discarded the medium, washed the cells with a solution of phosphate-buffered saline (PBS, 21-040-CV; Corning, USA), placed the cells in new serum-free media, and treated the cells with concentrations of 0.01, 0.025, 0.05. 0.10, 0.25, 0.50, 0.75, and 1.00% of the samples, and incubated the cells for 24 and 48 h. Then, we added 100 μL of MTT solution (0.5%, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H tetrazolium bromide) to each well and incubated the cells for another 2 hours. After removing the culture media, we added 100 μL of 100% dimethyl sulfoxide (DMSO), shook the cells for 10 minutes, and measured the absorbance at 590 nm with a microplate reader (Epoch 2 microplate reader, BioTek, USA).
4) Measurement of pro-collagen type I generation
The concentration of pro-collagen type I in the cell culture medium was measured using a commercially available ELISA kit (Procollagen type I C-peptide EIA Kit, MK101, TaKaRa Bio, Japan) according to the manufacturer's instructions. Each sample was analyzed in three replicates.
5) Measurement of mRNA expression levels using Quantitative
Real-Time polymerase chain reaction (qRT PCR) Cells were seeded at 1×105 cells/well on 6-well plates. HDFn cells were cultured for 24 h; then, the cells were synchronized with DMEM high glucose without FBS for 24 h. The medium was replaced with serum-free media and treated with concentrations of 0.05, 0.10, 0.25, 0.5, and 0.75% of the samples for 24 and 48 hours. RNA extraction was performed with the HDFn cells using an RNA extraction kit (TaKaRa MiniBEST Universal RNA extraction kit, 9767A, Takara Bio, USA), and samples were purified following the manufacturer's protocols. The isolated RNA (1 μg) was synthesized into cDNA using a T.R. reagent kit (PrimeScriptTM RT reagent Kit with gDNA Eraser, RR047A, Takara Bio, USA). Using a SYRB Green Realtime PCR Master Mix (Power SYBRTM Green PCR Master Mix, 4367659, Applied BiosystemsTM, USA) and QuantStudioTM 3 (QuanStudioTM 3 Real-Time PCR instrument, A28132, Thermo Fisher Scientific, USA) real-time PCR machine, the gene expression levels were standardized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers used in this study are presented in Table 2. The experiments were performed in duplicate for three independent repeats. Melting curve analysis was performed for each primer set.
6) Western blot analysis
Protein lysates from fibroblast cells were extracted with a lysis buffer (EzRIPA Lysis Kit, WSE-7420; ATTO Corporation, Japan). The Bradford method was used to quantify the proteins. Equal quantities of proteins were mixed with 5X sample buffer (TLP-102-01, TransLab, Korea) and denatured at 99℃ for 10 minutes. The proteins were separated by 10% SDS-PAGE at 150 V for 150 min and transferred to PVDF membranes (Clear Blot Membrane, 3322546, ATTO Corporation, Japan). The membranes were blocked with 0.5% bovine serum albumin (BSA, A4503-10G; Sigma) for 1 hour and washed with Tris-buffered saline that included Tween-20 (10X TBS with Tween 30, TR2007-100-74; Biosesang, Korea) three times; membranes were then incubated with primary antibodies against MMP-1 (Recombinant Anti-MMP1 antibody [EP1249Y], ab134184, Abcam, USA),β-Actin (Anti-β-Actin (N-tern) and PA0207 (Abfrontier, Korea)at 4℃ overnight. After rinsing, the membranes were treated with anti-rabbit IgG-horseradish peroxidase (HRP) (Anti-rabbit IgG, HRP-linked Antibody, #7074; Cell Signaling Technology,USA) as the secondary antibody for 1 hour. Chemiluminescence detection was performed using the ECL Prime western blotting detection reagent (GE Healthcare, USA) and the ATTO WSE-6100 LuminoGraph (ATTO Corporation, Japan). For quantitative analysis, the captured images were analyzed with Image software (C.S. analyzer 4, ATTO corporation, Japan).
7) Statistical analysis
Statistical analysis was performed using GraphPad Prism version 3.03 for Windows (GraphPad Software, USA). Results are presented as the mean±SD of 3 experiments. An unpaired Student's t-test was used, and p values <0.05 were considered statistically significant.
2. In vivo efficacy evaluation
1) Reagents and cosmetic products
The test products were skin lotion, essence cream, and eye cream; those creams contained 1% of the MHE as the active ingredient shown in Table 3.
2) Human participants
All volunteers agreed to the purpose and content of the test and signed written informed consent forms before starting the test. They were not allowed to use any other skincare products, and the test products were applied to the face in the order of skin-eye cream (only on the crow's feet)-essence cream-lotion cream. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Korea National Institute for Bioethics Policy (#P01-202108-13-001).
3) Efficacy test
Twenty-one female subjects aged between 45 and 55 (mean ±S.D. 50.81±2.86) years with normal skin participated in this study. Test products were applied twice daily (morning and evening) for 4 weeks. The participants visited the research center every 2 weeks, and the test sites were measured in the laboratory, where the ambient temperature and relative humidity were maintained at 22±2℃ and 50±5%, respectively. The participants acclimated for 20 min in the laboratory after washing their faces and were assessed for skin hydration, elasticity, texture, and crow's feet. Skin hydration was measured using a Corneometer® CM825 (C+K electronic GmbH, Cologne, Germany); elasticity was measured using a Cutometer® MPA 580 (C+K electronic GmbH, Cologne, Germany), and the texture and crow's feet were measured using a DermaTOP 3D-HE (EOTECH SAS, French).
4) Evaluation of adverse reactions of the skin
Adverse reactions were checked every visit by questionnaire and observation. In the evaluation, the subjective parameters were classified as itching, prickling, tickling, burning, stinging, stiffness, and tightening, and the objective parameters were classified as erythema, edema, scale, and papule.
5) Statistical analysis
Data were analyzed using the SPSS® software. The statistical significance of the differences between time points was determined using repeated measures analysis; a p-value<0.05 was considered statistically significant. The change rate (%) from the baseline value was calculated as follows:
Change rate=((after treatment-baseline)/baseline)×100%
