Antiwrinkle and antimelanogenesis activity of the ethanol extracts of Lespedeza cuneata G. Don for development of the cosmeceutical ingredients

Abstract To develop the ingredient with the cosmeceutical function, the antiwrinkle and antimelanogenesis effects of the ethanol extract of Lespedeza cuneata G. Don were investigated. DPPH radical scavenging activity was significantly increased with the extract of L. cuneata G. Don. Cell viability on CCD986Sk human fibroblast was also increased by the ethanol extract of L. cuneata G. Don. The inhibitory function of the extract of L. cuneata G. Don on collagenase, elastase, and tyrosinase was evaluated. Protein expression level of Claudin‐1, Occludin, and ZO‐1 was up‐regulated in HaCaT human keratinocyte by the extract of L. cuneata G. Don. In addition, the extract of L. cuneata G. Don inhibited melanin synthesis in B16F10 murine melanoma cells by decreasing MITF, TRP1, and TRP2 protein levels and increasing the phosphorylated Erk and Akt. Thus, these findings would be useful for developing the new cosmeceutical formulations based on the extract of L. cuneata G. Don.

Along with collagen, elastin is also another important protein component for the extracellular matrix and elastase is known to break down the elastin protein. Low protein expression level of collagen and elastin is the main cause for the wrinkle formation in skin (Xu, Ryoo, Kim, Choo, & Yoo, 2009). Thus, researcher has tried to develop the novel way to inhibit collagenase and elastase enzyme activity, which might help to provide skin tensile firmness and prepare the cosmeceutical formulation for antiwrinkle and antiaging properties (Apraj & Pandita, 2016).
Tight junction (TJ) is one type of cell junctions and usually can be mediated by the membrane-integrated proteins such as claudins, occludin, and junctional adhesion molecule (JAM) and the cytoplasmic zona occluden (ZO) proteins (Brandner & Schulzke, 2015;. Among these proteins, claudins have several types of isomers that are mainly responsible for TJ depending on the tissue types (Shigetomi & Ikenouchi, 2017). Especially, in skin keratinocytes, claudin-1 is known to be involved in regulation of TJ and its functional mechanism has been studied by several recent reports (Tokumasu, Tamura, & Tsukita, 2017;Tokumasu et al., 2016;Volksdorf et al., 2017). As TJ plays a significant role in skin barrier function, researcher have been considering the novel way for preventing and strengthening skin barrier by the development of various cosmetic materials to control TJ in skin.
In this study, we evaluate the possibility of the ethanol extracts of L. cuneata G. Don as a cosmeceutical material. The ethanol extracts of L. cuneata G. Don shows the antiwrinkle activity through inhibition of collagenase and elastase as well as induction of up-regulation of protein markers responsible for TJ in skin. In addition, the ethanol extracts of L. cuneata G. Don exerts the antimelanogenesis activity via inhibition of tyrosinase and regulation of the proteins related to controlling tyrosinase expression and activity. In conclusion, our current study might provide evidence that the ethanol extracts of L. cuneata G. Don can be applied to a potential agent for treatment of skin disorders by keeping skin tissue maintenance and regulating melanogenesis.

| Preparation of the ethanol extract of Lespedeza cuneata G. Don
Lespedeza cuneata G. Don was collected from the different geographical regions of Korea was rinsed carefully with the fresh water and dried in air. Then, dried L. cuneata G. Don was ground and sifted through a 30-mesh sieve (600 μm, particle size). The powder was extracted with 70% ethanol and the solvent was removed by rotary evaporation.

| Cell viability assay
Cell viability assay was performed with the water-soluble tetrazolium salts (WST-1) method. Aliquots of 200 μl of CCD986Sk cell culture (1 × 10 3 cells/well) were added to a 96-well plate and incubated for 18 hr. Then, L. cuneata G. Don extract at various doses (500, 250, 125, and 62.5 μg/ml) or vitexin (20 μM) were added into each well and cells were incubated for 48 hr. Control cultures were added with DMSO. After incubation, 20 μl of WST-1 solution (Daeillab Service, Seoul, Korea) was added to each well and cells were incubated for 4 hr. The absorbance of the solution was measured at 460 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
The plate was stored in the dark for 15 min and the absorbance was measured at 517 nm using the microplate reader.

| Collagenase inhibitory assay
Collagenase inhibitory assay was performed using an MMP-1 Human ELISA kit (Amersham, Little Chalfont, UK) with standard manufacturer's protocol. 4-phenylazobezyloxylcarbonyl Pro-Leu-Gly-Pro-Arg (the synthetic substrate) was dissolved in reaction buffer at 0.3 mg/ml and 250 μl of substrate was added in the reaction tube with 100 μl of various concentration of L. cuneata G. Don extract (500, 250, and 125 μg/ml) or vitexin (20 μM). Collagenase was dissolved in buffer at 0.2 mg/ml and 150 μl of enzyme was added in the reaction tube. After incubation at 25°C for 20 min, the reaction was stopped by addition of 6% citric acid. The reaction mixture was separated using 1.5 ml of ethyl acetate. The absorbance was measured at 320 nm using the microplate reader.

| Collagen type I synthesis assay
CCD986Sk human fibroblast cells (1 × 10 5 cells/well) were seeded to a 6-well plate and cultured for 24 hr. After incubation, the culture medium was changed to serum-free medium or L. cuneata G. Don extract-treated medium (500, 250, and 125 μg/ml) and incubated for 48 hr. The supernatant was collected from each well and the amount of procollagen type I C-peptide was measured with a procollagen type I C-peptide assay kit (Takara, Otsu, Japan) according to manufacturer's manual.

| Elastase activity assay
Elastase activity assay on L. cuneata G. Don extract was conducted The elastase activity was quantified by measuring light absorbance at 410 nm with the microplate reader.

| Tyrosinase activity assay
B16F10 cells were treated with L. cuneata G. Don extract (500, 250, and 125 μg/ml) or vitexin (20 μM) for 24 hr and were lysed with lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and then centrifuged at 18,000 g for 30 min at 4°C. The supernatant was collected and tested for the cell tyrosinase activity. The reaction mixture containing 60 mM phosphoric acid buffer solution (pH 6.8), 10 mM L-DOPA solution, and the supernatant was incubated at 37°C. After incubation, the dopachrome was monitored at 475 nm for 5 min.

| Wound healing assay
HaCaT cells (1 × 10 5 cells/well) were seeded in 6-well plates and cultured with serum-free medium for 18 hr. Then, an artificial wound was scratched into the confluent cell using a P200 pipette tip. Microscopy images (Leica DM IL LED; Leica, GmbH, Germany) were taken immediately for record of 0 hr status and then cell culture media was changed with DMEM supplemented with 1% FBS. Cells were treated with the vehicle control or L. cuneata G.

| Melanin content analysis
B16F10 cells (1 × 10 5 cells/well) were seeded into a 6-well plate and then incubated for 18 hr. The culture medium was removed and replaced with fresh medium containing various concentrations of L. cuneata G. Don extract (500, 250, and 125 μg/ml) or vitexin (20 μM) for 48 hr. After cells were harvested and washed with PBS, total melanin content in cell pellet was imaged with a light microscopy (Nikon1 J1; Nikon, Tokyo, Japan). Harbor, ME, USA). Chemiluminescence was detected using ECL (Gendepot, Barker, TX, USA).

| HPLC analysis of the ethanol extract of Lespedeza cuneata G. Don and vitexin
For comparing the retention time of vitexin standard with the crude 70% ethanol extract of L. cuneata G. Don, analytical highperformance liquid chromatography (HPLC) was adopted. Using WATERS SYMMETRY C18 column (WATERS, Seoul, Korea, 4.6 × 220 nm), vitexin standard with the crude 70% ethanol extract of L. cuneata G. Don were eluted and analyzed for their retention time. The flow rate of the solvent was 1 ml/min and a single absorption peak was identified at 250 nm. Then, for identification of the active chemical candidates, 15Tesla Fourier transform ion cyclotron resonance (15T FT-ICR) mass spectrometry analysis was performed.

| Statistical analysis
All experiments were performed in triplicate. The data are expressed as the mean ± standard deviation (SD). Significant differences between controls and L. cuneata G. Don extract-treated cells were determined using a Student's t test at p value <0.05.

| Effect of the ethanol extract of Lespedeza cuneata G. Don on the human fibroblast viability and DPPH radical scavenging activity
Antioxidant effect of the natural plant extracts is well-known standard to determine whether the extract can be used as a component of the cosmeceutical formulation or not. Antioxidant effect is primarily characterized by the relative amount of flavonoids in each plant . As shown in Figure 1a, it was demonstrated that the ethanol extract of L. cuneata G. Don showed DPPH radical scavenging activity with dose-dependent manner. Especially, 250 and 500 μg/ml of the ethanol extract of L. cuneata G. Don has the statistically significant (p value <0.05) DPPH radical scavenging activity. 500 μg/ml of the ethanol extract of L. cuneata G. Don has the similar DPPH radical scavenging activity with 1 μM of ascorbic acid that was used for the positive control.
Then, we investigated the cell growth effect of the ethanol extract of L. cuneata G. Don on CCD986Sk human fibroblast cells that are generally known to help skin to be maintained with the proper collagen expression level (Bhagavathula et al., 2009;Ravanti, Heino, López-OtıŃ, & Kähäri, 1999). We treated CCD986Sk cells with the ethanol extract of L. cuneata G. Don and measured the growth rate of CCD986Sk using the WST-1 assay. As shown in Figure 1b Don has the potential to provide more collagen around skin fibroblasts without any toxicity.

| Measurement of antiwrinkle activity of the ethanol extract of L. cuneata G. Don
The expression of matrix metalloproteinases (MMPs, also called collagenases) are significantly up-regulated by the external conditions such as UV irradiation and aging (Watanabe et al., 2004). Elastin is another component composing of the extracellular matrix in the connective tissue around skin dermis and can be degraded by elastase, a kind of proteinase enzyme (Meyer, Neurand, & Radke, 1981). Furthermore, hyper elastase activity is known to cause severe immune system-related diseases such as rheumatoid arthritis and lung fibrosis through the destruction of structural proteins (Dell'aica et al., 2007;Sartor et al., 2002). Therefore, regulation of elastase activity might protect skin connective tissue from losing tissue elasticity and be used for treating way of several diseases. For this purpose, we tried to examine whether the ethanol extract  (Seo et al., 2017). Collagen is derived from precursor procollagen that includes the extra-peptide sequences in N and C-terminus (Seo et al., 2017). After cleavage of this additional peptide sequence from procollagen by procollagen peptidase, collagens are synthesized as a kind of components for extracellular matrix proteins and incorporated into mesh network of extracellular fibrils (Lee, Hossaine, et al., 2016;. To examine the amount of collagen in CCD986Sk cell culture media treated by the ethanol extract of L. cuneata G. Don, the level of Type I collagen was measured by procollagen Type I C-peptide ELISA assay kit as the amount of procollagen secreted from CCD986Sk cell reflects the amount of collagen.
As shown in Figure

| Measurement of antimelanogenesis activity of the ethanol extract of Lespedeza cuneata G. Don
As the initial enzyme for melanin synthesis, tyrosinase plays a key role in pigmentation of skin and hair as well as in wound healing and immune response (Cabanes, Chazarra, & Garcia-Carmona, 1994;Fairhead & Thöny-Meyer, 2012). Tyrosinase is known to be induced by ionizing and UV radiation exposure to activate melanogenesis that takes place in melanosomes of melanocyte (Tsatmali, Ancans, & Thody, 2002). Excessive melanogenesis is generally associated with the pigmentation-related disorders such as hyperpigmentation and skin cancer (Costin & Hearing, 2007;Yoon et al., 2015). Tyrosinase inhibitors could be used for the preventing skin hyperpigmentation and the treatment of skin disorders (Ortonne & Passeron, 2005;Parvez et al., 2007). We tried to elucidate the effect of the ethanol extract of L. cuneata G. Don on tyrosinase activity. As shown in Error bars indicate the standard deviation (SD). The significance was determined by Student's t test (*p < 0.05). Experiments were performed in triplicate. (b) Evaluation of melanin content in B16F10 after treatment of the ethanol extract of L. cuneata G. Don with the indicated concentrations (500, 250, and 125 μg/ml) for 48 hr. After cells were harvested and washed with PBS, total melanin content in cell pellet was imaged with a light microscopy L. cuneata G. Don has the potential to be developed as cosmeceutical formulations to prevent the overproduction of melanin in skin.
TRP1 and TRP2 have about 40% homology with tyrosinase, are also responsible for melanogenesis of melanocytes, and transcriptionally modulated by MITF, a major transcription factor in melanogenesis (Slominski, Tobin, Shibahara, & Wortsman, 2004;Yasumoto, Yokoyama, Takahashi, Tomita, & Shibahara, 1997;Yavuzer et al., 1995). TRP1 is involved in regulation of tyrosinase activity by stabilizing of tyrosinase protein (Ghanem & Fabrice, 2011) and in affecting melanocyte proliferation by maintenance of melanosome structure (Sarangarajan & Boissy, 2001). TRP2 (dopachrome tautomerase) mediates cyclization of L-DOPA resulting in synthesis of dopachrome, one of the intermediates during the biosynthesis of melanin (Slominski et al., 2004). We further investigated whether the ethanol extract of L. cuneata G. Don on the reduction in MITF is related to the phosphorylation of Akt and Erk. The activation of Akt and Erk signaling pathways has been reported to inhibit melanogenesis (Englaro et al., 1998;Kim et al., 2003;Lee, Jung, Kim, & Park, 2007;Oka et al., 2000). Both of Akt and Erk activation can phosphorylate MITF followed by proteasome-mediated ubiquitination leading to decrease in MITF protein stability and activation of MITF protein degradation.

| Identification of vitexin from 70% ethanol extract of Lespedeza cuneata G. Don and evaluation of vitexin for antiwrinkle and antimelanogenesis activity
To show the bioactive components responsible for antiwrinkle and antimelanogenesis activity, we performed analytical HPLC analysis and 15Tesla Fourier transform ion cyclotron resonance (15T FT-ICR) mass spectrometry analysis. As shown in Figure 6a

| CON CLUS ION
Natural components derived from extracts of traditional herbal plant are of interest for cosmetic applications such as antiwrinkle and skin whitening. As the potential of the functional ingredients for cosmeceuticals, the novel activity of the ethanol extract of L. cuneata G. Don is not still fully understood and molecular mechanisms related to its activity remains to be discovered. For this purpose, we demonstrate that the ethanol extracts of L. cuneata G. Don on protein expression related to melanin synthesis in B16F10 cells. B16F10 cells were treated with the indicated concentration of the ethanol extract of L. cuneata G. Don (500, 250, and 125 μg/ml) for 48 hr. After harvesting cells, TRP1, TRP2, MITF, pAkt, Akt, pErk, and Erk protein expression was examined by Western blotting. βactin was used as a loading control in B16F10 melanoma cells through downregulation of MITF, TRP1, and TRP2. Thus, to the best of our knowledge, the current study provides the first evidences that as safe and effective ingredients, the ethanol extract of L. cuneata G. Don possess the protective effects against damage in skin connective tissues. Although we focused on the possibility for cosmetic agents using the ethanol extract of L. cuneata G. Don, as we mentioned in the introduction part, L. cuneata G. Don has been traditionally used as the medicinal herbal food in Asia for treating various diseases and symptom.
In this study, we performed in-vitro assays for showing the novel cosmeceutical activity such as antiwrinkle and antimelanogenesis and suggested L. cuneata G. Don, a kind of traditionally edible herbal plants, could be used for agents for cosmeceutical product.
Thus, we think that this study will contribute to the expansion of the application using the traditional herbal food to another industrial and scientific field. For the further studies, we plan to identify the more active compounds separated from the ethanol extract of L. cuneata G. Don and elucidate the detailed relationship between the candidate compounds and signaling pathways for collagen synthesis, TJ activation, and melanogenesis. In conclusion, our study could help to develop new cosmetic and pharmaceutical substances based on the ethanol extract of L. cuneata G. Don.

CO N FLI C T O F I NTE R E S T
The author declares no conflict of interest.

E TH I C A L S TATEM ENT
Neither animal nor human testing was not involved in this study.