Antimelanogenesis And Skin-protective Activities Of Panax Ginseng Calyx Ethanol Extract Part 2

3. Results 

3.1. Antimelanogenesis effect of Pg-C-EE 

We first measured the cytotoxicity of Pg-C-EE in B16F10 cells and showed no cytotoxicity at Pg-C-EE concentrations up to 400 mg/mL (Fig. 1A). Arbutin (1 mM) was used as a positive control for anti melanogenesis activity as it is known to effectively inhibit tyrosinase activity. The inhibitory effects of Pg-C-EE on melanin secretion and synthesis were examined in a-MSHetreated B16F10 cells. The levels of inhibition of melanin secretion were 10% for Pg-CEE 100 mg/mL, 28.5% for Pg-C-EE 200 mg/mL, 36.2% for Pg-C-EE 400 mg/mL, and 36.7% for arbutin 1 mM (Fig. 1B). The levels of inhibition of melanin content were 16.5% for Pg-C-EE 200 mg/mL, 50.2% for Pg-C-EE 400 mg/mL, and 49% for arbutin 1 mM (Fig. 1C). The photographs (upper graph) and graphs show that melanin secretion and synthesis were suppressed by Pg-C-EE treatment in a dose-dependent manner (Figs. 1B, 1C). To investigate the activity of enzymes involved in melanin production, we used mushroom tyrosinase. The inhibitory effect of Pg-C-EE on tyrosinase activity was 11.8% (100 mg/mL), 13.7% (200 mg/mL), and 21.9% (400 mg/mL), whereas kojic acid (300 mM) inhibited tyrosinase activity by up to 87.9% (Fig. 1D). In reverse transcription polymerase chain reaction analysis of the inhibitory effect of Pg-C-EE on the expression of genes related to melanin production (MITF, TYRP-1,2, tyrosinase, MLPH, MyoVa, and Rab27a), Pg-C-EE did not show a significant inhibitory effect on mRNA expression, but the mRNA expression of TYRP-2, tyrosinase, and MLPH has slightly decreased at Pg-C-EE 400 mg/mL (Fig. 1E). Although Pg-C-EE did not affect the mRNA expression of genes associated with melanin production, it showed a signifificant inhibitory effect at the level of protein expression. Especially, the protein expression of tyrosinase, TRP-1, and TRP-2 was dose-dependently decreased by Pg-C-EE treatment (Fig. 1F). Because Pg-C-EE inhibited the expression of melanin production-related proteins, we then examined whether Pg-C-EE blocked the upstream proteins regulating melanogenesis by measuring the phosphorylation of p38, ERK, and CREB at different time points (48e24 h). Pg-C-EE dose-dependently decreased levels of phosphorylated p38, ERK, and CREB that were stimulated under a-MSH conditions (Fig. 1G). To demonstrate that p38 and ERK inhibit melanin production, we examined the levels of melanin secretion and content in the presence of SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and U0126 (ERK inhibitor). Melanin production was suppressed with SB203580 and U0126 treatment similar to Pg-C-EE treatment but was not inhibited in the SP600125-treated group (Fig. 1H).

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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In addition, cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells. Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche. Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products. In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.

3.2. Skin-protective activity of Pg-C-EE against UVB irradiation

To investigate the skin-protective activity against UVB irradiation using HaCaT cells, the cytotoxicity of Pg-C-EE was measured by MTT assay. Pg-C-EE did not show cytotoxicity in HaCaT cells up to a concentration of 400 mg/mL (Fig. 2A). Images of cells captured with a camera attached to the microscope showed that Pg-C-EE protected against UVB-irradiated HaCaT cell damage (Fig. 2B). To elucidate the UVB-mediated skin damage protective mechanisms of Pg-C-EE, the expression levels of MMP-1, -2, -3, and -9 were measured under conditions of UVB irradiation, which is known to increase skin aging through upregulation of MMPs. In UVB-irradiated HaCaT cells, Pg-C-EE at 400 mg/mL reduced the mRNA expression of MMP-1, MMP-2, MMP-3, and MMP-9 (Fig. 2C). Pg-CEE also decreased the expression of inflflammation regulatory genes COX-2, IL-6, and IL-8. After UVB irradiation with 100 mJ/cm2, COX-2 expression was decreased in the Pg-C-EE-treated group, whereas Pg-C-EE did not affect the Sirt1 gene (Fig. 2D). After UVB irradiation with 30 mJ/cm2, Pg-C-EE reduced the gene expression of IL-6 and IL-8, the most important inflammatory genes in skin cells (Fig. 2E). To investigate the regulatory mechanisms of proteins involved in UVB-mediated cell damage, such as MMPs, COX-2, and IL-6, we performed the Western blot analysis of MAPK proteins (p38, JNK, and ERK), which are upstream of the activator protein 1 (AP-1) signal. Expression of phosphor-p38, phospho-JNK, and phosphorus decreased after Pg-C-EE treatment under UVB induction conditions (Fig. 2F). These results were confirmed by examining the expression of MMP-1 and MMP-9 in UVB irradiation conditions using MAPK inhibitors (SB203580, SP600125, and U0125). SP600125 and U0126 reduced the expression of MMP-1, and SB203580 decreased the expression of MMP-9, to the same extent as the Pg-C-EE treatment (Fig. 2G).

3.3. Skin-protective activity of Pg-C-EE against hydrogen peroxide 

To examine whether Pg-C-EE protects against H2O2-induced damage, HaCaT cells were treated with H2O2 with and without PgC-EE and photographed (Fig. 3A) The number of HaCaT cells under each condition was 202 for the untreated group, 49 for 1 mM H2O2, 91 for 1 mM H2O2 plus 100 mg/mL Pg-C-EE, 115 for 1 mM H2O2 plus 200 mg/mL Pg-C-EE, and 111 for 1 mM H2O2 plus 100 mg/mL Pg-C-EE (Fig. 3A, right panel). These data confirmed that Pg-C-EE inhibited H2O2-induced cell damage. In general, H2O2 is involved in the production of ROS in cells. ROS promotes skin aging through MMPs. Treatment with Pg-C-EE under H2O2-induced conditions decreased the expression levels of MMP-1, MMP-3, MMP-9, and heme oxygenase-1 in a dose-dependent manner. There was no change in the expression of nuclear factor 2 (Fig. 3B). To study the radical scavenging activity of Pg-C-EE, we conducted a DPPH assay using ascorbic acid (500 mM) as a radical-scavenging positive control. The scavenging levels of Pg-C-EE were 3.25% (50 mg/mL), 2.87% (100 mg/ mL), 6.72% (200 mg/mL), and 11.97% (400 mg/mL), whereas ascorbic acid showed a scavenging effect of 88.5% (Fig. 3C). To clarify previous results, the expression levels of MMP-3 and MMP-9 under H2O2 treatment conditions were measured in the presence of MAPK inhibitors (SB203580, SP600125, and U0125). SB203580, SP600125, and U0126 reduced the expression of MMP-3 to the same extent as the Pg-C-EE treatment. However, the expression of MMP-9 was inhibited only by SB203580 among the MAPK inhibitors (Fig. 3D).

3.4. Effect of Pg-C-EE on skin moisture retention activity and collagen synthesis 

A natural moisturizing factor (NMF) that enhances skin hydration is HA. HAS is widely known as a gene that synthesizes NMF. The mRNA levels of genes related to NMF production, filaggrin (FLG), transglutaminase-1, and HAS-1, 2, 3 were not altered by Pg-C-EE treatment (Fig. 4A). However, expression of hyaluronidase (HYAL), which is known to degrade HA, was decreased by Pg-C-EE. Especially, HYAL-4 was significantly reduced to the same extent as retinol (as a positive control) (Fig. 4B). Col1A1 (collagen, type I, alpha 1) had no effect on Pg-C-EE treatment; however, Col2A1 (collagen, type II, alpha 1) tended to increase the effects of Pg-C-EE (Fig. 4C).

3.5. Effect of Pg-C-EE on transcription factor regulation 

Melanogenesis and ROS-induced cell damage are associated with AP-1 signaling proteins, such as MAPKs. To support the aforementioned results, we measured luciferase activity using an AP-1-Luc plasmid construct. Interestingly, Pg-C-EE dose-dependently inhibited AP-1emediated luciferase activities triggered by PMA (100 nM) (Fig. 5A). CREB signaling associated with melanogenesis was analyzed using a CREB-Luc plasmid. The forskolin-triggered CREB-mediated luciferase activity was decreased by Pg-C-EE in a dose-dependent manner (Fig. 5B). Without stimulation, HEK293 cells were transfected with each luciferase plasmid [Nuclear factor-kB (NF-kB) and (Col1A1)] and treated with Pg-C-EE. NF-kB- and Col1A1-mediated luciferase activities showed no difference according to the absence or presence of Pg-C-EE (Figs. 5C, 5D).

4. Discussion

Skin cells are the cell type that is mostly affected by external environmental factors and are important at all ages throughout life. This study was carried out to examine skin cell protection and maintenance in a healthy state. External stimuli can be divided into physical or chemical stimuli. Among physical stimuli, UV irradiation is known to be involved in melanogenesis and wrinkle formation. Wrinkle formation is associated with the degradation of the ECM [23], and UV irradiation easily destroys the ECM. Among the UV spectrum, UVB with a wavelength of 280e315 nm is known to penetrate the upper dermis layer. The penetrative power of UVB is more effective than that of UVA of 315e400 nm wavelengths and UVC of 100e280 nm wavelengths [24]. UVB penetrates the dermis layer and degrades the ECM, resulting in photoaging of the skin. The photoaged skin layer shows various consequences of this damage, including inhibition of collagen synthesis, degradation of collagen, upregulation of MMP expression, DNA mutations, production of melanin, and local immunosuppression [25]. The DNA damage caused by UV irradiation leads directly to DNA single-strand breaks via the formation of thymidine dimers [26] and indirectly to ROS generation via the depletion of cellular antioxidants and antioxidant enzymes (superoxide dismutase, catalase) [27]. Consistent with the increase in MMPs with UV irradiation, MMPs function as collagenases, gelatinases, stromelysins, and membrane-type MMPs (mt-MMP) in skin cells. The MMPs can be subdivided according to functional differences. The collagenases are able to degrade triple-helical fibrillar collagens and include MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase), and MMP-13 (collagenase 3). The gelatinases are distinguished by the presence of an additional domain inserted into the catalytic domain and include MMP- 2 (gelatinase-A) and MMP-9 (gelatinase-B). The stromelysins, including MMP-3 (stromelysin 1), MMP-10 (stromelysin 2), and MMP-11 (stromelysin 3), show a broad ability to cleave extracellular matrix proteins; however, these enzymes are unable to cleave the triple-helical fibrillar collagens. Therefore, regulation of MMP expression is a very important factor in aged cells such as those with photoaging. UV irradiation increases ROS production and the expression of MMPs in the cells. The roles of ROS in the cells include host defense [28], wound repair, maintenance of blood homeostasis (to recruit platelets), damage of DNA or RNA, oxidation of amino acids, oxidative deactivation of specific enzymes, accelerated aging [29], and induction of cancer [30e32]. In addition, exogenous ROS can be produced from hydroxyl radicals, hydrogen peroxide, superoxide radicals, and ultimately oxygen. Above all, hydrogen peroxide is formed in mammals as a short-lived product in biochemical processes and is toxic to cells. Hydrogen peroxide can cause irritation and allergic reactions and in high concentrations can cause blisters, redness, and other skin damage. The cause of cytotoxicity is oxidative stress processes in skin cells. Oxidative stress is involved in many human diseases, such as heart disease and Alzheimer’s disease, in addition to the aging process. Cytotoxicity results from the oxidation of proteins, membrane lipids, and DNA by the peroxide ions. For these reasons, it is important to maintain antioxidant levels in skin cells.

UV irradiation-induced melanin production through the activation of melanocytes and ROS generation. Initiation of melanogenesis by exposure to UV irradiation causes skin tanning. Melanin is able to disperse approximately 99.9% of absorbed UV radiation [33]; therefore, melanin has skin-protective activity against UV radiation damage. However, melanin is associated with an increased risk of malignant melanoma, a cancer of melanocytes. The synthesis of melanin involves a chain of enzyme-catalyzed chemical reactions and non-enzyme-catalyzed reactions [34e36]. The main precursor of melanin is the conversion of L-tyrosinase to L-DOPA, which is catalyzed by the enzyme tyrosinase [36,37]. Different kinds of enzymes also involved in melanin synthesis include tyrosinase-related protein 1 (TRP-1 and TYRP1 and tyrosinase-related protein 2 (TRP-2 and TYRP2). According to Kegg pathways, melanogenesis is under complex regulatory control by multiple agents. The most important positive regulator of melanogenesis is the MC1 receptor with its ligands melanocortin peptides. MC1 receptor activates the CREB [38,39]. Increased expression of MITF and its activation by phosphorylation stimulate the transcription of tyrosinase (TYR), TRP-1, and dopachrome tautomerase, which produces melanin. Melanin synthesis occurs within intracellular organelles called melanosomes [40e42] and is regulated through this pathway. Melanin overproduction can cause skin spots and some diseases. Recently, women have shown considerable interest in skin whitening, and many companies are studying agents that inhibit melanin production.

Many studies have indicated that Korean ginseng and Korean Red Ginseng have beneficial activity [43]. However, to date, only inflammatory regulatory activity or phytochemical ingredients of Pg-C-EE have been studied. In this study, we examined the effects of Pg-C-EE on melanogenesis, photoaging, oxidative stress, moisture retention, and wrinkle formation using a-MSHeinduced B16F10 cells and hydrogen peroxide and UVB-induced HaCaT cells. We determined that Pg-C-EE can be used as a skin-protective agent because it suppresses melanin secretion and melanin synthesis as effectively as arbutin (Figs. 1B, 1C) [44]. We also determined that activities of mushroom tyrosinase, which is the most important enzyme in the production of melanin, were decreased in the Pg-C-EEetreated group to the same extent as by kojic acid (Fig. 1D). Then, we examined the mRNA levels of melanogenesis-related genes and found that mRNA levels were not significantly changed in the Pg-C-EEetreated group (Fig. 1E). We, therefore, confirmed the protein levels of tyrosinase, TRP-1, 2, and MITF under 48-h Pg-C-EE treatment conditions and phospho- and total p38, ERK, and CREB under 24-h Pg-C-EE treatment conditions. All protein levels were decreased dose-dependently in the Pg-C-EEe treated group (Figs. 1F, 1G). Therefore, we have confidence that Pg-C-EE has antimelanogensis activity in B16F10 cells. In our study, Pg-C-EE was found to have effective antioxidant and anti-photoaging activity. Interestingly, the mRNA levels of MMPs were attenuated dose-dependently in the Pg-C-EEetreated group under both UVB- and hydrogen peroxide induction conditions (Figs. 2C, 3B). Under UVB induction conditions, Pg-C-EE also reduced the expression of IL-6, the most important factor in inflammatory skin cells (Fig. 2E). Even more surprisingly, Pg-C-EE inhibited cell death due to hydrogen peroxide treatment (Fig. 3A). In the DPPH assay, Pg-C-EE showed similar antioxidative effects to other P. ginseng extracts (Fig. 3C) [45]. We determined that the mRNA levels of HYAL2 and HYAL4 in UVB induction conditions were attenuated in cells treated with Pg-C-EE (400 mg/mL) (Fig. 4B), indicating that Pg-C-EE can regulate moisture retention in skin cells. Surprisingly, we also found that Pg-C-EE can help the formation of collagen (Fig. 4C). We examined which transcription factors regulated the aforementioned process through the luciferase system. By transfection with NF-kB and Col1A1 luciferase, respectively, and treatment with Pg-C-EE, we confirmed that NF-kB- and Col1A1- driven luciferase activities were affected by Pg-C-EE (Figs. 5C, 5D). We also showed that AP-1 mediated luciferase activity with PMA induction and CREB-mediated luciferase activity with forskolin induction. Both luciferase activities were decreased with Pg-C-EE treatment (Figs. 5A, 5B). These results show that Pg-C-EE has a skin-protective effect through AP-1 and CREB. Several experiments carried out using AP-1 inhibitors (MAPK inhibitor; SB203580, SP600125, and U0126) [46e48] verified the results from Figs. 1e3 (Figs. 1H, 2G and 3D).

In summary, we found that Pg-C-EE exerted skin-protective effects through anti-melanogenesis activity in a-MSHeinduced B16F10 cells, antiphotoaging activity in UVB-irradiated HaCaT cells, and antioxidant activity in hydrogen peroxide-induced HaCaT cells. In addition, Pg-C-EE inhibited AP-1 and CREB transcription factors and their upstream activation pathway (MAPK and CREB). A brief summary of each activity is shown in Fig. 6. These results suggest that Pg-C-EE can be helpful as a skin-improving ingredient through the blockade of AP-1 and CREB signaling proteins.

Conflicts of Interest

Acknowledgments

This article was supported by Konkuk University in 2016.

Appendix A. Supplementary data

Supplementary data related to this article can be found.

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