Abstract: Ultraviolet (UV) exposure causes skin photoaging leading to skin wrinkling and sagging via the production of reactive oxygen species (ROS). For this reason, protection from photoaging is an important feature in cosmeceutical and dermatological products. Natural product-derived biomaterials are highly desired as future possible ingredients because these biomaterials are often safe and effective. In this study, we aimed to characterize the skin protective activity of Pradosia multisite, traditionally used to treat sunburn and erythema. We determined the free radical scavenging, anti-melanogenic, and moisturizing effects of a methanol extract of Pradosia multisite (Pm-ME) in keratinocytes (HaCaT cells), melanocytes (B16F10 cells), and fibroblasts (human dermal fibroblasts (HDFs)) at non-cytotoxic concentrations. Pradosia multisite methanol extract contains coumaric acid as a major component, and the extract exhibited protective activity against UVB- and H2O2-induced cytotoxicity. This extract also suppressed the expression of metalloproteinases (MMPs) and cyclooxygenase (COX)-2 in HaCaT cells. A reduction of Sirt-1 expression under UVB- and H2O2-treated conditions was recovered in HaCaT cells by Pm-ME. This extract displayed significant free radical scavenging activity according to the 2,20 -casino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assay. The Pm-ME also upregulated the expression levels of hyaluronic acid synthase (HAS) and transglutaminase-1 (TGM-1) in HaCaT cells, indicating a putative moisturizing activity. Interestingly, the expression of the collagen type 1 (Col1A1) gene and its promoter activity, as assessed by a reporter gene assay, were found to be increased in HDF and HEK293 cells. Similarly, Pm-ME helped recover collagen levels after UVB and H2O2 treatment in HDFs as well as decreased the synthesis and secretion of melanin from B16F10 melanoma cells, which may indicate a beneficial whitening cosmetic value. The p38 inhibitor SB203580 and the JNK inhibitor SP600125 suppressed MMP-9 and COX-2 expression in H2O2-treated HaCaT cells. Similarly, the ERK inhibitor U0126 inhibited HAS-2 in Pm-ME/H2O2-treated HaCaT cells. These findings suggested that inhibition of JNK and p38 and activation of ERK could be targeted by Pm-ME. Therefore, Pm-ME may exert anti-photoaging and anti-melanogenic properties via the regulation of mitogen-activated protein kinase, which could be beneficial in the cosmeceutical industry.

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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1. Introduction

Skin is the largest organ in the body, which serves as a barrier between the organism and the environment, and is responsible for maintaining skin homeostasis and ultimately determining the organism’s survival [1]. Skin is organized with the epidermis, dermis with adnexal structures, and subcutaneous fat [2]. In addition, skin is important for continuous communication with immune, neural, and endocrine systems [3], it mostly confers protection against pathogens, chemicals, physical injuries, and ultraviolet (UV) irradiation [4]. Ultraviolet spectra are divided into three zones, UVC (200 to 280 nm), UVB (280 to 320 nm), and UVA (320 to 400 nm) [5]. Of these, UVB is predominantly absorbed by the upper layers of the skin epidermis as well as papillary dermis, while UVA penetrates the reticular dermis with 1000 times lower efficiency at inducing various biological effects, compared to UVB [5]. Although UVC is very reactive and absorbed by the stratum corneum, it is mostly removed by the ozone layer and atmosphere. As a major UV irradiation source, UVB can lead to the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) via activation of ROS-generating enzymes including NADPH oxidase, xanthine oxidase, and D-amino acid oxidase [6–8] and the induction of skin photoaging, which leads to skin wrinkling and sagging [9,10]. In addition, the oxidative stress response has been reported to induce various cellular component damage such as cellular membrane lipids, proteins, and nucleic acids [11]. These damaged skin cells can initiate inflammatory responses leading to eventual damage manifested in chronically exposed skin [12].

Oxidative stress induces the activation of inflammatory and redox-sensitive transcription factors, nuclear factor (NF)-κB and activator protein (AP)-1, and their upstream signaling enzymes including mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinase (JNK) in the AP-1 pathway, or as an inhibitor of κBα (IκBα), IκBα kinase (IKKα/β), and AKT in the NF-κB pathway linked to the induction of inflflammation and wrinkle formation [13]. Extracellular signal-regulated kinase normally mediates cellular responses related to growth factors, JNK, and p38-mediated cellular responses related to cytokines and physical stress [14]. Mitogen-activated protein kinases can also induce the production of proteolytic matrix metalloproteinases (MMPs) [15], which induce collagen degradation thus decreasing skin elasticity [16]. Regarding these components, anti-oxidative components or extracts such as ginsenoside, curcumin, epicatechin, asiaticoside, ziyuglycoside I, magnolol, gallic acid, hydroxychavicol, hydroxycinnamic acids, glycyrrhizic acid, mangiferin, Mirko, rosmarinic acid, tectorigenin, tyrosol, BIOGF1K, and hydroalcoholic extract of Spartium junceum L. followers have been used for the development of anti-skin aging products [17–21]. Because oxidative stress is known as a major cause of human disease and the aging process which affects longevity, secondary bioactive metabolites in human diets with antioxidative properties are considered valuable ingredients [22,23].

Other photoaging-related genes include cyclooxygenase-2 (COX-2) and Sirt-1. Cyclooxygenase-2 is usually overexpressed in premalignant UV-induced skin lesions [24], and its inhibition can decrease malignant transformation in the epidermis [25]. Sirt-1 expression can prevent cell apoptosis and increase cell survival [26]. Another mechanism of protection against UVB radiation is the production of melanin, a pigment synthesized in melanocytes and further secreted to the keratinocytes in the epidermis layer [1]. Melanin is produced by the oxidation of L-tyrosine and its following conversion to L-dihydroxyphenylalanine (L-DOPA) [6] by catalyzing with the copper-dependent enzyme tyrosinase [27]. Despite its protective function, the excessive production of melanin can generate age spots [28], melisma, and hyperpigmentation [29]. Because of the current trend which considers light complexions as the beauty standard, skin whitening preparations that achieve either hyperpigmented lesions bleaching or skin whitening have become highly desirable in the pharmaceutical and cosmeceutical industries [29]. Thus, considerable effort has been directed toward the development of preparations that reduce melanin synthesis [30]. Because skin aging is associated with loss of skin moisture, an important consideration for maintaining healthy skin is adequate hydration [31]. Hyaluronic acid (HA), a high molecular weight glycosaminoglycan with hydrophilic properties, contributes to the hydration and plastic properties of the skin by regulating the expression of hyaluronic acid synthases (HASs) [32]. Another important gene in the skin is collagen, which provides support for epidermal structures [33], therefore being responsible for the strength and resilience of skin [34] and whose degradation leads to both skin sagging and wrinkling. In addition, transglutaminase-1 (TGM-1) is an epidermal constitutively expressed enzyme that catalyzes cornified epidermal cell envelope formation, helping to prevent water loss [35,36].

The Sapotaceae family is distributed mainly in the tropical and subtropical regions of Asia and Mesoamerica. Many Sapotaceae family species produce edible fruits of high economic value. These fruits have been used for medicinal purposes. In particular, the seeds are rich in nutrients, vegetable fats, proteins, and other beneficial compounds. For example, mamey oil has traditionally been used in skin care, to treat sunburns and erythema [37]. Pradosia music is a member of the Sapotaceae family whose oil has traditionally been used to cure skin scars [38]. However, its properties have yet to be scientifically proven. The aim of this research was therefore to determine the potential value of P. mutisii in skin care, cosmetology, and pharmacology.

2. Results

2.1. Pm-ME Characterization and its Effect on Cell Viability 

Pm-ME did not block viability up to 100 µg/mL, but slightly decreased viability at 200 µg/mL in HaCaT, B16F10, and human dermal fibroblasts (HDF) cells, according to the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 1a–c). Using ultrahigh-pressure liquid chromatography (UHPLC) and liquid chromatography (LC)/mass spectrometry, a major compound in Pm-ME was found to be coumaric acid at 4.27 min (Figure 1d). 

2.2. Protective Effect of Pm-ME against UVB and H2O2 damage

To determine whether Pm-ME protected HaCaT cells against damage caused by UVB- and H2O2-induced photoaging, cells were pretreated with different concentrations of Pm-ME (0–100 µg/mL) before exposure to UVB or H2O2. Figure 2a shows that UVB irradiation decreased the numbers of adherent HaCaT cells, while Pm-ME increased cell adherence. In addition, cellular damage triggered by H2O2 was recovered by Pm-ME at 100 µg/mL (Figure 2b). To elucidate whether Pm-ME attenuated UVB- and H2O2-mediated wrinkle formation, the expression levels of MMP-1 and MMP-9 were measured following UVB or H2O2 treatment in the presence or absence of Pm-ME. Pm-ME inhibited the expression of MMP-1 and MMP-9 under both conditions (Figure 2c,d). We next examined whether Pm-ME suppressed free radical-induced inflammatory responses by measuring mRNA levels of COX-2. Under UVB- or H2O2-treated conditions, mRNA levels of COX-2 were strongly suppressed by Pm-ME at 100 µg/mL (Figure 2e,f). We then measured mRNA levels of the anti-aging gene, Sirt-1, to determine its impact on photoaging. Both UVB and H2O2 reduced Sirt-1 expression, whereas Pm-ME remarkably restored its expression at 50 and 100 µg/mL (Figure 2g,h). Because Pm-ME suppressed certain molecular and cellular responses in HaCaT cells treated with UVB or H2O2, we next examined whether this extract had direct anti-oxidative activity using 2,2′-casino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS assay, in which Pm-ME showed dose-dependent free radical scavenging activity (Figure 2i).

2.3. Moisturizing and Collagen-Increasing Effects of Pm-ME

Pm-ME enhanced the expression of moisturizing-related genes such as HAS-2, and TGM-1 in HaCaT cells (Figure 3a). To determine if Pm-ME could enhance the expression of a collagen gene (Col1A1), we first treated HDF cells with Pm-ME (0–100 µg/mL) and then determined the expression level of Col1A1. As Figure 3b shows, an increased expression of the Col1A1 gene under Pm-ME treatment conditions was dose-dependently observed. These results were further confirmed using a reporter gene assay with the Col1A1-Luc construct transfected in HEK293 cells and HDF cells. Expectedly, Pm-ME increased the promoter activity of the Col1A1 gene in a dose-dependent manner in both cells (Figure 3c,d). In addition, Pm-ME was found to recover collagen gene levels which decreased under both UV irradiation and H2O2 treatment conditions (Figure 3e,f). 

2.4. Anti-Melanogenic Effect of Pm-ME 

Since our target concentration corresponded to 100 µg/mL, we determined whether Pm-ME was able to suppress the secretion of melanin and its cellular contents. To determine this, B16F10 melanoma cells were stimulated by α-melanocyte stimulating hormone (α-MSH) in the presence or absence of Pm-ME or arbutin (positive control). Levels of melanin were then measured. Pm-ME reduced the secretion of melanin up to 75% at 100 µg/mL (Figure 4a,b). Pm-ME also decreased the melanin contents in MSH-treated B16F10 cells up to 30% at 100 µg/mL, whereas melanin content at 50 µg/mL was not significantly reduced, compared to the positive control (α-MSH) (Figure 4c). Finally, to determine the effect of Pm-ME on the activity of melanin-producing enzymes, we performed a tyrosinase enzyme assay. Interestingly, Pm-ME (0–400 µg/mL) significantly decreased tyrosinase activity at concentrations higher than 200 µg/mL, while Kojic acid (KA), a controlled drug, strongly reduced the activity of tyrosinase (Figure 4d). 

2.5. Molecular Mechanisms of Pm-ME-Mediated Antiphotoaging and Moisturizing Effects 

3. Discussion

Because of their richness in nutrients and vitamins, different species from the Sapotaceae family have been used in traditional medicine [39]. However, the potential use of P. mutisii in the cosmetic and pharmaceutical industries has yet to be explored. For this reason, we studied the skin-protective activity of P. mutisii in keratinocytes and fibroblasts under skin-damaging conditions. We also evaluated its suitability for cosmetic preparations. Because toxicity testing to identify potential risks in humans is a necessary and critical step in both the drug and cosmetic industries [40], the cytotoxicity of P. mutisii was tested in HaCaT, B16F10, and HDF cells. Pradosia mutisii exhibited low toxicity until 200 µg/mL and no toxicity at 100 µg/mL in HaCaT, B16F10 (Figure 1a,b), and HDF cells (Figure 3c). Using UHPLC/MS analysis (Figure 1c), Pm-ME was shown to have a high concentration of coumaric acid, a phenolic acid [41,42]. Polyphenols are secondary metabolites of plants and are generally involved in defenses against UV radiation or infection by pathogens. In the human body, polyphenols have been shown to protect against the development of cancer, cardiovascular diseases, diabetes, osteoporosis, and neurodegenerative diseases [1]. Coumaric acid has been shown to have antioxidant and anti-inflammatory properties [43]. 

Ultraviolet irradiation is one of the main causes of free radical (e.g., ROS) production in the skin. Free radicals can lead to skin aging and cancer if they accumulate over long periods [44]. It is known that free-radical-generated oxidative stress and damage can compromise cell survival, proliferation, differentiation, and metabolism [45,46]. Consistent with previous reports [47,48], we also found that UVB irradiation-induced cellular damage in HaCaT cells. Damage included reduced proliferation, increased apoptosis, and molecular responses including the expression of genes related to inflflammation, wrinkle formation, and aging (Figures 2 and 3). Because UVB-induced radicals are major factors that damage cells, tissues, and organs, it was found that some endogenous compounds such as melatonin and its metabolite [49–52], and bilirubin [53] are involved in scavenging toxic radicals by exhibiting antioxidant, photo-protective, and anti-aging properties in our bodies. So far, these compounds are also developed as highly valuable biomaterials which can be applied to our bodies by pharmaceutical industries [54]. Nonetheless, vitamin C, coenzyme Q10, and α-tocopherol are becoming top global sales products for human health because of their signifificant anti-oxidative properties. Research laboratories at pharmaceutical and cosmetic companies continue to place a major emphasis on identifying and developing effective anti-oxidative drugs and natural compounds. In our study, we observed that Pm-ME had strong antioxidant activity when tested using the ABTS assay (Figure 2i). Similarly, Pm-ME reversed the free radical-induced suppression of cell adherence, the induction of nuclear damage, and the altered expression of genes such as MMP-1, MMP-9, and COX-2 under UVB and H2O2 conditions in a dose-dependent manner (Figure 2a–f). Notably, Pm-ME restored levels of the Sirt-1 gene (Figure 2g,h), which is involved in preventing apoptosis and increasing cell survival under oxidative stress conditions [26]. These results strongly implied that the aging of cells was linked to oxidative stress via the downregulation of Sirt1 expression. The anti-oxidative properties of Pm-ME allowed for the recovery of Sirt1 expression. This may result in protection from skin damage and aging under natural UVB irradiation conditions in vivo. In addition, there is a possibility that Pm-ME is also able to protect UVC-induced cellular responses since our UV irradiation conditions might be contaminated with UVC, although we used a filter system to clear out all wavelengths below 290 nm. The concept that anti-oxidative biomaterials can ameliorate cellular and molecular damage under oxidative stress conditions has driven the commercial production of various products such as BIOGF1K, ginsenoside Ro, EGCG, Fraxinus chinensis extract, and certain synthetic antioxidants. The anti-wrinkle and anti-photoaging effects of biomaterials in these and other products have been widely reported in the cosmetic industry [48,55,56]. 

Maintaining adequate skin hydration [31] and generating new collagen [57] are important processes for healthy skin. The expression of moisturizing (HAS2, TGM-1) (Figure 3a) and collagen (Col1A1) genes were remarkably increased by Pm-ME (Figure 3b–d), and helped to recover collagen levels after UVB exposure and H2O2 treatment (Figure 3e,f). Therefore, this suggests that Pm-ME can be an effective ingredient in the maintenance of healthy skin. The suppression of melanin by Pm-ME is also beneficial for its use in cosmetic preparations. Although melanin is valuable in protecting skin from UV irradiation, because many women prefer to lighten their skin, whitening by anti-melanogenic activity is a useful feature in cosmetic materials [29]. Pm-ME (100 µg/mL) displayed strong inhibitory activity during melanogenesis, as assessed by the determination of melanin levels from secreted medium and the decrease shown in melanin secretion levels (Figure 4a–c). Moreover, Pm-ME (200 µg/mL) significantly suppressed tyrosinase enzyme activity (Figure 4d), implying that higher concentration is directly effective to suppress enzyme activity, while lower concentration is involved in the regulation of melanin secretion. Therefore, because of its anti-oxidative, anti-photoaging, anti-wrinkle, moisturizer-stimulating, and anti-melanogenic activities, Pm-ME can be considered a good cosmeceutical candidate. To further explore this possibility, we plan to evaluate its clinical efficacy in future studies.

We performed studies to determine the molecular mechanisms by which Pm-ME exerted its anti-oxidative and moisturizing in keratinocytes. We focused on MAPK-related enzymes (ERK, JNK, and p38), because these enzymes play a critical role in the aging and melanogenesis of skin keratinocytes and melanocytes [58,59]. For example, the ERK pathway mediates cellular responses to transforming growth factor-β1 by increasing collagen [60] and HA synthesis pathways in keratinocytes [61]. JNK plays a key role in oxidative-stress-induced keratinocyte apoptosis [62]. The enzyme p38 is important in maintaining the homeostasis of human skin [63]. The JNK and p38 pathways both mediate cellular responses to cytokines and physical stress [14]. These pathways are activated by UVB- and ROS-induced stresses. These pathways can increase the production of proteolytic MMP and COX-2 genes [15], which are related to collagen degradation and skin inflflammation, respectively [64]. Notably, oxidative stress blocked the phosphorylation of ERK, while p38 and JNK were phosphorylated when treated with H2O2 (Figure 5a). In contrast, Pm-ME increased ERK phosphorylation and reduced the phosphorylation of p38 and JNK (Figure 5a), implying that these enzymes can differentially participate in Pm-ME-mediated pharmacological activities. To further understand the functional role of MAPK in the anti-photoaging, anti-melanogenic, and anti-inflammatory effects of Pm-ME in UVB-irradiated and H2O2-stimulated keratinocytes, specific inhibitors of MAPK were used. The results of Figure 5b strongly suggested that inhibition of p38 and JNK by Pm-ME resulted in anti-wrinkle and anti-inflammatory effects, because a p38 inhibitor (SB203580) and a JNK inhibitor (SP600125) blocked the expression of MMP-9 and COX-2, respectively. Moreover, Pm-ME-triggered ERK phosphorylation induced the expression of genes related to moisturizing because Pm-ME treatment triggered the expression of HAS-2, whereas U0126 suppressed it (Figure 5c). These results suggested that MAPK-related enzymes contributed to PM-ME-mediated anti-aging, anti-wrinkle, and anti-inflammatory activities. UVB irradiation is known to increase other signaling cascades, such as those linked to NF-κB and STAT3 [65,66]. Whether Pm-ME can regulate these signaling pathways must still be investigated.

In conclusion, we found that coumaric acid-rich Pm-ME exhibited anti-oxidative, anti-wrinkle, moisturizer-stimulating, and anti-melanogenic activities in skin cells irradiated by UVB or treated with H2O2. Pm-ME increased the phosphorylation of ERK in a dose-dependent manner but decreased the phosphorylation of p38 and JNK (Figure 6). Our results strongly suggested that Pm-ME may be an effective skin-protective biomaterial. Since Pm-ME exhibited very promising activities in both cancerous skin cells (HaCaT and B16F10 cells) and normal human keratinocytes during oxidative stress conditions, therefore, we propose the beneficial roles of Pm-ME’s use in cosmetic preparations. However, melanoma cells have abnormal features such as disrupted cell cycle control, aberrant gene expression patterns, and limited differentiation capacities [67], and additional work with primary melanocytes should be followed to exactly understand the anti-melanogenesis activity of Pm-ME and its molecular mechanism. Moreover, plants from the same family (Sapotaceae) or genus (Pradosia spp.) will be tested for similar skin-protective activities. 

Ask for more: david.deng@wecistanche.com  WhatApp:86 13632399501