Soluble Pearl Extract Provides Effective Skin Lightening By Antagonizing Endothelin

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Jing Wang MD1 | Zhixiong Chen MSc2 | Yaojia Lu BSc2 | Lihua Zhang BSc3 | Jiahuan Mo BSc2 | Fumin Cao BSc2 | Min Xie BSc3 | Xinzhong Shen BSc3 | Anquan Yang BSc3

Abstract

Background: The incidence of skin pigmentation disorders has been on the rise globally. This calls for safer and more effective topical skin lightening and freckle-removing products. In this study, we hypothesized that Soluble Pearl Extract (SPE) may possess endothelin antagonizing compounds with good skin whitening effects.

Objectives: (a) To determine the effect and mechanisms of SPE on ET-1-treated B16 melanoma cells. (b) To explore the cytotoxic effects of SPE on B16 melanoma cells.

Methods: CCK-8 assay was performed to determine how SPE and ET-1 affect the proliferation rate of B16 melanoma cells, the NaOH lysis assay was conducted to quantify the content of melanin while the tyrosinase activity was determined by DOPA oxidation test. The mRNA and protein expression levels of TYR and TRP-1 were determined by qRT-PCR assay and Western blot assay, respectively.

Results: We found that SPE at 0.1 and 1 μg/mL concentrations has no effect on the proliferation of the cells and 10 nmol/L ET-1 promoted B16 melanoma cells proliferation. Notably, B16 melanoma cells treated with 10 nmol/L ET-1 exhibited significantly higher melanin synthesis, tyrosinase activity, TYR, and TRP-1 mRNA expression levels compared with untreated cells. Of note, the effects of 10 nmol/L ET-1 treatment were abolished with SPE in a dose-dependent manner.

Conclusions: SPE inhibits endothelin thereby safely and effectively lightening lightens the skin by antagonizing endothelin. Moreover, SPE is safe and effective.

KEYWORDS: B16 melanoma cells, Endothelin-1, Soluble Pearl Extract, whitening

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1 | INTRODUCTION

In recent years, newer and effective skin whitening cosmetics for external use have been put on the market. This is attributed to economic development and changing lifestyle needs across people of all generations. The currently used skin whitening agents include hydroquinone, kojic acid, phospholipase D1, placenta, and vitamin A. Skin pigmentation disorders are mainly associated with the production of melanin. It is synthesized through a process initiated by the conversion of tyrosine to dopa catalyzed by tyrosinase. Dopa initiates a series of complex biochemical reactions leading to the synthesis of melanin. The whitening mechanism of dihydro asparagus ic acid,1 hydroquinone, and kojic acid involve inhibition of tyrosinase activity, which is the rate-limiting enzyme in melanin synthesis.2 In so-doing, these agents decrease the production of melanin resulting in skin whitening.3 By contrast, phospholipase D1, placenta, and retinoic acid act by reducing the amount of synthesis of tyrosinase.4 Although these agents achieve some level of skin whitening, their whitening mechanisms are not clear. Moreover, they are toxic and take longer to cause skin whitening.

It is therefore imperative to find safer and more effective skin whitening agents. Research on skin whitening has paid a high premium to the interaction between cytokines and melanocytes as an avenue to develop effective skin whitening agents. Keratinocytes secrete cytokines such as endothelin (ET) when exposed to Ultraviolet radiation. These cytokines influence the migration of melanocytes, dendritic development, and melanin production.5 As such, antagonizing the action sites of these cytokines will lead to whiter skin.6 Shi et al (2011) reported that resveratrol, a new endothelin antagonist, whitens the skin through a mechanism involving inhibition of tyrosinase activity in B16 melanocytes.7 Yang et al (2000) found that resveratrol targets melanocyte membrane. This implies that it only passes through the melanocyte membrane and the melanosome membrane to reach sites where it inhibits melanin production.8 Tyrosinase is located in the melanosome, and thus, its inhibitors must traverse four layers (the stratum corneum, the deep epidermis, the melanocyte membrane, and the melanosome membrane) to block its effect.9 Resveratrol only surpasses two layers (the stratum corneum and the deep epidermis) to inhibit tyrosinase activity.10 This shortens the period of whitening.

Harley (1993) found that pearls in Scottish freshwater, Australian, and Sri Lankan seawater were made of similar chemical composition, that is, calcium carbonate, 91.72%, organic matter, 5.94%, water, 2.23%, and other substances, 0.11%.11 Elsewhere, Zhang et al (2018) found that pearl extracts confer significant antioxidant effects in vitro.12,13 Yang et al (2016) found that pearl extracts can inhibit tyrosinase activity in B16F10 cells thereby reducing the production of melanin.14 This indicated that pearl extract may possess skin whitening effects, but the mechanism involved has not been studied. Herein, we postulated that SPE antagonizes endothelin altering the synthesis of melanin and tyrosinase activity of the B16 melanoma cells. Our results show that soluble pearl extracts antagonize endothelin causing skin whitening.

2 | MATERIALS AND METHODS

2.1 | Preparation and compositional analysis of soluble pearl extract (SPE)

Pearl powder was dissolved in lactic acid, and the pH of the mixture was adjusted to 7.0 with PBS. Enzymatic hydrolysis was initiated with the addition of protease, and the extract was filtered with 10 μm qualitative filter paper. The supernatant was obtained and lyophilized to prepare soluble pearl extract.

2.2 | Analysis of the composition of the lyophilized powder

Briefly, the moisture content of the SPE was measured with the atmospheric drying method. A specific amount of sample was heated in a drying oven at 95-105°C for 3 hours and weighed. The samples were repeatedly dried to obtain a constant weight. The moisture content was determined as the difference in mass before and after heating. The sample was burnt resulting in the formation of an inorganic substance termed ash. The burning weighing method was used to measure the quality of ash, in which the difference in quality before and after burning was considered as the ash content. The Kjeldahl method was employed to determine the protein content of the SPE. Peptide content was estimated as described before.15 The content of polysaccharides was determined using the anthranone-sulfuric acid method.

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2.3 | Cell culture

Murine B16 melanoma cells (kindly provided by Stem Cell Bank, Chinese Academy of Sciences) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin in an incubator (BB150 Thermo Fisher Scientific) set to 37°C and 5% CO2. After 3 days of culture, the cells were in a near-fused state and basically spread all over the dish. The cells were washed with phosphate buffer saline (PBS) and passaged by digesting with 0.25% trypsin. The cells were counted using a cell counting plate and cell suspensions prepared at appropriate concentrations. Cell suspensions were cultured in a cell incubator for subsequent experiments

2.4 | Detection of cell proliferation after treatment with SPE and ET-1 by CCK-8 assay

Cells in the logarithmic growth phase were cultured in 96-well plates at a concentration of 1 × 104 cells/mL in an incubator set at 37°C and 5% CO2 for 24 hours. Different concentrations of SPE (0, 0.01, 0.1, 1, and 10 μg/mL) were then added to the wells and left to stand for 24 hours. 10% of Cell Counting Kit-8 (CCK-8) reagent (Jian Cheng Bioengineering Co.) was added to each well to measure the proliferation rate of cells. The absorbance of each well was read at a wavelength of 490 nm using a microplate reader. Each group was tested as three replicates. The effect of ET-1 (0.1, 1, 10, and 100 nmoL) on cell proliferation was assessed following the same protocol. The cells were then divided into three groups: the control group (no treatment was given), the ET-1 group (cells were treated with 10 nmoL ET-1 for 24 hours), and the SPE group (cells were treated with 10 nmoL ET-1 for 24 hours and then treated with 0.1, 1 μg/mL SPE for 24 hours).

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2.5 | Tyrosinase activity test

10 μL of cells (differently treated) was seeded into each well of a 96-well plate at a cell density of (5-10) × 104 cells/mL, and 100 μL of the growth medium was added to each well. The cells were then pre-cultivated in an incubator set at 37°C and 5% CO2 for 24 hours. The plates were centrifuged at 2000 rotations per minute for 2 minutes, and the supernatant was discarded. The cells were washed thrice with PBS after, and 50 μL of 1% Triton X-100 solution (Jian Cheng Bioengineering Co.) was added to each well. The cells were immediately stored at −80°C for 30 minutes and then thawed at room temperature to achieve complete rupture of the cells. Cells were then preheated at 37°C for 5 minutes, and 10 μL of 1% L -DOPA solution was added. The cells were left to stand at 37°C for 2 hours, and the absorbance of the plate was measured at a wavelength of 490 nm using a microplate reader.

2.6 | Melanin content test

Cells were cultured in 6-wells plates at a density of (5-10) ×104 per well. Each well contained 2 mL of growth medium. The cells were incubated for 24 hours in an incubator set at 37°C and 5% CO2. The supernatant was discarded, and melanocytes were treated with 0.25% trypsin for 3 minutes. 2 mL 10% FBS-containing DMEM medium was subsequently added to stop the reaction. Melanocytes were collected into a 15 mL centrifuge tube, centrifuged at 1000 rotations per minute for 5 minutes, and the supernatant discarded. Melanocytes were washed with PBS and put in tubes containing 500 μL of ethanol/ether solution mixed in the ratio of 1:1 and mixed well by shaking. The mixture was left to stand at room temperature for 30 minutes and then centrifuged at 3000 rotations per minute for 5 minutes. The supernatant was discarded, and 300 μL of 1N NaOH (containing 10% DMSO) was added to the cells. This mixture was left to react in a water bath set at 80°C for 40 minutes to completely dissolve the melanin. 200 μL of the dissolved melanin was then transferred into a 96-well plate, and the absorbance was measured at a wavelength of 405 nm using a microplate reader.

2.7 | Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from cells of the three groups (Control, ET-1, and SPE groups) using the RNAiso Plus kit (TaKaRa Bio) and reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (TaKaRa Bio). the qRT-PCR assay was performed to detect the expression of β-actin in each sample. This was done using the SYBR green dye. The primer sequences16 used (TaKaRa Bio) are shown in Table 1. The PCR conditions were an initial denaturation temperature set at 95°C for 30 seconds, 40 cycles of denaturation, annealing temperature set at 95°C for 5 seconds, and 60°C for 34 seconds, respectively. This was carried out in a StepOne plus real-time PCR system (Applied Biosystems). The qRT-PCR results were analyzed and expressed as relative mRNA expression based on CT (threshold cycle) values that were later converted to fold-changes.

2.8 | Western blot analysis

Total proteins were extracted from the cells using Radio-Immunoprecipitation Assay (RIPA) (Solarbio Science & Technology Co., Ltd.). The concentration of the protein extracts was determined with the Bicinchoninic Acid Assay kit (BCA) (Solarbio Science & Technology Co., Ltd.). The proteins were then separated by Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), and the resultant bands electrophoretically transferred onto a Polyvinylidene Fluoride (PVDF) membrane. The membrane was then blocked with 5% skim milk powder and incubated with antibodies against TYR, TRP-1, and β-actin (Santa Cruz Biotechnology) at 4°C for 24 hours. Next, the membrane was incubated with the secondary antibody (Goat Anti-Mouse IgG Abmart) for 1 hour at room temperature. The protein bands were detected using the enhanced chemiluminescence (ECL) (Solarbio Science & Technology Co., Ltd.). The strip scanning analysis was performed using an Image J analysis system. β-Actin was used as the internal reference in the analysis of protein expression. Samples were run as independent replicates repeated thrice for each group.

2.9 | Statistical analysis

Statistical analyses were conducted using GraphPad prism8 software. Group differences were compared using one-way analysis of variance (ANOVA), and results are presented as means ± SD P<.05 was considered statistically significant.

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3 | RESULTS

3.1 | Compositional analysis of SPE

Chemical characterization showed that SPE contained a moisture content of 6.03 ± 0.16%, ash content of 1.55 ± 0.11%, peptide content of 7.52 ± 0.03%, protein content of 54.12 ± 0.18%, and polysaccharide content of 0.09 ± 0.005%. These results indicate that proteins were the most abundant in the organic matter of SPE.

3.2 | Effect of SPE on proliferation of B16 melanoma cells

After the B16 melanoma cells were treated with different concentrations (0, 0.01, 0.1, 1, and 10 μg/mL) of SPE for 24 hours, the cell proliferation was evaluated by the CCK-8 assay (Figure 1A). We found the proliferation of B16 melanoma cells treated with 0.01 μg/mL of SPE was higher than that of the blank group (P <.01). 0.1 and 1 μg/mL SPE had no effect on the proliferation of the cells. However, 10 μg/mL of SPE significantly inhibited the proliferation of B16 melanoma cells (P <.01). Thus, 0.1 and 1 μg/mL of SPE was used in subsequent experiments. On the basis of safety considerations, as a natural component of whitening cosmetics, SPE is not toxic to cells at these two concentrations; thus, it is safe to some extent.

3.3 | Effect of ET-1 on B16 melanoma cell proliferation

The B16 melanoma cells were treated with different concentrations of ET-1 for 24 hours, and the proliferation of the cells was evaluated by a CCK-8 assay. Notably, treatment of B16 melanoma cells with 0.1, 1, and 10 nmol/L of ET-1 promoted proliferation in a concentration-dependent manner, with 10 nmol/L of ET-1 producing the highest proliferation rate (P <.001). By contrast, 100 nmol/L of ET-1 decreased the proliferation of cells (Figure 1B). Therefore, 10 nmol/L was used in subsequent experiments.

3.4 | SPE inhibits Tyrosinase activity in B16 melanoma cells

To further explore the inhibitory mechanism of SPE on the synthesis of melanin, we analyzed its effect on the rate-limiting enzyme (tyrosinase) in the process of melanin synthesis. The activity of tyrosinase in cells was detected by the L-dopa oxidation method. B16 melanoma cells treated with 10 nmol/L ET-1 showed significantly higher intracellular tyrosinase activity compared to untreated cells (P < .01). However, the tyrosinase activity in cells treated with 0.1 μg/mL and 1 μg/mL SPE treatment was lower than in untreated cells (P < .05, P < .01). These results show that SPE inhibited tyrosinase activity in a concentration-dependent manner (Figure 2A).

3.5 | SPE inhibits melanin synthesis in B16 melanoma cells

We further tested whether the effects of SPE on melanin production in B16 melanoma cells were mediated by ET-1 using the NaOH lysis method. Results showed that melanin content in the B16 melanoma cells was significantly increased after treatment with 10 nmol/L of ET-1 compared to the control group (P <.01). However, the melanin content in cells treated with the 0.1 μg/mL and 1 μg/mL of SPE was significantly lower than in cells of the control group. Moreover, the inhibitory effects of SPE on melanin production were dose-dependent. The effects of SPE on melanin content and tyrosinase activity were similar (Figure 2B). Given that 0.1 μg/mL and 1 μg/mL of SPE did not alter the proliferation of B16 melanoma cells, the decrease in the melanin content at these two concentrations may occur through unknown mechanisms.

3.6 | Effect of SPE on TYR and TRP-1 mRNA levels

The mechanism by which SPE altered the effects of ET-1 on B16 melanoma cells was determined by measuring the mRNA levels of TYR and TRP-1 in cells that received different treatments. The mRNA levels of TYR and TRP-1 in B16 melanoma cells were significantly higher (P <.01 and P <.001, respectively) after treatment with 10 nmol/L ET-1 compared to cells of the control group. Interestingly, SPE treatment effectively inhibited the expression of TYR and TRP-1 mRNA (P <.05, P <.05 for 0.1 μg/mL, and P < .01, P < .01 for 1 μg/mL, respectively). Altogether, these results show that treatment with SPE inhibited the expression levels of TYR and TRP-1 mRNA in a concentration-dependent manner.

3.7 | Effect of SPE on TYR and TRP-1 protein levels in B16 melanoma cells

The protein expression of TYR and TRP-1 in B16 melanoma cells was detected by Western blot. We found higher protein levels of TYR and TRP-1 in B16 melanoma cells treated with 10 nmoL ET- 1 compared with the control group (P <.05 and P <.01, respectively). This indicated that 10 nmol/L ET-1 promoted the synthesis of melanin in the cells. This indicates that ET-1 may promote the synthesis of melanin by activating TYR and TRP-1 signaling pathways. In contrast, cells (treated with 10 nmoL ET-1 for 24 hours and then treated with SPE for 24 hours) had significantly lower protein levels of TYR and TRP-1 (P <.001 and P <.001, respectively) than cells treated with 10 nmol/L ET-1. This indicated that SPE antagonized the effect of ET-1 thereby inhibiting melanin synthesis (Figure 4).

4 | DISCUSSION

Endothelin (ET) is a natural bioactive peptide synthesized by endothelial cells in the human body. It comprises three isomers: ET-1, ET-2, and ET-3 that signal through three ET receptors: ETA, ETB, and ETC.17 ETA and ETB are the main receptors expressed in the human ET system.18 ET is released from keratinocytes when exposed to ultraviolet radiation B (UVB) irradiation. When ET binds to melanocyte receptors, it stimulates DNA synthesis and increases tyrosinase activity. This promotes the rapid proliferation of melanocytes and melanin synthesis.19 ET-l promotes melanocyte proliferation, dendritic formation, adhesion, and migration.20 Herein, we found that 0.1, 1, 10, and 100 nmol/L ET-1 significantly promoted cell proliferation. The proliferation was highest at 10 nmol/L ET-1 which is consistent with results reported by Geng et al (2011), who found that ET-1 enhanced the proliferation and melanin synthesis of cultured alpaca skin melanocytes. 21 TYR and TRP-1 genes play a role in melanin production. Indeed, a previous study found that effects of endothelin-1 on tyrosinase activity are mediated by ETA receptors.22 10 nmol/L of ET-1 treatment in B16 melanoma cells increased tyrosinase activity and melanin content. qRT-PCR results revealed that the mRNA levels of TYR and TRP-1 in the cells treated with 10 nmol/L ET-1 were higher than in the control group. This confirmed that ET-1 promoted melanin production.

In addition, SPE inhibited the effects of endothelin on B16 melanoma cells. At the concentration of 10 μg/mL, it exerted toxic effects and hence inhibited the proliferation of B16 melanoma cells. However, SPE did not alter the proliferation of cells at concentrations of 0.1 and 1 μg/mL. However, at a concentration of 0.01 μg/ mL, it significantly promoted cell proliferation when compared to the control group. These results suggested that SPE is safe at concentrations below 1 μg/mL. SPE inhibited ET-1-induced melanin production at concentrations of 0.1 and 1 μg/mL in a dose-dependent manner. Moreover, treatment with 0.1 and 1 μg/mL of SPE resulted in lower TYR and TRP-1 mRNA levels compared to the control group. Moreover, the inhibitory effect of SPE was concentration-dependent. Western blot analysis revealed similar effects of SPE at both concentrations (0.1 and 1 μg/mL) on protein levels of TYR and TRP-1.

In melanocytes, ET-1 binds to the Endothelin Receptor type B (EDNRB) receptor leading to the activation of phospholipase C (PLC).23 This further hydrolyzes phosphatidylinositol diphosphate (PIP2) and activates protein kinase C (PKC) thus changing the permeability of the ion channels.24 The change in permeability leads to the activation of voltage-dependent Ca2+ channels and phospholipase A (PLA).25 Eventually, this activates melanin synthesis.26 Herein, the mechanism by which SPE antagonized ET-1 to inhibit melanin production was only studied preliminarily. As such, expression of the EDNRB receptor as well as other aspects of melanin synthesis such as PKC, PKA, and KIT27 deserves further investigation.

The main chemical components in pearl powder are calcium carbonate and proteins, while others such as porphyrin and taurine are less abundant.28 The water extract of pearl powder mainly contains proteins. Hard keratin is the main protein in the water extract. However, the water extract may also contain a small number of trace elements.29 Modern pharmacological research suggests that pearl can promote the metabolic rate of the human body, increase the viability of epidermal cells, delay aging of cells, and inhibit the production of melanin as well as other pharmacological effects such as freckle whitening and detoxification.30 Chemical characterization revealed that proteins are the most abundant biomolecules in the content of organic matter of SPE. We thus hypothesized that the SPE would also have whitening effects in B16 melanoma cells. Our results confirmed this hypothesis.

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5 | CONCLUSION AND OUTLOOK

ET-1 promotes B16 melanoma cells proliferation and melanin synthesis by activating the TYR and TRP-1 signaling pathways. SPE at concentrations of 0.1 and 1 μg/mL inhibits melanin synthesis thereby antagonizing the effects of ET-1 without any cytotoxic effects on B16 melanoma cells. As such, SPE is a potential endothelin antagonist that can be used as a safe and effective skin-lightening agent.

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29. Wu WL, Zhang LP, Yang ZJ, et al. Components and ultrastructure with its formation of pear. J Mater Rep. 2011;025(021):79-85.

30. Shao DZ, Wang CK, Wang HJ, et al. Abstracts: comparison of hydration, tyrosinase resistance, and antioxidant activation in three kinds of pearl powders. Int J Cosmet Sci. 2010;32(5):396.