Paeoniflorin found in Paeonia lactiflora root extract inhibits melanogenesis by regulating melanin‐related signal transduction in B16F10 cells

Skin pigmentation is modulated by various processes, with melanogenesis playing a key role. Melanin is synthesized by the catalysis of melanogenesis‐related enzymes, such as tyrosinase and tyrosine‐related proteins TRP‐1 and TRP‐2. Paeoniflorin is the main bioactive component of Paeonia suffruticosa Andr., Paeonia lactiflora., or Paeonia veitchii Lynch and has been used for centuries for its anti‐inflammatory, anti‐oxidant, and anti‐carcinogenic properties.


| INTRODUC TI ON
The skin is the largest organ of the human body, accounting for 1.5-2.0m 2 of the total surface area.It has protective, sensation, and excretory functions, which aid in minimizing damage and UV-induced irritation.The skin consists of three layers-the epidermis, dermis, and hypodermis. 1 The basal layer of the epidermis comprises basal cells and melanocytes.Melanin-secreting melanocytes help prevent UV-induced skin damage.However, excessive production of melanin in the skin may cause hyperpigmentation and melanoma. 2This is a topic of great commercial interest to the cosmetic industry, warranting more research.
Melanin production is modulated by external factors, such as UV light exposure and drug consumption, as well as internal factors, such as the body's immune response and hormone signaling. 3Intense exposure to UV radiation can cause sunburns, DNA damage, and cancer as well as accelerate aging, with UVB being key in causing skin disorders.UV induces oxidative stress by generating reactive oxygen species, resulting in DNA and cellular damage. 4Under UV exposure, keratinocytes stimulate the secretion of alpha-melanocyte stimulating hormone (α-MSH) to enhance melanin biosynthesis in epidermal melanocytes. 58][9] Tyrosinase catalyzes the oxidation of L-tyrosine to 3,4-dihydroxyphenyl-L-alanine (L-DOPA), which is the first rate-limiting step of melanogenesis. 10,11TRP-1 and TRP-2 also catalyze key steps of melanin production. 12,13 Asian countries, women with fair skin are considered to be beautiful, and therefore, most people search for natural products to whiten their skin and reduce pigmentation, creating a greater demand for skin-whitening cosmetic products. 14,157][18] For instance, concentrations of kojic acid >1% can cause itchy skin and act as a carcinogen. 19Therefore, a safety natural antimelanogenic compound needs to be discovered.
1][22][23] Furthermore, paeoniflorin administration attenuates UVA-induced oxidative stress in human dermal fibroblasts by activating Nrf2 signaling pathways. 24A recent study showed that P. lactiflora and P. suffruticosa Andr.6][27] However, the effect of the main bioactive component "paeoniflorin" involved in the alleviation of skin hyperpigmentation has not been elucidated.Therefore, this study aimed to investigate the role of paeoniflorin in regulating α-MSH-induced melanogenesis in mouse melanoma B16F10 cells.

| Cell viability assay
assay was performed according to the manufacturer's instructions. 28ll viability was evaluated by MTT assay (475989, Sigma).The culture medium was replaced with 100 μL of MTT solution (0.5 mg/mL).
After 3 h of incubation at 37°C, the formazan crystals produced in the medium were solubilized in 100 μL DMSO.The absorbance was measured at 570 nm using a spectrophotometer, with cell viability indicated as percent with respect to the control.

| Measurement of melanin content
Melanin content in B16F10 cells was performed according to the manufacturer's instructions. 29B16F10 cells were cultured at a density of 2 × 10 5 cells/well in 6-well plates overnight, following treatment with different concentrations of α-MSH and paeoniflorin for 48 h.The cells were treated with 2 N NaOH (S5881, Sigma), followed by heating at 80°C for 30 min, and the melanin content was measured at an absorbance of 405 nm using a spectrophotometer.

| Cellular tyrosinase activity assay
Tyrosinase activity in B16F10 cells was performed according to the manufacturer's instructions. 29B16F10 cells were cultured at a density of 2 × 10 5 cells/well in 6-well plates overnight, following treatment with different concentrations of α-MSH and paeoniflorin for hyperpigmentation, melanoma, paeoniflorin, skin pigmentation, whitening agent 48 h.Following washing, the cells were treated with 1% Triton X-100 prepared in PBS.The cell lysate was incubated with freshly prepared 2.5 mM L-DOPA (333786, Sigma) in PBS for 1 h at 37°C, and tyrosinase activity was measured at an absorbance of 490 nm using an absorbance reader.The data were then normalized to the protein content of the cell lysates, and tyrosinase activity was indicated as tyrosinase activity (%) = (sample/control) × 100%.

| Western blot analysis
Western blot analysis was performed according to the manufacturer's instructions. 30

| Inhibitory effect of paeoniflorin on α-MSHinduced melanogenesis in B16F10 cells
A previous study demonstrated the anti-melanogenic activity of P. lactiflora dried root extract in human skin without elucidating the active ingredient responsible for this effect. 25Therefore, to characterize the anti-melanogenic effect of the key active ingredient paeoniflorin, B16F10 cells were treated with α-MSH (1 μM) for 24 h, followed by paeoniflorin (1, 5, 10 μM) treatment.Melanin content and tyrosinase activity increased upon α-MSH treatment, indicating melanogenesis.However, post paeoniflorin treatment, melanin content as well as tyrosinase activity decreased in a dosedependent manner, elucidating anti-melanogenic effect of paeoniflorin (Figure 3A,B).This was visualized by western blot analysis of the proteins purified post 48 h of incubation of B16F10 cells with different paeoniflorin concentrations (Figure 3C).

| DISCUSS ION
A previous study reported the ability of P. lactiflora root extract to cause skin depigmentation.However, not many studies have investigated the main active ingredient (paeoniflorin) responsible for the depigmentation effect. 25In this study, we characterized the antimelanogenic effects of paeoniflorin in α-MSH-induced hyperpigmented mouse melanoma (B16F10) cells.Briefly, our findings could be summarized as follows: Paeoniflorin has been reported to attenuate UVB-induced keratynocyte apoptosis and tumor necrosis factor-a-induced human dermal microvascular endothelial cell inflammation. 4,31Furthermore, the protective effects of paeoniflorin against UVA-induced human dermal fibroblasts aging through Nrf2/HO-1/NQ-O1 signaling pathway have been recently reported. 24Another study indicated that the administration of paeoniflorin enhanced foot wound healing in diabetic rats through Nrf2 signallings. 32This study showed that treating skin cells with paeoniflorin at micromolar (μM) concentration can attenuate stress-induced damage.In our study, we found that paeoniflorin did not cause cytotoxic effects at concentration ≤ 20 μM in B16F10 cells, demonstrating it is safety and efficacy as a potential natural anti-melanogenic agent.
There is mounting evidence for tyrosinase as an essential enzyme for melanogenesis.Tyrosinase catalyzes the oxidation of Ltyrosine to L-DOPA and then to dopaquinone.Dopaquinone was then used to synthesize eumelanin or pheomelanin.Therefore, agents possessing antioxidant and tyrosinase-inhibiting properties were able to reduce skin hyperpigmentation.Paeoniflorin, a monoterpene glucoside compound, showed antioxidant properties and could attenuate the activity of biphenolase and monophenolase, thus inhibiting tyrosinase activity and reducing melanin synthesis. 24,33However, another study showed contrary results, wherein paeoniflorin treatment enhanced human melanocyte proliferation and melanin biosynthesis as well as increased the number of hair follicles and melanin content in a monobenzoneinduced vitiligo mouse model. 34By comparing with our recent study (α-MSH-induced overproduction of melanin), this study showed that paeoniflorin possesses melanin-promoting effects for treating melanin deficiency-related diseases, such as vitiligo.
Approaches to develop anti-melanogenic agents mainly focus on inhibiting tyrosinase activity: (1) an inhibitor binding to the active site of tyrosinase can be designed to inactivate the enzyme and inhibit melanogenesis; (2) different molecules competing with the enzyme's cofactor copper can be added to bind to tyrosinase, resulting in enzyme inhibition. 35This study demonstrated the ability of paeoniflorin to inhibit α-MSH-induced melanin synthesis, tyrosinase activity, and melanogenesis-related signaling.In the future, F I G U R E 4 Schematic illustration of paeoniflorin attenuating α-MSH-induced B16F10 cells melanogenesis.
All experiments were performed in at least triplicates.The values are expressed as mean ± standard error.Differences in the effect of various treatments were compared using the Student's t-test or ANOVA.Statistically significant changes are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

F
I G U R E 1 α-MSH induced melanogenesis in B16F10 cells.Cells were treated with different concentrations of α-MSH for 48 h.(A) Cell viability and (B) levels of melanogenesis-related proteins were evaluated by MTT assay and western blotting, respectively.β-actin was used as a loading control.(C) Melanin content and (D) tyrosinase activity were also evaluated.Values indicate mean ± SD.Quantified results are indicated as (n = 3) *p < 0.05, **p < 0.01 versus untreated control cells.
(1) α-MSH induced CREB/MITF/tyrosinase signaling in a dose-dependent manner.(2) Melanin content and tyrosinase activity were upregulated in response to α-MSH administration in B16F10 cells.(3) α-MSH-induced melanogenesis was inhibited by paeoniflorin treatment.Recently, natural anti-melanogenisis products have attracted the attention in the development of cosmetic products against UV-induced hyperpigmentation because of their low toxicity.