Dermatologica Sinica

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 39  |  Issue : 1  |  Page : 33--40

Tanshinone IIA, a melanogenic ingredient basis of Salvia miltiorrhiza Bunge


Zhaojing Wang1, Huihao Tang1, Lili Yang2, Yiming Li1, Huali Wu1,  
1 Department of TCM Chemistry, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China
2 Department of Dermatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China

Correspondence Address:
Huali Wu
Department of TCM Chemistry, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai
China
Dr. Yiming Li
Department of TCM Chemistry, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai
China

Abstract

Background: The roots of Salvia miltiorrhiza Bunge (Lamiaceae) have been often used to treat vitiligo in clinical for many years. However, the main ingredient basis of efficacy has not been known. Objectives: We investigated whether the two main constituents of S. miltiorrhiza, major hydrophobic compound tanshinone IIA (Tan IIA) and the major hydrophilic compound salvianolic acid B (Sal B), had the same melanogenic activity. Methods: To testify the potential roles of Tan IIA and Sal B in pigmentation, tyrosinase (Tyr) activity, melanin synthesis ability, and the molecular mechanisms stimulating melanin production were determined in B16F10 melanoma cells. Results: Tan IIA promoted melanogenesis and enhanced Tyr activity at its maximum concentration (10 μM), whereas that of Sal B had no effect. Furthermore, the color of cell pellets and morphological observation of B16F10 cells were visibly darkened. Regarding molecular mechanisms, Western blot results showed that Tan IIA (1, 3, and 10 μM) dose dependently increased the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase MAPK, respectively. However, it decreased phosphorylation in extracellular signal-regulated protein kinase 1/2 MAPK signaling. Meanwhile, Tan IIA promoted the expression of microphthalmia-associated transcription factor and Tyr. Conclusion: Tan IIA might be a melanogenic ingredient basis of S. miltiorrhiza to increase the Tyr activity by activating the upstream MAPK signaling pathways, thereby contributing to pigmentary processing.



How to cite this article:
Wang Z, Tang H, Yang L, Li Y, Wu H. Tanshinone IIA, a melanogenic ingredient basis of Salvia miltiorrhiza Bunge.Dermatol Sin 2021;39:33-40


How to cite this URL:
Wang Z, Tang H, Yang L, Li Y, Wu H. Tanshinone IIA, a melanogenic ingredient basis of Salvia miltiorrhiza Bunge. Dermatol Sin [serial online] 2021 [cited 2021 Apr 20 ];39:33-40
Available from: https://www.dermsinica.org/text.asp?2021/39/1/33/311814


Full Text



 Introduction



Vitiligo is a progressive pigmentation disorder induced by multiple factors; despite there were new emerging researches on vitiligo, the pathogenesis of it is still unclear.[1],[2] The current most preferred therapy is narrow-band ultraviolet B treatment;[3] some oral and topical agents are also used to mitigate skin damage. However, most treatments have numerous limitations and side effects. Currently, more repigmentation agents from natural products are increasingly receiving scholarly attention.

Chinese herbs and herbal components have long been widely used in the treatment of skin diseases and pigmentation disorders.[4] Salvia miltiorrhiza, a widely used Chinese herb known as Danshen, and widely used to treat cardiovascular and cerebrovascular diseases,[5],[6] is one of the Chinese medicinal herbs most commonly prescribed for treating vitiligo. S. miltiorrhiza extract (SME) strongly promotes melanocyte adhesion and migration of melanocytes when used in the vitiligo treatment.[7] Moreover, in the experiment of S. miltiorrhiza extract activity, SME had a significant pigmentary effect in B16F10 cells. However, the main ingredient basis of melanogenic efficacy of S. miltiorrhiza remains unknown. The effective chemical components of S. miltiorrhiza can be classified into two groups: hydrophobic diterpenoid quinones and hydrophilic phenolic acids. Tanshinone IIA (Tan IIA), a diterpenoid quinone [Figure 1]a, is the most crucial fat-soluble ingredient of S. miltiorrhiza,[8] which accounts for about 0.1% ~ 0.9% of the total plants.[9] Tan IIA has been reported to mitigate cardiomyopathy and improve cardiac function.[10] Its anti-atherosclerotic,[11] anti-inflammatory, immunoregulatory,[12] and neuroprotective effects have also been established.[13] Accounting for 3%~5% of the herb's total dry weight, salvianolic acid B (Sal B) is one of the major water-soluble phenolic components of S. miltiorrhiza [Figure 1]b and is the highest-content component of the plant.[14] Sal B has been not only used to treat cardio–cerebral vascular diseases but also shows antioxidative activity.[15] The primary aim of the present study was to evaluate which ingredient is the melanogenic ingredient basis of S. mitiorrhiza in vitiligo therapy by determining tyrosinase (Tyr) activity and melanin content.{Figure 1}

Melanogenesis is ongoing in the melanocytes which located in the basal layer of the epidermis, and then the production melanin causes pigmentation in human eye, hair, and skin.[16] Evidence indicates that melanogenesis is controlled by numerous signaling pathways, most of which finally converge to the microphthalmia-associated transcription factor (MITF) pathway. During melanogenesis, MITF regulates various inherent mechanisms by integrating the upstream signaling of pathways mediated by p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated protein kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) and regulating the expression of downstream genes.[17] Tyr is another vital enzyme in melanogenesis, which located in the membrane of the melanosome. Its gene and protein expression are directly influenced by MITF.[18] Tyr is reported as the rate-limiting enzyme in the transformation of tyrosine into melanin pigment.[19] In the present study, we evaluated the melanogenic effects of Tan IIA and Sal B on B16F10 cells by detecting melanin content and Tyr expression and activity.

 Materials and Methods



Cell culture

The murine melanoma cell line B16F10 was acquired from the cell bank of the Chinese Academy of Sciences (Shanghai, China). B16F10 cells were cultured as a monolayer in Dulbecco's modified Eagle medium (Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Thermo Fisher Scientific). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Before the experiment, the B16F10 cells were cultured for <14 h after cell passage and then serum-starved for 72 h with compounds of various concentrations.

Reagents

S. miltiorrhiza was purchased from Shanghai Yutiancheng Traditional Chinese Medicine Co., Ltd (Shandong, China, batch number 2018092501). SME was acquired as follows: 30 g of S. miltiorrhiza (30 g) was extracted with 70% ethanol in an ultrasound bath (2 × 300 mL, 1 h and each). After solvent removal under reduced pressure, 9.3 g of extract was obtained. Tan IIA (purity ≥98%, high-performance liquid chromatography [HPLC], Chemical Abstracts Service [CAS]: 568-72-9) and Sal B (purity ≥98%, HPLC, CAS: 121521-90-2) were purchased from MACKLIN (Shanghai, China).

MTT assay

Tan IIA and Sal B were dissolved in dimethylsulfoxide (DMSO) before their addition to the medium, and the highest DMSO content(0.5 %) had no significant influence on drugs effect [Supplementary Material Figure 1]. Cells were seeded in 96-well plate at a density of 5 × 103 cells per well. After 24 h, the medium was suctioned out and the cells were treated with certain concentrations of Tan IIA and Sal B. Three wells were devoted to each concentration of the compounds. The plates were incubated for an additional 48 h before bring rewashed. Next, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in phosphate-buffered saline [PBS]) was added, and incubation was allowed to proceed for 4 h. After the MTT solution was suctioned out, 150 μL of DMSO was added. The mixture was shaken for 10 min to allow the formazan salt to dissolve. Finally, the absorbance was measured at 570 nm using a multifunctional microplate reader (BioTek Instruments, Winooski, VT, USA).

Melanin content assay

Melanin content was determined with the minor modified procedure described as previously report.[20] The harvested cells were washed twice with ice-cold PBS, then lysed by incubation in cell lysis buffer (1mM phenylmethylsulfonyl fluoride) at 4°C for 30 min. Next, the lysates were separated by centrifugation at 15,000 rpm for 10 min. Total melanin was dissolved in 100 μL of 1M NaOH containing 10% DMSO, and the mixture was then heated at 80°C for 1 h. Finally, melanin absorbance was determined at 405 nm using a multifunctional microplate reader. Melanin content was normalized to lysate total protein and was expressed as percentage change or fold change as compared with vehicle-treated controls.

Tyrosinase activity assay

Tyr activity was measured using a slightly modified spectrophotometric method.[21] After the protein content was quantified using a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology, Haimen, China), the volume of supernatant containing same concentration (10 μg) of total protein was set to 100 μL by 0.1 M PBS (pH 6.8). The mixture was then added to each well on a 96-well plate and mixed with 100 μL of 2 mg/mL levodopa (L-DOPA). After incubation at 37°C for 1 h, absorbance was measured at 475 nm using a multifunctional microplate reader. Tyr activity was normalized to lysate total protein and described as a percentage change as compared with vehicle-treated controls.

Western blot assay

The cells were lysed in cell lysis buffer (1 mM IP, Beyotime, Shanghai, China) at 4°C for 30 min; the lysates were separated by centrifugation at 10,000 rpm for 20 min at 4°C. The supernatant was collected to detect the protein concentration by BCA, and 25 μg of total protein from each sample was loaded on to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel in a loading buffer and then transferred to membrane (Millipore, USA) in transbuffer. The membrane was blocked with 5% bovine serum albumin (Beyotime, Shanghai, China), slowly shaken for 1 h, and then incubated overnight with primary antibodies: Tyr (product number SC7833, 1:1000, Abcam, Cambridge, UK), MITF (product number ab20663, 1:1000, Abcam, Cambridge, UK), P38 (product number ab8690, 1:1000, Abcam, Cambridge, UK), p-P38 (product number ab4511, 1:1000, Abcam, Cambridge, UK), JNK (product number CTS9252, 1:1000, Cell Signaling Technology Inc., MA, USA), p-JNK (product number CTS4668, 1:1000, Cell Signaling Technology Inc., MA, USA), ERK1/2 (product number CTS4696, 1:1000, Cell Signaling Technology Inc., MA, USA), p-ERK1/2 (product number CTS4370, 1:2000, Cell Signaling Technology Inc., MA, USA), as well as mouse polyclonal antibodies against β-actin (product number CTS3700, 1:1000, Cell Signaling Technology Inc., MA, USA). After reaction with the second antibody, the proteins were visualized with a chemiluminescent substrate (ELC, Millipore, USA) using a chemiluminescence imaging system (GE Healthcare, USA). The presented results are representative of at least three independent experiments.

Statistical analysis

All data were expressed as means ± standard error of the mean and analyzed using (GraphPad Software, San Diego, CA, USA). Statistical analysis was performed with a one-way analysis of variance followed by post hoc Tukey's test for correction of multiple comparisons. A P < 0.05 was considered statistically significant.

 Results



Effects of Salvia miltiorrhiza extract on melanogenesis in B16F10 cells

The melanin content assay revealed that SME promoted melanogenic activity in a dose-dependent manner. At the highest concentration (40 μg/mL), SME induced an approximately twofold increase in melanin content, resulting in melanin content being comparable to that achieved using the positive drug α-melanocyte-stimulating hormone (α-MSH). Furthermore, as shown in [Figure 2]a, visible darkening of the cell pellets of the B16F10 cells was observed. Microscopic observation of cell morphology confirmed greater pigment accumulation in the SME-treated cells than in the control cells [Figure 2]b. These results suggest that SME could promote melanogenesis in B16F10 cells.{Figure 2}

Effects of tanshinone IIA and Salvianolic acid B on cytotoxicity in B16F10 cells

To investigate the melanogenic ingredient basis of S. miltiorrhiza, we chose the major hydrophobic compound Tan IIA and the major hydrophilic compound Sal B, which have been widely applied in clinical. First, the MTT assay was used to determine the potential cytotoxic effect of different concentrations of Tan IIA and Sal B on B16F10 cells. As shown in [Figure 3]a for the treatment lasting 48 h, the vitality of the B16F10 cells decreased significantly as the concentration of Tan IIA was increased (from 1, 5, 10, and 20–40 μM; P < 0.001); thus, Tan IIA inhibited B16F10 viability in a dose-dependent manner. No significant cytotoxicity in B16F10 cells was observed at Tan IIA concentrations lower than 10 μM. No considerable differences in cell viability were observed as the concentration of Sal B was increased from 5, 10, 20, and 40–80 μM, indicating that these were all safe doses at which to administer Sal B treatment in future experiments [Figure 3]b.{Figure 3}

Effects of tanshinone IIA and Salvianolic acid B on melanogenesis and tyrosinase activity in B16F10 cells

Several natural chemical compounds can reverse hypopigmentation in vitiligo through mechanisms that vary according to the disease etiology. Herein, we mainly determined the effectiveness of Tan IIA and Sal B, representative compounds from S. mitiorrhiza, for stimulating pigmentation production in B16F10 cells and mechanisms underlying this process. Melanin content was quantified using a spectrophotometric method. Based on the MTT assay results, we selected concentrations of 1, 3, 5, 8, and 10 μM for Tan IIA and 10, 20, and 80 μM for Sal B in our investigation of the compounds' impacts on melanin. For Tan IIA, melanin synthesis was increased at Tan IIA concentrations of 3 and 10 μM, but a significant change was shown only for 10 μM. Sal B was not discovered to significantly affect melanin synthesis at any concentration. As shown in [Figure 4]a, Tan IIA increased the melanin content. This increase was significant at the maximum concentration (10 μM; P < 0.05) and corresponded to an approximately 23% increase in melanin content. Furthermore, microscopy revealed that Tan IIA significantly enhanced melanin synthesis [Figure 4]b. By contrast, melanin content was unchanged after Sal B treatment [Figure 4]c.{Figure 4}

As mentioned, Tyr is a rate-limiting enzyme in melanogenesis. It can convert L-DOPA into dopaquinone which exhibits substantial ultraviolet absorption at 475 nm in UV. Because Sal B showed no remarkable stimulation of melanin synthesis ability, we did not examine its effect on Tyr activity, only that of Tan IIA. To clarify the mechanism underlying the observed melanogenesis induced by Tan IIA, the Tyr activity of melanogenic enzyme was determined. The result of Tyr activity assay showed that Tan IIA can elevate Tyr activity which was same as the trend of melanin production. At 10 μM, the Tyr activity was increased by 21% [P < 0.05; [Figure 4]d].

Effects of tanshinone IIA on microphthalmia-associated transcription factor/mitogen-activated protein kinase signaling pathway in B16F10 cells

MAPKs are crucial upstream regulators of melanogenesis.[22] To investigate whether MAPK pathway was activated in Tan IIA-treated B16F10 cells, Western blot assay was employed to detect the effect of Tan IIA (1, 3, and 10 μM) on activation of the MAPK pathways (P38, JNK, and ERK1/2). As the result shown in [Figure 5]a, under Tan IIA treatment at 10 μM, the expression of p-P38 and p-JNK were significantly enhanced, and P38 and JNK phosphorylation were increased tenfold and twofold, respectively. ERK1/2 phosphorylation was decreased significantly (by 36%) only for the Tan IIA concentration of 10 μM, the maximum concentration [[Figure 5]b, [Figure 5]c and [Figure 5]d, P < 0.001]. Meanwhile, the total protein expression of P38, JNK, and ERK1/2 was unchanged.{Figure 5}

MITF regulates various inherent mechanisms during melanogenesis including the MAPK pathways. Tyr, which is the rate-limiting enzyme in melanogenesis, is the upstream of MITF. As the important transcription factor of the downstream of MAPK and essential enzyme, the protein expression levels of MITF and Tyr were also examined. As shown in [Figure 5]a, the protein expression of MITF was significantly enhanced after Tan IIA treated for 3 days. MITF expression in the B16F10 cells was two times higher than that in the control group for a Tan IIA concentration of 10 μM [[Figure 5]e; P < 0.01]. As the rate-limiting enzyme in melanogenesis, Tyr expression in the B16F10 cells was also enhanced after treatment with Tan IIA, but there was no statistical significance. At 10 μM, Tyr expression was 30% higher than that in the control group [Figure 5]f.

 Discussion



S. mitiorrhiza is a well-known traditional Chinese herb, widely used in clinical vitiligo treatment in China. Previous study has confirmed that SME can strongly enhance melanin production of B16F10 cells and Tyr activity which came from mushrooms.[23] Similarly, SME significantly promoted melanogenesis in B16F10 cells in the present study [Figure 2]. However, its active constituents and mechanism of action in melanogenesis are still unknown. There are three types of compound isolated from S. mitiorrhiza: diterpenoid quinones, hydrophilic phenolic acids, and essential oil constituents.[8] Chen et al.[23] discovered that danshensu and Sal B, the representative compounds of hydrophilic phenolic acids, showed attenuated effect on melanin production of α-MSH-stimulated B16F10 cells by inhibiting Tyr activity.[23] Herein, we examined the effect of Tan IIA as main diterpenoid quinones constituent and Sal B as main hydrophilic phenolic acids constituent in S. mitiorrhiza on melanin synthesis. In contrast to the findings of Chen et al.[23], Sal B was not found to significantly affect melanin synthesis. This discrepancy is most likely due to between-study differences in cell models. SME and Sal B showed totally different effects on melanogenic activity. With regard to the potential of Tan IIA to contribute to melanogenesis, the results are promising.

Tan IIA is an important kind of diterpenoid quinones and most widely studied as an anticardiovascular drug. A 2017 study, indicated that the standard anti-cardiovascular drug simvastatin is a potential therapeutic agent for vitiligo.[24] Tan IIA is also widely applied to clinical dyslipidemia. As illustrated in [Figure 4], Tan IIA slightly increased the melanin content of and Tyr activity in vitro in B16F10 cells. Whether the mechanisms of action of Tan IIA are similar to those of simvastatin is worthy of future investigation.

B16F10 cells share most similar melanogenic mechanisms with normal human melanocytes and thus are commonly used in in vitro assays of melanin synthesis.[25] Tyr activity and melanin content are screening indexes for screen repigmentation agents. The current study demonstrated that in the safe concentration range (1–10 μM), Tan IIA enhanced melanin synthesis by activating the rate-limiting enzyme Tyr and without having a cytotoxic effect in the B16F10 cells [Figure 4]. Because no significant differences were obtained between Tan IIA concentrations of 3, 5, and 8 μM, we selected the medium concentration of 3 μM to explore the potential molecular mechanism of Tan IIA. To further understand the molecular mechanisms of Tan IIA acting on melanogenesis in B16F10, we examined the protein expression level of important pathway MAPKs. Many studies have demonstrated the important role of MAPK signaling pathways in melanin synthesis, such as P38, JNK, and ERK1/2.[26],[27] Among them, after phosphorylation, p-P38 and p-JNK activate the expression of downstream MITF, whereas p-ERK1/2 induces MITF phosphorylation, which further leads to ubiquitin-dependent degradation of MITF.[28],[29] Our Western blot assay indicated that the phosphorylation levels of P38 and JNK were increased while the phosphorylation level of ERK1/2 was decreased. Therefore, the increased p-P38 and p-JNK could lead into more MITF protein production, and the decreased p-ERK1/2 could result in less MITF degradation. The final MITF protein level was greatly enhanced, contributing to increased Tyr expression [Figure 5]. Although the protein expression of Tyr is not significantly increased, the significant increased expression of MITF can reflect the increased melanin synthesis ability of Tan IIA, which is because many melanotic-related melanin synthesis signaling pathways eventually converge to MITF. Thus, we suggest that the activation of MITF/MAPK pathway after Tan IIA treatment increases melanin synthesis in the B16F10 cells by upregulation of MITF and Tyr expression [Figure 6]. In conclusion, our in vitro analysis results showed that the melanin content of B16F10 cells was significantly increased after treated with Tan IIA.{Figure 6}

Although Tan IIA showed the increased melanin synthesis ability on B16F10 cells at the maximum concentration in our experiment, whether it is the central substance of S. miltiorrhiza that prompts repigmentation warrants confirmation through future research. A 2019 study reported that thymoquinone-induced melanogenesis in cultured melanocytes and has potential for treating hypopigmentary disorders.[30] Tan IIA is a representative component of diterpenoid quinones in S. mitiorrhiza, considering the structure of compound, in the next phase of the study, other diterpenoid quinones ingredients in S. mitiorrhiza need to be confirmed on melanogenesis. In addition, MITF/MAPK signaling pathways and in vivo assay will be used to screen the melanogenic constituents of S. mitiorrhiza.

 Conclusion



Tan IIA might be a melanogenic ingredient basis of S. miltiorrhiza to increase the Tyr activity and MITF expression by activating the upstream MAPK signaling pathways like: P38, JNK and ERK1/2. As a representive compontent of diterpenoid quinones in S. mitiorrhiza, Tan IIA can real contribute to pigmentary processing and increase melanin synthesis.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (No. 81673570), the Excellent Academic Leaders Program of Shanghai (16XD1403500), and the programs of High Level University Innovation Team and Shanghai E-Research Institute of Bioactive Constituents in Traditional Chinese Medicine. This work was also supported by the Three-Year Action Plan to Further Speed Up the Development of Traditional Chinese Medicine in Shanghai, Construction and Cultivation Project of Dominant Diseases of Traditional Chinese Medicine (ZY(2018-2020)-ZYBZ-10), and the High-Level University Summit Project (Huimin Zhang, Summit Plateau Team).

Conflicts of interest

There are no conflicts of interest.

 Supplementary Material



In our experiment, Tan IIA and Sal B were dissolved in dimethyl sulfoxide, and the highest dimethyl sulfoxide content was 0.5 %. In order to avoid solvent disturbance, the effect of 0.5 % dimethyl sulfoxide on melanogenesis and tyrosinase activity in B16F10 cells was determined. Moreover, the result showed that 0.5 % dimethyl sulfoxide has no significant influence on both melanogenesis and tyrosinase activity [Figure 1].

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