| Background:Sinomenine, a monomer alkaloid extracted from menispermaceous plant Sinomenium actum Rehd.et wils., has similar structure to morphine. Sinomenium actum has been used to treat rheumatic diseases in East Asia for over 100 years,which shows traditional pharmacological effects, such as anti-inflammation and immunosuppression. However,reports regarding the effect of sinomenine on treating cutaneous inflammation are not available at present. Moreover, sinomenine also displays novel biological activities,including anti-tumor, alleviating neuropathic pain, relieving morphine and heroin addiction or withdrawal symptoms. Alkaloid drugs possess the binding site with melanin; in addition,the known action targets of sinomenine have partly overlapped with the related signal pathways for regulating the activity of tyrosinase, the rate-limiting enzyme of melanin synthesis. As a morphine analogue,whether sinomenine can play a role of whitening skin has not been reported.Objective:This paper aimed to determinate the effect of sinomenine on the existing melanin,melanin synthesis and tyrosinase activity. Moreover, to explore the antioxidant capacity of sinomenine based on the structural features of sinomenine, and to evaluate the therapeutic potential of sinomenine on inflammatory dermatosis. Sinomenine gels were prepared, so as to investigate whether the external preparation could reach the deep skin to exert its function as well as its safety on skin.Methods:1. Effects of various concentrations of sinomenine (1500-0.14 μg/mL) on proliferation of cells (B16-F10, HepG2, HaCaT, and Human dermal fibroblasts) were examined using CCK-8, so as to determine the optimal range of drug concentration in vitro.2. Effects of sinomenine within the concentration range of 10-100 μg/mL on melanin synthesis inhibition rate and eliminating rate of the existing melanin were investigated using sodium hydroxide pyrolysis assay, with B16-F10 cell as the model and arbutin as positive control (forskolin and 8-MOP induced B16-F10 cell modeling). Moreover, the inhibition rate of sinomenine on tyrosinase activity was detected through oxidation of L-Dopa.3 .The antioxidant ability of sinomenine within the concentration range of 33.3-300μg/mL was determined,with HepG2 as the model and ascorbic acid as positive control.4. Cytokines secreted by keratinocytes or demal fibroblasts under TNF-a induction were preliminarily screened out using ELISA. The detection indexes in the interventional experiment of sinomenine, together with TNF-a stimulation dose and time, were determined from the time-based and dose-based curves of keratinocytes and demal fibroblasts secreting cytokines upon TNF-α stimulation. Subsequently, interventional experiment of sinomenine was carried out.5. Sinomenine and sinomenine hydrochloride gel were prepared. A rapid and sensitive High Performance Liquid Chromatography assay method was established to determine the contents of sinomenine and sinomenine hydrochloride in vitro. The average penetration rates and accumulative penetration amount in 12h of both gels were examined through skin-penetrating capacity test in vitro.6. Sinomenine and sinomenine hydrochloride gel were prepared. The skin irritation effects of two gels were examined after single-dose and multi-dose, respectively, with New Zealand white rabbits as models (damaged skin and intact skin groups).Results:1.Cell survival rate of all cell lines or human fibroblasts reached over 85% when the concentration of sinomenine was below 300 μg/mL.2.The result suggested that sinomenine could effectively suppress melanin synthesis(68.2% at 100 μg/mL),eliminate the existing melanin (36.8% at 100 μg/mL) and inhibit tyrosinase activity (inhibition rate was 39.3% at 100 μg/mL)3.The gradient concentrations of sinomenine and vitamin C were 0, 33.3, 100 and 300,μg/ml. The average carbonylation inhibition rates corresponding to concentrations of sinomenine were 1.3%, 30.4%, 59.8% and 52.5% ; while those of vitamin C were 1.2%,31.1%, 58.3% and 65.1% respectively.4.The results of ELISA indicated that sinomenine and its hydrochloride could suppress the expression of IL-6, MMP-1 and MMP-9 secreted under TNF-a induction in skin cells (P<0.01), but both of them did not have any effects on IL-8 (P>0.05).5.Sinomenine gel had excellent transdermal penetration effect. Under the same prescription, the penetration rate and accumulative penetration amount of sinomenine were remarkably higher than those of sinomenine hydrochloride gel.6.Both sinomenine and sinomenine hydrochloride gel had little skin irritation and were safe.Conclusion:In all, sinomenine has the potential to relieve skin inflammation, improve and protect the complexion of skin, and promote skin homeostasis. Moreover, sinomenine hydrochloride gel has good percutaneous penetration effect and little skin irritation. |
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