The Preparation And Protective Effect Of Resveratrol-Loaded Nanostractured Lipid Carrier Sunscreen Gel

A large amount of ultraviolet(UV)irradiation leads to the lipid peroxidation in skin cells,which results in excessive production of reactive oxygen species(ROS)and DNA damage.Local supplementation of antioxidants can improve the antioxidative capacity of the skin and reduce the oxidative damage induced by UV.Resveratrol(RES),a natural polyphenol compound,exhibits significant antioxidative effect.Pretreatment guinea pig skin with resveratrol microemulsion almost completely prevented the erythema formation induced by UVB,and showed protective effects on oxidative damage induced by UV.However,RES is unstable under light and heat conditions,which results in the decrease of its activity and consequently limits its application in the pharmaceutical preparations of skin.Objective:The nanostructured lipid carrier(NLC)was composed of shea butter and capric triglyceride.The active component RES was encapsulated in NLC,and then dispersed in the sodium carboxymethylcellulose gel matrix.A nanostructured lipid carrier sunscreen gel(NLC-gel)was prepared to improve the stability of RES and increase the protective effect of RES on the skin against ultraviolet radiation.Methods:1.The shea butter,emulsifier,vitamin E(VE)and bisabolol were heated at 80 ?,and then 4-methylbenzylidene camphor(Parsol 5000),avobenzene(Parsol 1789)and ethylhexyl methoxycinnamate(Parsol MCX)were added.When the temperature of the mixture decreased to 40 ?,the solution of RES and capric triglyceride was added to form the oil phase.Allantoin,EDTA-2Na and deionized water were mixed and heated at 80 ? to form water phase.The nanostructured lipid carrier(NLC)was obtained by mixing aqueous phase and oil phase at 60 ? under the condition of ultrason.The nanostructured lipid carrier sunscreen gel(NLC-gel)was obtained by mixing the NLC with sodium carboxymethylcellulose gel.2.The absorbance value of NLC-gel was investigated by UV spectrophotometer at 280 nm,290 nm,300 nm,310 nm and 320 nm.The effects of the ratio of shea butter to capric triglyceride,the dosage of resveratrol,the amount of emulsifier,the shear speed,the shearing time,the ultrasonic power and the ultrasonic time on the absorbance value of NLC-gel were investigated.3.The experimental group was designed by JMP7 software to optimize the prescription composition and preparation process.According to the experimental design,NLC-gel was prepared,and its absorbance value was measured.The single variable linear model in SPSS software was used to analyse data,then the optimized prescription composition and preparation process were obtained.4.The particle size,zeta potential and morphology of NLC-gel were characterized by zeta potential and laser particle size analyzer and transmission electron microscope.The viscosity and pH of NLC-gel were measured by viscometer and pH meter,respectively.The sun protect factor(SPF)value of NLC-gel was determined by UV 2000 S in vitro analyzer.5.A high performance liquid chromatography(HPLC)method was established to simultaneously determine RES,Parsol 5000,Parsol 1789 and Parsol MCX.The stability of NLC-gel was investigated.6.The UVA and UVB damage model of HaCaT cells was established,and MTT method was used to detect the protective effect of NLC-gel on HaCaT cells.7.The skin irritation reaction of NLC-gel on rabbits was observed by repeatedly stimulative experiment.8.The accumulation of effective component of NLC-gel in the skin was observed by confocal laser scanning microscope(CLSM).9.The UVB damage mouse model was established.The protective effect of NLC-gel on mouse skin was observed by H&E staining.The effects of NLC-gel on catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GPx)in skin tissue of mice were detected by using kit.Results:1.Single factor analysis showed that the dosage of RES,the time of shearing and the time of ultrasound significantly affected the absorbance value of NLC-gel,and there were interaction between the dosage of RES and the time of ultrasound,the time of shearing and the time of ultrasound,respectively.2.The optimal formulation and preparation process were as follow: the ratio of capric triglyceride to shea butter was 1:5,the dosage of RES was 1%,the dosage of emulsifier was 4%,shearing velocity was 2000 r/min,shearing time was 5 min,ultrasonic power was 180 w,and ultrasonic time was 10 min.3.The NLC-gel was milky white gel,uniform and delicate,no stratification,no dry or liquefied,and good spreading.The particle size was(184±12)nm,the zeta potential was –(33±2)mV.The shape was spherical,and the viscosity was(16±1)Pa·s.The pH was 6.3±0.1,and the SPF value was 43±5.4.The newly established HPLC method was stable and accurate.The results of UV aging test showed that NLC obviously improved the stability of RES,Parsol 5000,Parsol 1789 and Parsol MCX.The content of RES,Parsol 5000,Parsol 1789 and Parsol MCX loaded in NLC-gel,the particle size,the viscosity and the pH of NLC-gel exhibited no obvious changes in the test on influence factor of stability,in the accelerated test of stability and in the long term test of stability.5.In the UVA and UVB damaged HaCaT cells models,NLC-gel significantly reduced the damage of HaCaT cells induced by UVA and UVB.6.NLC-gel was safe and not irritating to the skin of rabbits.7.CLSM experimental results indicated that effective component of NLC-gel accumulated in the epidermis of the skin.8.When UVB irradiated the back skin of mice for 2 h,the obvious erythema was observed after 24 h.In addition,the skin thickness increased after 7 days,and the surface of back skin was scab.The H&E staining showed that the structure of skin exhibited disorders,thickening of the epidermis,necrosis of most epidermis cells,and obvious infiltration of inflammatory cells in the model group.There was also a small number of inflammatory cells in the dermis in the model group.However,the skin structure was normal after NLCgel pretreatment,and there was no thickening of epidermis,no necrosis of epidermal cells,no obvious infiltration of inflammatory cells,and no inflammatory cells in the dermis in NLC-gel pretreatment group.Compared with normal control group,the activities of CAT,SOD and GPx in skin tissue were decreased in model group.Compared with model group,the activities of CAT,SOD and GPx in skin tissue significantly increased in NLC-gel pretreated mice.Conclusion:The properties of the prepared NLC-gel were stable and no irritating.NLC-gel effectively improved the stability of RES,increased the accumulation of RES on the epidermis,effectively protected mice skin from damage caused by acute UVB irritation,and subsequently enhanced the antioxidative effect of skin.NLC-gel has significant potential in application for sunscreen.