Study On The Whitening Effect Of Camellia Oleifera Shell Extract

China is the second largest cosmetics consumer in the world.In 2020,the size of Chinese cosmetics market reach 500 billion yuan,of which the skin care category is the largest at 265 billion yuan.With the increasing living standards of Chinese residents,the demand for cosmetics continues to expand,and skin care products with whitening function are very popular among consumers.Natural extracts derived from plants have various biological activities such as anti-oxidation,anti-tumor,hypoglycemic,and have low toxicity and easy availability of raw materials.So they have become a research hotspot in the development of whitening cosmetics at home and abroad.Camellia oleifera shell as the research object,determine the optimal extraction process for the active components,and further study the inhibitory effect and mechanism on tyrosinase.What’s more,cell and animal experiments were used to investigate the effect of Camellia oleifera shell extract on inhibiting tyrosinase activity and melanin production.Besides,Western blot technology was used to detect melanin production-related proteins to explore its mechanism.The main results are as follows:（1）Using Camellia oleifera shell as raw material and ethanol as extraction solvent,and taking the inhibition rate of tyrosinase activity as the investigation index,through single factor and orthogonal experiments,the optimal extraction process conditions for the active components of Camellia oleifera shell extract that inhibited tyrosinase activity were as follows:ethanol volume fraction was 60%,the extraction time was 3 h,the ratio of solid to liquid was 1:15（g/m L）,and the extraction temperature was 60℃.Under this process conditions,the yield of Camellia oleifera shell extract was 6.08%±0.04%,which contained 30.22%±1.02%total polyphenols and 13.52%±0.24%flavonoids.The tyrosinase activity inhibition rate of the Camellia oleifera shell extract with a mass concentration of 0.1 mg/m L could reach42.80%±1.37%.（2）L-tyrosine as the substrate,the IC₅₀of Camellia oleifera shell extract on tyrosinase monophenolase was 0.0711 mg/m L,and its inhibitory effect was produced by reducing enzyme activity and increasing lag time.L-DOPA as the substrate,the IC₅₀of Camellia oleifera shell extract on tyrosinase diphenolase was 0.2199 mg/m L,and the inhibition mechanism was further explored as anti-competitive and reversible inhibition,with the K_ISof 10.7487 mg/m L.（3）The antioxidant capacity of Camellia oleifera shell extract was determined.The IC₅₀of scavenging DPPH·and ABTS·⁺free radicals were 0.0271 mg/m L and0.2023 mg/m L and the total antioxidant capacity was 2.80 mmol/L,indicating its excellent antioxidant activity.Correlation analysis showed polyphenols and flavonoids may be the main substances inhibit tyrosinase activity in Camellia oleifera shell extract.（4）Taking human melanoma A375 cells as a cell model,the cell proliferation rate was measured by MTT method.When the concentration of Camellia oleifera shell extract was lower than 0.25 mg/m L,the cell proliferation rate exceeded 96%,indicating that it had no inhibitory effect on cell proliferation.Therefore,the maximum administration concentration of Camellia oleifera shell extract was set to0.25 mg/m L.When the concentration of Camellia oleifera shell extract was 0.15mg/m L,the inhibition rate of cellular tyrosinase activity was 53.17%±1.56%,and the effect was close to that of 0.1 mg/m L kojic acid（p>0.05）.The relative melanin content of cells at this concentration decreased to 35.74%±1.23%,which was better than that of 0.1 mg/m L kojic acid（p<0.05）.The experimental results showed that Camellia oleifera shell extract could significantly inhibit the tyrosinase activity and melanin content of A375 cells（p<0.05）.（5）Taking zebrafish as an animal model,the tyrosinase activity and melanin production in zebrafish were determined.Compared with the blank control group,the tyrosinase activity and melanin production in zebrafish treated with Camellia oleifera shell extract were significantly decreased（p<0.05）,and the inhibition rate of tyrosinase activity and the inhibition rate of melanin production were concentration-dependent with the concentration of Camellia oleifera shell extract.Western blot results showed that Camellia oleifera shell extract could significantly down-regulate the expression levels of melanin production-related proteins CREB,MITF and TYR in zebrafish（p<0.05）,thereby inhibiting melanin production in zebrafish.To sum up,Camellia oleifera shell extract had excellent tyrosinase inhibitory activity,antioxidant and other biological activities,which could be expected to be used as a substance with whitening function in cosmetic formulations,and provide a basis for the high-value utilization of Camellia oleifera shell.