Study On Extraction And Separation Of Longan Seed Active Lipids And Their Moisturizing Effect

In recent years,bio-skin care has created a new era of skin care products.The bio-active factors are adjusted at the molecular level to improve the skin growth environment,maintain the skin moisture content,and keep the skin elastic and hydrated and healthy.Therefore,the natural plant actives with green,safe and no side effeects have become hotspots in cosmetic research.Longan seeds are often discarded as a waste in the food processing industry,but it has certain application value because it contains active ingredients such as polysaccharides,flavonoids,phytosterols and sphingomyelin.In this paper,longan seeds active lipids were extracted and separated by ultrasound-assisted ethanol extraction,organic solvent extraction,silica gel and ODS column chromatography.The in vitro cell anti-drying injury test was used to study the components with high total sterol and ceramide content,and the moisturizing mechanism were studied.The main contents were as follows:(1)Active lipids were extracted from the longan seeds by ultrasound-assisted ethanol extraction.The total sterol content was the main evaluation index,and the yield of active lipid was the auxiliary evaluation index.The extraction conditions were optimized by single factor and orthogonal experiments.The optimal conditions were as follows:solid-liquid ratio of 1:15 g/mL,ultrasonic time of 40 min and temperature of 60℃.In this condition,the longan seeds active lipid yield was 10.86%,and the total sterol content was 5.28 mg/g,and it mainly contained triterpenoids,alkaloids,saponins,tannins,phenols,flavonoids and organic acids.(2)The chloroform phase(CEP)was obtained by fractional extraction method,and the active lipid yield was 34.62%,and the total sterol content was 191.95 mg/g.CEP was separated by Isolera silica gel column chromatography to obtain CEP-2,and the yield and total sterol content were 69.15%and 518.26 mg/g,respectively.The CEP-2 mainly contained triterpenoids,tannins,phenols and ceramide.The CEP-2 was further separated by Isolera ODS column chromatography to obtain 20 components CEP-2-1-20,which were analyzed by TLC and HPLC-ELSD.The content of ceramide in CEP-2-13 was 855.0 mg/g,and the total sterol content in CEP-2-17 was 946.32 mg/g.The CEP-2-13 was qualitatively studied by IR spectroscopy,TLC and UPLC-MS/MS,and speculated it mainly contained five ceramides,respectively named N-palmitoyl-D-sphingosine,N-octadecenooyl-D-phytosphingosine,N-arachiyl-D-sphingosine,N-tetracosenooyl-D-phytosphingosine,N-octadecenooyl-D-erythro sphingosine.The CEP-2-13 was qualitatively studied by TLC and HPLC-ELSD,and presumed it mainly contained stigmasteroi and P-sitosterol.(3)The moisturizing activity of active lipids were determined by weighing method.In the experiment,glycerol and CER-3 were used as controls.The results showed that the moisturizing effect of CEP-2-13 were stronger than CEP-2-1 7 but slightly lower than glycerol at RH=81%.However,when the relative humidity was 43%,the moisturizing activity as followed,CEP-2-13>glycerol>CER-3>CEP-2-17,which means that ceramide has better moisturizing effect under low humidity.The study on the body moisturizing activity showed that CEP-2-13 maintained the moisture content of the skin for a long time and was better than the moisturizing effect of CER-3 and CEP-2-17.Applied CEP-2-13 to the skin surface for 2 hours,the maximum increase of skin moisture content was 27.42%;after 6 h of skin exposure,the TEWL value was reduced to a minimum of 4.58 g/m2h.(4)CCK-8 assay cytotoxicity experiments showed that CEP-2,CEP-2-13 and CEP-2-17 had certain proliferation effects on HSF cells and HaCaT cells,and the proliferation of HaCaT cells was more obvious than HSF cells.The CEP-2-13 at concentration of 80 μg/mL had the strongest proliferation effect on HaCaT cells,and the cell survival rate was 125.93%(p<0.001).The CEP-2 and CEP-2-17 have the same protective and repairing effects on dry-damaged HaCaT cells.CEP-2-13 has the strongest protective and repairing effect,and the repairing effect was more obvious than the protective effect.(5)ELISA kit to determine the content of HA and AQP3 in dry-damaged HaCaT cells.The results showed that the ability of CEP-2-13 to induce dry damaged cells produce HA and AQP3 stronger than CEP-2-17,and CEP-2-13 up-regulates the ability of AQP3 in dry-damaged HaCaT cells to be greater than HA.When the mass concentration of CEP-2-13 was 100 μg/mL,the HA content increased by 91.68%and the AQP3 increased by 125.10%compared with the control group.