Extraction, Purification And Biological Activity Of Actinidia Arguta Essential Oil And Squalene

Actinidia arguta is known as the"king of the third generation fruit".It is highly sought after because of its sweet and sour taste and rich health care effect.and its related exploration and research is also an upsurge of research.Essential oil is a common natural product in plants,which is widely used in food,medical,beauty and other industries.It can be seen everywhere in daily life.Due to the great differences in composition and efficacy of different essential oils,the research on A.arguta essential oil is still rare.Squalene was found in A.arguta for the first time in the previous experiment,so this study explored it from the aspects of extraction,purification and biological activity of A.arguta essential oil and squalene from A.arguta.Progress has been made in the following aspects:1.In this study,the essential oil of A.arguta was extracted by water distillation method,compound organic solvent method and ultrasonic assisted method,respectively.The extraction rate of water distillation method was 1.04%,and the obtained essential oil was light in color and easy to volatilize;the extraction rate of compound organic solvent method was35.9%,and the color of essential oil was yellowish brown;The ultrasonic-assisted method has the maximum extraction rate of 9.02%under the conditions of material-liquid ratio of 1:5,ultrasonic power of 400 W and ultrasonic time of 180 mins.The color of essential oil is very light and almost transparent.The extraction rates of the three methods are quite different,and the intuitive properties are obviously different.2.The density,moisture content and other conventional physicochemical properties and compound composition of essential oil were determined.The density of essential oil by water distillation was 0.785 g/mL.A total of 36 components were identified,accounting for 87.82%of the total.The representative compounds are butyl acetate,1-dimethylbenzene,etc.,including esters,alkanes and aromatic compounds.The density of compound organic solvent method essential oil was 0.954 g/mL.60 compounds were analyzed,accounting for 95.15%of the total,including olefins and aliphatic compounds such as linoleic acid and squalene.The density of the essential oil by ultrasonic-assisted extraction was 0.916 g/mL.A total of 56compounds were identified,accounting for 82.09%of the total.The main compounds are long-chain alkanes,olefins and alcohols,including squalene,γ-sitosterol and so on.The differences in physical and chemical properties of the three essential oils can be reasonably explained from the differences in composition,which also shows that different extraction methods have a great influence on the composition and quality of essential oils.In addition,squalene was found in A.arguta essential oil for the first time.3.The bioactivity of essential oil was determined,and the antioxidant performance test results showed that the highest scavenging rates of water distillation essential oil against DPPH free radical,hydroxyl radical and superoxide anion radical were 61.28%,68.38%and51.97%,respectively;the highest scavenging rates of essential oil extracted by compound solvent method were 70.48%,79.03%and 60.44%,and those of ultrasonic-assisted essential oil were 67.21%,73.04%and 55.24%,respectively.The results of antibacterial test showed that the essential oil could not inhibit Gram-negative Escherichia coli,but was more effective against the other two Gram-positive bacteria.The minimum inhibitory concentration（MIC）of distilled essential oil against Staphylococcus aureus and Bacillus subtilis was 3.2 mg/mL,and the semi-inhibitory concentration(IC₅₀)were 0.601 mg/mL and 0.866 mg/mL,respectively.The MIC values of the essential oils obtained by the compound organic solvent method were3.2 mg/mL and 1.6 mg/mL,respectively.The lower IC₅₀ values were 0.469 mg/mL and 0.589mg/mL,respectively.The MIC values of the essential oils obtained by the ultrasound-assisted method against the two Gram-positive bacteria were both 3.2 mg/mL.The IC₅₀ values were slightly higher than that of the volatile oil by compound organic solvent method,which were 0.536 mg/mL and 0.677 mg/mL,respectively.The anti-tumor performance test results showed that both the concentration of essential oil and reaction time could affect the inhibition of tumor cells.The maximum inhibition rates of water distillation essential oil on HT-29 cells,PC-3 cells and A549 cells were 50.63%,57.37%and 55.12%,respectively,and the maximum inhibition rates of compound organic solvent essential oils were 78.63%,57.31%and 60.42%,respectively.The essential oils of ultrasonic-assisted method were 70.63%,65.31%and 63.42%.4.The crude squalene was isolated from A.arguta seed oil by saponification extraction method and purified by column chromatography.The sample was qualitatively determined by thin layer chromatography and high performance liquid chromatography,and then quantitatively detected by GC-MS.The results showed that the crude extraction rate of saponification method was 11%,the content of squalene in purified sample was 95.3%,and the content of squalene in A.arguta seed oil was 3.41%.5.The biological activity of squalene was tested,and the antioxidant results showed that the highest scavenging rates of squalene to the three free radicals were 75.66%,82.41%and84.1%,respectively.The results of antibacterial test were similar to those of essential oil,and showed good performance against Gram-positive bacteria.The minimum inhibitory concentrations of S.aureus and B.subtilis were 3.2 mg/mL,and the values of IC₅₀ were 0.663mg/mL and 0.713 mg/mL,respectively,but had no good effect on Gram-negative bacteria represented by E.coli.In the anti-tumor test,squalene had a certain inhibitory effect on SCG-7901 cells,A549 cells and SiHa cells,and the IC₅₀ values were 2.79 mg/mL,7.73mg/mL and 3.33 mg/mL,respectively.The inhibitory effect of squalene on A549 cells and SCG-7901 cells was better than that of SiHa cells.