Separation And Purification Of Squalene From The Deodorized Distillate Of Camellia Oil And Its Effect Of Glycometabolism In Cell

Squalene,which can be found in shark liver,plant seeds,certain microorganisms,microalgae,human sebum,etc.,is an unsaturated triterpenoid of six isoprenes.It is widely used in the cosmetics,medicine and food industries due to its ability to anti-oxidation,anti-radiation,regulation of cholesterol metabolism and inhibition of the growth of harmful microorganisms.Deodorized distillate of many plant oils contains plentiful squalene,as well as phytosterols,vitamin E and a small amount of phenolic substances.In this paper,the squalene was preliminarily extracted from Camellia Oil Deodorizer Distillate(CODD)through saponification.Gas Chromatography(GC)and Gas Chromatography Mass Spectrometry(GC-MS)were used to detect the non-saponifiable contents both qualitatively and quantitatively.After being purified by silica gel column,the squalene purification(SP)was tested its antioxidant activity in vitro and on lard,and the hypoglycemic effect of the insulin resistance(IR)in cell model was preliminarily investigated as well.The research conclusions are as follows:(1)Physical and chemical indicators of the deodorized distillate of Camellia oil were measured based on national standard,which lays the foundation for subsequent extraction and separation.The peroxidation value of CODD was 5.3 mmol/kg,the saponification value(in KOH)was 193.7 mg/g,the acid value was 78.4 mg/g,the iodine value was 85.2 g/100g,and the unsaponifiable content was 12.5%.The moisture and volatility content was 0.5%.The fatty acids are mainly composed of palmitic acid,stearic acid,oleic acid,linoleic acid and linolenic acid.The squalene content in the deodorized distillate obtained through the pressing and the solvent leaching was 535 mg/100g and 198 mg/100g respectively.In the precision detection,the recovery rate of the intra-day precision test is between 98.8%and 102.49%,and the RSD value is between 1.60 and 7.73.The recovery rate of the standard precision test is 98.4%?102.6%,RSD value ranges from 4.00 to 8.99,both of which comply with RSD<10%,indicating the test method is practical.(2)The squalene-rich unsaponifiable matter was preliminarily obtained by saponification method.Orthogonal test was used to determine the optimum conditions:alkali concentration 1.3 mol/L,ratio of material to liquid 1:6,reaction time 60 min,reaction temperature at 90 ?.With these condition,the yield of squalene was 35.2%and the extraction rate was 82.0%.Primary screening of eluent species and ratios by thin layer chromatography(TLC)and then the unsaponifiable matter was subsequently purified by silica gel column chromatography.Dry loading,200-300 mesh silica gel,eluent was n-hexane:ethyl acetate was 100:1(v/v),and the dropping rate was 3 mL/min.Under these conditions,the squalene yield was 95%and the purity was 90%.(3)Using two antioxidant evaluation systems(DPPH · and hydroxyl radical)to explore the antioxidant capacity of the SP.In DPPH · measurement,the SP scavenging effect increased with the concentration increased at low concentrations.concentration of 60 ?g/mL had the strongest free radical scavenging ability of 24.6%,and then stabilized.In the hydroxyl radical experiment,the SP scavenging effect also increased with the concentration increased at low concentrations.The highest scavenging rate was reached at 80 ?g/mL,which was 33.6%.The purified sample was also used to test the anti-oxidation on lard.0.02%SP had anti-oxidation effect on lard(P<0.05),and high concentration had certain pro-oxidation effect(P<0.05),and compared with other antioxidants,SP resistance Oxidation was relatively weak,but had synergistic effect with propyl gallate(PG)(P<0.05).(4)Using the IR cell model,the effect of SP on lowering blood glucose was preliminarily studied.The results showed that 200 ?mol of palmitic acid(PA)could successfully induce insulin resistance in HepG2 cells,whose viability was not damaged(P>0.05).Under this condition,SP of 50 ?g/mL,100 ?g/mL and 200 ?g/mL could increase cells' consumption of glucose(P<0.05),increasing the amount of glycogen synthesis(P<0.05).Each concentration increased the activity of glycogen synthase(GCS)and showed the best effect at 100 ?g/mL,consistenting with the results of glycogen synthesis;50 ?g/mL and 100 ?g/mL SP can significantly reduce the activity of Glucose-6-phosphatase(G6P),but the concentration of 200 ?g/mL was weak.The content of Gglycerin trilaurate(TG)was not significantly decreased(P>0.05),but it could improve the pathological condition of the cells in a sense;There were remarkable increase(P<0.05)in the contents of malondialdehyde(MDA)and superoxide dismutase(SOD)of the test group,indicating the anti-oxidation effect of SP in cells was more obvious.In summary,the squalene is initially extracted from CODD by saponification separation method,and then purified through silica gel column to effectively enrich squalene.Compared with supercritical CO2 extraction,molecular distillation and enzymatic extraction,the method in this paper is simpler and easier,the equipment investment and maintenance is relatively less,which provides a scientific guidance and theoretical basis for industrial production.Moreover,a certain concentration of SP can be used as an antioxidant or as a synergist for other natural or synthetic antioxidants in protecting and strengthening of oils.SP has a certain hypoglycemic effect on cells which have insulin resisitant,and may play a biological role by improving the vitality of key enzymes and oxidative stress.Though more experiments are needed.