Extraction Of Triterpene Acids From Loquat Leaves Via Deep Eutectic Solvent

Loquat leaf is the leaf of the evergreen tree loquat(Eriobotrya japonica(Thunb.)Lindl.)in the family Rosaceae,also known as Ba Ye,which is a traditional Chinese medicine rich in a wide variety of functional active ingredients.Triterpene acid is the main functional active ingredient of loquat leaf,which has broad application prospects in the fields of medicine,cosmetics and others.At present,loquat leaf triterpene acid components are mainly obtained by volatile organic solvent extraction,and the extraction process usually has disadvantages such as high cost,high pollution and low efficiency.In this thesis,hydrophobic deep eutectic solvents(HDES)were structured and screened via COSMO-SAC model combined with experimental validation for green and efficient extraction of loquat leaf triterpene acid.Then,single-factor and response surface experimental disigns were conducted for optimizing the HDES extraction process.Finally,the enrichment of loquat leaf triterpene acid and the recovery of HDES from HDES extraction solution were perfomed,respectively,based on C18 reversed-phase silica gel column chromatography.Meanwhile,the in vitro antioxidant activity of the prepared loquat leaf triterpene acid samples was investigated.The main results were as follows:(1)The COSMO-SAC model could accurately predict the solubilization ability of HDESs on ursolic acid.The COSMO-SAC model prediction combined with experimental validation results showed that HDES composed of thymol/benzyl alcohol(Thy/BA)(2:1,molar ratio)had better extraction effect on loquat leaf triterpene acid.(2)Using the extraction yield of ursolic acid,the main component of loquat leaf triterpene acid,as evaluation index,the optimized HDES extraction condition of loquat leaf triterpene acid was established by single-factor and response surface tests,which was liquid to material ratio 14(g/g),extraction temperature 59°C and extraction time 64 min.Under this condition,the yield of ursolic acid in loquat leaf triterpene extract was 17.79 ± 0.45mg/g,which was significantly higher than that(11.42 ± 0.23 mg/g)of ursolic acid in loquat leaf triterpene extract via 80%(v/v)ethanol extraction.(3)C18 reversed-phase silica gel low-pressure column could effectively recover HDES and enrich the crude loquat leaf triterpene acid from the HDES extraction solution,respectively.The optimized chromatographic separation conditions of the C18 column were as follows,that is,60%(v/v)ethanol,80%(v/v)ethanol and 100%(v/v)ethanol were sequentially used for gradient elution,and the elution volume of each gradient elution was 5column volume(Bed volume,BV).The crude triterpene acid sample was enriched in 80%(v/v)ethanol eluate with a purity of 81.18%,in which the mass ratio of ursolic acid,oleanolic acid,corosolic acid and maslinic acid was 53:10:26:11.The recovered HDES Thy/BA was used for the extraction of triterpene acid from loquat leaves,and its extraction capacity was comparable to that of the freshly prepared Thy/BA.The prepared triterpene acid samples showed strong antioxidant activity.To sum up,COSMO-SAC model prediction combined with experimental validation can effectively screen suitable HDES for loquat leaf triterpene acid extraction with the advantages of high efficiency and low cost.HDES Thy/BA(2:1)can efficiently extract loquat leaf triterpene acid with better extraction effect than traditional solvents.C18reversed-phase silica gel can achieve the recovery of HDES and the enrichment of loquat leaf triterpene acid under gradient elution conditions.This study expands the application of COSMO-SAC model to solvent screening and provides new idea and theoretical basis for HDES recovery and efficient preparation of loquat triterpene acids based on chromatographic column chromatography separation,which is of great significance for the development and utilization of loquat triterpene acids in pharmaceuticals,cosmetics and functional foods.