Optimization of Extraction Process and Biological Activities of Flavonoid Aglycones from Ribes meyeri Stem

In order to optimize acid hydrolysis extraction process of 5 flavonoid aglycones(luteolin,quercetin,kaempferol,isorhamnetin and apigenin)from Ribes meyeri stem and evaluate their antioxidant activity and α-glucosidase inhibitory activity in vitro,the content of 5 flavonoid aglycones were determined simultaneously by UPLC-MS/MS. The acid hydrolysis extraction process of flavonoid aglycones of Ribes meyeri stem was optimized by single factor experiments and orthogonal test. The antioxidant and hypoglycemic activities of acid hydrolysis extract and 5 flavonoid aglycones from Ribes meyeri stem were evaluated by DPPH,ABTS free radical scavenging and α-glucosidase inhibitory activity assays. The results showed that the limit of detection and limit of quantification about the five flavonoid aglycones were 0.8~1.6 μg/L and 2.9~5.4 μg/L. The five flavonoid aglycones had good linear relationship(R²≥0.9962)within 3.2~207.0 μg/L. The precision,repeatability and stability of the method were good for the 5 flavonoids(RSD≤4.4%). The optimal extraction process of flavonoid aglycones from Ribes meyeri stem was established as follows:90% methanol solution(including 5% hydrochloric acid),the extraction temperature was 70 ℃,the extraction time was 100 min. In this optimized extraction conditions,the extraction rate of total flavonoid aglycones reached 4.06 mg/g(RSD<5%),which indicated the extraction method was stable and feasible. The acid-hydrolysis extract of Ribes meyeri stem and the 5 flavonoid aglycones showed significant α-glucosidase inhibitory activity and antioxidant activity. The α-glucosidase inhibitory activities of methanol acid-hydrolysis extract and 5 flavonoid aglycones were higher than the positive control,acarbose(IC₅₀:(53.84±2.41) mg/L). Quercetin had the best DPPH free radical scavenging activity(IC₅₀:(2.94±0.18) mg/L),while luteolin and kaempferol had higher ABTS free radical scavenging activity(IC₅₀:(5.34±0.10),(5.55±0.17) mg/L,respectively),and their antioxidant activities were higher than the positive control V_C. The established UPLC-MS/MS method is sensitive,reliable and accurate,which can be used for the simultaneous quantitative analysis of 5 flavonoid aglycones. The optimal acid hydrolysis process can effectively improve extraction rate of flavonoid aglycones from Ribes meyeri stem.