天山茶茎黄酮苷元提取工艺优化及其生物活性

为了优化天山茶藨茎5种黄酮苷元（木犀草素、槲皮素、山奈酚、异鼠李素、芹菜素）的酸水解提取工艺，评价其体外抗氧化和α-葡萄糖苷酶抑制活性，本文利用UPLC-MS/MS法测定天山茶藨茎5种黄酮苷元的提取率，通过单因素实验和正交试验优化天山茶藨茎黄酮苷元酸水解提取工艺。利用DPPH、ABTS自由基清除实验测定天山茶藨茎提取物及5种黄酮苷元的抗氧化活性，并测定了其α-葡萄糖苷酶抑制活性。结果表明，5种黄酮苷元的检出限、定量限分别在0.8~1.6、2.9~5.4 μg/L，在3.2~207.0 μg/L范围内具有良好的线性关系（R2≥ 0.9962），方法的稳定性、精密度和重复性良好（RSD≤4.4%）。确定最佳酸水解提取工艺为:90%甲醇溶液（含5%盐酸）、提取温度:70℃、提取时间:100 min，该条件下，总黄酮苷元提取率为4.06 mg/g，RSD<5%，表明正交试验优化的提取条件稳定可行。天山茶藨茎提取物及5种黄酮苷元均具有一定的α-葡萄糖苷酶抑制活性和抗氧化活性，其中甲醇盐酸提取物与5种黄酮苷元的α-葡萄糖苷酶抑制活性均高于阳性对照阿卡波糖（IC₅₀:53.84±2.41 mg/L），槲皮素对DPPH自由基的清除活性最好（IC₅₀:（2.94±0.18） mg/L），木犀草素、山奈酚对ABTS自由基清除活性较好（IC₅₀分别为（5.34±0.10）、（5.55±0.17） mg/L），它们的抗氧化活性高于阳性对照V_C。本研究建立的UPLC-MS/MS方法灵敏、精确、高效，可以对天山茶藨茎5种黄酮苷元化合物同时进行定量分析。优化的酸水解提取工艺能有效提高天山茶藨茎黄酮苷元的提取率。

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 andα-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 andα-glucosidase inhibitory activity assays.The results showed that the limit of detection and limit of quantification about the five flavonoid aglycones were 0.8~1.6μg/L and 2.9~5.4μg/L.The five flavonoid aglycones had good linear relationship(R 2≥0.9962)within 3.2~207.0μg/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 significantα-glucosidase inhibitory activity and antioxidant activity.Theα-glucosidase inhibitory activities of methanol acid-hydrolysis extract and 5 flavonoid aglycones were higher than the positive control,acarbose(IC 50:(53.84±2.41)mg/L).Quercetin had the best DPPH free radical scavenging activity(IC 50:(2.94±0.18)mg/L),while luteolin and kaempferol had higher ABTS free radical scavenging activity(IC 50:(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.