超声辅助双酶法提取紫苏叶花色苷的工艺优化及抗氧化活性研究

Ultrasound-assisted Dual-enzyme Extraction Condition Optimization and Antioxidant Activity Evaluation of Anthocyanins from Perilla Leaves

摘要:

【目的】综合开发紫苏，提高其利用价值。【方法】以紫苏叶为原料，采用超声辅助双酶法提取紫苏叶中的花色苷，研究不同料液比、乙醇体积分数、超声时间、酶解时间对紫苏叶中花色苷质量分数的影响，采用响应面法优化超声辅助双酶法提取紫苏叶花色苷的最佳工艺条件。并测定紫苏叶花色苷的抗氧化活性和酚类化合物质量分数，通过色谱法分析主要酚类化合物。【结果】紫苏叶花色苷的最佳提取工艺为料液比1 g∶16 mL、乙醇体积分数74%、超声时间23 min、酶解时间92 min。在此条件下，紫苏叶中花色苷质量分数为252.87 mg/100 g，其DPPH自由基清除率为48.88%，总抗氧化能力为51.12%，羟自由基的清除率为34.99%。紫苏叶酚类化合物质量分数为4.81 mg/g，含飞燕草素-3-O-葡萄糖苷（0.43±0.04） mg/g、矢车菊素（0.86±0.07） mg/g、芍药花素-3，5-O-二葡萄糖苷（1.41±0.03） mg/g，同品种不同批次酚类化合物质量分数差异较小。此外，紫苏叶花色苷清除DPPH自由基、羟自由基能力强，芍药花素-3，5-O-二葡萄糖苷等能高亲和力地结合KEAP1的Nrf2结合口袋，抑制KEAP1-Nrf2结合，促进Nrf2核转位及血红素氧合酶-1（HO-1）、醌氧化还原酶1（NQO1）的表达。【结论】双酶联用质量比、双酶联用总酶量、温度、料液比、乙醇体积分数、超声时间和酶解时间等因素会对紫苏叶花色苷的质量分数产生不同的影响，且紫苏叶酚类质量分数与原料、贮藏条件相关；紫苏叶花色苷可通过直接清除自由基与激活KEAP1-Nrf2/抗氧化反应元件（ARE）通路发挥抗氧化作用，超声辅助双酶法可高效提取高纯度紫苏叶花色苷。该研究为紫苏叶花色苷在食品加工中的应用提供了理论依据。

Abstract:

[Objective]This study aims to comprehensively develop perilla and improve its utilization value.[Method]Using perilla leaves as raw material, the effects of solid-liquid ratio, ethanol volume fraction, ultrasonic time, and enzymatic hydrolysis time on the mass fraction of anthocyanins in perilla leaves were studied. The ultrasound-assisted dual-enzyme extraction conditions for anthocyanins from perilla leaves were optimized by the response surface method. The antioxidant activity and phenol mass fraction of anthocyanins from perilla leaves were determined, and the main phenols were analyzed by chromatography.[Result]The optimal extraction conditions of anthocyanins from perilla leaves were solid-liquid ratio of 1 g∶16 mL, ethanol volume fraction of 74%, ultrasonic time of 23 min, and enzymatic hydrolysis time of 92 min. Under these conditions, the mass fraction of anthocyanins from perilla leaf was 252.87 mg/100 g, with a DPPH radical scavenging rate of 48.88%, a total antioxidant capacity of 51.12%, and a hydroxyl radical scavenging rate of 34.99%. The phenols in perilla leaf anthocyanins showed the mass fraction of 4.81 mg/g, containing (0.43±0.04) mg/g delphinidin-3-O-glucoside, (0.86±0.07) mg/g cyanidin, and (1.41±0.03) mg/g paeoniflorin-3,5-O-diglucoside. The mass fraction of phenols in different batches of the same variety had little difference. In addition, perilla leaf anthocyanins had a strong ability to scavenge DPPH and hydroxyl free radicals. Paeoniflorin-3,5-O-diglucoside bound to the Nrf2 pocket of KEAP1, inhibited KEAP1-Nrf2 binding, and promoted the nuclear translocation of Nrf2 and the expression of hemeoxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1).[Conclusion]Factors such as the mass ratio of dual enzymes, total enzyme dosage, temperature, solid-liquid ratio, ethanol volume fraction, ultrasonic time, and enzymatic hydrolysis time have different effects on the mass fraction of anthocyanins in perilla leaves, and the mass fraction of phenols in perilla leaves is related to the raw materials and storage conditions. Perilla leaf anthocyanins can exert antioxidant effects by directly scavenging free radicals and activating the KEAP1-Nrf2/antioxidant response element (ARE) pathway. Ultrasound-assisted dual-enzyme method enables efficient extraction of high-purity anthocyanins from perilla leaves. This study provides a theoretical basis for the application of perilla leaf anthocyanins in food processing.