月桂酰基谷氨酸钠辅助微波萃取杜仲叶总多酚及提取物活性研究

Extraction of total polyphenols from Eucommia ulmoides oliver leaves assisted by sodium lauroyl glutamine-microwave and activities of the extracts

中文关键词:  杜仲叶  总多酚  月桂酰基谷氨酸钠  抗氧化  酪氨酸酶活性  微波

英文关键词:Eucommia ulmoides Oliver leaves  total polyphenols  sodium lauroyl glutamate  antioxidation  tyrosinase activity  microwave

中文摘要:

      目的 优化氨基酸表面活性剂月桂酰基谷氨酸钠(lauroyl glutamate sodium, LGS)辅助微波萃取杜仲叶总多酚的提取工艺, 并比较不同提取工艺得到的提取物(水提物、LGS水提物、醇提物)的体外抗氧化能力及对酪氨酸酶的作用。方法 采用单因素试验结合响应面法, 以总多酚得率为评价指标, 对杜仲叶LGS辅助微波萃取总多酚进行工艺优化; 采用高效液相色谱法(high performance liquid chromatography, HPLC)对不同工艺得到的提取物进行活性成分对比分析, 并对不同提取物的抗氧化活性及对酪氨酸酶的作用进行比较。结果 杜仲叶LGS辅助微波萃取的最佳工艺为: LGS浓度2.4×10-2 mol/L、料液比1:27 (g/mL)、静置时间78 min、微波功率200 W、微波时间2.6 min、提取2次, 得到的LGS提取液总多酚得率为(60.20±0.65) mg/g, 显著优于水提液[(45.75±0.38) mg/g, P<0.05], 更接近醇提液[(76.62±0.52) mg/g]。杜仲叶多酚特征成分绿原酸在提取物中的含量为醇提液[(13.18±0.33) mg/g]>LGS水提液[(12.40±0.24) mg/g]>水提液[(11.18±0.27) mg/g], 与总多酚提取得率一致。3种提取物对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)、2,2-联氮-二-3-乙基-苯并噻唑-6-磺酸[2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS]、羟基(hydroxyl, OH)、3-氧代-2-苯基-4,4,5,5-四甲基咪唑啉-1-氧[(4,4,5,5-tetramethyl-2-phenyl-2-imidazoline 3-oxide)-1-yloxy, PTIO]具有一定的清除效果, 均随其质量浓度升高而增强。3种提取物对酪氨酸酶的促进作用表现为: 醇提物>LGS水提物>水提物, 与总多酚/绿原酸提取得率保持一致。分子对接结果显示绿原酸能增强酪氨酸酶与左旋多巴的结合力, 改变左旋多巴与酪氨酸酶的结合位点, 从而推动左旋多巴氧化进程。结论 所选取的提取工艺合理、可行, LGS辅助微波提取可提高杜仲叶总多酚得率, 得到的提取物具有良好的体外抗氧化能力, 并可提高酪氨酸酶活性。

英文摘要:

      Objective To optimize the extraction process of total polyphenols from Eucommia ulmoides leaves by microwave extraction assisted with amino acid surfactant sodium lauroyl glutamate sodium (LGS), and compare the in vitro antioxidant capacities of different extraction process obtained extracts (LGS extract, water extract, and ethanol extract) as well as their effects on tyrosinase activities. Methods Single factor test combination with response surface methodology was used to optimize the extraction process of total polyphenols from Eucommia ulmoides leaves by LGS assisted microwave extraction, and the yield of total polyphenols was selected as evaluation index. High performance liquid chromatography (HPLC) was used to analyze the active components of the extracts, and the antioxidant capacities and tyrosinase activities under these extracts treatments were determined. Results The optimum condition for LGS assisted microwave extraction total polyphenols of Eucommia ulmoides leaves was as follows: The LGS concentration was 2.4×10?2 mol/L, the solid-liquid ratio was 1:27 (g/mL), the standing time was 78 min, the microwave power was 200 W, the microwave time was 2.6 min, and the extraction times was twice. Under these conditions, the yield of total polyphenols of LGS extracting solution was (60.20±0.65) mg/g, which was significantly higher than that of the water extracting solution [(45.75±0.38) mg/g, P<0.05], and closer to the ethanol extracting solution [(76.62±0.52) mg/g]. The content of the characteristic component of Eucommia ulmoides leaves (chlorogenic acid) in the extracting solution was ranked as ethanol extracting solution [(13.18±0.33) mg/g]>LGS extracting solution [(12.40±0.24) mg/g]>water extracting solution [(11.18±0.27) mg/g], which was consistent with the extraction rate of total polyphenols. The 3 kinds of extracts had certain scavenging rates for 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), hydroxyl (OH), and (4,4,5,5-tetramethyl-2-phenyl-2-imidazoline 3-oxide)-1-yloxy (PTIO), and their clearance rate increased along with their mass concentration elevation. The promoting effect of the three extracts on tyrosinase ranked as ethanol extract>LGS extract>water extract, which was in agreement with the extraction yield of total polyphenols and chlorogenic acid. Molecular docking further showed that chlorogenic acid could enhance the binding energy as well as change the binding site between levodopa and tyrosinase, so that facilitate the oxidation process of levodopa. Conclusion The selected extraction process is reasonable and feasible. LGS assisted microwave extraction can improve the yield of total polyphenols extracted from Eucommia ulmoides leaves. The obtained extract reveal excellent in vitro antioxidant capacity and can accelerate the activity of tyrosinase.