紫花地丁总香豆素提取工艺优化及其抗氧化和抗肿瘤活性考察
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篇名: 紫花地丁总香豆素提取工艺优化及其抗氧化和抗肿瘤活性考察
TITLE: Optimization of the extraction technology of total coumarin from Viola yedoenis and its antioxidant and anti-tumor activities
摘要: 目的 优化紫花地丁总香豆素的提取工艺,并评价其纯化物的体外抗氧化活性和抗肿瘤活性。方法在单因素实验的基础上,以紫花地丁总香豆素得率为响应值,以液料比、提取时间、乙醇体积分数为变量,采用响应面法优化紫花地丁总香豆素的提取工艺并验证。采用1,1-二苯基-2-三硝基苯肼(DPPH)法、2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)法和2-苯基-4,4,5,5-四甲基咪唑啉-3-氧代-1-氧(PTIO)法评价紫花地丁总香豆素纯化物的抗氧化活性。采用CCK-8法检测其对人宫颈癌细胞HeLa、人胆管细胞型肝癌细胞HCCC-9810、人肝癌细胞HepG2、人乳腺癌细胞MDA-MB-231和人非小细胞肺癌细胞A549的抑制作用;采用Hoechst33258染色法检测其对HCCC-9810细胞凋亡形态的影响,并测定细胞中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)含量。结果最优提取工艺为液料比25∶1(mL/g)、提取时间1.9h、乙醇体积分数68%;3次验证实验的紫花地丁总香豆素平均得率为(21.10±0.17)mg/g,RSD为0.80%(n=3),与预测值(20.79mg/g)的相对误差为1.49%。紫花地丁总香豆素纯化物对DPPH和ABTS自由基的半数抑制浓度(IC50)分别为21.54、17.11μg/mL,略高于抗坏血酸的15.21、12.45μg/mL;对PTIO自由基的IC50为1422.01μg/mL,远高于抗坏血酸的37.20μg/mL;对上述5种细胞的IC50分别为(113.18±15.97)、(51.17±1.72)、(134.15±11.75)、(171.83±15.27)、(93.38±4.65)μg/mL;能促进HCCC-9810细胞凋亡,增加其LDH含量并降低SOD含量。结论成功优化了紫花地丁总香豆素的提取工艺;所得提取物的纯化物具有抗氧化活性和抗肿瘤活性。
ABSTRACT: OBJECTIVE To optimize the extraction technology of total coumarin from Viola yedoensis and to evaluate its in vitro antioxidant and anti -tumor activities of its purified product . METHODS On the basis of single factor experiments ,the extraction technology of total coumarin from V. yedoensis was optimized by the response surface methodology ,using yield of total coumarin from V. yedoensis as response value ,solid-liquid ratio ,extraction time and ethanol volume fraction as variables . The verification tests were conducted . The antioxidant activity of purified product of total coumarin from V. yedoensis was evaluated by 1,1-diphenyl-2-picrylhydrazyl(DPPH)method,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic)acid ammonium salt (ABTS) method and 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl(PTIO)method. CCK-8 assay was used to determine the inhibitory effect of it on human cervical cancer cells HeLa ,human intrahepatic bile duct cancer cells HCCC -9810,human hepatocellular carcinoma cells HepG 2,human breast cancer cells MDA -MB-231 and human non -small cell lung cancer cells A 549. The apoptosis morphology of HCCC -9810 cells was detected by Hoechst 33258 staining,and lactate dehydrogenase (LDH)and superoxide dismutase (SOD)content were determined . RESULTS The optimal extraction technology included solid -liquid ratio was 25∶1(mL/g),extraction time was 1.9 h,ethanol volume fraction was 68%. Average yield of total coumarin from V. yedoensis was (21.10±0.17)mg/g,and RSD was 0.80%(n=3),relative error of which with predicted value (20.79 mg/g)was 1.49%. The median inhibitory concentrations (IC50)of purified product of total coumarin from V. yedoensis to DPPH and ABTS free radicals were 21.54 and 17.11 μg/mL,which were slightly higher than that of ascorbic acid (15.21,12.45 μg/mL). The IC 50 to PTIO free radicals was 1 422.01 μg/mL,which was far higher the that of ascorbic acid (37.20 μg/mL). The IC 50 values of total coumarin from V. yedoensis to above 5 cells were (113.18±15.97),(51.17±1.72),(134.15±11.75),(171.83±15.27)and(93.38±4.65)μg/mL,respectively. Total coumarin from V. yedoensis induced the apoptosis of HCCC -9810 cells and increased the content of LDH ,as well as decreased the content of SOD . CONCLUSIONS The extraction technology of total coumarin from V. yedoensis is optimized successfully ;the purified product of obtained extract has good antioxidant and anti -tumor activities.
期刊: 2022年第33卷第20期
作者: 陈仁强,刘桢,胡慧玲,李林锋,张春
AUTHORS: CHEN Renqiang,LIU Zhen,HU Huiling,LI Linfeng,ZHANG Chun
关键字: 紫花地丁;总香豆素;提取工艺;抗氧化作用;抗肿瘤作用
KEYWORDS: Viola yedoensis ;total coumarin ;extraction technology ;antioxidant effect ;anti-tumor effect
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