黄秋葵多糖提取纯化及其体外结合胆酸能力和抑制肿瘤活性分析
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摘要
黄秋葵是一种具有较高营养价值的蔬菜,具有很高的开发利用潜力。本课题以新鲜蔬菜黄秋葵嫩果为原料,对黄秋葵多糖的提取、分离纯化、体外胆酸结合能力以及抑制肿瘤细胞增殖能力等生物活性进行了较为系统的初步研究。主要的研究内容与结论如下:
探讨黄秋葵多糖的超声提取工艺。选定时间、水料比和温度作为研究对象,以黄秋葵粗多糖提取率为评价指标。在单因素试验的基础上,通过3因素3水平Box-Behnken中心组合实验,建立多糖提取率的二次多项式回归方程,经响应面回归分析得到优化组合条件。结果表明:提取工艺条件为提取时间20min、水料比44:1(mL/g)、提取温度52℃、提取1次时,多糖提取率达到最大值。该条件下多糖提取率预测值为27.82%,验证值为27.75%。
提取分离黄秋葵多糖组分,分析其体外胆酸结合能力。通过水提醇沉提取黄秋葵粗多糖(raw polysaccharide,RPS),经DEAE-纤维素阴离子交换层析柱分离得到3种多糖洗脱组分E1、E2和E3;比较粗多糖与多糖洗脱组分的体外结合胆酸能力,结果显示:粗多糖具有较强的胆酸结合能力14.13%(以消胆胺为阳性对照);纯化获得的组分E1和E2的胆酸结合能力分别为10.82%和10.60%。推测多糖分离过程活性组分损失从而影响其体外胆酸结合的能力,而多糖与胆酸的结合与其自身的物化结构及与胆酸的活性结合位点有关。
进一步探讨黄秋葵多糖组分对人体卵巢癌细胞(OVCAR-3)、乳腺癌细胞(MCF-7)、宫颈癌细胞(Hela)、胃腺癌细胞(MCG-803)增殖的抑制作用。取不同质量浓度黄秋葵多糖(RPS、E1、E2和E3),分别作用于人体肿瘤细胞72h,采用MTT法对其抑制肿瘤细胞增殖活性进行分析。结果显示:RPS和E1组分可抑制OVCAR-3细胞的生长,随质量浓度的增加细胞存活率降低,且呈剂量依赖关系,其IC50值分别为1981.18μg/mL、819.74μg/mL;E3组分可以抑制Hela和MCG-803细胞的生长,抑制作用呈剂量依赖关系,其IC50值分别为1195.61μg/mL、1337.65μg/mL。另外,E3组分可抑制MCF-7细胞增殖,当浓度为200μg/mL时,细胞存活率为63.90%,未显示剂量依赖关系。总之,该研究为进一步开发黄秋葵成为降脂和抗癌食品提供参考。
The fruit of okra, Abelmoschus esculentus (L.)Moench, is a vegetable of high nutrition deserving further investigation. The extraction and purification of polysaccharides from okra, and analysis of their in vitro bile acid-binding capacity and antitumor activity were investigated in this study.
Ultrasonic extraction of polysaccharides from okra was optimized by response surface analysis. According to the single-factor experiments, three levels of the factors including extraction time, liquid/solid ratio and extraction temperature were selected for the Box-Behnken factorial design to establish a quadric regression equation for describing the yield of polysaccharide. Results showed that the optimum extraction conditions were obtained as follow: extraction time of 20min, liquid/solid ratio at 44:1 and extraction temperature at 52℃, which led to estimated and observed values of maximal yield of polysaccharides of 27.82 % and 27.75 %, respectively.
In order to investigate active components having the ability to bind bile acid from okra, raw polysaccharide (RPS) was obtained from the fresh fruits of okra by water extraction and ethanol precipitation. RPS was further purified by DEAE-cellulose anion exchange chromatography to obtain fractions E1, E2 and E3. Their bile acid binding capacities in vitro were determined. The comparison of the ability to bind bile acid demonstrated that RPS could bind bile acid as 14.13% (compared with cholestyramine). E1 and E2 presented different bile acid-binding capacities, which accounted for 10.82% and 10.60% of that of cholestyramine, respectively. And the E3 fraction had no bile acid binding capacity. These results indicated that the ability to bind bile acid of polysaccharide was affected by the purification process, which may change the physical and chemical structures of purified fractions or the active binding sites.
The inhibitory effect of polysaccharides (RPS, E1, E2 and E3) extracted from okra, was investigated on different human cancer cell lines, including OVCAR-3, MCF-7, Hela and MCG-803 cells. These cancer cells were treated with different concentrations of okra polysaccharides for 72 h and their proliferation were analyzed by MTT assay. RPS and E1 had a significant inhibition effect on the proliferation of OVCAR-3 cells in a dose-dependent manner, and their IC50 were 1981.18μg/mL、819.74μg/mL, respectively. E3 had an obvious inhibition effect on Hela and MCG-803 cells in a dose-dependent manner, with the IC50 were 1195.61μg/mL、1337.65μg/mL, respectively. In addition, E3 inhibited the proliferation of MCF-7 cell; cell viability was 63.90% at 200μg/mL.
In conclusion, investigations here provide references for exploring and developing okra as cholesterol lowering food and anti-tumor functional food in the furture.
探讨黄秋葵多糖的超声提取工艺。选定时间、水料比和温度作为研究对象,以黄秋葵粗多糖提取率为评价指标。在单因素试验的基础上,通过3因素3水平Box-Behnken中心组合实验,建立多糖提取率的二次多项式回归方程,经响应面回归分析得到优化组合条件。结果表明:提取工艺条件为提取时间20min、水料比44:1(mL/g)、提取温度52℃、提取1次时,多糖提取率达到最大值。该条件下多糖提取率预测值为27.82%,验证值为27.75%。
提取分离黄秋葵多糖组分,分析其体外胆酸结合能力。通过水提醇沉提取黄秋葵粗多糖(raw polysaccharide,RPS),经DEAE-纤维素阴离子交换层析柱分离得到3种多糖洗脱组分E1、E2和E3;比较粗多糖与多糖洗脱组分的体外结合胆酸能力,结果显示:粗多糖具有较强的胆酸结合能力14.13%(以消胆胺为阳性对照);纯化获得的组分E1和E2的胆酸结合能力分别为10.82%和10.60%。推测多糖分离过程活性组分损失从而影响其体外胆酸结合的能力,而多糖与胆酸的结合与其自身的物化结构及与胆酸的活性结合位点有关。
进一步探讨黄秋葵多糖组分对人体卵巢癌细胞(OVCAR-3)、乳腺癌细胞(MCF-7)、宫颈癌细胞(Hela)、胃腺癌细胞(MCG-803)增殖的抑制作用。取不同质量浓度黄秋葵多糖(RPS、E1、E2和E3),分别作用于人体肿瘤细胞72h,采用MTT法对其抑制肿瘤细胞增殖活性进行分析。结果显示:RPS和E1组分可抑制OVCAR-3细胞的生长,随质量浓度的增加细胞存活率降低,且呈剂量依赖关系,其IC50值分别为1981.18μg/mL、819.74μg/mL;E3组分可以抑制Hela和MCG-803细胞的生长,抑制作用呈剂量依赖关系,其IC50值分别为1195.61μg/mL、1337.65μg/mL。另外,E3组分可抑制MCF-7细胞增殖,当浓度为200μg/mL时,细胞存活率为63.90%,未显示剂量依赖关系。总之,该研究为进一步开发黄秋葵成为降脂和抗癌食品提供参考。
The fruit of okra, Abelmoschus esculentus (L.)Moench, is a vegetable of high nutrition deserving further investigation. The extraction and purification of polysaccharides from okra, and analysis of their in vitro bile acid-binding capacity and antitumor activity were investigated in this study.
Ultrasonic extraction of polysaccharides from okra was optimized by response surface analysis. According to the single-factor experiments, three levels of the factors including extraction time, liquid/solid ratio and extraction temperature were selected for the Box-Behnken factorial design to establish a quadric regression equation for describing the yield of polysaccharide. Results showed that the optimum extraction conditions were obtained as follow: extraction time of 20min, liquid/solid ratio at 44:1 and extraction temperature at 52℃, which led to estimated and observed values of maximal yield of polysaccharides of 27.82 % and 27.75 %, respectively.
In order to investigate active components having the ability to bind bile acid from okra, raw polysaccharide (RPS) was obtained from the fresh fruits of okra by water extraction and ethanol precipitation. RPS was further purified by DEAE-cellulose anion exchange chromatography to obtain fractions E1, E2 and E3. Their bile acid binding capacities in vitro were determined. The comparison of the ability to bind bile acid demonstrated that RPS could bind bile acid as 14.13% (compared with cholestyramine). E1 and E2 presented different bile acid-binding capacities, which accounted for 10.82% and 10.60% of that of cholestyramine, respectively. And the E3 fraction had no bile acid binding capacity. These results indicated that the ability to bind bile acid of polysaccharide was affected by the purification process, which may change the physical and chemical structures of purified fractions or the active binding sites.
The inhibitory effect of polysaccharides (RPS, E1, E2 and E3) extracted from okra, was investigated on different human cancer cell lines, including OVCAR-3, MCF-7, Hela and MCG-803 cells. These cancer cells were treated with different concentrations of okra polysaccharides for 72 h and their proliferation were analyzed by MTT assay. RPS and E1 had a significant inhibition effect on the proliferation of OVCAR-3 cells in a dose-dependent manner, and their IC50 were 1981.18μg/mL、819.74μg/mL, respectively. E3 had an obvious inhibition effect on Hela and MCG-803 cells in a dose-dependent manner, with the IC50 were 1195.61μg/mL、1337.65μg/mL, respectively. In addition, E3 inhibited the proliferation of MCF-7 cell; cell viability was 63.90% at 200μg/mL.
In conclusion, investigations here provide references for exploring and developing okra as cholesterol lowering food and anti-tumor functional food in the furture.
引文
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