西兰花芽苗菜中莱菔硫烷的分离纯化及抗癌活性的测定
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摘要
莱菔硫烷(Sulforaphane,简称SF)又称“萝卜硫素”,是硫代葡萄糖苷经黑芥子酶酶解或酸水解产生的一类异硫代氰酸盐,莱菔硫烷是迄今为止在蔬菜中发现的抗癌活力最强的活性物质之一,相对分子质量为177.29,分子式为C6H11S2NO。有研究表明它对人乳腺癌细胞、结肠癌细胞、人膀胱癌细胞等有很好的抑制作用并能诱导癌细胞凋亡。
硫代葡萄糖苷(硫苷)广泛存在于十字花科蔬菜中,尤其是西兰花芽苗菜,硫苷的含量是其成熟时期的40~100倍,这些硫苷经体内的黑芥子酶酶解成莱菔硫烷。本课题以西兰花芽苗菜为原料,通过对其种植条件的研究,确定了适合西兰花芽苗菜的种植条件为:温度25℃;相对湿度60;上架时间2.5d;浇水频率4次/天;见光时间为芽高3~4cm。通过对西兰花芽苗菜随生长天数的增加莱菔硫烷含量的变化趋势的研究,确定了最佳的提取时间为生长到第7天的芽苗菜。通过对提取工艺技术的摸索,找到了超声波法提取莱菔硫烷的技术参数;通过四元二次回归正交旋转组合实验设计对硫苷酶解条件的优化,确定了提取莱菔硫烷的实验方案为:将生长至第七天的新鲜的西兰花芽苗菜,经鼓风低温烘干后粉碎,过100目筛。称取10g,加入0.0189mg/g的Vc水溶液并用盐酸调pH到5.34,加入0.1g硫化钠,料液比1:3,振荡酶解, 8h+40min(28℃、180r)。冷冻干燥得粉末,粉末中加入二氯甲烷,设定超声温度设定为40℃,超声功率为320W,超声频率设定为70KHz,超声时间45 min,进行超声波处理,抽虑收集滤液。滤渣再用二氯甲烷浸提,超声条件相同,进行超声波处理。合并两次提取液,滤纸过滤,旋转蒸发浓缩后,冷冻干燥得莱菔硫烷的粗品。
通过对于莱菔硫烷粗品的分离纯化的研究,首先用硅胶柱层析初步分离,然后再用Sephadex LH-20凝胶柱层析纯化粗样,采用L9(33)正交实验对流速、填料高度及进样浓度这三个影响因素进行优化,确定最佳分离条件为:流速为30 s/滴、填料高度为70 cm、进样浓度为40 mg/mL。通过验证实验,HPLC分析纯度,得出莱菔硫烷的纯度为90.4%,已经达到了预期的实验效果。
通过MTT体外抗癌实验,测定制备莱菔硫烷样品对人前列腺癌细胞系生长的抑制以及诱导其凋亡的作用。测定对照组和加药组的OD值得出在添加24μg/mL剂量的莱菔硫烷的条件下,人前列腺癌细胞生长至第五天受到较为明显的抑制,根据生长抑制率计算公式求得细胞在第五天的生长抑制率达到62.30%,抑制作用明显。
本研究为对西兰花芽苗菜中硫苷酶解成莱菔硫烷的提取分离方法进行了有益的探索,并成功得到了高纯度的莱菔硫烷样品,为大规模从西兰花芽苗菜中提取分离莱菔硫烷奠定了基础,也为今后进一步开发抗癌新药提供了科学依据。
Sulforaphane can be also called raphanin which is a kind of isothiocyanate. Glucoraphanin can be enzymolysised or acidolysised by myrosin into sulforaphane (abbreviation for SF). Sulforaphane is one of the best anticancer substance discovered in vegetables so far, and its relative molecular weight is 177.29, and molecular formula is C6H11S2NO. Researches has shown that sulforaphane has a good inhibition effect on breast cancer cells, colon cancer cells and bladder cancer cells of human beings,and it also can induce apoptosis of cancer cells.
Sulforaphane can be enzymolysised from Glucoraphanin by myrosin, it widely exists in cruciferae vegetables,expecially in broccoli sprouts,and its content is 40 to 100 times than its maturation. This research was based on broccoli sprouts as raw materials, through the study of planting condition, the optimum planting conditions were as follows: temperature was 25℃, relative humidity was 60, upper time was 2.5 day and the frequency of irrigation was 4 times per day. The optimum time for broccoli sprouts to see the lights was when broccoli sprouts was 3 to 4 centimeters. The optimum extraction time was when the broccoli sprouts grew to the seventh day by investigating the tends of sulforaphane content. Ultrasonic wave extraction parameteres of sulforaphane were obtained by studying extraction technology.The optimum enzymolysis conditions was selected by composite rotatable experiment design, and the optimum programs were as follows:
The seventh day’s growth broccoli sprouts were shattered and dried by low-temperature and then 100 mesh into powder. Ten grams of powder was added into vitamin C solution, which concentration was 0.0189mg/g, and the solution pH value was adjusted to5.34 by hydrochloric acid. 0.1g sodium sulfide was added into the solution, the proportion of dosage liquor was 1 to 3,and then the solution was enzymolysised under the conditions of time 8 hours and 40 minutes, temperature 28℃and the convolution of rocking bed 180 per minute.Then the solution was freezed drying into powder. Dichloromethane as lixiviation solvent was added into powder, then treated by ultrasonic wave. Ultrasonic wave conditions were as follows:temperature 28℃,power 320W, frequency 70KHz and time 45 minutes. The ultrasonic waved solution was filtered and filter residue was ultrasonic waved one more time under the same conditions.The extracting solution was merged together and condensed by rotary evaporation after filtration, freezed drying into impurity sulforaphane.
For the study on separating and purifying, the impurity sulforaphane was firstly separated by silica gel column chromatography, and then purified the impurity one by Sephadex LH-20. L9 (33) orthogonal experiment on optimizing the conditions of the velocity, height and the concentration were studied, and the optimum separating condition were as follows: the velocity was 30s per drop, filling height was 70cm and the injecting concentration was 40mg/mL. The purity of sulforaphane was 90.4 percent by HPLC analysis and verification experiment, which had achieved the expect results of the experiment.
The effect of prepared sulforaphane on inhibiting growth of PC-3 and inducement cancer cells apoptosis was determined by MTT extraorgan anticancer experiment. We came to the conclusion that carcinoma of prostate cells were apparently inhibited by the fifth day under the dosage of 24μg/mL sulforaphane, and the fifth day’s inhibition ratio was 62.3 percent according to the formula of growth inhibition ratio.
A beneficial exploration for extracting, separating and purifying sulforaphane from broccoli sprouts has been made and the highly purified sulforaphane was successfully obtained. We obtained a good foundation for extracting sulforaphane in a large scale, and also provided a scientific basis for further developing new anticancer drugs.
硫代葡萄糖苷(硫苷)广泛存在于十字花科蔬菜中,尤其是西兰花芽苗菜,硫苷的含量是其成熟时期的40~100倍,这些硫苷经体内的黑芥子酶酶解成莱菔硫烷。本课题以西兰花芽苗菜为原料,通过对其种植条件的研究,确定了适合西兰花芽苗菜的种植条件为:温度25℃;相对湿度60;上架时间2.5d;浇水频率4次/天;见光时间为芽高3~4cm。通过对西兰花芽苗菜随生长天数的增加莱菔硫烷含量的变化趋势的研究,确定了最佳的提取时间为生长到第7天的芽苗菜。通过对提取工艺技术的摸索,找到了超声波法提取莱菔硫烷的技术参数;通过四元二次回归正交旋转组合实验设计对硫苷酶解条件的优化,确定了提取莱菔硫烷的实验方案为:将生长至第七天的新鲜的西兰花芽苗菜,经鼓风低温烘干后粉碎,过100目筛。称取10g,加入0.0189mg/g的Vc水溶液并用盐酸调pH到5.34,加入0.1g硫化钠,料液比1:3,振荡酶解, 8h+40min(28℃、180r)。冷冻干燥得粉末,粉末中加入二氯甲烷,设定超声温度设定为40℃,超声功率为320W,超声频率设定为70KHz,超声时间45 min,进行超声波处理,抽虑收集滤液。滤渣再用二氯甲烷浸提,超声条件相同,进行超声波处理。合并两次提取液,滤纸过滤,旋转蒸发浓缩后,冷冻干燥得莱菔硫烷的粗品。
通过对于莱菔硫烷粗品的分离纯化的研究,首先用硅胶柱层析初步分离,然后再用Sephadex LH-20凝胶柱层析纯化粗样,采用L9(33)正交实验对流速、填料高度及进样浓度这三个影响因素进行优化,确定最佳分离条件为:流速为30 s/滴、填料高度为70 cm、进样浓度为40 mg/mL。通过验证实验,HPLC分析纯度,得出莱菔硫烷的纯度为90.4%,已经达到了预期的实验效果。
通过MTT体外抗癌实验,测定制备莱菔硫烷样品对人前列腺癌细胞系生长的抑制以及诱导其凋亡的作用。测定对照组和加药组的OD值得出在添加24μg/mL剂量的莱菔硫烷的条件下,人前列腺癌细胞生长至第五天受到较为明显的抑制,根据生长抑制率计算公式求得细胞在第五天的生长抑制率达到62.30%,抑制作用明显。
本研究为对西兰花芽苗菜中硫苷酶解成莱菔硫烷的提取分离方法进行了有益的探索,并成功得到了高纯度的莱菔硫烷样品,为大规模从西兰花芽苗菜中提取分离莱菔硫烷奠定了基础,也为今后进一步开发抗癌新药提供了科学依据。
Sulforaphane can be also called raphanin which is a kind of isothiocyanate. Glucoraphanin can be enzymolysised or acidolysised by myrosin into sulforaphane (abbreviation for SF). Sulforaphane is one of the best anticancer substance discovered in vegetables so far, and its relative molecular weight is 177.29, and molecular formula is C6H11S2NO. Researches has shown that sulforaphane has a good inhibition effect on breast cancer cells, colon cancer cells and bladder cancer cells of human beings,and it also can induce apoptosis of cancer cells.
Sulforaphane can be enzymolysised from Glucoraphanin by myrosin, it widely exists in cruciferae vegetables,expecially in broccoli sprouts,and its content is 40 to 100 times than its maturation. This research was based on broccoli sprouts as raw materials, through the study of planting condition, the optimum planting conditions were as follows: temperature was 25℃, relative humidity was 60, upper time was 2.5 day and the frequency of irrigation was 4 times per day. The optimum time for broccoli sprouts to see the lights was when broccoli sprouts was 3 to 4 centimeters. The optimum extraction time was when the broccoli sprouts grew to the seventh day by investigating the tends of sulforaphane content. Ultrasonic wave extraction parameteres of sulforaphane were obtained by studying extraction technology.The optimum enzymolysis conditions was selected by composite rotatable experiment design, and the optimum programs were as follows:
The seventh day’s growth broccoli sprouts were shattered and dried by low-temperature and then 100 mesh into powder. Ten grams of powder was added into vitamin C solution, which concentration was 0.0189mg/g, and the solution pH value was adjusted to5.34 by hydrochloric acid. 0.1g sodium sulfide was added into the solution, the proportion of dosage liquor was 1 to 3,and then the solution was enzymolysised under the conditions of time 8 hours and 40 minutes, temperature 28℃and the convolution of rocking bed 180 per minute.Then the solution was freezed drying into powder. Dichloromethane as lixiviation solvent was added into powder, then treated by ultrasonic wave. Ultrasonic wave conditions were as follows:temperature 28℃,power 320W, frequency 70KHz and time 45 minutes. The ultrasonic waved solution was filtered and filter residue was ultrasonic waved one more time under the same conditions.The extracting solution was merged together and condensed by rotary evaporation after filtration, freezed drying into impurity sulforaphane.
For the study on separating and purifying, the impurity sulforaphane was firstly separated by silica gel column chromatography, and then purified the impurity one by Sephadex LH-20. L9 (33) orthogonal experiment on optimizing the conditions of the velocity, height and the concentration were studied, and the optimum separating condition were as follows: the velocity was 30s per drop, filling height was 70cm and the injecting concentration was 40mg/mL. The purity of sulforaphane was 90.4 percent by HPLC analysis and verification experiment, which had achieved the expect results of the experiment.
The effect of prepared sulforaphane on inhibiting growth of PC-3 and inducement cancer cells apoptosis was determined by MTT extraorgan anticancer experiment. We came to the conclusion that carcinoma of prostate cells were apparently inhibited by the fifth day under the dosage of 24μg/mL sulforaphane, and the fifth day’s inhibition ratio was 62.3 percent according to the formula of growth inhibition ratio.
A beneficial exploration for extracting, separating and purifying sulforaphane from broccoli sprouts has been made and the highly purified sulforaphane was successfully obtained. We obtained a good foundation for extracting sulforaphane in a large scale, and also provided a scientific basis for further developing new anticancer drugs.
引文
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