小球藻抗肿瘤多肽的分离及其微囊化研究
摘要
小球藻(Chlorella)是一类普生性单细胞绿藻，营养丰富，具有非常多的活性成分，具有显著的药理和营养保健作用，是很好的保健食品及药品来源，极具广阔的开发前景。本文通过响应面优化了小球藻蛋白质的提取工艺，并通过酶法制备小球藻抗肿瘤多肽，同时采用微囊技术达到抗肿瘤多肽活性保护及缓释的目的，为小球藻多肽作为抗肿瘤药物的开发提供了理论依据。研究结果如下：
(1)采用低温超高压连续流细胞破碎机对小球藻蛋白质进行提取，考察液料比、压力、循环次数对小球藻蛋白质的提取率的影响。通过响应面设计确定最佳工艺参数为：液料比21:1，压力170MPa，循环次数4，在此优化工艺条件下，实际测得的小球藻蛋白质提取率为45.78%。
(2)选择木瓜蛋白酶、胰蛋白酶及Alcalase2.4L三种蛋白酶分别对小球藻蛋白质进行酶解，在考察水解液pH值、酶与底物浓度比、水解温度和水解时间对水解反应的影响的基础上，采用正交试验设计对酶解条件进行了优化分析。木瓜蛋白酶的最优酶解条件为：酶与底物浓度比(E/S)为2%，温度为70℃，pH为6.0，反应时间为4h，该条件下水解度为14.33%；胰蛋白酶的最优酶解条件为：酶与底物浓度比(E/S)为4%，温度47°C，pH7.0，反应时间为6h，该条件下水解度为8.47%； Alcalase2.4L的最优酶解条件为：酶与底物浓度比(E/S)为2%，温度50°C，pH8.0，反应时间为10h，该条件下水解度为18.31%。
(3)根据酶解条件优化结果，各酶种分别在最优条件下对小球藻蛋白质进行酶解，各酶解液及小球藻蛋白质原液经超滤，共得到十二种产物，评价各产物抑制肝癌细胞HepG2的能力，结果表明木瓜蛋白酶酶解液3KD-5KD对HepG2细胞的抑制能力最强。通过离子交换色谱和凝胶过滤色谱两种分离技术，分别对小球藻木瓜蛋白酶酶解液3KD-5KD进行逐级分离，评价了各级分离产物抑制肝癌细胞HepG2的能力，并最终得到了小球藻多肽。
(4)MTT结果显示，小球藻多肽和氟尿嘧啶对肝癌细胞HepG2表现出抑制作用，存活率随药物浓度的增加而增大，呈明显的量效关系；其IC50值分别为426μg/mL和314μg/mL。小球藻多肽浓度梯度(0.1-0.5mg/mL)处理HepG2细胞，倒置显微镜分析结果显示了典型的凋亡特征。
(5)得到的小球藻抗肿瘤肽，用微囊化的方法对其活性进行保护。通过复凝聚法和离子凝胶法两种工艺制备小球藻抗肿瘤多肽微囊，其包埋率分别为74.5%、30.1%；载药率分别为12.7%、12.3%。两种微囊在胃液中的释放率均较低，在肠液中可继续释放，达到了缓释的目的。包埋后的微囊抗肿瘤活性与未包埋时活性相比，损失不是很大，说明微囊化适合作为小球藻抗肿瘤多肽保护的方法。
Chlorella, a kind of single-celled algae bloom, has many active ingredients and remarkablepharmacology and nutrition health care function. As very good health food and drug source, ithas extremely broad development prospects. This article optimized extraction of protein fromchlorella by the response surface, prepared peptides by enzymic method, at the same time toachieve protection of antitumor activity and the purpose of the slow release throughmicroencapsulation technology. The main contents and results were as follows:
(1)Effects of ratio of liquid-solid, extraction pressure and broken numbers on total proteinsextraction were determined individually. By response surface design, the results showed thatoptimum conditions: ratio of liquid-solid21:1, extraction pressure, broken numbers4, theextraction yield of proteins was45.78%.
(2) Three kinds of enzyme (Papain, Trypsin, Alcalase2.4L) were chose for hydrolysis. Basedon single-factor tests analysis and orthogonal experimental design, the conditions of eachenzymatic reaction were determined and optimized. Optimum enzymatic conditions of Papainwere E/S2%, temperature70°C, pH6.0, time4h, the degree of hydrolysis was14.33%.Optimum enzymatic conditions of Trypsin were E/S4%, temperature47°C, pH7.0, time8h,the degree of hydrolysis was8.47%. Optimum enzymatic conditions of Alcalase2.4L wereE/S2%(V/V), temperature50°C, pH8.0, time3h, the degree of hydrolysis was18.31%.
(3)Each enzyme respectively hydrolyzed chlorella proteins under each optimum enzymeaticconditions. Three kinds of hydrolysate after wultrafiltration were obtained nine products andthe result showed that the3KD-5KD of hydrolysate by Papain had the strongest inhibitoryability on HepG2cell. Chlorella peptide was finally got after the ion exchangechromatography and gel chromatography separation technology on the basis of the inhibitoryability on HepG2cell.
(4) The MTT assay results showed that, chlorella peptide and fluorouracil couldinhibit HepG2cell in vitro and proliferate of HepG2cell in a dose-dependentmanner. Its IC50were426μg/mL and314μg/mL. After treated with differentconcentrations of Chlorella peptide (0.1-0.5mg/mL) respectively, HepG2cells whichwere observed by inverted microscope manifested typical Characteristic of apoptosis.
(5) The activity of antitumor Chlorella peptide that was gained and its activity wasprotected by microencapsulation. Microballoons of Chlorella antitumor peptide wereprepared by complex coacervation and ionotropic gelation in this experiment. The embedding rate were74.5%and30.1%, dosage were12.7%and12.3%. The vitro testcertified: the release rate of two kinds of microballoons was both low in the gastricfluid, but continued to release in the intestinal juice for the purpose of delayed release.Comparing the activity of antitumor embedment with not embedment, activity ofEmbedment had a little loss. The test showed that the activity of antitumor peptidewas protected by microencapsulation..
(1)采用低温超高压连续流细胞破碎机对小球藻蛋白质进行提取，考察液料比、压力、循环次数对小球藻蛋白质的提取率的影响。通过响应面设计确定最佳工艺参数为：液料比21:1，压力170MPa，循环次数4，在此优化工艺条件下，实际测得的小球藻蛋白质提取率为45.78%。
(2)选择木瓜蛋白酶、胰蛋白酶及Alcalase2.4L三种蛋白酶分别对小球藻蛋白质进行酶解，在考察水解液pH值、酶与底物浓度比、水解温度和水解时间对水解反应的影响的基础上，采用正交试验设计对酶解条件进行了优化分析。木瓜蛋白酶的最优酶解条件为：酶与底物浓度比(E/S)为2%，温度为70℃，pH为6.0，反应时间为4h，该条件下水解度为14.33%；胰蛋白酶的最优酶解条件为：酶与底物浓度比(E/S)为4%，温度47°C，pH7.0，反应时间为6h，该条件下水解度为8.47%； Alcalase2.4L的最优酶解条件为：酶与底物浓度比(E/S)为2%，温度50°C，pH8.0，反应时间为10h，该条件下水解度为18.31%。
(3)根据酶解条件优化结果，各酶种分别在最优条件下对小球藻蛋白质进行酶解，各酶解液及小球藻蛋白质原液经超滤，共得到十二种产物，评价各产物抑制肝癌细胞HepG2的能力，结果表明木瓜蛋白酶酶解液3KD-5KD对HepG2细胞的抑制能力最强。通过离子交换色谱和凝胶过滤色谱两种分离技术，分别对小球藻木瓜蛋白酶酶解液3KD-5KD进行逐级分离，评价了各级分离产物抑制肝癌细胞HepG2的能力，并最终得到了小球藻多肽。
(4)MTT结果显示，小球藻多肽和氟尿嘧啶对肝癌细胞HepG2表现出抑制作用，存活率随药物浓度的增加而增大，呈明显的量效关系；其IC50值分别为426μg/mL和314μg/mL。小球藻多肽浓度梯度(0.1-0.5mg/mL)处理HepG2细胞，倒置显微镜分析结果显示了典型的凋亡特征。
(5)得到的小球藻抗肿瘤肽，用微囊化的方法对其活性进行保护。通过复凝聚法和离子凝胶法两种工艺制备小球藻抗肿瘤多肽微囊，其包埋率分别为74.5%、30.1%；载药率分别为12.7%、12.3%。两种微囊在胃液中的释放率均较低，在肠液中可继续释放，达到了缓释的目的。包埋后的微囊抗肿瘤活性与未包埋时活性相比，损失不是很大，说明微囊化适合作为小球藻抗肿瘤多肽保护的方法。
Chlorella, a kind of single-celled algae bloom, has many active ingredients and remarkablepharmacology and nutrition health care function. As very good health food and drug source, ithas extremely broad development prospects. This article optimized extraction of protein fromchlorella by the response surface, prepared peptides by enzymic method, at the same time toachieve protection of antitumor activity and the purpose of the slow release throughmicroencapsulation technology. The main contents and results were as follows:
(1)Effects of ratio of liquid-solid, extraction pressure and broken numbers on total proteinsextraction were determined individually. By response surface design, the results showed thatoptimum conditions: ratio of liquid-solid21:1, extraction pressure, broken numbers4, theextraction yield of proteins was45.78%.
(2) Three kinds of enzyme (Papain, Trypsin, Alcalase2.4L) were chose for hydrolysis. Basedon single-factor tests analysis and orthogonal experimental design, the conditions of eachenzymatic reaction were determined and optimized. Optimum enzymatic conditions of Papainwere E/S2%, temperature70°C, pH6.0, time4h, the degree of hydrolysis was14.33%.Optimum enzymatic conditions of Trypsin were E/S4%, temperature47°C, pH7.0, time8h,the degree of hydrolysis was8.47%. Optimum enzymatic conditions of Alcalase2.4L wereE/S2%(V/V), temperature50°C, pH8.0, time3h, the degree of hydrolysis was18.31%.
(3)Each enzyme respectively hydrolyzed chlorella proteins under each optimum enzymeaticconditions. Three kinds of hydrolysate after wultrafiltration were obtained nine products andthe result showed that the3KD-5KD of hydrolysate by Papain had the strongest inhibitoryability on HepG2cell. Chlorella peptide was finally got after the ion exchangechromatography and gel chromatography separation technology on the basis of the inhibitoryability on HepG2cell.
(4) The MTT assay results showed that, chlorella peptide and fluorouracil couldinhibit HepG2cell in vitro and proliferate of HepG2cell in a dose-dependentmanner. Its IC50were426μg/mL and314μg/mL. After treated with differentconcentrations of Chlorella peptide (0.1-0.5mg/mL) respectively, HepG2cells whichwere observed by inverted microscope manifested typical Characteristic of apoptosis.
(5) The activity of antitumor Chlorella peptide that was gained and its activity wasprotected by microencapsulation. Microballoons of Chlorella antitumor peptide wereprepared by complex coacervation and ionotropic gelation in this experiment. The embedding rate were74.5%and30.1%, dosage were12.7%and12.3%. The vitro testcertified: the release rate of two kinds of microballoons was both low in the gastricfluid, but continued to release in the intestinal juice for the purpose of delayed release.Comparing the activity of antitumor embedment with not embedment, activity ofEmbedment had a little loss. The test showed that the activity of antitumor peptidewas protected by microencapsulation..
引文
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