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芹菜籽和鹰嘴豆中多肽成分的分离及其生物活性研究
马庆苓
学位类型博士
导师阿吉 ; 阿不力米提
2011-06-03
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业有机化学
关键词芹菜籽 鹰嘴豆 多肽 生物活性 提取
摘要1. 在维吾尔药中,芹菜籽主要用于治疗高血压、关节炎、类风湿关节炎、肾脏等疾病。研究芹菜籽中的活性多肽及其抗氧化活性,为全面解析芹菜籽的药理作用提供了理论依据。 采用单因素实验(提取温度、提取固液比、提取时间和提取溶液pH对提取多肽的量的影响)和正交试验,确定了芹菜籽多肽最佳提取工艺条件:即pH=9.0,物料比为1:5的条件下,超声提取30 min;通过对多肽进行清除ABTS自由基作用的研究,结果表明利用此提取工艺条件并没有改变所提取的芹菜籽多肽清除ABTS自由基的能力。 利用磷酸盐缓冲溶液提取、硫酸铵沉淀芹菜籽中的多肽类化合物,采用阴阳离子交换树脂相结合的方法分离提取的多肽类物质,并采用HW-55F分子筛、Sephadex G-75凝胶柱层析、HPLC制备色谱分离以及超滤等进行纯化提取的多肽,共得到三个单体多肽;其中一个中性多肽,其分子量为5.005 kDa, N-端氨基酸序列为:H2N-Ala-Arg-Asp-Glu-Asn-Thr-Ala-Asp-Ala-Tyr-Arg-Val-Pro-Pro- Val;一个碱性多肽,分子量为11.39 kDa,其N-端氨基酸序列为:H2N- Ala-Ser-Lys-Glu- His-Lys-Glu-Arg-Gln-Pro;一个酸性多肽,分子量为6.5 kDa。 对芹菜籽多肽进行抗氧化活性分析,结果表明芹菜籽酸性多肽组分清除ABTS自由基的作用最强,其IC50的值为8.23 μg/mL;三个纯化多肽IC50值分别为182.0615 μg/mL(QCZCMW)、158.7 μg/mL(QCZDE)和193.2 μg/mL(QCZCM-2)。 2. 鹰嘴豆是维吾尔医药食两用植物,具有降血糖、降血脂的作用。鹰嘴豆发芽后,其营养成分发生了变化,大分子蛋白质发生降解。研究鹰嘴豆和发芽后的鹰嘴豆中的多肽及其生物活性,对于解析鹰嘴豆发芽过程中大分子降解过程以及研究鹰嘴豆及其药理活性提供了科学依据。 利用芹菜籽多肽提取、分离、纯化的方法,从脱脂的鹰嘴豆原豆得到两个多肽:碱性多肽YZDCM的分子量为5.483 kDa,氨基酸序列为H2N-Ala-Cys-Cys-Glu-Asn-Lue-Ala-Asp-Thr-Tyr- Arg-Gly-Pro-Cys-Phe,此多肽属于防御素一族;酸性多肽YZDDEAE的分子量约8.0 kDa。 利用芹菜籽多肽提取、分离、纯化的方法,从发芽后的鹰嘴豆豆瓣得到三个单体多肽:YZDBCM-1分子量为9.086 kDa,N端氨基酸序列为H2N-Ala-Ile-Thr- Cys-Gly-Arg-Val-Ser-Ala-Ala-Leu-Ala-Pro-Pro-Leu.,是一个新型的、富含丙氨酸的、新型碱性多肽,是由鹰嘴豆发芽过程中大分子蛋白质降解产生的;YZDBCM-2分子量为5.411 kDa,YZDBCM-3分子量为4.688 kDa。 生物活性筛选结果表明,发芽后的鹰嘴豆豆瓣中提取的多肽的总体来说要比原豆中的多肽强,且碱性多肽的活性普遍要比中性和酸性多肽的活性强;其中豆瓣YZDB-50%C对人结肠腺癌细胞的抑制作用最强,其半数抑制浓度(IC50)为3.8 μg/mL,而纯化的鹰嘴豆碱性多肽YZDCM对人结肠腺癌细胞的半数抑制浓度(IC50)为1.74 μg/mL。鹰嘴豆豆瓣YZDBCM-1对细菌和人结肠腺癌细胞均无抑制作用,但是有较强的清除ABTS自由基的作用,其IC50的值为156.2 μg/mL(即17.20 μmol/L)。
其他摘要1. The seeds of Apium graveolens L. (Celery) as a Uyghur traditional medicine, are used to treat hypertensive, arthritis. Using single factor and orthogonal experiments, bioactive peptide was extracted by phosphate buffered solution with assisstance of ultrasonic from the seeds of Apium graveolens L. (Celery) from Xinjiang of China,and the peptide by (NH4)2SO4 (80%)precipitation was used as an index to determine the amount of peptides.The optimum extraction conditions were as follows:pH 9.0,time of extraction by supersound 30 min,ratio of solid to solution 1:5.And the radical-scavenging activity of the peptides was determined with ABTS assay. Three peptides were isolated and purified from seeds of the the seeds of celery with a procedure that involved these steps of ion exchange chromatography on DEAE-cellulose and KM-TSK-650M, ultrafiltration, gel filltration on HW-55F and Sephadex G-75 and HPLC. The neutral peptide (QCZCMW) was 5.005 kDa. The partial N-terminus sequence was determined by the Edman method as: Ala-Asp-Asn-Ala- Ala-Arg-Pro-Val-Arg-Glu-Thr-Asp-Ala-Val-Pro. The basic peptide (QCZCM-2) with molecular weight 11.39 kDa, and the partial N-terminus sequence was determined as: H2N- Ala-Ser-Lys-Glu- His-Lys-Glu-Arg-Gln-Pro. The acidic peptide (QCZDE) was 6.5 kDa. A study of the antioxidant activity of the peptides showed that both the acidic proteins and peptides had the strongest inhibiting effect on ABTS free radical. Three peptide had the strong inhibiting effect on ABTS free radical, and their IC50 value were 182.0615 μg/mL (the neutral peptide), 158.7 μg/mL (the acidic peptide) and 193.2 μg/mL (the basic peptide). 2. Chickpea (Cicer arietinum L.) is used in the treatment of various diseases including bronchitis, hyperlipemia, diabete. Two peptides were isolated and purified from seeds of the Kabuli chickpea (Cicer arietinum L.).The basic peptide (YZDCM) with molecular weight of 5.483 kDa was a kind of plant defensins, and its N-terminus sequence was determined as: H2N-Ala-Cys-Cys- Glu-Asn-Lue-Ala-Asp-Thr-Tyr-Arg-Gly-Pro-Cys-Phe. Another was acidic peptide with MW of 8.0 kDa. Three basic peptides were isolated from the sprout of chickpea (Cicer arietinum L.) . The N-terminus sequence of YZDBCM-1 with MW of 9.086 kDa was determined as H2N-Ala-Ile-Thr-Cys-Gly-Arg-Val-Ser-Ala-Ala-Leu-Ala-Pro-Pro-Leu. It was product of protein degradation during srpouting of chickpea. The second is a peptide with MW of 5.411 kDa, and the third with MW of 4.688 kDa. A study of the biological activity of the crude and purified peptides showed that the peptides from the sprout had the stronger biological activity than those from the seeds; the total peptides YZDB-50%C had the strongest biological activity among all the crude peptides. The IC50 value of YZDB-50%C and pudified peptide YZDCM for human colon caner Caco-2 cells inhibiting activity were 3.8 μg/mL and 1.74 μg/mL, respectively. YZDBCM-1 had no inhibiting effect on bacterium and Caco-2. But it had strongest inhibiting effect on ABTS free radical, and IC50 value was 156.2 μg/mL (17.20 μmol/L).
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4447
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
推荐引用方式
GB/T 7714
马庆苓. 芹菜籽和鹰嘴豆中多肽成分的分离及其生物活性研究[D]. 北京. 中国科学院研究生院,2011.
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