葵花籽中非竞争型胰蛋白酶抑制剂的发现和纯化
摘要
胰蛋白酶抑制剂广泛存在于动物、植物、微生物中,并且具有重要的生理功能。胰蛋白酶抑制剂主要应用于治疗急性胰腺炎、体外循环手术、预防早产和治疗不孕、抗肿瘤、抑制脑水肿等方面。因此,寻找一种高效、天然的胰蛋白酶抑制剂对于其基础研究及药用价值研究都有着实际的意义。
目前,已经从许多种天然植物尤其是种子中提取出胰蛋白酶抑制剂,如大豆、菠菜种子、花生、南瓜子等。国外已有文献报道,从葵花籽中分离纯化出一种有强抑制活性的胰蛋白酶抑制剂,但在国内还未见报道。本研究选用吉林省所产的葵花籽为原料,烘干后粉碎,经石油醚回流提取除脂,酸提并对粗提物进行微滤、超滤、通过离子交换色谱进行分离纯化,得到了一种胰蛋白酶抑制剂。应用反相高效液相色谱对其纯度进行测定,表明仍需要进一步纯化。经过Lineweaver-Burk作图法分析,发现F3-Ⅲ为非竞争型胰蛋白酶抑制剂,与以往报道的天然产物中提取的竞争型胰蛋白酶抑制剂不同,它可能避免竞争型抑制剂造成的体内其它同样以丝氨酸为活性中心的正常蛋白酶失活而引起的不良反应,因此,可能为临床研究提供一个药物前体。
Protease inhibitors, as a kind of physiological active substances, play important role in the fields, such as anti-infectivity, anti-virus, anti-tumor, and prevention of premature delivery. In the recent years, the trypsin inhibitors which competitively inhibit serine proteases, i.e. aprotinin, urinary trypsin inhibitor, and soybean protease inhibitor, etc. were widely used. A variety of human serine proteases share the active center with almost identical primary structure and special structure; therefore, the normal functions of other serine proteases are usually changed when the trypsin competitive inhibitors are used. It was the typical example that State Food and Drug Administration of China decided to suspend aprotinin injection in the sale and use. Consequently, it is necessary to search the new trypsin inhibitors.
The powder of dried sunflower seeds which planted in Jilin Province was defatted by using petroleum ether reflux, and then was extracted by 0.001mol/L HCl. The supernatant was microfiltrated and ultrafiltrated to 4 fractions, which were Fraction1 (F1 , microfiltration: > 0.2μm), Fraction 2 (F2, microfiltration: < 0.2μm ~ ultrafiltration: 6kDa), Fraction 3 (F3, ultrafiltration: 6kDa ~ ultrafiltration: 3kDa), and Fraction 4 (F4, ultrafiltration: <3kDa). Based on gelatin plate method, F3 with trypsin inhibitory activity (the inhibition activity ratio is 14.21) was determined, and it was further confirmed by using azocasein method.
4 fractions (F3 -Ⅰ, F3 -Ⅱ, F3 -Ⅲ, F3-Ⅳ) were isolated and collected by using ion exchange chromatography. F3-Ⅲ, as interest ingredient, was quantitatively determined (inhibition activity, 8719.35U/mg). 4 peaks shown in RP-HPLC for F3-Ⅲindicates that it is not the non-pure component and needs further purification. The F3-Ⅲinhibitor type which was conducted by Lineweaver-Burk mapping shows that it is non-competitive trypsin inhibitor.
Taken together, a new non-competitive inhibitor which molecular weight ranges from 3K to 6KDa was discovered and isolated. Based on the gelatin plate method, casein protein assay and BAPNA assay, an evaluation technology portfolio of trypsin inhibition effectiveness was established. For a new trypsin inhibitor of research and development, development and application lay the foundation.
目前,已经从许多种天然植物尤其是种子中提取出胰蛋白酶抑制剂,如大豆、菠菜种子、花生、南瓜子等。国外已有文献报道,从葵花籽中分离纯化出一种有强抑制活性的胰蛋白酶抑制剂,但在国内还未见报道。本研究选用吉林省所产的葵花籽为原料,烘干后粉碎,经石油醚回流提取除脂,酸提并对粗提物进行微滤、超滤、通过离子交换色谱进行分离纯化,得到了一种胰蛋白酶抑制剂。应用反相高效液相色谱对其纯度进行测定,表明仍需要进一步纯化。经过Lineweaver-Burk作图法分析,发现F3-Ⅲ为非竞争型胰蛋白酶抑制剂,与以往报道的天然产物中提取的竞争型胰蛋白酶抑制剂不同,它可能避免竞争型抑制剂造成的体内其它同样以丝氨酸为活性中心的正常蛋白酶失活而引起的不良反应,因此,可能为临床研究提供一个药物前体。
Protease inhibitors, as a kind of physiological active substances, play important role in the fields, such as anti-infectivity, anti-virus, anti-tumor, and prevention of premature delivery. In the recent years, the trypsin inhibitors which competitively inhibit serine proteases, i.e. aprotinin, urinary trypsin inhibitor, and soybean protease inhibitor, etc. were widely used. A variety of human serine proteases share the active center with almost identical primary structure and special structure; therefore, the normal functions of other serine proteases are usually changed when the trypsin competitive inhibitors are used. It was the typical example that State Food and Drug Administration of China decided to suspend aprotinin injection in the sale and use. Consequently, it is necessary to search the new trypsin inhibitors.
The powder of dried sunflower seeds which planted in Jilin Province was defatted by using petroleum ether reflux, and then was extracted by 0.001mol/L HCl. The supernatant was microfiltrated and ultrafiltrated to 4 fractions, which were Fraction1 (F1 , microfiltration: > 0.2μm), Fraction 2 (F2, microfiltration: < 0.2μm ~ ultrafiltration: 6kDa), Fraction 3 (F3, ultrafiltration: 6kDa ~ ultrafiltration: 3kDa), and Fraction 4 (F4, ultrafiltration: <3kDa). Based on gelatin plate method, F3 with trypsin inhibitory activity (the inhibition activity ratio is 14.21) was determined, and it was further confirmed by using azocasein method.
4 fractions (F3 -Ⅰ, F3 -Ⅱ, F3 -Ⅲ, F3-Ⅳ) were isolated and collected by using ion exchange chromatography. F3-Ⅲ, as interest ingredient, was quantitatively determined (inhibition activity, 8719.35U/mg). 4 peaks shown in RP-HPLC for F3-Ⅲindicates that it is not the non-pure component and needs further purification. The F3-Ⅲinhibitor type which was conducted by Lineweaver-Burk mapping shows that it is non-competitive trypsin inhibitor.
Taken together, a new non-competitive inhibitor which molecular weight ranges from 3K to 6KDa was discovered and isolated. Based on the gelatin plate method, casein protein assay and BAPNA assay, an evaluation technology portfolio of trypsin inhibition effectiveness was established. For a new trypsin inhibitor of research and development, development and application lay the foundation.
引文
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