当归饮片蛋白质的提取与活性分析

Extraction and activity analysis of the protein in the decoction pieces of Chinese Angelica

关键词:  当归饮片, 蛋白质, 稳定性, DPPH自由基, 细胞增殖

Key words:  decoction pieces of Chinese Angelica, protein, stablity, DPPH free radicals, cell proliferation

摘要:

当归是传统的补血中药,其所含的多糖与小分子已有大量的研究,然而当归中的蛋白质组成与功效仍无人知晓。该研究通过0.05 mol·L^-1 Tris-HCl(pH=8.0)缓冲液浸提和组织匀浆得到当归饮片粗提液,结合硫酸铵沉淀和透析法去除粗提液中的多糖及还原糖等小分子成分,得到当归饮片总蛋白质,并首次对其组成和生物活性进行了研究。结果表明:当归饮片蛋白含量较高,分子量为17.5～90.7 kDa,其中 17.5 kDa的蛋白含量最高,达47%。饮片蛋白在pH 5～11范围内较为稳定,pH为3时,仅余少量17.5 kDa的蛋白。当归饮片蛋白质中至少有3种蛋白在80 ℃内稳定存在,其中热稳定性最好的是17.5 kDa的蛋白,在热处理温度达到100 ℃时,仍然稳定存在,但随着处理时间的延长,该蛋白有部分单体发生了交联反应。当归饮片蛋白质具有清除DPPH自由基的能力,且该能力随着热处理温度及热处理时间的增加而增加,pH处理会影响该能力,pH为5.0时,清除能力最高,5.0两侧清除能力均下降。此外,当归饮片蛋白质对细胞有很强的选择性,表现为对正常肝细胞L-02有显著的增殖作用(1.0～4.0 mg·mL^-1,P<0.01),对白血病细胞K562则表现出显著的抑制作用(0.5～1.5 mg·mL^-1,P<0.01),当归饮片蛋白浓度为1.0 mg·mL^-1时,可使L-02细胞增值率达550%(P<0.01),而K562细胞的抑制率达18.3%(P<0.01)。综上所述,当归饮片饮片蛋白具有重要的生物活性,可望从中开发出具有保肝作用的药物蛋白。

Abstract:

Chinese Angelica is one of the herbs most commonly used by Traditional Chinese Medicine(TCM)practitioners with the ability of “enrich the blood”. A large number of studies have been done on the polysaccharides and small molecules of Chinese Angelica while the protein composition and of Chinese Angelica are still barely known. In this research, crude extract in the decoction pieces of Chinese Angelica was obtained by 0.05 M Tris-HCl(pH = 8.0)buffer extraction and tissue homogenates. Protein of the decoction pieces of Chinese Angelica was obtained by combination with ammonium sulfate precipitation and dialysis which could remove the polysaccharides and other small molecules such as reducing sugars in crude extract. The composition and the biological activity of the protein of Chinese Angelica were studied for the first time to evaluate their potential for development and application. The results showed that Angelica Pieces is high in protein. These protein's molecular weight range from 17.5 kDa to 90.7 kDa and the 17.5 kDa one was the highest at content, amounting to 47%. Almost all proteins were stable under the condition of pH 5-11 while only a few proteins with the molecular weight of 17.5 kDa survived under acidic conditions (pH 3). At least three proteins were stable at 80 ℃. Among those protein, the 17.5 kDa protein showed the best thermostability, which was stable on heat treatment(100 ℃). But some of the monomers of 17.5 kDa protein seemed to be cross linked to from an emerging one with the Mw bigger than 116 kDa during the process of boiling water bath. Protein of the decoction pieces of Chinese Angelica was proved to have the ability to scavenge DPPH radicals which was increased with the temperature and time of heat treatments. The highest ability was presented under pH conditions of 5.0 and declined under other pH conditions. In addition, protein of the decoction pieces of Chinese Angelica was proved to have effects on normal cells and tumor cells. Results in vitro experiments showed that the proliferation of human normal hepatic cell line L02 cells was promoted significantly(1.0 - 4.0 mg·mL^-1,P<0.01)while that of human erythromyeloblastoid leukaemic cell line K562 was inhibited observably by these proteins(0.5-1.5 mg·mL^-1,P<0.01). With 1.0 mg·mL^-1 pretreatment of the protein, the proliferation of L-02 cells was enhanced to 550%(P<0.01), while the inhibitory rate of K562 cells reached 18.3%(P<0.01). In conclusion, the important biological activity of proteins of decoction pieces of Chinese Angelica was confirmed and a pharmaceutical protein with hepatic protective effect would be developed from these proteins.