摘要
构成革兰阴性菌(Gram-negative,G-)外膜的主要成分内毒素(lipopolysaccharide,LPS),是介导细菌脓毒症的重要病原分子,LPS由O-侧链多糖、核心多糖和脂质A(Lipid A)三部分组成,其中Lipid A是LPS的生物学活性中心。研究证实,LPS在脓毒症发生过程中具有重要作用,因此,寻求能拮抗LPS的药物是预防和治疗G-细菌介导的脓毒症的重要手段;传统中草药在脓毒症领域的应用具有悠久的历史。现代医学实践已证明,许多中药在体内外都具有拮抗内毒素的作用。然而由于中药成份复杂,在分离纯化的过程中缺少有效的活性跟踪手段,使对具有拮抗内毒素活性中药的深入研究受到限制。本课题利用本实验室建立的生物传感器筛选和跟踪平台,对与Lipid A具有较高结合活性的栀子进行拮抗内毒素活性组分的定向分离,以期获得具有拮抗内毒素活性的单体化合物,为脓毒症的防治研究提供一种可能的手段。
方法:(1)依托课题组前期已经建立的生物传感器筛选平台,选择60种药物与Lipid A进行结合活性的检测,选择具有高结合活性的药物进行研究(2)应用与Lipid A的结合活性测定及对脓毒症模型动物的保护作用作为分离过程中的活性跟踪手段,采用大孔吸附树脂、萃取、聚酰胺层析、HPLC等技术对栀子进行活性组分的定向分离,以期最终得到具有拮抗LPS活性的单体化合物(3)对所得化合物进行HPLC、薄层色谱及熔点测定等技术分析。(4)活性评价:在体外应用鲎试验观察化合物对LPS的中和作用,并通过细胞培养观察化合物对LPS介导的RAW264.7活化的抑制作用;在体内观察其在小鼠体内对LPS的中和作用。
结果:(1)对60种清热解毒中草药进行结合活性测定,筛选出22种与Lipid A具有较高结合活性的中药,并以其中的栀子作为研究对象(2)从栀子水提取物中定向分离出一个与Lipid A具有较高结合活性的化合物CJ-1(3)经HPLC、薄层色谱及熔点测定等技术分析,初步确定CJ-1为京尼平苷(C17H24O10)(4)活性评价:京尼平苷在体外对LPS具有较好的中和作用,并呈现剂量效应关系;能显著抑制LPS诱导的RAW264.7细胞释放TNF-α;在小鼠体内对LPS也具有显著的中和作用。
结论:利用大孔吸附树脂层析、聚酰胺层析、HPLC等技术从栀子中分离出化合物京尼平苷,京尼平苷在体内外对LPS均具有较好的拮抗作用。
LPS, one major component of the outer membrane in gram negative bacteria, is the most important pathogen recognition molecule for sepsis. There are three basic parts in LPS, lipid A, O-specific polysaccharide chain and core polysaccharide, among which lipid A is the core structure. LPS plays an important role in sepsis according to research findings. As a result, the way to find anti-LPS drugs is also an important way to prevent and treat sepsis. Traditional Chinese Medicines have a long history in septic therapy. Clinical experiences and studies in vitro and in vivo also suggest that many herbs possess anti-LPS activity. However, complicated chemical constitutes in herbs and lack of effective specific target and screening methods hinder further study on these herbs. The biosensor technology set up in our laboratory is applied in this study to screen and guide the isolating process of Gardenia jasminoides Ell, an traditional Chinese herb with high binding affinity to lipid A. anti-LPS monomers were expected to be isolated in order to provide a new approach to septic therapy research.
Methods: (1)aqueous extracts of sixty herbs were tested with the help of the biosensor platform to screen those with higher binding ability for further study; (2)binding affinity to Lipid A and protective effect on animal models of sepsis were tested to screen the bioactive fractions. Macroporous resin, abstraction, polyamide laminar analysis and high performance liquid chromatography(HPLC) were applied to isolate the compound. (3) HPLC, thin-layer chromatography (TLC) and fusion point measurement were used to analyze the purity and quality of the isolated compound. (4) anti-LPS activities of the fractions and compound included neutralization of LPS (imulus Amebocyte Lysate (LAL) test) and inhibition of TNF-αrelease from LPS-stimulated RAW264.7 cells in vitro and protection of mice challenged by leather dose of heat killed E coli. in vivo.
Results: (1) Anti-endotoxin activities of forty-two Chinese herbs were screened using lipid A as target with biosensor technique and ten possessed components with higher binding ability to lipid A, among which Gardenia jasminoides Ell was chosen for further study; (2) fraction CJ-1 was isolated from the aqueous extracts of Gardenia jasminoides Ell, which possessed high binding ability to lipid A; (3) HPLC, thin-layer chromatography (TLC) and fusion point measurement proved CJ-1 to be Geniposide (C17H24O10); (4) Geniposide showed significant endotoxin-neutralizing effect in vivo and in vitro, And could markedly inhibited TNF-αrelease in RAW264.7 cells induced by LPS.
Conclusions: (1) Geniposide has high binding capability to lipid A and possesses significant endotoxin- neutralizing capacity in vitro and in vivo.
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