定志小丸化学成分及体外活性、代谢的相关研究
文献类型：学位论文
| 作者 | 唐诗瑶 |
| 学位类别 | 硕士 |
| 答辩日期 | 2015-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 中国科学院长春应用化学研究所 |
| 导师 | 刘志强 |
| 关键词 | 阿尔茨海默病 定志小丸 化学成分 PC12细胞 肠内菌代谢 |
| 中文摘要 | 阿尔茨海默病 (Alzheimer’s disease, AD) 是一种中枢神经系统退行性疾病，神经病理特征主要为血管及细胞外的老年斑和神经元内神经纤维缠结。临床表现为记忆减退、认知障碍、人格变性。如今AD已经成为仅次于心脏病、脑卒中和肿瘤的第四大死亡疾病。AD发病机制复杂，因此针对不同的病因学说其防治药物也有多种。但各药均为针对单一发病机制发挥治疗作用，尚未能彻底治愈AD。而传统中药组成具有化学成分复杂的特点，治疗时能够通过多种途径和多个靶点。尤其是中药复方，包含不同味中药，各味药相辅相成，因此中药复方对AD的治疗具有独特的优势。 定志小丸一方源于唐代孙思邈的《备急千金要方》，由人参、茯苓、远志和石菖蒲四味药按照3:3:2:2的比例组成，该方是治疗健忘的基本方，其已在临床上应用多年，用于治疗阿尔茨海默病、帕金森氏病、抑郁、焦虑、神经衰弱及神经官能症等。 本文采用乙酸乙酯，正丁醇，95% 醇，水四种溶剂连续分级提取的方式，使定志小丸中的不同极性成分得到充分提取。建立了HPLC-IT-MSn和HPLC-Q-TOF-MS两种检测分析方法，根据精确分子质量、串联质谱信息及一些人参皂苷标准品对照首次对定志小丸中的化学成分进行了分析鉴定，负离子模式下共分析鉴定了64种化合物，包括16种茯苓三萜类，14种远志皂苷类，10种寡糖酯类，6种蔗糖酯类，2种口山酮糖苷类及16种人参皂苷类。本次研究所得结果填补了定志小丸化学成分研究的空白，为保证定志小丸质量控制及对其生物活性的透彻研究提供了物质基础。 通过谷氨酸诱导PC12细胞损伤，建立AD细胞模型。在利用谷氨酸造模前应用定志小丸对细胞进行预处理，并采用尼莫地平作为阳性药进行对照，通过MTT实验，观测细胞存活率。结果发现浸膏量30-240 μg/ml的定志小丸总水提物预处理PC12细胞0.5 h，而后加入20 mM谷氨酸诱导损伤细胞24 h，细胞存活率较模型组有明显的提高。说明定志小丸水提物对PC12细胞具有保护作用。为定志小丸用于治疗AD等神经系统疾病提供了理论依据。 将定志小丸水提物与大鼠肠内菌共同厌氧培养，不同时间点取样，利用HPLC-IT-MSn 液质联用技术对定志小丸的代谢变化进行检测分析，并利用质谱碎裂信息对产物进行了鉴定。结果发现3个远志皂苷，8个远志寡糖酯，及13个人参皂苷均发生了明显代谢，代谢途径主要为去糖基化作用。说明肠内菌的苷键水解酶系具有水解药物化合物苷键的能力，定志小丸中部分化合物可被肠道菌群代谢分解，以利于人体对药物的吸收。 |
| 英文摘要 | Alzheimer’s disease is a common disease of the central nervous system. The prominent neuropathological characteristics of the degenerative disease are senile plaques and neurofibrillary tangles (NFT). And the main clinical manifestations of AD are hypomnesia, cognitive disorder and personality change. Now heart disease, stroke and cancer are the diseases with high rates, and they can lead to death. Unfortunately, AD has become the fourth largest death disease. Duing to the pathogenesis of AD is complicated, there are a variety of drugs against different causes. However, all the drugs are symptomatic treatments and can’t completely cure AD. As we all know, ingredients in Chinese medicines are very complex, especially in the Chinese herbal formula. So the therapy characteristics of Chinese medicines are the multiple pathways and targets, and the Chinese herbal formula for the treatment of AD has a unique advantage. Ding-Zhi-Xiao-Wan (DZXW) was originally recorded in the Chinese classical prescription book “Bei-Ji-Qian-Jin-Yao-Fang” (means precious formulas for emergency) which was written by Sun Si-miao in Tang dynasty. DZXW is composed of four herbs, Ginseng Radix, Poria, Polygala Radix, and Acori Tatarinowii Rhizoma, with the mass ratio of 3:3:2:2. It is a renowned Chinese herbal formula that can help strengthen mental function. The formula has been clinically used to treat emotional disease for many years, for instance, Alzheimer’s disease, Parkinson's disease, depression, anxiety, neurasthenia and neurosis. In this paper, four solvents, ethyl acetate, n-butanol, 95% ethanol and deionized water were used to extract DZXW sequentially, in order to acquire the different polarity components adequately, as well as an HPLC-IT-MSn and an HPLC-Q-TOF-MS method were successfully established to provide the possibility for observing the chemical pro?ling. On the basis of the speci?c and accurate mass, tandem MS information, and some ginsenosides reference standards, we analyzed and identified the chemical constituents of DZXW prescription for the first time. Ultimately, 64 components including 16 triterpenoids, 14 Polygala saponins, 10 oligosaccharide esters, 6 sucrose esters, 2 xanthone C-glycosides and 16 ginsenosides were identi?ed in negative ion mode. The results could be used for quality control of DZXW as well as offered chemical basis for investigation on the biological activity of DZXW. In our experiments, we used glutamate to induce toxicity of PC12 cells to establish AD cell model. Before adding glutamate to the cells, we pretreated PC12 cells with the total water extract of various final concentrations (6-240μg/mL) with nimodipine (18 μM) as a reference group for 30min, then the cell proliferation was measured by MTT assay. Finally, we found that the groups of final concentrations 30-240μg/mL had higher cell proliferation than the model group. That is the total water extract of DZXW is biocompatible and show protecting effect against glutamate-induced injury of PC12 cells. These results are consistent with the usage of DZXW in clinical treatment and provide theoretical basis for the treatment of AD. In this work, the total water extract of DZXW was incubated with rat intestinal bacteria in vitro. The samples were gotten at different time points, then we studied the biotransformation and metabolites of the extract of DZXW by HPLC-IT-MSn method. The results showed that 3 Polygala saponins, 8 oligosaccharide esters, and 13 ginsenosides were all metabolized by rat intestinal bacteria, the metabolic pathway was mostly deglycosylation. The results demonstrated that the intestinal bacterial glycosides hydrolysis enzyme system can hydrolyze the compounds with glycosidic bond, and some of the compounds in DZXW could be metabolized by the intestinal bacteria, which would be good for the body to absorb the drug. |
| 语种 | 中文 |
| 公开日期 | 2016-05-03 |
| 源URL | [http://ir.ciac.jl.cn/handle/322003/64458]  |
| 专题 | 长春应用化学研究所_长春应用化学研究所知识产出_学位论文 |
| 推荐引用方式 GB/T 7714 | 唐诗瑶. 定志小丸化学成分及体外活性、代谢的相关研究[D]. 中国科学院长春应用化学研究所. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：长春应用化学研究所
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