鹧鸪茶多酚提取纯化及防治去势大鼠骨质疏松的研究

发布时间：2018-12-14 21:46

【摘要】：目的：探讨鹧鸪茶多酚的提取纯化工艺并对其进行质量控制研究；研究鹧鸪茶多酚防治去势大鼠骨质疏松的作用，并初步探讨其作用机制；建立鹧鸪茶药材的指纹图谱，并进行模式识别研究。 方法：以鹧鸪茶多酚提取率为指标考察提取工艺，采用单因素和正交实验确定鹧鸪茶多酚的最佳提取工艺；通过AB-8、NKA-9、DM-130、HPD-600、HPD-826五种树脂的静态实验优选最佳纯化树脂，通过动态实验确定最佳的吸附、解吸条件。 对鹧鸪茶多酚进行质量控制。采用薄层色谱法对鹧鸪茶多酚进行定性鉴别，并采用一测多评方法进行没食子酸与表儿茶素的含量测定。 探讨鹧鸪茶多酚防治去势大鼠骨质疏松的作用。将大鼠随机分为6组，即假手术组、模型组、鹧鸪茶多酚低、中、高剂量组和戊酸雌二醇阳性对照组。除假手术组大鼠外，其他组大鼠均行卵巢切除术，手术4w后开始给药。给药8w后，10%水合氯醛麻醉，，腹主动脉取血，按试剂盒方法测定E2、SOD、MDA、ALP（碱性磷酸酶）；并计算子宫指数、胸腺指数、脾脏指数、股骨骨密度等指标。 建立鹧鸪茶药材的指纹谱，并进行模式识别研究。采用戴安Acclaim120C18色谱柱（250mm×4.6mm，5μm）；以乙腈-0.03%三氟乙酸梯度洗脱；检测波长：280nm；流速1.0ml/min；柱温30℃；信号采集时间70分钟。结果：提取工艺确定为：取鹧鸪茶药材适量，粉碎，加15倍量40%乙醇回流提取3次，每次45min，合并滤液，过滤，减压回收乙醇并浓缩至相对密度约1.1（60℃），喷雾干燥（进口温度105℃，出口温度95℃，蠕泵转速7r/min，风机转速1900r/min），即得鹧鸪茶提取物。 通过静态试验选出AB-8大孔吸附树脂为最佳树脂，纯化工艺确定为：取鹧鸪茶提取物过AB-8大孔吸附树脂，上样量为12mg/ml树脂，上样流速为1BV/h，上样药液pH值为3。先用水洗至洗脱液无色，再用4BV80%乙醇洗脱，洗脱流速为2BV/h，收集醇洗脱液，减压回收乙醇并浓缩至相对密度约1.0（60℃），低温冷冻干燥(-50℃，400Pa)，得到鹧鸪茶多酚。 建立了鹧鸪茶多酚的质量标准草案：采用薄层色谱法鉴别出儿茶素、EGCG、ECG、EGC、没食子儿茶素、没食子酸等6种成分，斑点清晰，重现性好，可作为鉴别方法。利用HPLC采用一测多评法测定没食子酸与表儿茶素含量，该方法准确可靠，重现性好，可作为鹧鸪茶多酚的质控方法。 鹧鸪茶多酚可以用于防治去势大鼠骨质疏松。可增加骨密度、升高血清SOD、E2含量，降低MDA、ALP含量，升高子宫指数等指标。 在建立的鹧鸪茶药材HPLC指纹图谱中，共标记15个共有峰，指认出没食子酸、EGC、儿茶素、EC、EGCG、ECG等6种成分；并进行聚类分析与主成分分析，最终将鹧鸪茶药材分为3类。 结论：鹧鸪茶多酚提取纯化工艺简便可行，其质量稳定可控，可以用于防治去势大鼠骨质疏松症；建立鹧鸪茶药材的指纹图谱，并进行了模式识别研究，为开发这一民间药用资源提供了科学依据。
[Abstract]:Objective: to study the extraction and purification of polyphenols from Partridge tea and its quality control, to study the effect of tea polyphenols on preventing and treating osteoporosis in ovariectomized rats, and to explore its mechanism. To establish the fingerprint of Partridge tea and to study the pattern recognition. Methods: the extraction process of polyphenols from Partridge tea was investigated by single factor and orthogonal experiment. The best purified resin was selected by static experiment of five kinds of AB-8,NKA-9,DM-130,HPD-600,HPD-826 resins, and the optimum conditions of adsorption and desorption were determined by dynamic experiment. The quality control of tea polyphenols from Partridge was carried out. The content of Gallic acid and epigallocatechin was determined by TLC. To investigate the effect of tea polyphenols on preventing and treating osteoporosis in ovariectomized rats. Rats were randomly divided into six groups: sham-operated group, model group, partridge tea polyphenol group, middle and high dose group and estradiol valerate positive control group. Ovariectomies were performed in all rats except sham-operated rats. After 8 weeks of administration, 10% chloral hydrate was used to anesthetize abdominal aorta, blood was collected from abdominal aorta, ALP (alkaline phosphatase) was determined by kit method, and the indexes of uterus index, thymus index, spleen index and bone mineral density of femur were calculated. The fingerprint spectrum of Partridge tea was established and the pattern recognition was studied. A Acclaim120C18 column (250mm 脳 4.6 mm ~ 5 渭 m);) with acetonitrile-0.03% trifluoroacetic acid gradient elution was used. The detection wavelength was 280 nm, the flow rate was 1.0 ml / min, the column temperature was 30 鈩

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