【作者】 孙延芳；

【导师】 梁宗锁； Helmut Viernstein；

【作者基本信息】 西北农林科技大学 ， 药用植物学， 2010， 博士

【摘要】 酸枣(Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou)为鼠李科枣属落叶植物,广泛分布在世界的热带和亚热带地区。在中国酸枣是一种分布十分广泛的野生果树,即使在干旱的黄土高原地区也能生长良好。其种子酸枣仁,是一种抗焦虑失眠的传统名贵中药。其果实具有净化血液和助消化的特性,且具有养颜美容的功效。而且富含可溶性糖、有机酸、矿质元素、类胡萝卜素和各种维生素,并有“维生素C王”之称,具有重要的药用和营养价值。为了加强对野生酸枣果进行全面的开发利用,丰富我国野生资源天然库,本文主要对野生酸枣果进行质量评价,活性成分及其药理活性进行研究,主要的研究结果如下:1.由于药材成分复杂性,建立一种规范化的质量标准评价体系,可以对药材进行辨别真伪和质量的评价。本研究采用高效液相蒸发光指纹图谱方法来获得更多的化学指纹信息,从而对10个地区的酸枣果进行全面的质量评价。Waters液相色谱系统,匹配ELSD-UM 3000蒸发光检测器,Trisep-2003数据采集软件。采用梯度洗脱,流动相A为色谱级乙腈,流动相B为0.3%乙酸。洗脱程序为:20%A (0 min)-30%A (10 min)-60%A (20 min)-100%A (50 min);蒸发管温度为60℃,气体流速为2.5 min/L,可达到最好的信噪比。采用化学计量学上的相似度评价和聚类分析对获得的指纹图谱进行归类,相似度评价得出不同地区之间酸枣果相似度可达0.99以上,聚类分析显示辽宁和河北地区的酸枣果归为一类,山东归为一类,其他的归为一类。依据中国药典对酸枣果进行TLC检测,总灰分、酸不溶性灰分以及醇浸物和水浸物进行了测定。以上得出的结果表明本研究建立的指纹图谱方法可实现对酸枣果进行质量评价,从而为酸枣果入选《中国药典》起草可靠全面地质量评价方案。2.本研究构建TOPSIS评价模型,综合考虑评价不同地区酸枣果天然抗氧化产物和抗氧化活性之间的差异,结果显示不同地区酸枣果天然抗氧化物和抗氧化活性依次为:宁夏>甘肃>陕北>山西>四川>陕西>河南>山东>河北>辽宁。根据抗氧化活性得出的结论,可验证TOPSIS法可以对不同地区酸枣果进行有效地综合评价,得出的结果客观可靠,为酸枣果原料优选提供了科学依据。相关性分析显示酸枣果抗氧化活性与其天然抗氧化产物存在着不同程度上的显著和极显著关系,说明酸枣果抗氧化活性是由其天然抗氧化产物含量决定的。环境因子尤其海拔高度和年降雨量对天然抗氧化物和抗氧化活性有着显著的影响,是直接的环境相关因子。本研究得出生长在干旱高海拔地区的酸枣果比其他地区可产生较多的天然抗氧化产物,从而表现较强的抗氧化活性。据我们所知,目前的研究还没有类似的报道,该结论还需要在其他的药用植物和水果上作进一步验证。3.酸枣果三萜化合物分离鉴定具体操作步骤为:干燥酸枣果粉经石油醚回流脱脂,96%乙醇提取浓缩,得到的浸膏用饱和的正丁醇溶液萃取,浓缩干法上大孔树脂柱,依次用去离子水、30%乙醇、70%乙醇和96%乙醇洗脱,收集70%乙醇组分(Fr-C)干法上硅胶柱,氯仿:甲醇(20:1-0:1)洗脱,得到组分Fr-H,经化学检验为三萜皂苷。Fr-H浓缩后用丙酮沉淀,干燥后得淡黄色粉末,然后用2M三氯乙酸氮气封管水解12h,分别用氯仿和水萃取。氯仿层浓缩,TLC显色为紫红色斑点,经鉴定为三萜,命名为TS,继续通过制备液相分离纯化,得到化合物K(9.8 mg)。化合物K经波谱分析(TLC、UV、ESI-MS、1H-NMR、13C-NMR、DEPT)鉴定为五环三萜苷元熊果酸。4.通过抑菌,抗氧化和抗乳腺癌细胞增殖活性等体外试验来研究酸枣果三萜TS的药理活性。微孔稀释法测定三萜TS对8种病菌抑菌活性,结果表明三萜TS具有广谱抗菌效果,对实验所用的致病菌有较强的抑制作用。对金黄色葡萄球菌、乙型溶血性链球菌和炭疽杆菌的MIC可达0.078 mg/mL,尤其对绿脓假单胞菌和白色假丝酵母菌显示明显的抑制作用。通过清除DPPH自由基、铁还原-抗氧化能力法、降低能量、硫代巴比妥酸法和抑制β-胡萝卜素漂白等体外抗氧化反应,并以人工合成的抗氧化剂BHT作对照,研究TS抗氧化活性,结果表明TS具有显著的抗氧化效应。MTT法来检测TS对人类乳腺癌细胞MCF-7抑制作用,结果表明具有明显的增殖抑制作用并呈浓度剂量依赖性,其半抑制率浓度为23.119μg/mL;流式细胞术分析得出TS主要作用于G1期细胞;Annexin-V/PI双染法检验对细胞的诱导作用,结果表明可诱导MCF-7早期凋亡;RT-PCR和Western blotting方法分别从基因和蛋白水平上,进一步验证乳腺癌细胞凋亡的机理,得出Bax基因和蛋白的表达随着TS浓度增大而增大,而Bcl-2基因和蛋白的表达随着TS浓度增大而减少,证明酸枣果三萜TS对乳腺癌细胞MCF-7具有浓度剂量型的凋亡作用。5.根据我们以上的研究结果可知,尽管酸枣果三萜TS表现出很好的药理活性,但其水溶性差。为了提高TS在临床应用上的利用率,本研究采用β-环糊精对其进行微囊化,来提高TS在水中的溶解度。采用相溶解度法来研究TS水溶解性,相溶解度图显示二者的溶解曲线为AL型,证明TS与β-CD包合比为1:1,包合后TS的水溶解性大大提高。通过紫外光谱和扫描电镜对包合物进行鉴定,结果显示形成包合物后,TS在210 nm的紫外吸收峰消失;扫描电镜观察包合物在外观形态上发生了很大的变化,证明形成TS-β-CD包合物。研发以TS-β-CD包合物为主要活性成分的药妆品,表现出稳定的流变学特性,具有显著的抗衰老美白效果。更多还原

【Abstract】 Sour jujube [Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou] is a thorny, rhamnaceous deciduous plant widely distributed throughout the tropical and subtropical regions of the world. In China, sour jujube is a hardy fruit tree that grows well in wild areas. It adapts to dry and hot climates even in the arid regions of loess plateaus where other fruits cannot be grown. Its dried seeds named suanzaoren (Zizyphi Spinosi Semen) in Chinese, have a thousand-year history of use in traditional Asian medicine as sedative for treating mild anxiety, nervousness and sleep-related problems. Its fruits have long been used in folk medicine and are widely utilized in many herbal preparations for over one thousand years that claim to purify the blood and aid digestion. The fruits are also believed to promote anti-aging effects for women. Moreover, the fruits which are rich in nutrients such as sugar, organic acids, mineral, carotenoids and vitamins, have important pharmacological and nutritional functions. To strengthen comprehensively utilization of sour jujube fruit as natural antioxidants and other functional product, the systematical study on the quality control, chemical components and pharmacology functions is necessary. The major accomplishments and innovations in the paper as follows:1. Due to the complexity of ingredients in herbals, established a quality standard system is an important way to distinguish different Chinese medical herbs resources. In this study, a chromatographic fingerprint analysis was developed using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) to gain more chemical information to create a representative fingerprint to quality assessment of sour jujube. The HPLC system (Waters, Milford, MA, USA) and Waters SunFire column (250×4.6 mm, 5μm), equipped with a binary pump and an ELSD-UM 3000. Data were acquired and processed by Trisep-2003 software. HPLC analysis was carried out by gradient elution with solvent A (acetonitrile) and solvent B (water containing 0.3% acetic acid, pH 2.8) at a flow rate of 0.8 mL/min. The percentage of acetonitrile (A) in the mobile phase was programmed as follows: 20% (0 min)-30% (10min)-60% (20min)-100% (50min). The optimized parameters of ELSD are 60℃for the evaporator tube temperature and 2.5 min/L for the nebulizing gas flow rate to obtain the best signal to noise (S/N) ratios. In order to compare and analysis the differernce of HPLC fingerprint, the chemometric methods including similarity evaluation (SA) and hierarchical cluster analysis (HCA) were applied to classify ten batches of sour jujube according to their cultivated origins. In addition, sour jujube was investigated referring to the qualitive TLC examination; total ash, acid-insoluble ash as well as water soluble extraction, alcohol extraction according to Chinese Pharmacopoeia. So the quality requirement of sour jujube was suggested to add that the contents of total ash and acid-insoluble ash were 1.0263-2.6409%, 0.0200-0.1874%, and water soluble extraction and alcohol extraction were 12.8790-14.5603%, 5.5652-8.0278%, respectively. All the results indicated that this reliable method was suitable for comprehensively quality control of sour jujube, and this methods established would lay a foundation for the quality evaluation of sour jujube.2. Technique for Order Performance by Similarity to Ideal Solution (TOPSIS) method was used to comprehensively evaluate total natural antioxidants and antioxidant activities in Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin. Correlations between natural antioxidants, antioxidant activities and geographical conditions were investigated. sour jujube fruits from Ningxia, Gansu, Shaanbei and Shaanxi, all of which are found the fruits from the semi-arid regions of loess plateaus, demonstrated significantly higher antioxidant effects. TOPSIS analysis revealed the following order of natural antioxidants and antioxidative properties of the fruits from different regions, according to the highest amount: Ningxia > Gansu > Shaanbei > Shanxi > Sichuan > Shaanxi > Henan > Shandong > Hebei > Liaoning. The results demonstrated that TOPSIS method is an efficient ranking method that can be efficiently utilized in the assessment of total natural antioxidant content and quality of sour jujube fruits. Consequently, manufacturing companies can obtain sour jujube fruits from locations known to enhance the antioxidant activities of these fruits. Growing conditions appear to have a direct impact on natural antioxidant content and activities. Correlation coefficients analyses showed that the altitude and annual precipitation exert significant influences on the fruit. Thus, fruits grown in arid harsh, and high-altitude areas can produce a larger amount of natural antioxidants and therefore exhibit higher antioxidant activities compared to fruits grown in other areas. While studies have yet to clearly confirm this, other herbs and fruits are underway.3. The power of sour jujube fruits was extracted with Pe, then extracted with 96% EtOH and concentrated. The residue was partitioned with saturated BuOH, eluted by microporous resin D101 adsorption technology and get four fractions (Fr-A, Fr-B, Fr-C, Fr-D). The Fr-C was further isolated and purified with silica gel and get five fractions (Fr-E, Fr-F, Fr-G, Fr-H, Fr-I). The Fr-H was precipitated with acetone and hydrolysed by 2M trifluoroacetic acid, purified with semi-HPLC to yield compound K (9.8 mg). The structure of compound K was established on the basis of extensive spectral evidence (TLC, UV,ESI-MS,1H-NMR,13C-NMR,DEPT), and identified was ursolic acid (3-β-hydroxy-urs-12- en-28-oic acid).4. The pharmacological potentials of TS from sour jujube fruits were evaluated by antimicrobial, antioxidant and antiproliferative activities: In the case of antimicrobial activity, the results possessed broad and remarkable antimicrobial activity against the microorganisms tested containing 8 bacteria by microplate bioassay. The minimal inhibitory concentration (MIC) value for bacteria was 0.078 mg/mL. Antioxidant activity was employed by in vitro test systems namely DPPH radical scavenging activity, ferric reducing antioxidant power (FRAP), reducing power, inhibition of lipid peroxidation, and bleaching ability ofβ-carotene in linoleic acid system. The results were found to be similar activity pattern with synthetic antioxidant BHT. In antiproliferative activity, we first examined the growth-inhibitory effect of TS on human breast cancer MCF-7 cells by the MTT assay. In order to explore the underlying mechanism of cell growth inhibition, cell cycle and apoptosis were studied by flow cytometry. The expression of Bax and Bcl-2 gene were analysis by RT-PCR and Bax and Bcl-2 protein were further assayed by Western blotting. TS inhibited the growth of MCF-7 cell in a dose-dependent manner. The IC50 value was 23.119μg/mL after 72h treatment, TS arrested the cell cycle in the G1 phase and induced apoptosis. RT-PCR and Western blotting analysis showed that TS inhibited Bcl-2 gene/protein expression and induced Bax gene/protein expression. To the best of our knowledge, the cytotoxic activity of sour jujube fruit towards MCF-7 was studied for the first time in the present study. Our studies encourage the evelopment of novel, efficient and less toxic natural product from sour jujube fruits for cancer chemotherapy.5. Although TS expressed excellent pharmacological potentials according to our test, it is very poorly water-soluble. In order to improve the utilization of TS in clinical practication, the inclusion complex between TS-β-CD was prepared by blending method. The solubilization of TS in aqueous solution ofβ-CD was investigated by the solubility method, and the solid inclusion complex was further identified by ultraviolet and scanning electron microscopy (SEM). The solubility of TS in water increased as the concentrations ofβ-CD, showing an AL type phase solubility diagram. The curvature closely corresponded to the simulation curve which was calculated on the basis of the 1:1 (guest:host) complexation model. The results indicated that the formation of a TS-β-CD inclusion complex is more water soluble than TS alone. New cosmetic formulation with TS-β-CD inclusion complex as cosmeceuticals was prepared and possessed good rheological properties, anti-aging and skin whitening effect.更多还原