【作者】 徐晓云；

【导师】 谢笔钧； 潘思轶；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2007， 博士

【摘要】 沙棘（Hippopha（?） rhamnoides）是我国蒙医、藏医的习用药材，已有千余年的历史，大量的研究结果表明：沙棘在其果子、叶片、种子、树皮中含有多种营养活性成分，具有抗癌、抗氧化、抗辐射、抗血栓、增强肌体活力和免疫力、消炎、抑菌、止痛和促进组织再生的功效。人们对于沙棘中维生素C、类胡萝卜素、黄酮、脂肪和多糖等成分在生理和药理方面的活性研究较多。然而，沙棘作为原花青素的新资源，无论是其化学结构还是其生物活性的系统研究均鲜见报道，因此沙棘中原花青素的深入研究将对沙棘资源的深度开发和利用具有重要的理论和实际意义。本课题综合运用了色谱及波谱等现代分离、分析技术和化学分析方法，对沙棘茎皮中原花青素的结构进行了分析与鉴定，同时采用现代医学、生物学技术和手段，着重研究了沙棘原花青素的体外抗氧化活性和体内抗胃溃疡作用，并对其作用机制进行了探讨。主要研究结果如下：1．沙棘中原花色素的含量测定和提取纯化对沙棘各部分原花色素含量进行了测定，结果显示沙棘籽和茎皮的含量较高，其中沙棘籽的含量高达9.67％，茎皮中的含量为4.42％。以沙棘籽为原料，比较了提取剂、温度、pH值等因素对沙棘籽原花色素（以下简称SBPAs）提取效果的影响，在单因素实验的基础上，通过正交实验优化，确定了最佳提取条件：以65％的乙醇水溶液作为提取剂，提取温度21℃，pH5.1，料液比为1∶10，提取三次，每次90min。提取得率为5.10％。通过对16种树脂的动态吸附与静态吸附试验的比较，以D3520大孔吸附树脂用于沙棘籽原花色素提取物的纯化为宜，30％乙醇洗脱液中原花色素的含量达到96％以上，回收率为30％。2．沙棘茎皮中原花青素B3的结构鉴定对沙棘茎皮提取物经D3520大孔吸附树脂纯化得到的30％乙醇洗脱液（SBPC）进行NP-HPLC和RP-HPLC-MS分析，结果表明，SBPC中含有原花青素单体（儿茶素和表儿茶素）以及至少含有三种二聚体和三聚体，其对应分子量分别为290，578，866。采用Toyopearl HW-40S凝胶色谱以100％甲醇洗脱SBPC，得到四个组分（Ⅰ，Ⅱ，Ⅲ和Ⅳ）。其中组分Ⅰ经NP-HPLC和RP-HPLC-MS分析为一单峰，因此命名为CompoundⅠ，DAD分析显示其具有典型的原花青素特征光谱图。负离子模式分析得到的准分子离子的m／z为577，对应的二级质谱碎片为289和425，为典型的B型二聚体的特征碎片。通过过量间苯三酚存在条件下的酸水解，得到的末端单元经与标准品比对为儿茶素，进一步采用一维和二维核磁共振波谱的分析，鉴定该化合物是2个儿茶素通过C4-C8键连结的原花青素二聚体B3。3．SBPC和B3的抗氧化活性研究及比较本文通过体外抗氧化实验，研究了SBPC对羟自由基、红细胞溶血、肝脏与肝线粒体中的脂质过氧化以及肝线粒体肿胀度的影响，结果发现SBPC能够有效地清除羟自由基，抑制脂质过氧化，保护生物膜，减少红细胞溶血，减轻肝线粒体肿胀程度，提示SBPC具有很强的抗氧化能力。采用CuSO₄-Phen-VC-H₂O₂化学发光体系、邻苯三酚—鲁米诺发光体系、H₂O₂-鲁米诺发光体系和CuSO₄-Phen-VC-H₂O₂-DNA发光体系分别测定了SBPC和B3对羟自由基、超氧阴离子自由基、H₂O₂的清除效果及对DNA的保护作用，结果显示二者均具有极强的抗氧化能力，进一步提纯后得到的单一组分B3的抗氧化活性高于混合物SBPC。4．SBPC对大鼠应激性胃溃疡的预防作用研究以大鼠浸水束缚应激建立急性胃溃疡模型，造模前灌胃不同剂量的SBPC（50mg，100，150mg／kgbw）或Ranitidine阳性药物（30mg／kgbw）。测定溃疡指数、大鼠胃液中胃酸含量和胃蛋白酶活性、大鼠的血浆中血浆皮质醇（Cor）、促肾上腺皮质激素（ACTH）和大鼠血浆表皮生长因子（EGF）及NO水平等生化指标；通过免疫组化的方法分析了胃粘膜组织中iNOS的表达；表皮生长因子受体（EGFR）及细胞核增殖抗原PCNA的表达；采用原位末端标记法检测了胃粘膜上皮细胞的凋亡。透射电镜观察和分析了大鼠胃粘膜的超微细胞结构。与对照组相比，高剂量组SBPC（150mg／kgbw）的溃疡指数明显降低（P＜0.01）。SBPC能抑制胃酸的分泌（P＜0.01），降低胃蛋白酶活性（P＜0.01），及血浆中Cor、ACTH、NO的水平（P＜0.01），同时能抑制粘膜中iNOS的表达，及显著升高血浆EGF的水平（P＜0.05），降低血浆中NO的浓度（P＜0.01）；及NOS和iNOS的活力（P＜0.05），使粘膜中的iNOS的表达降低（P＜0.01），增强EGFR和PCNA的表达分别达到显著和及显著水平（P＜0.01，P＜0.05），凋亡指数也显著降低（P＜0.05）。研究显示预先给予急性胃溃疡大鼠150mg／kgbw SBPC，可以有效缓解应激引发的胃粘膜损伤。这种作用的机理是复杂的，可能是SBPC通过调控ACTH、Cor的分泌来影响胃酸的分泌，这与超微细胞结构的观察相一致，也有可能是SBPC通过调节EGF及EGFR和NO水平促进大鼠的细胞增殖抑制细胞凋亡来实现的，这一结果也从PCNA的表达上调得到支持。5．SBPC对大鼠乙酸性胃溃疡愈合的促进作用通过浆膜下注射乙酸建立慢性胃溃疡模型，造模后灌胃不同剂量的SBPC（50mg，100，150mg／kgbw）或（Ranitidine阳性药物，30mg／kgbw）。治疗7天后、14天后分别处死动物。通过测定溃疡指数和大鼠的血浆中EGF以及免疫组化的方法测定第七天胃粘膜溃疡组织周围EGFR、PCNA的表达，以及粘液中PAS染色阳性的比率，观察了SBPC对大鼠乙酸性胃溃疡的促进作用。结果显示，与对照组相比，SBPC各剂量组的溃疡指数明显降低，均能迅速提高血浆中EGF的水平，同时SBPC高剂量组（150mg／kgbw）大鼠胃粘膜EGFR及PCNA的表达也显著增强（P＜0.01，P＜0.05），且中性粘液的分泌量增加（P＜0.01）。提示SBPC对大鼠乙酸性胃溃疡的愈合促进作用可能是通过提高EGF的浓度、促进EGFR的表达来实现对上皮细胞的增殖的促进作用，同时SBPC有增加粘液细胞分泌中性粘液的作用，从而达到增加胃粘膜的保护强度。更多还原

【Abstract】 Sea buckthom （Hippopha rhamnoides L.） is a conventional herb with a long history of application （more than 1000 years） in Tibetan and Mongolian medicines in the treatment of various diseases. Numerous nutritional and physiological activities have been reported in most part of sea buckthom including the berry, leaves, seed and bark. Many of which have bioactivities such as anticancer, antiradiation, anti-atherosclerotic, enhancing body energy and immunity, anti-inflammation, antiblastic, acesodyne and accelerating regeneration. So far, there has been a great amount of research about the effects of its composition, including vitamine C, carotenoids, flavonoid, fat and polysaccharides etc. Although sea buckthom is a resource of procyanidins, neither the systematic researches of procyanidins structure indentification nor its bioactivities has been reported at home and abroad. Therefore it has very important academic and plactical significance to make comprehensive research on procyanidins of sea buckthorn for its deep exploitation and use.In this study, modem separate and analytical technologe of chromatogram, spectrum and the chemical analysis are used to analysis the component and identify the structure of procyanidins in sea buckthorn bark. While some advanced technologies of modern medical and molecular biological technology were used to study the antioxidation activity in vitro and antigastric activities and its mechanism in vivo. The results are as follows:1. Extraction, purification and determination of content of proanthocyanidinsThe analytical results of proanthocyanidins in different part of sea buckthorn showed that the content of seed and bark is 9.67% and 4.42%, respectively. The extraction rations of proanthocyanidins from sea buckthorn seed was compared using different solvent, temperature, pH value and so on. After investigating the single effect factor and orthorhombic analysis, the optimal parameters of extraction were obtained: 65%ethanol/water （v/v）,temperature （21℃）,pH（5.1）,the ratio of material to extract solvent （1:10, v/v）,extract time （90min,×3） .The yield was 5.10%.In order to purify proanthocyanidins from sea buckthom seed by resin, the behavior of static adsorption, dynamic dasoption and desorption of sixteen kinds of resins was compared. D3520 resin is the optimum for proanthocyanidins purification; the optimum elution fraction in the 30% ethanol aqueous get 96.5% proanthocyanidins purity and 30.4% rate of recover.2. Identification and chemical structure characteristics of procyanidins B3 in sea buckthorn barkSBPC, 30% ethanol fraction of sea buckthorn bark procyanidins extract which eluted from D3520 resin, was analyzed by NP-HPLC and RP-HPLC. The results showed that it consisted of monomer, at least three dimmers and trimer of procyanidins. Their molecular weights are 290,578,866 respectively. SBPC was separated into 4 fractions （Ⅰ,Ⅱ,ⅢandⅣ） with100% methanol eluted from Toyopearl HW-40S gel chromatogram. FractionⅠwas neamed as compoundⅠby the reason that both NP-HPLC and RP-HPLC chromatograms of fractionⅠpresented single peak. And the spectrum of compoundⅠprovided by DAD totally in character with spectrogram of procyanidins. ESI-MS spectrogram indicated that m/z of pseudomolecular was 577 and the fragment ions were 289 and 425 in negative ion spectum, which accorded with fragment formation of procyanidins B3. When compoundⅠwas acid-catalyzed in the presence of excess phloroglucinol, the terminal subunit is catechin via reference standards. The identification of compoundⅠwas further confirmed using 1D NMR and 2D NMR technique, and the result indicated that compound was procyanidins B3 composed of two catechin units linked through C4-C8 bonds.3. The antioxidative activity of SBPC and B3 in vitroThis paper investigated the antioxidative activityof SBPC in vitro including the effects of SBPC on hydroxyl radical （’OH）, red blood cells （RBC） hemolysis, liver mitochondria swelling and lipid peroxidation in liver homogenate and liver mitochondria. The results showed that SBPC could inhibit .OH and lipid peroxidation. It could protect the biomembrane, reduce RBC hemolysis and lighten the degree of liver mitochondria swelling. So SBPC had very strong antioxidation activities.Effects of SBPC and B3 on scavenging hydroxyl radical （OH） and hydrogen perocide, removing super oxygen-anion free radical and preventing DNA were determined in CuSO₄-Phen-VC-H₂O₂ system, Luminol- H₂O₂ system, Pyrogallol-luminol system and CuSO₄-Phen-VC-H₂O₂-DNA chemiluminescence system respectively. The results showed that SBPC and B3 possessed a good potency to scavenge reactive oxygen （ROS） in different systems. However, the antioxidation activities of B3 were stronger than that of SBPC.4. Prevention effects of SBPC on gastric ulcer in stress ratesThe rat model of acute gastric ulcer was induced by water immersion and restraint stress （WRS）. Rats were orally administrated with different dose of SBPC （50,100, 150mg/kgbw） or ranitidine （positive drug, 30mg/kgbw ） before stressed, Gastric lesion index, acidity and pepsin activity of gastric juice, Cortisol, ACTH, epidermal growth factor （EGF）, NO in plasma were determined. The expression of epidermal growth factor receptor （EGFR）, iNOS and proliferating cell nuclear antigen （PCNA） around ulcer were detected by immunohistochemical method. The apoptotic cells in the gastric mucosa were examined through the methods of terminal deoxynucleatidyl transferase mediated DUTP nick and labeling （TUNEL）. The ultrastructural change of parietal cells was observed by. transmission electron microscope. Compared with the control group, the ulcer index, the level of [H⁺] and pepsin activity of gastric juice of SBPC high-dosed group significantly decreased（P〈0.01 ）,and the level of Cortisol, ACTH, NO in plasma of SBPC high-dosed group significantly decreased （P〈0.01） , In plasma, the EGF level was dose dependently increased, and inducible Nitric oxide synthase （iNOS） expression in gastric mucosa in high-dose group was significantly decreased （P〈0.01） . The expression of EGFR protein and the PCNA were increased significantly （P〈0.01, P〈0.05, respectively）, meanwhile the apoptosis index （AI） was significantly decreased （P〈0.05）.The results above Showed that gastric lesion could be depressed in rat when orally administrated with SBPC beforehand. The mechanisms of which are complicated: one of mechanisms is that SBPC could regulate the level of ACTH and Cor which effect the gastric juice secretion. And this assumption could be proved by the observation of the ultrastructural change of parietal cells. Another possible mechanism is SBPC could up-regulated the EGF and EGFR and down regulated NO to promote cell proliferation and inhibit cell apoptosis, which could be accorded with the up-regulator of expression of PCNA simultaneously.5. Effects of SBPC on healing of acetic acid-induced gastric ulcer in ratesChronic gastric ulceration was induced by injecting acetic acid in the subserosa of stomach. Different concertrations of SBPC were orally administrated to gastric ulcers rats. After treatment 7d and 14d, rats were sacrificed respectively. The ulceration was measured by ulcer index （UI）. The level of EGF in plasma and PAS position area in gastric mucosal was determined.The expression of EGFR and PCNA around ulcer was detected by immuohistochemical method after treatment 7d. SBPC was found to reduce the size of the ulcers at day 7 and 14 in a dose-dependent manner. Compared with the control, the UI of SBPC group was significantly decreased （P〈0.01） and the level of EGF in the plasma of SBPC group increased significantly （P〈0.01 ）, meanwhile the expression of EGFR and PCNA around ulcer in high-dose SBPC stomach were enhanced （P〈0.05）. The results implied that SBPC plays an important role in healing of acetic acid-induced gastric lesions possibly by the acceleration of the mucosal repair. Meanwhile SBPC could enhance the defence mechanism by promotion mucous secretion.更多还原