【作者】 张作法；

【导师】 时连根；

【作者基本信息】 浙江大学 ， 特种经济动物科学， 2008， 博士

【摘要】 生物体的内环境无时无刻不在生成自由基,自由基在生物体内很容易与蛋白质、不饱和脂肪酸反应,引发氧化修饰作用,成为机体衰老及与衰老相关疾病的主要诱因。同时,随着年龄的增长,机体的抗氧化防御能力却在逐渐减弱,与衰老相关的疾病如肿瘤、心血管疾病、糖尿病、白内障、老年性痴呆等发病机会大大增加,所以自由基与衰老相关疾病发生机制之间的关系,成为了研究的新热点。为了最大限度地降低自由基对机体的损害,人们开始寻求抗氧化剂,但人工合成的抗氧化剂具有致癌性等很大的毒副作用。植物体富含黄酮类化合物等活性成分,具有很强的抗氧化活性,是天然、安全的抗氧化剂来源物。为此,植物抗氧化剂的开发与应用,成为了人们关注的研究领域。桑枝是桑科植物桑Morus alba L.的干燥嫩枝。《中华人民共和国药典》记载桑枝具有祛风湿、利关节、行水之功效,适用于风寒湿痹、四肢拘挛、脚气浮肿之病症。临床上主要用于治疗肩臂关节及手足酸痛麻木、风湿痹痛、瘫痪等多种疾病。现代药理学研究发现,桑科桑属植物具有降血糖、降血压、抗癌、抗菌、抗病毒、抗炎、镇痛、抑制花生四烯酸代谢等药理活性,其药理活性与其所含有活性成分密切相关。我国桑枝资源非常丰富,但对其利用主要局限于以桑枝药材原料为主的初级开发,查明桑枝药用活性成分和药理作用,对发展天然中药制剂等产品的深度开发,蕴藏有巨大的发展空间。本课题以桑枝抗氧化性为中心,采用测定、分离、纯化、鉴定等方法,对其中的活性物质进行分析,将所分离纯化的活性物质进行一系列的药理学实验,探明其活性物质的药理作用机理,从而为桑枝深度开发提供科学依据。主要研究结果如下：1、对桑枝中黄酮类化合物提取和测定条件进行优化,确立了桑枝过160目,以80%乙醇作为溶剂,超声波提取20 min,在80℃下回流30 min,重复提取3次,为桑枝黄酮类化合物提取的最佳实验方案。建立了桑枝黄酮类化合物含量测定的分光光度法,该方法精密度、稳定性、重现性和回收率均符合测试要求。2、采用正交实验法对桑枝多糖的测定条件进行优化,确立了桑枝过160目,超声提取40min,在100℃水浴中提取90 min,操重复2次,为最佳提取方案。对桑枝多糖进行了精制,得到桑枝多糖与葡萄糖对照品的换算因子为4.96。桑枝中富含多糖,其含量在桑枝品种间存在显著差异,高低相差约1.7倍；在同一品种的不同生长季节和老嫩程度间,春季新生嫩桑枝中多糖含量>冬季一年生老桑枝中多糖含量>春季老桑枝中多糖含量。3、DNJ本身没有发色基团,不能用液相色谱直接测定。本研究采用9-芴基氯甲酸甲酯标记、配有荧光检测器的反相高效液相色谱法,建立了可检测大数量桑枝样品中DNJ含量的快速、可靠方法。利用正交实验法,确立了在30℃、pH7下反应50 min为最佳衍生化条件。DNJ含量在桑品种间存在显著差异,高低相差2.9倍,在同一品种的不同生长季节和老嫩程度间,冬季桑枝中含量最高,春季老枝中含量次之,春季新生嫩枝中含量最低,表明DNJ在桑枝中具有一定的累积作用。4、采用蛋白快速分离纯化仪与示差检测器相结合的方法,分离纯化桑枝中的多糖,用液相色谱测定其分子量,用离子色谱分析其单糖组成,用红外光谱进行结构鉴定。研究结果表明,桑枝多糖是α-构形吡喃醛糖,分子量约为6.5×104,主要由鼠李糖、树胶醛糖、半乳糖、葡萄糖和木糖组成,它们的摩尔比分别为9.1：24.1：44.1：12.22：7.8。5、对桑枝多糖进行硫酸化、磷酸化、乙酰化和苯甲酸化,得到四种衍生物,研究其结构与抗氧化活性之间的关系。结果发现,衍生化后的多糖的抗氧化活性显著提高,温度和酸碱度显著影响其抗氧化活性,抗氧化活性随pH值升高而增高,随着温度升高而降低。6、根据DPPH的液相色谱特点,对桑枝乙醇提取液中的主要抗氧化活性物质进行了快速分离纯化,确定了桑枝乙醇提取液中的主要抗氧化活性物质为氧化芪三酚。此方法简单、快速、方便,避免了传统方法的反复操作和活性物质的分解。7、采用在线液相色谱法和液相色谱-电喷雾质谱联用法,鉴定桑枝中抗氧化活性成分,确立氧化芪三酚在品种间的分布规律。桑枝富含有氧化芪三酚,其含量在桑品种间存在显著差异,高低相差2.3倍。此实验结果对于推动桑枝及其制品的开发利用,具有一定的意义。8、对从桑枝中分离到的四种活性物质----氧化芪三酚、二苯乙烯苷、桑色素和芸香苷,进行抗氧化活性检验,确立它们的结构与抗氧化活性之间的关系。结果显示,氧化芪三酚分子中的4和3’位上的羟基,对于清除超氧离子、羟基自由基和DPPH具有重要作用；桑色素分子中3位上的羟基对于清除超氧离子和羟基自由基有重要的作用,芸香苷分子中的3位上的糖基对其还原力有重要的作用。9、用ICR小鼠的血清和肝脏中的生化指标、组织病理学和细胞因子表达,研究桑枝乙醇提取液对四氧嘧啶引起的肝损伤的保护作用。研究结果表明,桑枝乙醇提取液具有护肝作用,其作用机理主要有以下三点：(1)桑枝提取液作为一种抗氧化剂,直接清除四氧嘧啶产生的自由基；(2)桑枝提取液作为一种金属络合剂,通过络合金属来抑制自由基的产生,从而降低自由基的损害；(3)桑枝提取液作为糖苷酶的抑制剂,通过降低血糖浓度来抑制抗氧化活性酶的糖基化,从而提高其活性来降低损害。10、DPPH可以通过提高转氨酶活性、MDA含量和NO水平,并伴随GSH水平和SOD、CAT、GPx活性的降低,对机体肝脏产生损害,组织病理学观察和炎症因子表达的研究结果进一步证实了DPPH的此毒性。此研究结果对于建立DPPH的急性肝损伤模型,并以此模型来研究药物对氧化压力的相关疾病的治疗作用,具有积极的意义。11、利用硫代乙酰胺的肝损伤模型,研究桑枝中芸香苷的护肝作用。结果表明,桑枝中芸香苷具有明显的护肝作用,其作用机理有以下四个方面：(1)通过促进核糖体RNA的合成来促使肝细胞再生；(2)作为一种抗氧化剂来清除和调节细胞内谷胱甘肽的含量；(3)通过稳定细胞膜和调节通透性来阻止有毒物质的进入；(4)抑制星状肝细胞向纤维原细胞的转变,从而来阻止肝硬化的发生。12、利用酒精引起的肝损伤模型,调查桑枝中氧化芪三酚及其糖苷的护肝作用。结果表明,氧化芪三酚和二苯乙烯苷能够通过提高抗氧化活性酶活性和GSH的含量、降低MDA水平、削弱TNF-α表达以及改善小鼠肝组织状况等,对酒精引起的肝损伤起保护作用；氧化芪三酚的护肝作用显著高于二苯乙烯苷的护肝作用,这两种活性物质的护肝作用与抗氧化活性密切相关。13、通过桑枝提取液组分中抗炎活性的筛选,从抗炎活性最强的组分中分离到氧化芪三酚和二苯乙烯苷两种活性物质。采用醋酸引起的扭体实验、福尔马林引起的舔足实验、皮肤毛细管通透性实验、角叉胶引起足跖肿胀实验以及局部PGE2含量,检验桑枝中的这两种活性物质的抗炎、镇痛活性,结果二苯乙烯苷表现出很强的抗炎、镇痛活性,此物质可能是桑枝中起抗炎镇痛作用的主要物质之一。更多还原

【Abstract】 There is overwhelming evidence to indicate that the inner circumstance of the organism can produce the free radicals all the time and free radicals cause oxidative damage to lipids, proteins, and nucleic acids. Free radicals may lie at the heart of the etiology or natural history of a number of diseases, including cancer, heart, vascular, diabetes and neurodegenerative diseases. At the same time, the ability of the protective system of the organism was wear off with the age increasing, the chance of the disease such as diabetes, cancer, heart disease, arteriosclerosis and cataract in corrective with the aging was increasing. So the mechanism of the relation with the free radical and the disease with aging has become the hotpot of research.In order to minimize the oxidative damage largely, the people begin to search for the antioxidant, synthetic antioxidants are known to have toxic and carcinogenic effects on human, Thus, with increasing consciousness of consumers with regard to safety of food additives and the higher manufacturing cost and lower efficiency of natural and safer sources of food antioxidant is created. Therefore, search for natural antioxidants, especially of plant origin, has notably increased in recent years.Sangzhi (Ramulus mori) is the branch of Morus alba L., family Moraceae. According to theories of traditional Chinese medicine, it is slightly bitter in taste, mild in nature, and attributive to the liver meridian. In traditional Chinese medicine,its pharmacological actions are to expel wind, dredge the meridians, and ease joint pain. In the clinic, it was used to cure the disease of joint of shoulder and arm, ache and anaesthesia of hand, rheumatism and paralysis. In modern pharmacology, it has the activities of lowering the blood sugar and press, anticancer, antibacterium, antivirus, anti-inflammation and easing pain. There is consanguineous correlation between the activity of pharmacology and the chemical composition. In our country, there is rich resource of Ramulus mori, the utilization of it was mostly limited to the primary exploitation. So it is important to find out the main medical composition and the pharmacological function of Ramulus mori for developing the depth exploitation of the natural preparation of Chinese traditional medicine. The present study focus on the antioxidant activity of Ramulus mori, analysis the active compound with the modern methods of measurement, separation, purification, and identification, carry out a series of pharmacological experiments with the isolated compound, prove up the mechanism of pharmacological function, and try to provide the scientific basis for the further exploitation of Ramulus mori. The main results as follows: 1. The experiment optimized the condition of the extraction and determination the flavonoid in Ramulus mori. The flavonoid content could be exactly and stably measured with ultraviolet spectrophotometer after it was treated with the process:it was sifted with 160 item, use the solvent of 80% ethanol, extracted with the ultrasonic for 20 minuates and the circumfluence for 30min at 80℃for three times.2. The present study optimized the condition of the determination of the polysaccharide in Ramulus mori with the orthogonal methods. The content of polysaccharide could be exactly and stably measured with spectrophotometer after it was treated with the following process:it was sifted with 160 item, extracted with the ultrasonic for 40 minuates and the extraction for 90 min at 100℃in water for twice. The present study isolated and refined the polysaccharide of Ramulus mori too, and got the conversion factor between the polysaccharide and glucose (4.96). There was rich polysaccharide in Ramulus mori and the content of polysaccharide were significantly different to the mulberry varieties. In the same variety, the order of the content was new Ramulus mori in spring> Ramulus mori in winter > one year old Ramulus mori in spring.3. The DNJ could not be determined directly because there was no chromogenic group in its structure, the present study marked it with 9-fluorenylmethyl chloroformate for detecting the content of DNJ. The optimum condition of DNJ derivation including reaction for 50 min at 30℃with pH7 potassium borate buffer was established with orthogonal design. There was rich DNJ in Ramulus mori, and the content significantly different according to the mulberry varieties. In the same variety, the order of the content was one year old Ramulus mori in spring> Ramulus mori in winter> new Ramulus mori in spring. The DNJ can be accumulated in Ramulus mori.4. The polysaccharide of Ramulus mori was purified with the combination of the protein rapid separated appearance and detection, the molecular weight was determined by HPLC, the monose composition of the polysaccharide in Ramulus mori was measured with ICS2500 ion chromatography and its structure was determined with infrared spectral. The results showed that the polysaccharide is a-configuration and mainly composed of five monoses:rhamnose, arabinose, galactose, glucose and xylose and the ratio of composition of isrhamnose:arabinose:galactose:glucose:xylose is 9.12:24.12:44.09:12.22:7.18.5. The preliminary research found that the polysaccharide showed very poor antioxidant activity, in the present study, different polysaccharide derivatives were prepared to give sulfated, phosphorylated, acetylated and benzoylated polysaccharide. The antioxidant activity of the polysaccharide and its derivatives in vitro was determined, including scavenging activity against DPPH, superoxide and hydroxyl radical, reducing power, and chelating ability. Among the five samples, the polysaccharide derivatives showed stronger antioxidant activity than the polysaccharide in Ramulus mori. The activities of the tested samples varied with pH and temperature. The antioxidant activity in the linoleate emulsion improved with increasing pH from 3 to 11, while it decreased with ascending storage temperature between 20 and 90℃.6. Based on the character of DPPH in HPLC, in the present study, the result of HPLC was used as direction to isolate the main antioxidant compound in the ethanol extract of Ramulus mori. The main antioxidant compound was identified as oxyresveratrol. This method is simple, rapid, accurate, sensitive and avoiding the repeating operation and decomposing of the active compounds.7. On-line HPLC method and liquid chromatography-electrospray ionization tandem mass spectrometry was used to separate and identify the main antioxidant compound in Ramulus Mori, and the result of the above method was used as a reference to isolate and identify the main antioxidant compound, which is oxyresveratrol. The content of oxyresveratrol in different mulberry varieties were also performed by high performance liquid chromatography. The method is simple, quick and sensitive, the result is satisfactory.8. To investigate the antioxidant effect of compounds isolated from Ramulus mori structure-activity relationship, the present study determined the antioxidant activities of rutin, morin, oxyresveratrol and cis-mulberroside A and establish the important role of 4 and 3’hydroxyl groups in oxyresevertrol for eliminating superoxide radical, hydroxyl radial and DPPH. The 3 hydroxyl group in morin was important for riding the superoxide radical and hydroxyl radial and the 3 glucose in rutin was important for its reducing power.9. The present study revealed that alloxan could induce the liver damage through the elevation of transaminases activities and MDA accompanied by significant reductions in GSH level and SOD and CAT activities. The treatment with ethanol extract of Ramulus mori can bring those changes to near normal level. The protective effect of it was further supported by the histopathological observation and the expression of pro-inflammatory cytokines. Considering the mechanism of alloxan-induced damage and the above result, we can conclude that the protective effect of EER primarily involves three mechanisms:(1) acts as a free radical scavenger, directly scavenges the free radical caused by alloxan, (2) acts as a metal chelator, inhibits radical generations by stabilizing transition metals, consequently reducing free radical damage, and (3) acts as a a-glucosidase inhibitor, through lowering the blood glucose concentration to inhibit the glycation of antioxidant enzyme, which can elevate the activity of antioxidant enzyme.10. Based on the character of DPPH, the model of liver injured was established. Considering that oxidative stress plays a role in most of clinical conditions, the findings of this experiment will be very useful for evaluating oxidative stress mechanisms and as an experimental model for testing the benefits of therapeutic substances on oxidative stress-related diseases.11. Rutin treatment could protected against the acute TAA-induced liver injury in mice was investigated, Form the results, we can predict the possible mechanisms of the prevention of the rutin from Ramulus Morus in four different ways:(A) stimulate the regeneration of the liver through promoting the synthesis of ribosomal RNA; (B) act as an antioxidant through scavenging and regulating the intracellular content of glutathione; (C)prevent hepatotoxic agents from entering hepatocytes throught stablizing cell brane and regulating the permeability; (D) inhibit the tansformation of stellate hepatocytes into myofibroblasts, which is responsible for the deposition of collagen fibres leading to cirrhosis. The key mechanism that ensures hepatoprotection appears to be free radical scavenging.12. Oxyresveratrol and its glucoside treatment could protect against the ethanol-induced liver injury in mice as demonstrated by increasing the activities of SOD, CAT, GPx and GST and the content of GSH as well as decreasing the content of MDA. The protective function was further supported by the histopathological observation and the expression of TNF-a. The result showed that the protective efficiency of oxyresveratrol was more pronounced than its glucoside. Considering their antioxidant activities in vitro and the mechanism of the metabolism of ethanol, their protective effect may mainly be related to the antioxidant properties.13. The present research isolated the active compounds from Ramulus mori through bioassay method and investigated their anti-inflammatory and anticoncept through acetic acid-induced writhing response, formalin-induced licking, carrageenan-induced air pouch, acetic acid-induced vascular permeability and the content of PGE2. cis-mulberroside A exhibited high anti-inflammatory and anticoncetipt activities. So cis-mulberroxide A may be the main active compound with the anti-inflammatory activity in Ramulus mori.更多还原