【作者】 李俶；

【导师】 倪永年；

【作者基本信息】 南昌大学 ， 食品科学， 2007， 博士

【摘要】 槲寄生为桑寄生科（Loranthaceae）植物槲寄生（Viscum coloratum（Kom．）Nakai）的干燥茎、枝。槲寄生是我国常见的传统中草药，使用记载的历史始于秦汉时期，古代称桑上寄生，为《神农本草经》上品。但槲寄生V．coloratum（Kom．）Nakai和桑寄生Loranthus parasiticus（L．）Merr．用名较为混乱，而且由于两者功效相似及用药习惯的沿袭，临床上两者一直是混用的。从《名医别录》到《本草纲目》的一些著作中，关于桑上寄生一项大都是指槲寄生，直至近代。《中国药典》1985年版已将桑寄生和槲寄生分列开来，在其中仅收载槲寄生Viscum coloratum（Kom．）Nakai一种。我国北方诸省产的槲寄生品种单一，均为药典记载的槲寄生Viscum coloratum（Kom．）Nakai。它是以干燥的带叶茎枝入药，主要功效在于祛风湿、补肝肾、强筋骨、降血压、安胎等。现代药理研究发现，槲寄生具有抗肿瘤、抑菌、抗衰老以及免疫调节等功效。槲寄生的主要有效成分是黄酮类化合物，生物碱、粘毒素及凝集素等，但临床应用主要还是以本草或者粗提物的形式为主，影响了槲寄生的有效利用，使得我国的资源优势无法得到充分的发挥应用，故提取分离纯化槲寄生的有效成分并明确其化学结构显得尤为重要。目前，对槲寄生的研究主要集中在有关抗癌蛋白活性成分，即槲寄生毒肽和凝集素及其它们的作用机理。对槲寄生中的其它化学成分及其生物活性的研究报道比较少，到目前为止，我国北方产的槲寄生中的生物碱和黄酮类化合物的提取、分离、纯化以及产品活性的研究未见有系统的报道。因此，对我国北方产的槲寄生中的黄酮和生物碱等成分展开研究，旨在可丰富槲寄生的应用范围，为充分利用我国北方的这一丰富药材资源，开发我国自主知识产权的新药提供科学依据。本文对槲寄生主要的活性成分黄酮及生物碱均做了较为详细的研究，为槲寄生综合利用和新药开发的研究奠定了相关的理论和应用基础。主要研究内容与所得结果如下：1．应用现代食品分析技术，对槲寄生的基本营养成分进行了较为全面的分析。结果如下（g／100g，干重）：水分含量为9.03，灰分含量为6.07，粗纤维含量为19.65，脂肪含量为14.69，总糖含量为5.53，还原糖含量为1.98，蛋白质含量为11.29，维生素E含量为127.63μg／g。采用ICP-AES法同时测定槲寄生的微量元素，结果表明，槲寄生中含有丰富的K、Ca元素，Na、Mg的含量也相对丰富；一次煎煮液和二次煎煮液中的微量元素含量变化不大，故就微量元素而言，完全可以只煎煮一次得槲寄生煎煮液即可。2．在对槲寄生的有效成分生物碱的研究中，采用重量法测定其含量，研究比较了酸水回流提取法、超临界CO₂萃取法、超声波辅助浸提法等不同提取方法，并对实验条件进行了正交设计试验优化，结果表明：超声波辅助浸提法中总碱的得率高于超临界CO₂萃取和传统的酸水提取法。通过正交试验确定提取的最佳工艺参数为：乙醇体积分数90％，超声时间30min，固液比1∶20，总碱的提取率可达1.73％。相比之下，超声波辅助浸提的方法简单易行，不失为一种有效的提取方法。3．对总生物碱作了进一步分离纯化的探讨，与离子交换相比，大孔吸附树脂更适合于本体系生物碱的分离与纯化。通过对四种不同极性的大孔吸附树脂的筛选，优选出D101型大孔吸附树脂。在优化条件（上样液浓度0.288mg／mL，流速控制在4BV／h和pH10）下，总生物碱吸附率可达到3.15mg／g干树脂以上；在解吸优化条件（乙醇／水为50／50，解吸液流速控制在2BV／h）下，总生物碱解析率达到94％。4．重点对槲寄生中主要活性成分黄酮进行了研究。采用铝盐显色可见分光光度法测定其含量，回收率为99.73％。研究比较了传统提取法、超临界CO₂萃取法、超声波辅助浸提法等不同提取方法对槲寄生总黄酮的提取，并进行了正交设计和二次旋转回归设计试验优化，对试验结果进行了统计分析或响应面分析，得到其最高得率和最优条件组合。实验结果发现，超声波辅助提取优于常规提取方法，其最佳提取条件为乙醇浓度84.72％、固液比1∶32、超声时间32.78min，在此条件下的黄酮得率为1.32％。5．在获取黄酮粗提液后，对其纯化方法进行了探索，筛选出AB-8大孔吸附树脂对槲寄生黄酮进行分离纯化，并对AB-8树脂解吸剂的选择、上样浓度和pH值、进样速度、洗脱速度进行了研究，结果表明：当初始样液浓度为5.2mg／mL，pH值4左右时，以4BV40％乙醇作洗脱剂，上样速度2BV／h，洗脱流速1BV／h为最佳纯化条件。纯化后的样品纯度由原来的12.16％提高到69.63％，提高了5.73倍，回收率可达81.36％。6．经显色反应和紫外光谱扫描后，初步鉴别槲寄生中黄酮类化合物的类型可能为黄酮和黄酮醇。7．通过利用AB-8型大孔吸附树脂对提取后的总黄酮进行分步梯度洗脱，总黄酮得到了初步的分离，10％～30％乙醇洗脱液中的化合物比较多，彼此间分离效果不佳，但40％～60％乙醇洗脱液中的化合物有所减少，且分离效果尚可。对其中40％～60％乙醇洗脱液中部分黄酮类化合物进一步分离研究，采用制备型高效液相色谱，改变了两次的流动相体系，分离得到了黄酮类化合物A和B。与其他方法相比较，省时，省力，方便快捷。该两化合物分别经UV、IR、¹H NMR／¹³C NMR以及MS相关波谱鉴定，A为高圣草素-7-O-β-D-葡萄糖甙-4’-O-β-D-芹菜糖甙，B可能为高圣草素-7-O-β-D-葡萄糖甙。8．通过四种不同体系（油脂过氧化、DPPH自由基、超氧阴离子、羟基自由基）研究了槲寄生黄酮的抗氧化性能。结果表明：在油脂体系中，一定浓度的槲寄生黄酮提取物对豆油的抗氧化作用强于一定浓度的Vc和BHT，与增效剂柠檬酸、Vc有良好的协同作用，且Vc的增效作用强于柠檬酸；在自由基体系中，采用分光光度法和化学发光法测定了纯化样品清除DPPH·、·OH、O₂^-·的能力。结果显示，槲寄生清除自由基性能良好，清除率与浓度成正量效关系，清除能力大小关系为：DPPH·＞·OH＞O₂^-·。9．通过高圣草素-7-O-β-D-葡萄糖甙-4’-O-β-D-芹菜糖甙对抗血小板活化因子（PAF）的作用，首次发现该化合物对PAF诱导的血小板聚集有显著抑制作用，并且呈剂量依赖关系。更多还原

【Abstract】 Mistletoe, the dried culms of Viscum coloratum（Kom.） Nakai of Loranthaceae family, is a well-known tradition herbal, medicine in our country. It is used from the Qin and Han Dynasties. But the name usage of V. coloratum（Kom.）Nakai and Loranthus parasiticus（L.）Merr, are comparatively disordered. Furthermore, due to their efficacy and usage being similar, both of these are confusedly used at clinic. According to the Chinese Pharmacopeia （1985）, Loranthaceae and Viscum coloratum（Kom.）Nakai are separated, and only Viscum coloratum （Kom.） Nakai is embodied. The species of mistletoes are single in the northern provinces of our country and are Viscum coloratum （Kom.） Nakai. Traditionally, it is considered that mistletoe has an effect on dispelling chronic rheumatism, nourishing liver and kidney, boosting up physique, reducing blood pressure, and protecting fetus. In recent years, it is considered that mistletoe has an effect on anti-cancer, anti-bacteria, anti-consenescence, therapy cardiovascular disease and immunoregulation.The main effective components of mistletoe are flavonoids, alkaloid, lectins and viscotoxins, but only its herbal or rough extract is used at clinic. In this way, its utilization is limited. It is very important that effective components in mistletoe are extracted, purified while their structures are characterized. At present, the researches on the effective anti-cancer protein component and its action mechanism are always one of the hotspots in the correlative field. Up to now, the alkaloid and flavonoids of mistletoe are studied, no systematic and detailed studies are found on its extraction, separation, purification and activity. Our focus in this dissertation is on the studying of flavonoids and alkaloid, which can enrich application of mistletoe. It offers scientific data for the ulterior study on mistletoe. The main contents and conclusions in this dissertation are summarized as follows:1. Using modern analytical technologies, the nutrients of mistletoe are analyzed. The results are shown as follows （g/100g, dry mass）:①water 9.03, ash 6.07, coarse fibers 19.65, fats 14.69, total sugar 5.53, reduced sugar 1.98, protein 11.29,②Vitamin E （μg/g） 127.63. Trace elements of mistletoe are simultaneously determined by ICP-AES. The results showed that K, Ca, Mg and Na are rich in the mistletoe. The content change of trace elements is a little between once decoction solution and twice decoction solution. So, once decoction solution is satisfied to trace elements.2. During the study of alkaloid of mistletoe, the weight method is used to determine the content. Mistletoe alkaloid is extracted by reflux, SFE, ultrasonic methods. These methods are compared and the experimental conditions are optimized by orthogonal design. The results indicated that ultrasonic extraction is better than reflux and SFE methods. Orthogonal test is used to evaluate the influence factors, that 90% ethanol, extracting 30min, the rate of solid and liquid 20:1 are the optimum extraction conditions. The rate of total alkaloid is 1.73%. Therefore, ultrasonic extraction is an easy, simple and effective method.3. The purification technique of alkaloid is studied. The macroporous resin is much fitter for the separation and purification of alkaloid than ion exchange resin. Four macroporous resins are used for the separation of total alkaloid. Adopting the sorption and desorption, the different macroporous resins and its capacity are studied. The results showed that D 101 resin possesses much higher adsorption and desorption capacity. Under optimum condition （initial solution 0.288mg/mL, 4BV/h and pH10）, the ratio of adsorption is more that 3.15mg/g dry resin. Under desorption optimal condition （50%ethanol, 2BV/h）, the ratio of desorption can reach 94%.4. Mistletoe flavonoids are main active components. It is studied amply in this dissertation The spectrophotometry is used to analyze flavonoids content. The recovery is 99.73%. Flavonoids are extracted by conventional extraction, SFE and ultrasonic extraction. Meanwhile, several experimental designs, such as the orthogonal experimental design and the orthogonal & rotable experimental design, together with Statistical analysis and response surface graph analysis are also performed to get the upmost extraction efficiency and optimal condition. It is indicated that ultrasonic extraction is better than others. Its optimal extraction conditions are as follows: the concentration of alcohol is 84.72% （V/V）, the ratio of material and alcohol is 1:32 （W/V）, and the time need of ultrasonic extraction is 32.78 min. The ratio of flavonoids is 1.32% under this condition.5. The purification of flavonoids is studied after extracting it from mistletoe, and AB-8 resin is selected to separate and purify flavonoids. Based on the research of eluent solvent, concentration and pH of initial solution, the velocity of initial solution and eluent, the optimal conditions are as follows: when the concentration of initial solution is 5.21mg/mL, pH is 4.0, the sample solution speed is 2.0BV/h, the elution solvent 4.0BV of 40% ethanol, the flow rate is 1.0BV/h, and the purity of flavonoids could reach the height of 69.63%, which is approximately 5.73 times higher than the previous 12.16%, while the yield of flavonoids could reach 81.36%.6. The flavonoids from mistletoe detected by color reaction and UV spectrum analysis belong to flavone and flavonol.7. Flavonoids get primary separation after grads desorption by AB-8 resin. In the outflow of 10% ethanol to 30% ethanol, there are a lot of compounds and the separation effect is dissatisfactory. But the eluent is 40% ethanol to 60% ethanol, and the compounds of outflow separate satisfactorily. The flavonoids components of fraction of 40% ethanol eluent are further investigated. Two fractions are collected after the flavonoids are further separated and purified by Prep-HPLC. Analyzed by UV, IR, ¹H NMR/¹³C NMR and LC/M, then their molecular structures have been determined. Monomer A is homoeriodictyol-7-O-β-D-glycoside-4’-O-β-D-apiosyl and B may be homoeriodictyol-7-O-β-D-glycoside respectively.8. Finally, the antioxidant activity of mistletoe flavonoids is studied by four different systems （oil, DPPH·, O₂^·and·OH）. The result showed that the purified samples have satisfying the antioxidant activity in the oil system.. Their antioxidant activity is enhanced with the adding of concentration. At certain concentration, their antioxidant activity reduced. In the system of oil, citric acid and Vc have synergic anti-oxidative activities for purified samples. In the system of radicals, the abilities of scavenging DPPH·, O₂^-·and·OH of the purified samples are detected by UV spectrophotometry and chemiluminescence. The results showed that the antioxidant activity of the extract is excellent. The order of scavenging abilities is DPPH·＞·OH＞O₂^·.9. Homoeriodictyol-7-O-β-D-glycoside-4’-O-β-D-apiosyl shows good activities against Platelet Activiting Factor （PAF） for the first time. The inhibition effect on PAF-induced rabbit platelet aggregation is dose-dependent.更多还原