【作者】 汪春牛；

【导师】 刘平怀；

【作者基本信息】 海南大学 ， 生物化工， 2012， 硕士

【摘要】 琼榄[Gonocaryum lobbianum (Miers) Kurz]具有清热解毒、散郁结的药用功能,海南民间用其治疗黄疸型肝炎、胸肋闷痛等。琼榄作为一种重要的民间药用植物,其化学物质基础及生物活性却鲜见报道,而对同属的台湾琼榄[Gonocaryum calleryanum (Baill) Becc]也仅是分离得到三萜、环烯醚萜、黄酮苷类化合物的报道,且未对其生物活性进行过研究。为挖掘琼榄民间药用价值、充分利用其药用植物资源,本文首次研究了琼榄多糖与油脂的提取工艺及其生物活性物质,初步探明了琼榄的药效物质基础。本研究首先对琼榄根、茎、叶部位进行了化学成分的预试验,结合预试结果,重点对总黄酮、多糖分别采用Al(NO3)3-NaNO2-NaOH比色法和硫酸-苯酚法进行了含量测定,并对其进行了方法学的考察。随即对琼榄多糖及油脂提取工艺进行了研究,比较了水浴回流、超声辅助、微波辅助等提取方法提取多糖的提取率,结合响应面法Box-Benhnken中心组合试验优化超声提取琼榄多糖,确定了最佳的提取工艺条件；采用索氏、超声辅助、快速溶剂、微波辅助、超临界CO2提取法提取琼榄油,比较得油率并采用正交试验确定最佳提取工艺。结合GC-MS法对其脂肪酸鉴定,并对油脂理化指标进行测定。最后结合体外抗氧化及抗菌活性模型跟踪分离纯化乙醇浸提物,得到有效部位或化合物,并对其进行结构鉴定。得出如下结论：(1)化学成分预试验结果表明各部位中含有多糖、氨基酸、蛋白质、黄酮、甾体及萜类、生物碱、香豆素、内酯、挥发油等,均可能含有有机酸；此外根、叶中含有酚类、强心苷等,茎中则是可能含有酚类、强心苷；各部位均不含皂苷等。总黄酮、多糖含量均以叶部位为最高,分别为0.033%和16.35%。RSD值均小于2%,方法学考察表明结果稳定可靠、重现性好、精密度高。(2)由琼榄多糖的提取率可知超声辅助提取法最优,结合响应面法优化对其工艺条件优化,结果发现水料比26mL/g,超声时间50min,超声温度70℃,超声功率296W条件下琼榄叶多糖提取率达15.28%,与预测值基木一致。由琼榄油得率可知采用超声辅助提取为佳,其在提取率、油脂表观特征、油脂所含脂肪酸成分、理化指标上均优于其他4种方法,正交试验优化超声提取琼榄油的工艺条件为料液比1：9、超声时间10min、超声功率400W、超声温度70℃。此条件下,油得率可到达31.32%。GC-MS分析表明5种提取方法所提琼榄油中不饱和脂肪酸分别占总量的80.61%、78.68%、79.88%、76.75%、75.91%。(3) DPPH及FRAP法抗氧化活性实验得出琼榄叶的乙酸乙酯部位的IC50和FRAP值分别为154.1μg/mL、1729.2±68.3μmol/L,显示其为抗氧化活性最强部位,抑菌活性测试同样得出琼榄叶的乙酸乙酯部位最强,对此部位进行分离纯化后得到12个化合物,结合波谱学手段鉴定出其中的6个化合物,分别是p-谷甾醇、hopan-3p,22-diol、乌苏酸、齐墩果酸、羽扇豆醇、β-胡萝卜昔,均首次从该植物中分离得到,其余6个化合物结构正在鉴定之中。文献表明此6个化合物均有一定的抗氧化、抗菌等生物活性,尤其以齐墩果酸、乌苏酸对肝损伤有保护作用,因此推断琼榄具有治疗黄疸型肝炎的药理活性可能与其所含齐墩果酸、乌苏酸这类物质有关,初步揭示了其药效物质基础。更多还原

【Abstract】 Gonocaryum lobbianum (Miers) Kurz, has some medicinal function such as heat-clearing and detoxifying and sattering stagnation. It used by the folk to cure jaundice hepatitis and stemocostal dull pain in Hainan. As an important medicinal plant, the chemical composition and pharmacological activity of G. lobbianum (Miers) Kurz were few reported. Even in Taiwan which have G. calleryanum (Baill) Becc, the research was only about separating of tritepenoid, secoiridoid and flavonoid glycoside, but not go further to research Biological activity. In order to make the best use of G. lobbianum (Miers) Kurz in the area folk medicion, this main purpose of this study is to research the extraction process of G. lobbianum (Miers) Kurz polysaccharide and oil and its biologically active substances. Through this project, the pharmacodynamic material basis of G. lobbianum (Miers) Kurz will be explored.The first step of this experiment was to conduct preliminary test on the composition root, stem, leaves of G. lobbianum (Miers) Kurz. Based on the pre-test result, the next step was focused on measuring the content of flavonoids and polysaccharide by using sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method and sulfuric acid-phenol method respectively, and investigating the reliability of these methods. Then, attention will move forward to the study of extraction technology on polysaccharide and oil. Comparing the extraction rate of polysaccharides employing water bath reflux, ultrasonic-assisted extraction, microwave-assisted extraction and combining response surface methodology central combination experiment, the optimum conditions could be got. In tenns of fat-soluble substance, oil would be taken as a research object and soxhlet extraction, ultrasonic-assisted extraction, accelerated solvent extraction, microwave-assisted extraction and supercritical CO2extraction would be used to extract oil. The optimum ultrasonic processing conditions should be achieved by oil yied and orthogonal experiments. The component of fatty acid and the relative mass fraction would be analysed by using GC-MS and four parameters of the oil would be measured by using national standard method. Finally, alcohol-soluble substance of G. lobbianum(Miers) Kurz would be studied. The ethanol extracts of the roots, stem, leaves of G. lobbianum(Miers) Kurzwere dispersed with water and then extracted by using petroleumether, ethyl acetate, and n-butyl alcohol. From the screening of all parts by using antioxidant and antimicrobial model in vitro, the parts which had strongest biological activity would be got. After that, these parts would be isolated and purified and the structure would be identified. The following conclusions could be obtained:1. The result of the preliminary test showed that the different parts of G. lobbianum (Miers) Kurz contain sugar, protein, flavonoids, sterides, terpenoids, alkaloids, cumarin, latones, volatile oils, may contain organic acids. Moreover, the root and leaves contain phenols, anthraquinone, cardiac glycosides, while stem may contain phenols, anthraquinone, cardiac glycosides. However, saponins were not existed in each part, based on the result of the experiment, we can get the conclusion like that leaves of G. lobbianum (Miers) Kurz have the highest total flavonoids and polysaccharides, which are0.033%and16.35%respectively. RSD values were less than2%, Methods used in this research are investgated to enjoy a advantage in its reliablity. good reproducibility, high precision.2. The best one among these extraction methods mentioned before in extracting polysaccharides is ultrasonic-assisted process and this method would be optimized with reponse surface methodology. The result indicated that the optimum conditions are that water material ration is26mL/g, ultrasonic time is50min, ultrasonic temperature is10℃, ultrasonic power is296W. Under these conditions, the polysaccharides yield of leaves is up to15.28%, which is the same as predicted values.Based on the oil yield, apparent characteristics, the fatty acid compositions, physical and chemical indicators, ultrasonic-assisted extraction method is better than the other four. The optimal conditions were got by using orthogonal design to conduct this experiment which should1:9material-liquid ratio,10min extraction time,400W ultrasonic power and70℃extraction temperature and the oil yield could get31.21%. GC-MS analysis showed that the amount of unsaturated fatty acids are80.61,78.68,79.88,76.75,75.91%.3. Though DPPH and FRAP antioxidant activity experiment, the values of IC50and FRAP are1541.1μg/mL,1729.2±68.3μmol/Lof L-EtOAc of G. lobbianum (Miers) Kurz, which is strongest antioxidant activity part. Similar with antioxidant activity, the antibacterial activity tests showed L-EtOAc of G. lobbianum (Miers) Kurz is strongest activity part. Then the attention would be taken into isolating and purifying this part and12compounds were got in this step. With spectroscopic method,6compounds were identified which are (3-sitosterol, hopan-3[3,22-diol, ursolic acid, oleanolic acid and β-daucosterol. These compounds were first isolated in this plant. Some studies indicated that oleanolic acid and ursolic acid have protective action on liver damage. Therefore, the treatment of jaundice hepatitis may be related to oleanolic acid, ursolic acid.更多还原