【作者】 杨俊；

【导师】 蒋惠娣；

【作者基本信息】 浙江大学 ， 生药学， 2003， 硕士

【摘要】 本实验旨在确定杭白菊（Chrysanthemum morifolium Ramat.）总黄酮的最佳提取工艺，并观察杭白菊不同成分（总黄酮、绿原酸、游离木犀草素、总糖、氨基氮、挥发油）含量在（2001年和2002年）采摘期中的变化趋势，初步建立杭白菊提取物的质量标准，并分离符合制订杭白菊提取物质量标准用的化学对照品。 采用正交试验法，考察提取温度、提取时间、乙醇浓度及提取次数四个因素对提取液中总黄酮含量的影响，确定最佳提取条件。结果显示，在所考察的四个因素中，对杭白菊黄酮提取影响程度依次为：乙醇浓度＞提取温度、提取次数＞提取时间，杭白菊黄酮最佳提取条件为：75％乙醇，100℃水浴回流提取3次，每次1.5hr。依据确立的最佳提取条件，对同一种植区域，不同时间采摘的杭白菊进行总黄酮含量测定。两年采摘期中，总黄酮含量都随采摘周期推迟呈下降趋势。 在药典方法基础上，对HPLC法测定绿原酸含量的方法进行改进，采用梯度洗脱法测定不同采摘期杭白菊中绿原酸含量。结果表明：采摘期中绿原酸含量未见明显变化。 在杭白菊游离木犀草素的含量测定中，样品用乙酸乙酯索氏提取，采用ODS柱，以pH2.50磷酸盐缓冲液-甲醇-四氢呋喃-异丙醇（65∶7.8∶11.7∶15.5）为流动相，流速0.6ml·min^-1，检测波长360nm，测定杭白菊中游离木犀草素的含量。结果显示，在整个采摘期内，杭白菊游离木犀草素以第一次采摘的含量最低，第二次采摘的含量最高，其余变化不显著。 硫酸-苯酚比色法测定杭白菊乙醇提取物中总糖含量，甲醛滴定法测定杭白浙江大学硕士论文中药CMR的质量研究及成分分离菊水浸出物中的氨基氮，水蒸气蒸馏法提取并测定杭白菊挥发油含量，并检测不同采摘期中各个成分的变化趋势。氨基氮含量在整个采摘周期中无明显变化;总糖含量在2001年采摘前期变化不明显，而在2001年采摘后期却明显升高，在2002年采摘期中总糖含量变化不明显;挥发油含量在2001年采摘期中随采摘周期推迟呈下降趋势，而在2002年整个采摘周期中无明显变化。 在杭白菊提取物质量标准研究中，我们对杭白菊提取物进行了鉴别检查，含量测定。鉴别检查中进行了黄酮营峰面积积分值比值测定，理化鉴别，薄层鉴别。作为鉴别检查项目之一，HPLC法测定杭白菊提取物中芹菜素一7一p一D一葡萄糖营峰面积积分值与木犀草素一7一p一D一葡萄糖营峰面积积分值之比，该值为1.42一1.83。通过盐酸一镁粉、莫氏反应、三氯化铝反应三种理化鉴别，表明杭白菊提取物中含有黄酮类和昔类成分。运用乙酸乙酷一甲醇、氯仿一甲醇两种极性梯度的分离体系，薄层鉴别杭白菊提取物在对照品木犀草素一7一p一D一葡萄糖普、芹菜素一7一p一D一葡萄糖普相应位置有相同颜色的斑点。含量测定项则测定了总黄酮含量和指标成分一木犀草素一7一p一D一葡萄糖普含量。亚硝酸钠一硝酸铝一氢氧化钠显色法测定杭白菊提取物中的总黄酮，其含量为63.9%一70.0%。采用ODs柱，以pH2.00磷酸盐缓冲液一90ry0乙氰为流动相梯度洗脱，流速1.oml.min一’，检测波长338lun测定杭白菊提取物中木犀草素一7一p一D一葡萄糖普的含量。杭白菊提取物中木犀草素一7一p一D一葡萄糖普的含量为4.89一6.84%。 通过硅胶、聚酞胺柱层析从杭白菊提取物中分离得到两个黄酮昔类成分，木犀草素一7一p一D一葡萄糖普和芹菜素一7一p一D一葡萄糖营（进一步的纯化和结构鉴定工作正在进行中）。 上述研究显示:杭白菊总体质量与采摘时间有一定关系;提取物质量标准初步研究将为杭白菊产品的质量控制提供依据。更多还原

【Abstract】 The first aim of the present study is to optimize the extraction condition for flavonoids from Chrysanthemum morifolium Ramat, and observe the various trends of different components (flavonoids, chlorogenic acid, free-luteolin, total sugar, amino-nitrogen, chlorogenic acid and essential oil ) in Chrysanthemum morifolium Ramat. during collection time. The second aim is to investigate the quality of extract from Chrysanthemum morfolium Ramat. The third aim is to obtain purified apigenine-7-p-D-glucoside and luteolin-7-p-D-glucoside for standard.Optimum conditions (bath temperature, extraction hours, ethanol concentration and extraction times) were determined by the orthogonal experiment guided by the flavonoids content in the extract. The order of factors to affect the flavonoids extracion was ethanol concentration > bath temperature, extraction times > extraction hours. The optimum extraction conditions were 75% ethanol, refluence for 1.5 hr at 100 water bath for 3 times. The flavonoids in Chrysanthemum morifolium Ramat. was extracted under the optimum condition and measured by Spectrophotometery. As the collection time defered, the contents of flavonoids descend obviously.Chlorogenic acid was extracted 2 hours by methanol under refluence in water bath and determinated by HPLC in gradient elution of sodium hydrogen phosphate (pH2.70, 0.01M) and methanol. No significant changes were found in the content of chlorogenic acid.In order to measure luteolin content, ODS column was used, the mobile phase was methanol-tetratetrole-isopropyl alcohol (65:7.8:11.7:15.5) at a flow rate of 0.6 ml-min-1, the UV detection wavelength was 360nm. No significant difference of luteolin content was observed among the different Chrysanthemum morifolium Ramat. in 2001 , except that the sample collected first time of 2001 showed the lowest content of luteolin.The content of total sugar in Chrysanthemum morifolium Ramat. extracted by enathol were mesured by Spectrophotometery. Amino-nitrogen was extracted by water at room temperature and tested by formaldehyde’s potentiometric titration. Essential oid extracted by steam distillation was weighted. In the collection prophase of 2001, total sugar did not change, but it obviously ascended later.The total sugar did not change markedly in early collection but ascended obviously in later, in contrast, essential oil content did not vary, but reduced in later collection during 2001. However, no obviously variety was showed in content of essential oil and total sugar in 2002. No significant changes were found in the content of amino-nitrogen.On research of the quality of extract from Chrysanthemum morfolium Ramat., identification and determination were proceeded. The ratio of area of flavonoid glucosides, properties and TLC were identified respectively. The ratio of area between apigenin-7-D-glucoside and luteolin-7-p-D-glucoside were analyzed by HPLC, the value were 1.42 to 1.83. The physiochemical properties for extract from Chrysanthemum morifolium Ramat. were identified in the three system ( HCl-Mg, Molish, AlCl3). Flavonoids and sugar were identified according to physiochemical properties of extract. The TLC for extract were also identified in the two solvent system (ethyl acetate-methanol=2:l,chloroform-methanol=4:l).Luteolin-7-p-D-glucoside and apigenin-7-D-glucoside of extract could be identified by TLC. Quantitative analysis, including the content of total flavonoids and index component were tested. Total flavonoids content were analyzed by sodium nitrite-aluminium nitrate-sodium hydroxide and thick sulphuric acid-phenolcolorimetric methods. The flavonoids content of extract were 63.9% to 70.0%. The content of index component in extract was measured by HPLC. ODS column was used, the mobile phase was sodium hydrogen phosphate-90% acetonitrile at a flow rate of 1ml-min-1, the UV detection wavelength was 338nm. The content luteolin-7-p-D-glucoside of different extract were 4.89 % to 6.84%.Two components, luteolin-7-p-D-glucoside and apigenin-7-更多还原