【作者】 冯青；

【导师】 吴新荣； 李茹冰；

【作者基本信息】 华南理工大学 ， 制糖工程， 2014， 硕士

【摘要】 桑叶为桑科植物桑Morus alba L.的干燥叶，始载于《神农本草经》，具有疏散风热、清肺润燥、清肝明目的功效。桑叶中含有多糖、黄酮、生物碱、挥发油成分、维生素、氨基酸等活性物质，具有降血糖、降血脂、清除氧自由基等药理活性。桑叶中化学成分的种类与含量受种属与种植环境条件的影响，从而影响桑叶及其产品的质量。为加强桑叶药材资源的开发利用与质量控制，本研究以高效液相色谱（HPLC）指纹图谱结合多指标成分定量分析的方法，对21批次不同产地桑叶药材进行HPLC特征指纹图谱研究，构建准确可靠的桑叶色谱指纹图谱。通过超声波辅助提取法，优化桑叶黄酮的最佳提取工艺，通过试凑试验法建立桑叶的HPLC检测条件，并建立了桑叶的HPLC指纹图谱。结合一些主要有效成分的定量检测，对桑叶的质量标准进行了补充和完善，为桑叶质量控制标准提供一定的参考。本文主要内容如下：（1）桑叶总黄酮的最佳提取工艺：桑叶中黄酮为主要的活性化学成分，桑叶的提取条件对其含量具有较大的影响，为提高总黄酮提取率，系统研究了不同提取溶剂、提取方式、温度、时间等因素对提取率的影响，采用单因素优化、正交试验，优化桑叶总黄酮的提取工艺。结果表明：以70%乙醇溶液为提取剂，料液比为1:25（W/V），超声波提取2次，每次120min为黄酮提取率最高的提取工艺。（2）桑叶的HPLC指纹图谱研究：本研究对采集自各地药店的21批次桑叶进行HPLC指纹图谱研究，优化了色谱条件为Agilent1100型高效液相色谱仪，二极管阵列检测器，Agilent TC-C18色谱柱（250mm×4.6mm，5μm），流动相A：0.1%磷酸水溶液，流动相B：甲醇，采用二元梯度洗脱程序，0-14-28-35-50-65-70min，15-30-40-45-60-70B%；流速：1.0mL·min-1；检测波长340nm；柱温25℃；进样量10μL；方法学验证该法建立的桑叶HPLC指纹图谱专属性强、稳定性好，能比较真实全面的反映桑叶的特征。（3）不同批次的桑叶HPLC指纹图谱研究：运用已建立的桑叶指纹图谱的方法对21批次的桑叶进行分析研究，结果显示，21批次桑叶共有25个共有峰，且占所有峰面积的80%，与黄酮标准品对照，可确定绿原酸、芦丁、槲皮素色谱峰。对21批桑叶的特征指纹图谱进行相似度计算，结果表明21批样品中，相似度均在0.70以上，相似度最高为0.991，相似度大于0.90的有17个样品；相似度在0.85-0.90之间的有1个样品；相似度小于0.85的有3个样品。（4）桑叶性状观察与主要活性成分含量分析研究：通过对不同批次桑叶进行性状观察，均符合中国药典中性状鉴别项中描述，但性状对比后，不同产地桑叶在颜色、叶片大小与薄厚、叶柄粗细等均有明显差异；采用紫外-可见分光光度法、HPLC-DAD、HPLC-ELSD法对桑叶黄酮中绿原酸、槲皮素、芦丁、1-脱氧野尻霉素（DNJ）进行含量测定，分析活性化学成分含量与性状的相关性，不同批次桑叶中各活性成分含量差异较大，与性状没有明显的关联性，在药材种植期，其气候、土壤及采摘时间等均对药材性状、活性成分含量具有较大的影响。（5）桑元止渴胶囊桑叶原药材与中间品指纹图谱的研究：桑元止渴胶囊为广州军区广州总医院药学部研发的单方制剂，已开发为军队医疗机构非标准制剂，通过指纹图谱技术对控制胶囊中间品、成品质量，对生产工艺进行评价与规范，也是从整体上系统控制并提高桑元止渴胶囊质量的有效途径。对中间品、成品的特征指纹图谱进行相似度计算结果表明，胶囊制备工艺稳定可行；桑叶药材与胶囊中间品及成品的相关性分析结果表明，桑叶药材中主要化学组分的色谱峰在中间品图谱中有所体现，胶囊制备过程中化学成分损失少，是对成品制备提供了良好的保证。本论文的意义在于系统的研究了不同产地桑叶的区别，建立了可靠的分析方法，为以桑叶为主要原料的保健产品、中药制剂提供了良好的质量控制标准参考，有助于桑叶药材构效研究和药材的全面开发。更多还原

【Abstract】 Mulberry leaves are dried leaves from Morus alba L., recorded in the " Shen Nong’sHerbal Classic", with the effects of wind-heat evacuation, clearing the lungs and moistening,liver eyesight. Mulberry leaves contain polysaccharides, flavonoids, alkaloids, volatile oilcomponents, vitamins, amino acids and other active substances, with pharmacologicalactivities of hypoglycemic, lipid-lowering, scavenging oxygen free radicals and others. Typeand content of the chemical compositions of mulberry leaves are affected by species andplanting environmental conditions, thus they are affecting the qualities of mulberry leaves andrelevant products.In order to enhance the development and utilization of resources and control the qualityof mulberry leaf, this study mainly focused on fingerprint of21batches of different originMulberry herbs using the method of high performance liquid chromatography (HPLC)combined fingerprint and quantitative analysis of multi-component index, then an accurate andreliable chromatographic fingerprints was built. Optimizing the process of flavonoidsextraction from mulberry leaves by ultrasonic assisted extraction method; establish HPLCfingerprint of mulberry leaves and its conditions by trial and error experimentation. Combinedwith quantitative detection of some of the main active ingredients, the quality standards ofmulberry leaves was supplemented and improved, it may provide a reference for the controlstandards of mulberry quality. The main contents are as follows:(1)The optimum extraction of flavonoids Mulberry: flavonoids are the main activechemical ingredients in mulberry leaf; the method of extraction has a greater impact on theircontents. In order to improve the extraction rate of flavonoids, it takes a systematic studyabout the different effects of extraction, such as solvent, temperature, time and other factors.Using single-factor optimization and orthogonal experiment, it is to optimize the process offlavonoids extraction from mulberry leaves. The results showed that: the best extraction is that70%ethanol as extraction solvent,1:25(W/V) as solid-liquid ratio, using ultrasonic extraction,twice, each time120min.(2) The study of HPLC fingerprint of Mulberry leaves: The21batches of mulberry leaves were collected from pharmacy. The chromatographic conditions were optimized: Agilent1100high-performance liquid chromatography combined diode array detector, Agilent TC-C18column (250mm×4.6mm,5μm), mobile phase A:0.1%phosphoric acid, mobile phase B:methanol, using a binary gradient elution program:0-14-28-35-50-65-70min,15-30-40-45-60-70B%; Flow rate is1.0mL·min-1; the detection wavelength is340nm;column temperature is25℃; Injection volume is10μL. The result of methodology showedthat the method of mulberry leaves HPLC fingerprint is specific, stable, and it can be morefully reflect the characteristics of mulberry leaves.(3) The study of21batches of Mulberry leaves HPLC fingerprint: the result of21batchesof mulberry fingerprints using established method showed that21batches mulberry leaveshave25common peaks, and the common peaks’ areas are accounted for80%of all peak areas.Meanwhile, compared with flavonoid references, it can determine the peaks of Chlorogenicacid, Rutin, Quercetin. The result of HPLC fingerprint similarity calculation showed thatsimilarity of21batches of samples are more than0.70, the highest similarity is0.991.17samples’ similarity are greater than0.90, one is between0.85-0.90, there are three samplessimilarity of less than0.85.(4) The observation of mulberry leaves’ characters and analysis of the main activeingredients: traits of different batches of mulberry leaves are in line with description ofcharacter identification in Chinese Pharmacopoeia, but the traits have significant differences incolors, mulberry leaves size and thinness, thickness of petiole, etc. The contents ofChlorogenic acid, Quercetin, Rutin,1-deoxynojirimycin (DNJ) were determined by usingmethods of ultraviolet spectrophotometry, HPLC-DAD, HPLC-ELSD, then to analyze thecorrelation between the content of active chemical ingredient and the character of each of theactive ingredients. The results showed that there is a large difference between different batchesof mulberry leaves, with no apparent correlation with the traits. In the period of planting, thefactors such as the climate, soil, plucking time have a greater impact on medicinal propertiesand contents of active ingredients.(5) The fingerprint of raw herbs and intermedial of Sangyuan Zhike Capsule: Sangyuan Zhike Capsule is developed by General Hospital of Guangzhou Military Command of PLA,department of pharmacy; it is a unilateral formulation which is the non-standard preparationsin the military medical institution. Controlling the quality of the intermediate and evaluatingthe process using the method of fingerprint technology are the effective way to improve thequality of Sangyuan Zhike Capsule in the whole system. The result of similarity calculation ofintermediate and preparation’s fingerprints showed that the process of capsule is stable andfeasible. The result of correlation between mulberry herbs, capsuable intermediate andpreparation showed that the peaks of main chemical components in mulberry leaves arereflected in chromatogram of intermediate, and there is a less loss of chemical compositionduring the preparation process, it provides a good grarantee for finished product.The significance of this thesis is to study the differences between different origins ofmulberry leaves. A reliable analytical method has been established and it provides a qualitycontrol reference for health care products and Chinese herbal preparation which raw materialis mulberry leaves. At the same time, the result is also benefit for structure-activity researchand development comprehensively of mulberry leaves.更多还原