【作者】 赵强；

【导师】 余四九；

【作者基本信息】 甘肃农业大学 ， 动物医学工程， 2013， 博士

【摘要】 红茂草[Dicranostigma Leptopodum（Maxim.）Fedde,DLF]，又名秃疮花、秃子花、勒马回（陕西），为罂粟科秃疮花属二年生或多年生草本植物，生于海拔400～1300m的丘陵、山坡、路边、农田、草地、墙上等处，耐旱、耐瘠薄，我国全产，主要分布在甘肃、陕西、河南、山西、青海、宁夏、四川、云南、西藏等省区，尤其以在甘肃省陇东南地区的秦岭南北、渭河流域分布十分广泛。本实验以甘肃省陇东南地区特色药用植物红茂草为研究对象，从药材的人工栽培与田间规范化种植、药物活性成分检测提取与分离鉴定、以及生物活性等方面做了系统性研究，为红茂草药物资源的开发和利用提供一定的参考依据。1.以三年为期限，采用调查研究与实验研究相结合、室内测试分析与室外观察相结合、实验设计与统计分析相结合的技术路线开展工作，基本完成了对红茂草植物学和生物学特性、红茂草不同生长期生物碱含量变化监测、人工栽培技术与规范化种植等的初步研究。研究表明，红茂草对生长条件要求相对不高，人工栽培条件下其物候表现和抗逆性表现与自然野生状态下基本一致，易于在甘肃省陇东南地区人工驯化及大面积推广种植。2.采用植物生物碱系统提取法对红茂草干草粉末进行水提和醇提处理，经化学成分系统预试验发现其中以生物碱沉淀反应现象最为明显。对95%乙醇提取的红茂草药液，以95%乙醇︰冰乙酸︰水︰浓氨水（15︰0.5︰2.5︰0.5）为展开剂，分离效果最好，斑点清晰。以异紫堇碱为标准品，在波长415nm、pH=4.0的枸橼酸-磷酸氢二钠缓冲溶液、1.5mL0.1%溴甲酚绿染色剂作用下，测定红茂草生物碱最大吸光值，得到其生物碱含量为1.386%，从而建立了酸性染料比色法对红茂草中总生物碱含量的测定。采用色谱柱Eclipse×DB–C18柱（4.6mm×250mm），流动相甲醇–1%冰醋酸=60∶40（v/v）；紫外检测波长：254nm，流速：1.0mL/min，进样量20μL，柱温：30℃，对红茂草中提取的精制黄酮类化合物（芦丁）进行HPLC检测，其含量均值为0.106%。3.以1mg/mL红茂草生物碱溶液与360μL1%钼酸钠-浓硫酸试剂在70℃水浴20min，生成浅蓝色物质，经紫外分光光度计扫描该物质得最大吸收峰为310nm，再用紫外吸收法测定红茂草胶囊中生物碱含量，检测生物碱含量均值在18.65～18.75%之间，最后通过用1mg/mL异紫堇碱标准品对此方法进行验证，得检测值0.493mg略小于实际值0.50mg，从而建立了一种检测红茂草胶囊中生物碱含量的新方法。4.利用火焰原子吸收光谱对红茂草各成分中Cu、Zn、Fe、Mn、Co、Ni、Cr、Cd、As、K、Na、Ca、Mg、Li、Sr、Al、Pb、Se、Hg等19种微量金属元素含量进行了测定，对结果运用SPSS17.0进行统计学分析，发现红茂草中K、Ca、Mg、Al四种金属元素含量最高，对于单个成分中K元素含量的大小为：花＞茎＞根＞叶＞豆荚＞全草＞种子。采用CO2超临界法萃取红茂草中挥发油，得挥发油3.16g，出油率0.63%，对其进行GC-MS分析，得到红茂草挥发油总离子流色谱图，共检出201个色谱峰，采用计算机对各峰质谱图进行NIST02标准谱库检索，并根据质谱裂解规律进行核对，鉴定出46个化合物（相对含量在0.20%以上），其占挥发油总量的96.59%。利用普通硅胶柱色谱对红茂草生物碱进行提取、分离和纯化，并利用超导核磁共振等现代光谱和波谱技术鉴定化合物结构，最终确定为异紫堇碱、紫堇碱、原阿片碱和白屈菜碱。通过紫外光谱、红外光谱、差热-热重技术对红茂草中提取的黄酮类化合物（芦丁）结构及稳定性进行了检测。5.以红茂草中主要生物碱异紫堇碱为指标，采用单因素水平筛选，按照L₂₅（5⁶）正交表进行正交实验设计，并用SPSS17.0软件对实验结果进行统计学分析，确定红茂草生物碱最佳提取工艺条件为：乙醇体积分数65%、超声波作用时间20min、液料比15ml/g、回流时间2.5h、浸提液pH=8、浸泡时间4h，在此条件下红茂草提取液中生物碱含量为0.928%；以响应面实验设计法对红茂草生物碱提取工艺进行研究，结果表明其主要影响因素为：液料比15mL/g、超声时间35min、回流时间2.5h、乙醇浓度65%，在此条件下红茂草生物碱含量为0.933%，从而优选建立了红茂草生物碱正交设计和响应面法最佳提取工艺条件。运用真空冷冻干燥法提取红茂草生物碱，以HPLC检测红茂草中两种最主要生物碱（异紫堇碱和原阿片碱）的含量，结果表明，经真空冷冻干燥后，红茂草生物碱冻干率为44.26%，RSD为0.21%。HPLC检测所得的两种样品线性关系良好，异紫堇碱R2=0.9998，原阿片碱R2=0.9996，，此法专属性强，可为红茂草药物品质监测及质量控制提供参考标准。6.通过对正交设计和响应面法优化提取的红茂草提取物、CO2超临界法萃取的红茂草挥发油等对羟自由基（·OH）和超氧阴离子自由基（O-2·）的清除能力进行测定，结果表明，红茂草生物碱在25.0和1.8mg/mL时对·OH和O-2·清除效果显著，其清除率为58.70和49.26%，IC50为23.50和1.75mg/mL；当各样品溶液的浓度为0.45ug/mL时，其清除·OH的能力大小为：Vc>异紫堇碱>芦丁>红茂草提取液>挥发油；当各溶液的浓度达到0.50ug/mL时，其清除·O-2的能力大小为：Vc>芦丁>挥发油>异紫堇碱>红茂草提取液。7.以大肠埃希氏杆菌、金黄色葡萄球菌、白色念珠菌和绿脓杆菌为实验供试菌，进行了细菌生长曲线测定、纸片法、MIC、MBC、IC50、大肠埃希氏杆菌膜通透性和形态测定、透射电镜超微观察等实验，结果表明，红茂草生物碱提取液对大肠埃希氏杆菌、金黄色葡萄球菌、白色念珠菌和绿脓杆菌四种供试菌的MIC分别为：0.28、0.20、0.20和0.30g/mL，MBC分别为：0.26、0.10、0.10和0.28g/mL，IC50分别为：0.2162、0.1805、0.1805和0.2604g/mL。红茂草生物碱提取液作用于大肠埃希氏杆菌后，细胞通透性与空白对照组相比显著提高，提取液能使菌体形态改变，最终使菌体缢裂，其抑菌效果显著。利用玻碳电极支撑的磷脂双层膜（s-BLM）作为生物膜模型，以Fe（CN）3-/4-6为探针分子，利用循环伏安（CV）与扫描电化学显微镜（SECM）实验研究了红茂草中黄酮类化合物（芦丁）对磷脂双层膜电化学行为的影响，发现s-BLM与芦丁之间可发生比较强烈的相互作用，红茂草中黄酮类化合物（芦丁）对卵磷脂双层膜电化学行为存在一定的影响，为进一步研究红茂草药材的生物活性及药物疗效机理提供了参考。8.将红茂草水溶性外用制剂作用于感染传染性化脓性皮肤炎的羊只，从治疗结果观察，红茂草对羊只的传染性化脓性皮肤炎有良好的疗效。将不同剂量红茂草生物碱提取物接种BALB/c小鼠，在不同时间段采集心脏、肝脏、脾脏、肺脏、肾脏和注射部位肌肉组织，经HE染色镜检，各种组织中未发现有明显的病理变化，表明红茂草生物碱提取物对小鼠作用比较安全，为红茂草临床用药提供了实验依据。通过上述一系列实验和研究，丰富和拓展了红茂草药物研究的内容，为甘肃省陇东南地区特色药用植物红茂草资源的进一步深层次开发和利用提供了重要的实验和理论依据。更多还原

【Abstract】 Dicranostigma leptopodum(Maxim.)Fedde,(DLF), also named “Tuchuangflower, Tuzi flower, and Lemahui” in Chinese, a biennial or perennial herbagebelonging to Dicranostigma in papaveraceae and growing in hills, braes, roadsides,fields, meadows, etc, in latitude of400～1300m, is characterized by resistance todrought and barren. DLF mainly distributes in Gansu, Shanxi, Henan, Shanxi,Qinghai, Ningxia, Sichuan and Tibet, particularly in the south and north of QinlingMountains in southeast region of Gansu province and Weihe River Basin.In the present study, DLF in the southeast of Gansu was used to investigate itsartificial and field standard cultivation, to extract and identify its active ingredientsand to determine the bioactivities in order to further exploitation and utilization.1. By means of investigation combining experiment, the indoor test combiningoutdoor observation, the experimental design combining statistical analysis in a3-year term study, the preliminary results of the botanical and biologicalcharacterization of DLF was obtained, including the monitoring of the variation ofalkaloids content in different periods of growth and the technology of artificial andstandard cultivation. The results showed that DLF did not need high standard growingcircumstances and its phonological performance and resistance in the artificialcultivated condition were generally consistent with that in the natural conditions. It issuitable to grow widely in an artificial cultivation condition in southeast region ofGansu province.2. The DLF dry powder was extracted with water and ethanol using the alkaloidsystematic extracts method. We found the alkaloid precipitation was most obvious.The thin-layer chromatographic analysis showed that the95%ethanol extract of DLFusing the ice acetic acid: water: strong aqua (15:0.5:2.5:0.5) as the developing solventhave a good repeat, strong speciality and stability. We built a special acid dyecolorimetry method to determine the total alkaloid content of DLF. In this method, thesample in pH4.0citric acid-sodium hydrogen phosphate buffer were stained by1.5mL0.1%bromcresol, then measured the optical density at415nm using isocorydine as the standard, the content of alkaloid is1.386%. Then the refinedflavonoids (rutin) were measured by HPLC. The HPLC condition arechromatographic column Eclipse×DB–C18(4.6mm×250mm), mobile phase ofmethanol:1%glacial acetic acid=60∶40(v/v); The UV wavelength at254nm;flowrate in1.0mL/min, injection volume at20μL; column oven temperature at30℃;HPLC result showed the content of alkaloid is0.106%.3. Firstly,1mg/mL alkaloid of DLF and360μL1%sodium molybdate-oil ofvitriol reagent was incubated at70℃water for20min, a light blue substancegenerated, the maximum absorbance was measured at310nm. The alkaloids averagecontent was18.65～18.75%. Comparing with the positive control of1mg/mLisocorydine, the0.493mg is little less than the actual value of0.50mg.4.The content of19trace metals in DLF including Cu, Zn, Fe, Mn, Co, Ni, Cr,Cd, As, K, Na, Ca, Mg, Li, Sr, Al, Pb, Se, Hg were determined by Flame AtomicAbsorption Spectrometry. The statistical analyze with SPSS17.0software showedthat the contents of K, Ca, Mg and Al were higher than others. The content of K fromdifferent tissue followed the order: flower> stem> root> leave> pod> herb> seed.3.16g volatile oils were extracted by supercritical CO2with0.63%oil yield rate.201chromatographic peaks were found in the GC-MS spectrum, then retrieve them withNIST02standard fragmentation in the warehouse,46compounds are recognized,which account for96.59%of total volatile oils. The alkaloids of DLF were furtherseparated and purified using silica gel column chromatographic and identified itsstructure by superconducting nuclear magnet resonance, isocorydine, corydine，protopine and chelidonine were obtained. Finally, the structure and stability offlavonoid (Rutin) extracted from DLF was tested by ultraviolet spectrum, infraredspectrum, and DTA-TG.5. All analyses were conducted by the single factor and L625(5) orthogonalmethod by using SPSS software. The optimal condition of extraction is65%ethanol,ultrasonic for20min, liquid to solid ratio:15:1ml/g, reflux2.5h, pH8extractingsolution, macerate4h. The alkaloids content in DLF extraction is0.928%under thisoptimized condition. Using response surface analysis the technique of alkaloids extraction from DLF, the result showed the main influencing factors are: Liquid tosolid ratio is15ml/g, the treatment time of ultrasonic is35min, reflux time is2.5h, andconcentration of ethanol is65%. Under this condition the alkaloids content is0.933%.The alkaloids of DLF were extracted by vacuum freeze drying, the two main alkaloids(isocorydine and protopine) content were determined by HPLC. The results showedthat the freezing rate of alkaloids is44.26%, RSD is0.21%. The isocorydine andprotopine showed good linear relation, the R2value of isocorydine is0.9998, the R2value of protopine is0.9996respectively. This method has a good specificity andcould provide reference standard for the monitoring and controlling the quality ofDLF drugs.6. The hydroxyl radical(·OH) and superoxide anion radical (O-2·), scavengingability of the extract of DLF using the optimization condition and the volatile oils ofDLF using supercritical CO2were determined.25.0mg/mL and1.8mg/mL alkaloidsof DLF showed the58.70%hydroxyl radical and49.26%superoxide anion radicalscavenging activity respectively, which IC50value is23.50mg/mLand1.75mg/mLrespectively. Comparing the hydroxyl radical scavenging activity of these extract at0.45ug/mL, they showed the following order: Vc> isocorydine> Rutin> extract ofDLF> volatile oils. When these extracts at0.50ug/mL, their hydroxyl radicalscavenging activity exhibited a different order: Vc> Rutin> volatile oils>isocorydine> extract of DLF.7. Anti-bacteria activity of alkaloids extraction of DLF was determined onEscherichia coli, Staphylococcus, Candida albicans and Pseudomonas aeruginosausing the bacteria-grow-curve test, slip method, MIC, MBC, IC50, the permeability ofEscherichia coli and morphology, transmission electron microscope observation. Theresults showed that the MIC of alkaloids extraction of DLF on Escherichia coli,Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa are0.28g/mL,0.20g/mL,0.20g/mL and0.30g/mL respectively, the IC50are0.2162g/mL,0.1805g/mL,0.1805g/mL and0.2604g/mL respectively. After treatment with thealkaloids extraction of DLF, the cells permeability of E.coli were obvious improved,making its morphological change.Finally, the thalli were splited.all these showed that this extract has a good anti-bacteria activity. The effect of flavonoid (Rutin) extractedfrom DLF on electrochemical behavior of lipid bilayer membranes was investigatedby Cyclic Voltammetry (CV) and SECM using s-BLM supported by Pt/C electrode asbio-membrane model, Fe(CN)3-/4-6as probe. We found that there are stronginteractions between s-BLM and rutin; meanwhile the flavone compounds have someextent effect on the electrochemical behavior of bilayer lecithin membrane. Theseresults provide some theory for further understanding the bioactivity of DLF and itsmechanism as drugs.8. The water-soluble external preparation of DLF exhibited the good effect onthe goat with orf. After treatment with different doses of alkaloids extraction, the heart,the liver, the spleen, the lung, the kidney and the muscular tissue in injection wereharvested at different periods, then stain all the tissue with HE, we found it presentedobvious acute inflammation and in injection part of muscular tissue, however thereare no obvious pathological changes in all the tissues. All these showed that theextract of DLF is safe for goat and mice and provideed some evidence for the clinicalpractice.All the results provided some important experimental and theoretical supports forfurther and deeper exploitation and utilization of DLF as the special medicinal plantin southeast region of Gansu province.更多还原