肉苁蓉、草苁蓉活性成分和指纹图谱研究
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摘要
肉苁蓉（Cistanche deserticola Ma）和草苁蓉（Boschniakia rossica）均是我国传统著名中药，二者同为列当科药用植物，分属肉苁蓉属和草苁蓉属，均具有补肾壮阳、保护肝脏、润肠通便和滋补强身等功效。但是，大量现代研究表明，肉苁蓉和草苁蓉中化学成分的组成和含量均有较大差别。由于近年来对草苁蓉的开发力度不足、重视不够，使草苁蓉的研究明显滞后于对肉苁蓉的开发。而对肉苁蓉功效的过度宣传，使得国内外市场需求急剧增加，出现极为严重的乱挖滥采现象，导致肉苁蓉野生资源几近丧失殆尽。《中国药典》（2010年版）中收载的肉苁蓉质量标准指标单一且缺乏专属性，不能真正反映其内在质量；而对于草苁蓉并未被收录。五味子是传统的抗肝损伤中药材，药理作用明确，疗效显著，广为熟知。
本文将以肉苁蓉多糖和草苁蓉多糖为研究对象，对其提取方法进行优化筛选、所得粗多糖进行精制纯化、并测定精制多糖的理化性质以及其在体内外的药理活性；以五味子木脂素成分作为阳性对照药材，以评价肉苁蓉药材和草苁蓉药材的生物学活性。此外，本文将采用光谱手段、色谱手段和DNA分子标记技术对肉苁蓉和草苁蓉建立全面的中药指纹图谱库，为二者的评价和质量控制提供更全面、科学、有效的方法。
本研究获得国家科技部重大专项课题-重大新药创制“中药药效物质基础及物质资源库研究关键技术”资助，主要研究内容如下：
1.通过对不同提取方式的比较，最终采用热回流的方式对肉苁蓉和草苁蓉中粗多糖进行提取。在采用回流提取的基础上，使用单因素实验考察和[L9（34）]正交实验设计对回流过程中涉及的影响条件进行优化，通过对结果的分析以及优化结果的验证，最终得到二者多糖组分的最佳提取工艺。肉苁蓉多糖的最佳提取工艺：回流提取，料液比为1:12，石油醚提取2次，每次1小时，80%乙醇溶液回流提取2次，每次1小时；将两次回流所得提取物过滤，所得滤渣使用蒸馏水再次回流提取2次，每次2小时；90%终浓度的乙醇进行沉淀反应。最终肉苁蓉粗多糖得率为12.35%。草苁蓉多糖的最佳提取工艺：回流提取，料液比为1:10；石油醚提取2次，每次1小时；80%乙醇回流提取2次，每次1小时；两次回流所得残渣蒸馏水回流提取3次，每次1.5小时；90%终浓度的乙醇进行沉淀反应。最终草苁蓉粗多糖得率达7.95%。通过对回流法提取肉苁蓉粗多糖和草苁蓉粗多糖的优化，该方法很适合在实验室中操作，并具有设备简单、操作方便、提取率较高的优点。该提取工艺的建立为后续的粗多糖纯化获得精制多糖以及进行肉苁蓉多糖和草苁蓉多糖的药理实验奠定了坚实的物质基础。优化筛选五味子木脂素的最佳提取工艺，同样选定回流的提取方式，对提取条件进行了优选：分别选择提取溶剂浓度、溶媒倍数、提取次数、提取时间四个因素，进行L9[(34)]正交实验设计，通过正交分析和方差分析确定各因素对五味子木脂素提取率的影响。确定了最佳提取条件为：使用12倍的95%的乙醇回流提取3次，每次1.5h，并对优化方案进行了实验验证。
2.对肉苁蓉多糖、草苁蓉多糖同五味子木脂素成分的抗急性肝损伤活性比较。以CC14所致急性肝损伤小鼠为模型，以上述多糖和木脂素对CCl4致急性肝损伤小鼠进行治疗，观察正常小鼠同肝损伤小鼠的肝脏和血液中生物指标的不同。实验过程中，不同剂量的肉苁蓉多糖、草苁蓉多糖和五味子木脂素对小鼠血液中ALT和AST的含量有不同程度的降低作用，并可不同程度降低肝组织中MDA的含量，降低肝组织中NO的活性，提高肝组织中SOD的活性。通过对小鼠肝组织HE染色切片观察可以发现，多糖可以减轻肝细胞肿胀、炎细胞浸润的程度。综上这些指标数据，与传统的抗肝损伤中药材五味子相比较，均提示肉苁蓉多糖和草苁蓉多糖对CC14致急性肝损伤有一定的抵抗作用。
3.肉苁蓉粗多糖和草苁蓉粗多糖的纯化。首先，对多糖中的蛋白质成分进行清除，本实验中采用Sevag法脱蛋白，在反复考察萃取次数的基础上，获得去除蛋白质成分的纯化肉苁蓉和草苁蓉粗多糖。其次，通过多种层析色谱柱相结合的方式，对粗多糖逐步纯化。肉苁蓉粗多糖经DEAE-cellulose柱层析和Sepharose CL-6B柱层析，最终获得肉苁蓉多糖CDPS-A1和CDPS-A2，得率分别为1.25%和2.79%。草苁蓉多糖经DEAE-Sepharose和Sepharose CL-6B柱层析，终得草苁蓉多糖BRPS-A1、BRPS-B1和BRPS-B2，得率分别为1.75%，1.97%，0.89%。
4.精制多糖理化性质的测定。使用高效体积排阻色谱（HPSEC）法测定各精制多糖的纯度和分子量分布，结果显示，肉苁蓉精制多糖CDPS-A1和CDPS-A2，草苁蓉精制多糖BRPS-A1、BRPS-B1和BRPS-B2在HPSEC色谱图中色谱峰呈单一对称色谱峰，这表明所得5份精制多糖纯度较高，且分子量分布均一，其分子量(Mw)分别为：4.52×10~4Da、1.1×10~5Da、1.2×10~4Da、3.0×10~4Da和2.1×10~5Da。对五种分子量均一的精制多糖理化性质进行测定，首先测定多糖中糖醛酸含量，结果为CDPS-A2和BRPS-A1中不含有糖醛酸成分，CDPS-A1、BRPS-B1和BRPS-B2中含有的糖醛酸含量分别为0.82%，23.2%和25.3%。其次，使用气相色谱法测定精制多糖的单糖组成，结果为肉苁蓉多糖CDPS-A1由鼠李糖（Rha）、阿拉伯糖（Ara）、半乳糖（Gal）和葡萄糖（Glu）以12:15:37:26组成；CDPS-A2由阿拉伯糖（Ara）、半乳糖（Gal）和葡萄糖（Glu）以20:38:41的比例组成。草苁蓉多糖BRPS-A1由阿拉伯糖（Ara）、甘露糖（Man）、半乳糖（Gal）、葡萄糖（Glu）和木糖（Xyl）以13:22:26:30:10的比例组成；BRPS-B1由甘露糖（Man）、半乳糖（Gal）、葡萄糖（Glu）和葡萄糖醛酸（Glu acid）以8:20:50:21组成；BRPS-B2由半乳糖（Gal）、葡萄糖（Glu）和葡萄糖醛酸（Gluacid）以23:52:24组成。再次，对五种精制多糖中的蛋白质百分含量进行测定，结果CDPS-A1、CDPS-A2、BRPS-A1、BRPS-B1和BRPS-B2的蛋白含量分别为3.2%、1.8%、6.0%、3.8%和5.5%。经查询相关文献比对，肉苁蓉多糖CDPS-A1、CDPS-A2和草苁蓉多糖BRPS-A1、BRPS-B1、BRPS-B2均为首次从肉苁蓉和草苁蓉中分离纯化得到的产物。
5.肉苁蓉和草苁蓉多糖的抗氧化活性。将多糖CDPS-A1、CDPS-A2、BRPS-A1、BRPS-B1和BRPS-B2进行体外抗氧化作用测试，测试项目包括：对DPPH、超氧阴离子和氢自由基的清除作用。测定结果显示各种多糖组分均具有一定的抗氧化活性，但抗氧化能力有一定差异。肉苁蓉多糖和草苁蓉多糖明显的清除自由基作用，有可能是其在动物体内保护受损肝脏组织的生物学基础。
6.建立了肉苁蓉和草苁蓉红外（IR）指纹图谱，并用该方法对共计16批样品（肉苁蓉药材10批，草苁蓉药材6批）进行指纹图谱分析。以各批次药材的平均光谱作为红外参考光谱（R），使用“OPUS”软件进行相似度分析。10批肉苁蓉药材指纹图谱与参考图谱（R）相似度范围为（98.09~99.85%），6批草苁蓉药材指纹图谱与参考图谱（R）相似度为（99.45~99.92%），两种药材的参考图谱进行相似度比较，相似度为89.43%。可见红外指纹图谱可有对肉苁蓉药材和草苁蓉药材进行有效区分。同时，应用聚类分析法对各批次药材进行分析，两种药材被相应分为两大类，这更加说明了红外指纹图谱法可快速、有效对肉苁蓉和草苁蓉药材进行区分鉴别。
7.建立了肉苁蓉和草苁蓉HPLC指纹图谱。对肉苁蓉的研究，共确定了10个共有峰为特征色谱峰，指认了其中3种成分，用所建立的方法对10批药材进行相似度分析，相似度范围在85.39~98.63%之间。对草苁蓉的研究，共确定了6个共有峰为特征色谱峰，指认了其中2种成分，用所建立的方法对6批药材进行相似度分析，相似度范围在97.64~99.98%之间。
8.应用随机扩增DNA多态性分析（RAPD）技术对3批肉苁蓉药材和3批草苁蓉药材样品进行DNA指纹分析，从40条引物中筛选出4条重复性好、扩增条带多且清晰的引物。其中引物S19扩增结果多态性丰富、扩增条带特异性强，可区分肉苁蓉和草苁蓉药材。
综上所述，本课题以肉苁蓉和草苁蓉为主要研究对象，对二者中主要的活性成分，即多糖组分进行研究。利用急性肝损伤动物模型，通过传统经典抗肝损伤中药五味子中木脂素成分作为阳性对照，评价二种多糖组分的预防和治疗急性肝损伤的能力。在确定其中多糖组分的最佳提取分离及纯化工艺的基础上，针对获取的精制多糖组分，研究了它们的理化性质及相关药理活性，证实所得肉苁蓉多糖和草苁蓉多糖均为首次发现，且这些多糖在体内具有保护肝脏的作用，在体外具有抗氧化活性。对肉苁蓉和草苁蓉药材建立相应指纹图谱，将两种易混淆药材进行快速、有效的鉴别，以便获取品质优秀的药材进行成分研究和活性测定。本研究为建立中药药效物质基础及物质资源库提供了肉苁蓉和草苁蓉药材的多糖资料，为深入开发这两种药材多糖组分成为保肝新药物及抗氧剂提供了理论依据；对我国传统中药肉苁蓉、草苁蓉的系统、深入开发研究，提供了一定的理论参考。
Chinese medicine in China, both for the orobanchaceae medicinal plants, belong todesertliving Cistanche genus and Boschniakia, having kidney strong sun, runchangpurge and nourishing strength etc.However, a large number of modern research showsthat c.deserticola and boschniakia have significant difference between thecomposition and content of the chemical composition.Because in recent years toBoschniakia development dynamics is insufficient, and the attention is not enough,make Boschniakia research significantly lags behind the development ofc.deserticola.And efficacy of c.deserticola excessive publicity, make domestic andinternational market demand has increased dramatically, appear very seriousdisorderly dig coyoting phenomenon, lead to wild resources of c.deserticola nearlylost."Chinese pharmacopoeia (2010edition), which contains quality of c.deserticolasingle standard index and lack of specificity, can't really reflect its intrinsic quality.ForBoschniakia not included.This paper will use the spectrum method andchromatographic method and DNA molecular marker techniques of c.deserticola andBoschniakia traditional medicine fingerprint library, establish a comprehensiveevaluation and quality control for both to provide a more comprehensive, scientificand effective method.In addition, this paper will with polysaccharide cistanchedeserticola and Boschniakia polysaccharide as the research object, the extractionmethod was optimized screening, the crude polysaccharides were refined purification,and determination of physical and chemical properties of purified polysaccharide andits pharmacological activities in the body.This research obtains the national ministryof science and technology major special project-significant drug discovery"traditional Chinese medicine medicinal material base and material database keytechnology research", the main research content is as follows:
1.By comparing different ways of extract, eventually adopt the way of hot refluxof c. deserticola and Boschniakia coarse polysaccharide in the extraction. In by reflux extraction, on the basis of using the single factor experiment investigation and [L9(34)] orthogonal design of experiment involving in the reflow process conditions wereoptimized, the influence of by the results of the analysis and the optimization resultsof validation, finally got the best extraction technology of polysaccharide composition.The best extraction technology of polysaccharide cistanche deserticola: refluxextraction, ratio of feed liquid1:12, petroleum ether extract2times, each time1hour,80%ethanol solvent reflux extraction2times, each time1hour; Will return twice theextract filter, the filter residue with distilled water reflux extraction again2times,each time2hours; Final concentration of90%ethanol precipitation reaction. C.deserticola eventually crude polysaccharide yield of12.35%. Boschniakia: the bestextraction process of polysaccharides extracted, and solid-liquid ratio of1:10;Petroleum ether extract2times, each time1hour;80%ethanol reflux extraction2times, each time1hour; Two return the residual distilled water reflux extracting3times,1.5hours every time; Final concentration of90%ethanol precipitation reaction.Eventually Boschniakia coarse polysaccharide yield of7.95%. By reflux extraction ofcistanche deserticola crude polysaccharide and Boschniakia crude polysaccharidesoptimization, this method is very suitable for operation in the laboratory, and has theadvantages of simple equipment, convenient operation, high extraction yield. Theestablishment of the extraction process for subsequent coarse polysaccharide purifiedto obtain refined sugar and polysaccharide cistanche deserticola and Boschniakiapolysaccharide of pharmacological experiment laid a solid material foundation.Optimum extraction technology of optimization screening wooden fat element alsoselected reflux extraction method, the optimum extraction conditions: concentrationof extraction solvent, selected solvent ratio and extraction times, extracting time, fourfactors, carries on the L9(34) orthogonal experiment design, determined by orthogonalanalysis and variance analysis on various factors mixed ZiMu influence the extractionrate of fat. The best extraction conditions were determined as follows: using12times95%ethanol reflux extraction3times, each time1.5h, and experiments have beencarried out to verify the optimization scheme.
2. C. deserticola polysaccharide, polysaccharide Boschniakia and mixed graincomposition of ZiMu fat comparing activity against acute liver damage. Is model withacute liver injury in mice caused by CC14, polysaccharide and wooden fat element intreatment of CCl4induced acute liver injury in mice, and observe the normal mice with liver injury in mice liver and blood biological indicators. Experiments, differentdoses of c. deserticola polysaccharide, polysaccharide Boschniakia and tasty ZiMu fatin mice blood ALT and AST levels have different degrees of lower effect, and canreduce MDA content in liver tissue in varying degrees, reducing the activity of livertissues NO, improve the activity of SOD in the liver tissue. Based on mice liver HEstaining observation can be found that the polysaccharide can reduce the degree ofliver cell swelling, inflammatory cells infiltration. All these indicators data, comparedwith the traditional Chinese medicinal materials of anti liver injury fructusschisandrae, suggests that c. deserticola and Boschniakia polysaccharide with CC14toacute liver injury has certain resistance.
3. C. deserticola coarse polysaccharide and Boschniakia coarse polysaccharidepurified. First of all, to remove the protein in crude polysaccharide composition, thisexperiment adopts the Sevag method protein, in repeatedly inspected extractionnumber of times, on the basis of removing proteins purification of c. deserticola andBoschniakia coarse polysaccharide. Secondly, through a variety of chromatographiccolumn chromatography is a combination of, the coarse polysaccharide purificationstep by step. C. deserticola crude polysaccharides by DEAE cellulose columnchromatography and Sepharose CL-6b column chromatography, and eventually getpolysaccharide cistanche deserticola CDPS-A1and CDPS-A2, yield of1.25%and2.79%respectively. Boschniakia polysaccharide by DEAE Sepharose and Sepharosecolumn chromatography CL-6b, she gains Boschniakia polysaccharide BRPS-A1,BRPS-B1and BRPS-B2, rate were1.75%,1.97%,0.89%.
4. The refined polysaccharide content determination of physical and chemicalproperties. Using efficient size exclusion chromatography (HPSEC) method fordetermining the purity and molecular weight distribution of the purifiedpolysaccharide, results showed that c. deserticola CDPS refined polysaccharides A1and CDPS-A2, Boschniakia BRPS refined polysaccharides A1, B1and BRPS BRPS-B2in HPSEC chromatographic peak in the chromatogram is single symmetricalchromatographic peak, which suggests that the five refined polysaccharide purity ishigher, and uniform molecular weight distribution, its molecular weight (Mw)respectively as follows:4.52×104Da、1.1×105Da、1.2×104Da、3.0×104Da和2.1×105Da. The physical and chemical properties of five kinds of molecular weight ofuniformly refined polysaccharide were determined, the determination of uronic acid in polysaccharide content in the first, the results of CDPS-BRPS-A1and A2does notcontain uronic acid composition, CDPS-A1, B1and BRPS BRPS--B2containuronic acid content was0.82%,23.2%and25.3%. Secondly, the use of gaschromatography determination of monosaccharide composition of polysaccharidepurified results of polysaccharide cistanche deserticola CDPS-A1composed ofrhamnose (Rha), Arabian sugar (Ara), galactose (Gal) and glucose (Glu) to12:15:37:26; CDPS-A2by Arab (Ara), sugar galactose (Gal) and glucose (Glu) inproportion of20:38:41. BRPS Boschniakia polysaccharides A1by Arab (Ara) sugar,mannose and galactose (Man)(Gal), glucose (Glu) and xylose (Xyl) to13:22:26:"theproportion of composition; BRPS-B1by mannose, galactose (Man)(Gal), glucose(Glu) and glucuronic acid (Glu acid) with8:20:50:21; BRPS-B2by galactose (Gal),glucose (Glu) and glucuronic acid (Glu acid) to23:52:24composition. Again, proteinpercentage of five kinds of refined polysaccharides were determined, the results ofCDPS-A1, the CDPS-A2, BRPS-A1, B1and BRPS BRPS--B2protein were3.2%,1.8%,6.0%,3.8%and5.5%. The query literature, polysaccharide cistanchedeserticola CDPS-A1, the CDPS-BRPS-A1, A2and Boschniakia polysaccharidesBRPS-B1, BRPS-B2were first isolated from c. deserticola and Boschniakiapurified product.
5. C. deserticola and Boschniakia polysaccharide antioxidant activity.Polysaccharide to CDPS-A1, the CDPS-A2, BRPS-A1, B1and BRPS BRPS--B2in vitro antioxidant effect test, test items include: on DPPH, superoxide anion andhydrogen free radical scavenging effect. Measurement results show that all kinds ofall have certain antioxidant activity of polysaccharide component, but antioxidantcapacity has a certain difference. C. deserticola polysaccharide and Boschniakiaobvious scavenging free radicals, may be in the damaged liver tissue the biologicalbasis of animal protection.
6. Set up c. deserticola and Boschniakia infrared (IR) fingerprints, using themethod of a total of16samples (c. deserticola10batches of herbs, Boschniakia herbs6batch) fingerprint analysis. With an average spectrum of each batch herbs asinfrared reference spectra (R), use the "OPUS" software for similarity analysis.10batches of c. deserticola herbs fingerprint reference map (R) and similarity range(98.09~99.85%),6batch Boschniakia herbs fingerprint reference map (R) andsimilarity of (99.45~99.92%), two kinds of medicinal materials of reference map compare similarity, similarity of89.43%. Visible infrared fingerprint can have to thedistinction between c. deserticola medicinal materials and Boschniakia medicinalmaterials effectively. At the same time, the application of clustering analysis methodto analyze each batch herbs, corresponding to two kinds of medicinal materials weredivided into two categories, the more shows the infrared fingerprint method canquickly and effectively to distinguish identify c. deserticola and Boschniakiamedicinal materials.
7. C. deserticola and Boschniakia HPLC fingerprint is established. Studies of c.deserticola, determines the characteristics of10common peak peak, identified threekinds of ingredients, using the method of10batches of herbs for similarity analysis,similarity range between85.39~98.63%. Boschniakia research, has identified sixcommon peak characterized chromatographic peaks, identifying the two kinds ofingredients, using the method of the6batch of similarity analysis, similarity rangebetween97.64~99.98%.
8. Application of random amplified polymorphic DNA analysis (RAPD)technique on3batches of c. deserticola medicinal materials and2batch Boschniakiamedicinal materials samples for DNA fingerprinting, selected from40primer5repeatability and the clear primer amplification bands. With S28amplification resultsrich, amplification bands polymorphic primers specificity is strong, can distinguishbetween cistanche deserticola and Boschniakia medicinal materials.
To sum up, this topic in cistanche deserticola and Boschniakia as the mainresearch object, the main active ingredient of the two, namely the polysaccharidecomponents were studied. With acute hepatic injury animal model, through thetraditional classic anti liver damage wooden fat element compositions in Chinesetraditional medicine fructus schisandrae as positive control, evaluation of twopolysaccharide components of the ability of prevention and treatment of acute liverdamage. In determining the optimum extraction and separation and purification ofpolysaccharide composition, on the basis of to obtain refined polysaccharidescomponents, their physical and chemical properties was studied and the relatedpharmacological activity, confirmed that the polysaccharide cistanche deserticola andBoschniakia polysaccharide were found for the first time, and the polysaccharides inthe protective effect of liver in vivo and in vitro antioxidant activity. Of c. deserticolaand Boschniakia medicinal materials to establish fingerprint, corresponding to two kinds of confusing in the identification of medicinal materials for rapid, effective, inorder to obtain good quality of medicinal materials studied the composition andactivity determination. This study in order to establish Chinese medicine medicinalmaterial base and material resource provides the cistanche deserticola andBoschniakia herbs polysaccharides material, for the further development of these twokinds of medicinal herbs polysaccharides components become a new drugs andantioxidants protect liver to provide a theoretical basis; To our country system andindepth development of the traditional Chinese medicine cistanche deserticola,Boschniakia research, provides certain theoretical reference.
本文将以肉苁蓉多糖和草苁蓉多糖为研究对象，对其提取方法进行优化筛选、所得粗多糖进行精制纯化、并测定精制多糖的理化性质以及其在体内外的药理活性；以五味子木脂素成分作为阳性对照药材，以评价肉苁蓉药材和草苁蓉药材的生物学活性。此外，本文将采用光谱手段、色谱手段和DNA分子标记技术对肉苁蓉和草苁蓉建立全面的中药指纹图谱库，为二者的评价和质量控制提供更全面、科学、有效的方法。
本研究获得国家科技部重大专项课题-重大新药创制“中药药效物质基础及物质资源库研究关键技术”资助，主要研究内容如下：
1.通过对不同提取方式的比较，最终采用热回流的方式对肉苁蓉和草苁蓉中粗多糖进行提取。在采用回流提取的基础上，使用单因素实验考察和[L9（34）]正交实验设计对回流过程中涉及的影响条件进行优化，通过对结果的分析以及优化结果的验证，最终得到二者多糖组分的最佳提取工艺。肉苁蓉多糖的最佳提取工艺：回流提取，料液比为1:12，石油醚提取2次，每次1小时，80%乙醇溶液回流提取2次，每次1小时；将两次回流所得提取物过滤，所得滤渣使用蒸馏水再次回流提取2次，每次2小时；90%终浓度的乙醇进行沉淀反应。最终肉苁蓉粗多糖得率为12.35%。草苁蓉多糖的最佳提取工艺：回流提取，料液比为1:10；石油醚提取2次，每次1小时；80%乙醇回流提取2次，每次1小时；两次回流所得残渣蒸馏水回流提取3次，每次1.5小时；90%终浓度的乙醇进行沉淀反应。最终草苁蓉粗多糖得率达7.95%。通过对回流法提取肉苁蓉粗多糖和草苁蓉粗多糖的优化，该方法很适合在实验室中操作，并具有设备简单、操作方便、提取率较高的优点。该提取工艺的建立为后续的粗多糖纯化获得精制多糖以及进行肉苁蓉多糖和草苁蓉多糖的药理实验奠定了坚实的物质基础。优化筛选五味子木脂素的最佳提取工艺，同样选定回流的提取方式，对提取条件进行了优选：分别选择提取溶剂浓度、溶媒倍数、提取次数、提取时间四个因素，进行L9[(34)]正交实验设计，通过正交分析和方差分析确定各因素对五味子木脂素提取率的影响。确定了最佳提取条件为：使用12倍的95%的乙醇回流提取3次，每次1.5h，并对优化方案进行了实验验证。
2.对肉苁蓉多糖、草苁蓉多糖同五味子木脂素成分的抗急性肝损伤活性比较。以CC14所致急性肝损伤小鼠为模型，以上述多糖和木脂素对CCl4致急性肝损伤小鼠进行治疗，观察正常小鼠同肝损伤小鼠的肝脏和血液中生物指标的不同。实验过程中，不同剂量的肉苁蓉多糖、草苁蓉多糖和五味子木脂素对小鼠血液中ALT和AST的含量有不同程度的降低作用，并可不同程度降低肝组织中MDA的含量，降低肝组织中NO的活性，提高肝组织中SOD的活性。通过对小鼠肝组织HE染色切片观察可以发现，多糖可以减轻肝细胞肿胀、炎细胞浸润的程度。综上这些指标数据，与传统的抗肝损伤中药材五味子相比较，均提示肉苁蓉多糖和草苁蓉多糖对CC14致急性肝损伤有一定的抵抗作用。
3.肉苁蓉粗多糖和草苁蓉粗多糖的纯化。首先，对多糖中的蛋白质成分进行清除，本实验中采用Sevag法脱蛋白，在反复考察萃取次数的基础上，获得去除蛋白质成分的纯化肉苁蓉和草苁蓉粗多糖。其次，通过多种层析色谱柱相结合的方式，对粗多糖逐步纯化。肉苁蓉粗多糖经DEAE-cellulose柱层析和Sepharose CL-6B柱层析，最终获得肉苁蓉多糖CDPS-A1和CDPS-A2，得率分别为1.25%和2.79%。草苁蓉多糖经DEAE-Sepharose和Sepharose CL-6B柱层析，终得草苁蓉多糖BRPS-A1、BRPS-B1和BRPS-B2，得率分别为1.75%，1.97%，0.89%。
4.精制多糖理化性质的测定。使用高效体积排阻色谱（HPSEC）法测定各精制多糖的纯度和分子量分布，结果显示，肉苁蓉精制多糖CDPS-A1和CDPS-A2，草苁蓉精制多糖BRPS-A1、BRPS-B1和BRPS-B2在HPSEC色谱图中色谱峰呈单一对称色谱峰，这表明所得5份精制多糖纯度较高，且分子量分布均一，其分子量(Mw)分别为：4.52×10~4Da、1.1×10~5Da、1.2×10~4Da、3.0×10~4Da和2.1×10~5Da。对五种分子量均一的精制多糖理化性质进行测定，首先测定多糖中糖醛酸含量，结果为CDPS-A2和BRPS-A1中不含有糖醛酸成分，CDPS-A1、BRPS-B1和BRPS-B2中含有的糖醛酸含量分别为0.82%，23.2%和25.3%。其次，使用气相色谱法测定精制多糖的单糖组成，结果为肉苁蓉多糖CDPS-A1由鼠李糖（Rha）、阿拉伯糖（Ara）、半乳糖（Gal）和葡萄糖（Glu）以12:15:37:26组成；CDPS-A2由阿拉伯糖（Ara）、半乳糖（Gal）和葡萄糖（Glu）以20:38:41的比例组成。草苁蓉多糖BRPS-A1由阿拉伯糖（Ara）、甘露糖（Man）、半乳糖（Gal）、葡萄糖（Glu）和木糖（Xyl）以13:22:26:30:10的比例组成；BRPS-B1由甘露糖（Man）、半乳糖（Gal）、葡萄糖（Glu）和葡萄糖醛酸（Glu acid）以8:20:50:21组成；BRPS-B2由半乳糖（Gal）、葡萄糖（Glu）和葡萄糖醛酸（Gluacid）以23:52:24组成。再次，对五种精制多糖中的蛋白质百分含量进行测定，结果CDPS-A1、CDPS-A2、BRPS-A1、BRPS-B1和BRPS-B2的蛋白含量分别为3.2%、1.8%、6.0%、3.8%和5.5%。经查询相关文献比对，肉苁蓉多糖CDPS-A1、CDPS-A2和草苁蓉多糖BRPS-A1、BRPS-B1、BRPS-B2均为首次从肉苁蓉和草苁蓉中分离纯化得到的产物。
5.肉苁蓉和草苁蓉多糖的抗氧化活性。将多糖CDPS-A1、CDPS-A2、BRPS-A1、BRPS-B1和BRPS-B2进行体外抗氧化作用测试，测试项目包括：对DPPH、超氧阴离子和氢自由基的清除作用。测定结果显示各种多糖组分均具有一定的抗氧化活性，但抗氧化能力有一定差异。肉苁蓉多糖和草苁蓉多糖明显的清除自由基作用，有可能是其在动物体内保护受损肝脏组织的生物学基础。
6.建立了肉苁蓉和草苁蓉红外（IR）指纹图谱，并用该方法对共计16批样品（肉苁蓉药材10批，草苁蓉药材6批）进行指纹图谱分析。以各批次药材的平均光谱作为红外参考光谱（R），使用“OPUS”软件进行相似度分析。10批肉苁蓉药材指纹图谱与参考图谱（R）相似度范围为（98.09~99.85%），6批草苁蓉药材指纹图谱与参考图谱（R）相似度为（99.45~99.92%），两种药材的参考图谱进行相似度比较，相似度为89.43%。可见红外指纹图谱可有对肉苁蓉药材和草苁蓉药材进行有效区分。同时，应用聚类分析法对各批次药材进行分析，两种药材被相应分为两大类，这更加说明了红外指纹图谱法可快速、有效对肉苁蓉和草苁蓉药材进行区分鉴别。
7.建立了肉苁蓉和草苁蓉HPLC指纹图谱。对肉苁蓉的研究，共确定了10个共有峰为特征色谱峰，指认了其中3种成分，用所建立的方法对10批药材进行相似度分析，相似度范围在85.39~98.63%之间。对草苁蓉的研究，共确定了6个共有峰为特征色谱峰，指认了其中2种成分，用所建立的方法对6批药材进行相似度分析，相似度范围在97.64~99.98%之间。
8.应用随机扩增DNA多态性分析（RAPD）技术对3批肉苁蓉药材和3批草苁蓉药材样品进行DNA指纹分析，从40条引物中筛选出4条重复性好、扩增条带多且清晰的引物。其中引物S19扩增结果多态性丰富、扩增条带特异性强，可区分肉苁蓉和草苁蓉药材。
综上所述，本课题以肉苁蓉和草苁蓉为主要研究对象，对二者中主要的活性成分，即多糖组分进行研究。利用急性肝损伤动物模型，通过传统经典抗肝损伤中药五味子中木脂素成分作为阳性对照，评价二种多糖组分的预防和治疗急性肝损伤的能力。在确定其中多糖组分的最佳提取分离及纯化工艺的基础上，针对获取的精制多糖组分，研究了它们的理化性质及相关药理活性，证实所得肉苁蓉多糖和草苁蓉多糖均为首次发现，且这些多糖在体内具有保护肝脏的作用，在体外具有抗氧化活性。对肉苁蓉和草苁蓉药材建立相应指纹图谱，将两种易混淆药材进行快速、有效的鉴别，以便获取品质优秀的药材进行成分研究和活性测定。本研究为建立中药药效物质基础及物质资源库提供了肉苁蓉和草苁蓉药材的多糖资料，为深入开发这两种药材多糖组分成为保肝新药物及抗氧剂提供了理论依据；对我国传统中药肉苁蓉、草苁蓉的系统、深入开发研究，提供了一定的理论参考。
Chinese medicine in China, both for the orobanchaceae medicinal plants, belong todesertliving Cistanche genus and Boschniakia, having kidney strong sun, runchangpurge and nourishing strength etc.However, a large number of modern research showsthat c.deserticola and boschniakia have significant difference between thecomposition and content of the chemical composition.Because in recent years toBoschniakia development dynamics is insufficient, and the attention is not enough,make Boschniakia research significantly lags behind the development ofc.deserticola.And efficacy of c.deserticola excessive publicity, make domestic andinternational market demand has increased dramatically, appear very seriousdisorderly dig coyoting phenomenon, lead to wild resources of c.deserticola nearlylost."Chinese pharmacopoeia (2010edition), which contains quality of c.deserticolasingle standard index and lack of specificity, can't really reflect its intrinsic quality.ForBoschniakia not included.This paper will use the spectrum method andchromatographic method and DNA molecular marker techniques of c.deserticola andBoschniakia traditional medicine fingerprint library, establish a comprehensiveevaluation and quality control for both to provide a more comprehensive, scientificand effective method.In addition, this paper will with polysaccharide cistanchedeserticola and Boschniakia polysaccharide as the research object, the extractionmethod was optimized screening, the crude polysaccharides were refined purification,and determination of physical and chemical properties of purified polysaccharide andits pharmacological activities in the body.This research obtains the national ministryof science and technology major special project-significant drug discovery"traditional Chinese medicine medicinal material base and material database keytechnology research", the main research content is as follows:
1.By comparing different ways of extract, eventually adopt the way of hot refluxof c. deserticola and Boschniakia coarse polysaccharide in the extraction. In by reflux extraction, on the basis of using the single factor experiment investigation and [L9(34)] orthogonal design of experiment involving in the reflow process conditions wereoptimized, the influence of by the results of the analysis and the optimization resultsof validation, finally got the best extraction technology of polysaccharide composition.The best extraction technology of polysaccharide cistanche deserticola: refluxextraction, ratio of feed liquid1:12, petroleum ether extract2times, each time1hour,80%ethanol solvent reflux extraction2times, each time1hour; Will return twice theextract filter, the filter residue with distilled water reflux extraction again2times,each time2hours; Final concentration of90%ethanol precipitation reaction. C.deserticola eventually crude polysaccharide yield of12.35%. Boschniakia: the bestextraction process of polysaccharides extracted, and solid-liquid ratio of1:10;Petroleum ether extract2times, each time1hour;80%ethanol reflux extraction2times, each time1hour; Two return the residual distilled water reflux extracting3times,1.5hours every time; Final concentration of90%ethanol precipitation reaction.Eventually Boschniakia coarse polysaccharide yield of7.95%. By reflux extraction ofcistanche deserticola crude polysaccharide and Boschniakia crude polysaccharidesoptimization, this method is very suitable for operation in the laboratory, and has theadvantages of simple equipment, convenient operation, high extraction yield. Theestablishment of the extraction process for subsequent coarse polysaccharide purifiedto obtain refined sugar and polysaccharide cistanche deserticola and Boschniakiapolysaccharide of pharmacological experiment laid a solid material foundation.Optimum extraction technology of optimization screening wooden fat element alsoselected reflux extraction method, the optimum extraction conditions: concentrationof extraction solvent, selected solvent ratio and extraction times, extracting time, fourfactors, carries on the L9(34) orthogonal experiment design, determined by orthogonalanalysis and variance analysis on various factors mixed ZiMu influence the extractionrate of fat. The best extraction conditions were determined as follows: using12times95%ethanol reflux extraction3times, each time1.5h, and experiments have beencarried out to verify the optimization scheme.
2. C. deserticola polysaccharide, polysaccharide Boschniakia and mixed graincomposition of ZiMu fat comparing activity against acute liver damage. Is model withacute liver injury in mice caused by CC14, polysaccharide and wooden fat element intreatment of CCl4induced acute liver injury in mice, and observe the normal mice with liver injury in mice liver and blood biological indicators. Experiments, differentdoses of c. deserticola polysaccharide, polysaccharide Boschniakia and tasty ZiMu fatin mice blood ALT and AST levels have different degrees of lower effect, and canreduce MDA content in liver tissue in varying degrees, reducing the activity of livertissues NO, improve the activity of SOD in the liver tissue. Based on mice liver HEstaining observation can be found that the polysaccharide can reduce the degree ofliver cell swelling, inflammatory cells infiltration. All these indicators data, comparedwith the traditional Chinese medicinal materials of anti liver injury fructusschisandrae, suggests that c. deserticola and Boschniakia polysaccharide with CC14toacute liver injury has certain resistance.
3. C. deserticola coarse polysaccharide and Boschniakia coarse polysaccharidepurified. First of all, to remove the protein in crude polysaccharide composition, thisexperiment adopts the Sevag method protein, in repeatedly inspected extractionnumber of times, on the basis of removing proteins purification of c. deserticola andBoschniakia coarse polysaccharide. Secondly, through a variety of chromatographiccolumn chromatography is a combination of, the coarse polysaccharide purificationstep by step. C. deserticola crude polysaccharides by DEAE cellulose columnchromatography and Sepharose CL-6b column chromatography, and eventually getpolysaccharide cistanche deserticola CDPS-A1and CDPS-A2, yield of1.25%and2.79%respectively. Boschniakia polysaccharide by DEAE Sepharose and Sepharosecolumn chromatography CL-6b, she gains Boschniakia polysaccharide BRPS-A1,BRPS-B1and BRPS-B2, rate were1.75%,1.97%,0.89%.
4. The refined polysaccharide content determination of physical and chemicalproperties. Using efficient size exclusion chromatography (HPSEC) method fordetermining the purity and molecular weight distribution of the purifiedpolysaccharide, results showed that c. deserticola CDPS refined polysaccharides A1and CDPS-A2, Boschniakia BRPS refined polysaccharides A1, B1and BRPS BRPS-B2in HPSEC chromatographic peak in the chromatogram is single symmetricalchromatographic peak, which suggests that the five refined polysaccharide purity ishigher, and uniform molecular weight distribution, its molecular weight (Mw)respectively as follows:4.52×104Da、1.1×105Da、1.2×104Da、3.0×104Da和2.1×105Da. The physical and chemical properties of five kinds of molecular weight ofuniformly refined polysaccharide were determined, the determination of uronic acid in polysaccharide content in the first, the results of CDPS-BRPS-A1and A2does notcontain uronic acid composition, CDPS-A1, B1and BRPS BRPS--B2containuronic acid content was0.82%,23.2%and25.3%. Secondly, the use of gaschromatography determination of monosaccharide composition of polysaccharidepurified results of polysaccharide cistanche deserticola CDPS-A1composed ofrhamnose (Rha), Arabian sugar (Ara), galactose (Gal) and glucose (Glu) to12:15:37:26; CDPS-A2by Arab (Ara), sugar galactose (Gal) and glucose (Glu) inproportion of20:38:41. BRPS Boschniakia polysaccharides A1by Arab (Ara) sugar,mannose and galactose (Man)(Gal), glucose (Glu) and xylose (Xyl) to13:22:26:"theproportion of composition; BRPS-B1by mannose, galactose (Man)(Gal), glucose(Glu) and glucuronic acid (Glu acid) with8:20:50:21; BRPS-B2by galactose (Gal),glucose (Glu) and glucuronic acid (Glu acid) to23:52:24composition. Again, proteinpercentage of five kinds of refined polysaccharides were determined, the results ofCDPS-A1, the CDPS-A2, BRPS-A1, B1and BRPS BRPS--B2protein were3.2%,1.8%,6.0%,3.8%and5.5%. The query literature, polysaccharide cistanchedeserticola CDPS-A1, the CDPS-BRPS-A1, A2and Boschniakia polysaccharidesBRPS-B1, BRPS-B2were first isolated from c. deserticola and Boschniakiapurified product.
5. C. deserticola and Boschniakia polysaccharide antioxidant activity.Polysaccharide to CDPS-A1, the CDPS-A2, BRPS-A1, B1and BRPS BRPS--B2in vitro antioxidant effect test, test items include: on DPPH, superoxide anion andhydrogen free radical scavenging effect. Measurement results show that all kinds ofall have certain antioxidant activity of polysaccharide component, but antioxidantcapacity has a certain difference. C. deserticola polysaccharide and Boschniakiaobvious scavenging free radicals, may be in the damaged liver tissue the biologicalbasis of animal protection.
6. Set up c. deserticola and Boschniakia infrared (IR) fingerprints, using themethod of a total of16samples (c. deserticola10batches of herbs, Boschniakia herbs6batch) fingerprint analysis. With an average spectrum of each batch herbs asinfrared reference spectra (R), use the "OPUS" software for similarity analysis.10batches of c. deserticola herbs fingerprint reference map (R) and similarity range(98.09~99.85%),6batch Boschniakia herbs fingerprint reference map (R) andsimilarity of (99.45~99.92%), two kinds of medicinal materials of reference map compare similarity, similarity of89.43%. Visible infrared fingerprint can have to thedistinction between c. deserticola medicinal materials and Boschniakia medicinalmaterials effectively. At the same time, the application of clustering analysis methodto analyze each batch herbs, corresponding to two kinds of medicinal materials weredivided into two categories, the more shows the infrared fingerprint method canquickly and effectively to distinguish identify c. deserticola and Boschniakiamedicinal materials.
7. C. deserticola and Boschniakia HPLC fingerprint is established. Studies of c.deserticola, determines the characteristics of10common peak peak, identified threekinds of ingredients, using the method of10batches of herbs for similarity analysis,similarity range between85.39~98.63%. Boschniakia research, has identified sixcommon peak characterized chromatographic peaks, identifying the two kinds ofingredients, using the method of the6batch of similarity analysis, similarity rangebetween97.64~99.98%.
8. Application of random amplified polymorphic DNA analysis (RAPD)technique on3batches of c. deserticola medicinal materials and2batch Boschniakiamedicinal materials samples for DNA fingerprinting, selected from40primer5repeatability and the clear primer amplification bands. With S28amplification resultsrich, amplification bands polymorphic primers specificity is strong, can distinguishbetween cistanche deserticola and Boschniakia medicinal materials.
To sum up, this topic in cistanche deserticola and Boschniakia as the mainresearch object, the main active ingredient of the two, namely the polysaccharidecomponents were studied. With acute hepatic injury animal model, through thetraditional classic anti liver damage wooden fat element compositions in Chinesetraditional medicine fructus schisandrae as positive control, evaluation of twopolysaccharide components of the ability of prevention and treatment of acute liverdamage. In determining the optimum extraction and separation and purification ofpolysaccharide composition, on the basis of to obtain refined polysaccharidescomponents, their physical and chemical properties was studied and the relatedpharmacological activity, confirmed that the polysaccharide cistanche deserticola andBoschniakia polysaccharide were found for the first time, and the polysaccharides inthe protective effect of liver in vivo and in vitro antioxidant activity. Of c. deserticolaand Boschniakia medicinal materials to establish fingerprint, corresponding to two kinds of confusing in the identification of medicinal materials for rapid, effective, inorder to obtain good quality of medicinal materials studied the composition andactivity determination. This study in order to establish Chinese medicine medicinalmaterial base and material resource provides the cistanche deserticola andBoschniakia herbs polysaccharides material, for the further development of these twokinds of medicinal herbs polysaccharides components become a new drugs andantioxidants protect liver to provide a theoretical basis; To our country system andindepth development of the traditional Chinese medicine cistanche deserticola,Boschniakia research, provides certain theoretical reference.
引文
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