Study On The Active Constituent And Fingerpirnt Of Cistanche Deserticola Ma And Boschniakia Rossicaa

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�'�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.