| Rhubarb is a traditional Chinese herbal medicine variety,the major producing areas mainly inlcude Gansu,Qinghai,Shaanxi,Xizang,etc.Zhenba County,Shaanxi Province,is the largest rheum officinale planting base in China,but there is little research on quality standard and quality evaluation.This paper conducts the systematic research into the component analysis of Rheum officinale from Zhenba County.The strains of biodegradable Conjugated Anthraquinone compounds are found,and the technical parameters in the biotransformation are further optimized,providing a new green process for the enrichment and preparation of Free Anthraquinone monomers.This thesis also probes into the separation and purification of the glycosidase involved in the biotransformation and the basic characteristics of the enzyme.Major experiments and the results are listed as follows:?1?The polysaccharide content obtained through phenol-vitriolic colorimetry is 7.04%,the protein content obtained through which is obtained through coomassie brilliant blue staining is 0.35%,and the ash content that obtained according to GB 5009.4-2016 is 3.78%.The content of heavy metal lead,arsenic and arsenic in Rheum officinale obtained in the ICP emission spectrometer are 1.94,0.56,1.76 mg·kg-1,respectively.The content of the total anthraquinone and Free Anthraquinone are 1.79%and 1.07%respectively.Therefore,the result of the experiment shows that major indicators of the heavy metal in Rheum officinale conform to the quantity limitations that listed in the Pharmacopoeia of the People's Republic of China.?2?The main free and Conjugated Anthraquinone compounds are purified by high performance liquid chromatography?HPLC?.Five Free Anthraquinone monomers are prepared,i.e.aloe emodin?99.26%?,rhein?92.31%?,emodin?98.65%?,chrysophanol?99.75%?and emodin methyl ether?98.47%?.Two Conjugated Anthraquinones were prepared and compared with the standard samples,i.e.emodin-8-O-?-D-glucoside?97.65%?and rhein-8-O-?-D-glucoside?98.32%?.?3?The strains which can grow in Rheum officinaleare found in the natural environment,and three pure strains are obtained by using plate drawing method to purify and culture the dominant strains with vigorous growth.The three seperated strains are identified as Aspergillus aculeatus,Galactomyces and Aspergillus flavus by morphological observation and ITS sequence analysis.The results of preliminary biotransformation test shows that the it is feasible to transform the Conjungated Anthraquinone into Free Anthraquinone by the three strains,among which Aspergillus flavus promote the biotransformation rate to the highest level of above 60%.?4?The experiment of biotransformation by Aspergillus flavus is conducted.In this experiment,the biotransformation rate of the Conjungated Anthraquinone to the Free Anthraquinone is taken as an indicator to evaluate the impacts of time,temperature and inoculum amount on the biotransformation.On the basis of single factor experiment,the technical parameters in the biotransformation process are optimized through the response surface methodology.It is showed in the results that the optimum biotransformation can be obtained with inoculum volume of 8.4%under the temperature of 29.3?in 5 days.In such case,the aveage biotransformation rate of Free Anthraquinone can be as high as 73.2%.?5?The glucosidase is separated and purified by dextran gel column chromatography.The molecular weight of the enzyme is 68 kDa measured by SDS-polyacrylamide gel method,and the properties of the enzyme are studied.The optimum pH value and temperature of enzyme reaction are 7.0 and 35.0?,respectively.In vitro enzyme-catalyzed rhubarb and emodin-8-O-?-D-glucoside,it is shown in the experiment that the enzyme can hydrolyze the cleavage-binding anthraquinone sugar chain to form Free Anthraquinone.It is further proved that Aspergillus flavus produced anthraquinone from rhubarb-bound anthraquinone to Free Anthraquinone by secreting glycosidase to hydrolyze sugar side chain. |
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