| Achyranthes bidentata is the dried root of Achyranthes bidentata BL.It has the functions of removing blood stasis and dredging meridians,tonifying liver and kidney,strengthening muscles and bones,diuresis and draining blood.After processing,it can enhance the functions of removing blood stasis and dredging meridians,relieving pain and so on.Achyranthes bidentata is a kind of Chinese herbal medicine with high medicinal value.It has the specifications of Achyranthes bidentata,Achyranthes bidentata,Achyranthes bidentata,Achyranthes bidentata,Achyranthes bidentata,Achyranthes bidentata charcoal and so on.Among them,raw Achyranthes bidentata and Achyranthes bidentata are the main specifications.At present,the research on Achyranthes bidentata mainly focuses on raw Achyranthes bidentata,and there is still a lack of related research on processed products,and the inconsistency and uncertainty in processing technology,quality specification,production demand of decoction pieces and clinical use demand lead to the lack of unity in production technology and quality standard of Achyranthes bidentata,and great differences in quality.Achyranthes bidentata polysaccharide is one of the higher active components in Achyranthes bidentata,and the polysaccharide has obvious changes after processing.In this paper,raw Achyranthes bidentata and wine Achyranthes bidentata were taken as the research objects,and the extraction,separation and purification,chemical composition research and quality evaluation of Achyranthes bidentata polysaccharide after wine roasting were studied,so as to provide reference value for the comprehensive development and utilization of Achyranthes bidentata.Experimental methods:1.AHP,CRITICAL and AHP-CRITICAL mixed weighting method were used to determine the weight,and UV spectrophotometry was used to determine the content of polysaccharide,uronic acid and protein.Box Behnken response surface methodology was used to optimize the extraction conditions of Achyranthes bidentata polysaccharides.2.Based on the 2020 edition of Chinese Pharmacopoeia,the identification,inspection,extract,content determination of Achyranthes bidentata and wine Achyranthes bidentata were determined,and the contents of polysaccharide,uronic acid and protein were added.The results were analyzed by normal analysis and variance analysis,the content limit was re established,and the influence of origin and processing method on Achyranthes bidentata was discussed.3.Establish the infrared and HPLC fingerprints of Achyranthes bidentata before and after wine processing,and improve the quality evaluation system of Achyranthes bidentata and its processed wine tasting Achyranthes bidentata by using the methods of cluster analysis,principal component analysis（PCA）,partial least squares regression（PLS）.4.Cation Anion tandem chromatography and DEAE cell-52 Ion exchange fiber column chromatography was used to separate and purify the total polysaccharides of Achyranthes bidentata.After the purity of the components was identified by HPLC,the molecular weight of the homogeneous polysaccharide was determined.The content of free protein and nucleic acid in the homogeneous polysaccharide was determined by UV spectrophotometry.The functional group of the polysaccharide was determined by infrared spectrophotometry to determine whether it contained the characteristic peak of the polysaccharide.The PMP pre column derivatization was determined by HPLC The composition of monosaccharide in polysaccharide.Experimental result:1.Taking the comprehensive evaluation of polysaccharide,uronic acid and protein content as the index,combined with the results of single factor analysis,box Behnken response surface method was used to further optimize the extraction conditions of Achyranthes bidentata polysaccharides.The results showed that the optimal extraction conditions were as follows:the extraction times were 2,the solid-liquid ratio was 1:15g/m L,and the extraction time was 3 h.2.The identification,inspection,extract,content determination and other inspection items of Achyranthes bidentata before and after wine roasting all meet the requirements of Chinese Pharmacopoeia（2020Edition）,and the quality of the medicinal materials is qualified.Through normal analysis,the content indexes of water,extract,β-ecdysterone,polysaccharide,uronic acid and protein before and after wine roasting of Achyranthes bidentata were found to follow normal distribution.On this basis,the content limits of water,extract,β-ecdysterone,polysaccharide,uronic acid and protein before and after wine roasting of Achyranthes bidentata were re established.The water content limit of Achyranthes bidentata before and after wine roasting was set at no more than 12.07%The content limit ofβ-ecdysterone should not be less than 0.033%,the content limit of Achyranthes bidentata extract should not be less than6.75%,the content limit of Achyranthes bidentata extract should not be less than 7.98%,the content limit of polysaccharide should not be less than 26.03%,the content limit of glycolic acid should not be less than23.64%,and the content limit of protein should not be less than 1.0%2%。Through the analysis of variance,it can be seen that there is no significant difference in water content,extract content,polysaccharide content,uronic acid content and protein content among different producing areas;different processing methods have no significant effect on polysaccharide content and uronic acid content;after wine roasting,water content decreases,extract content increases and protein content decreases.For protein content,the content of Achyranthes bidentata from Henan and Hebei increased after wine processing,but the content of Achyranthes bidentata from Shanxi had no effect;forβ-ecdysterone content,the content of Achyranthes bidentata from Shanxi was higher than that from Hebei and Henan,and after processing,the content of Achyranthes bidentata from Shanxi was higher than that from Hebei and Henan The amount of water has decreased.3.The infrared and HPLC fingerprints of Achyranthes bidentata before and after wine frying were established.The similarity results combined with stoichiometric analysis showed that the similarity of Achyranthes bidentata before and after processing was high,and the chemical composition had little difference.After wine frying,the content of Achyranthes bidentata increased,indicating that the chemical composition of Achyranthes bidentata from different producing areas and processing methods had good similarity,but the content had great difference4.The crude polysaccharides of Achyranthes bidentata were purified step by step by Cation Anion tandem chromatography and DEAE cellulose-52 ion exchange fiber column chromatography.After purity determination,six homogeneous polysaccharides were obtained,named Tn-5,otn-5,otn-6,ftn-5,ttn-6,ttn-7,with molecular weights of 5.96×107,3.32×106,2.44×106,1.07×106,5.02×106,5.94×106 da.UV detection showed that the six homogeneous polysaccharides had no absorption at the wavelength of 260 and 280cm-1,and did not contain protein,nucleic acid and other components;infrared detection showed that there were multiple characteristic absorption peaks of polysaccharides in the region of 4000-400cm-1,which proved that they were polysaccharide compounds.The results of PMP derivatization showed that FTN-5,TTN-6 and TTN-7 were all composed of 10monosaccharides,including mannose,rhamnose,ribose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose,xylose and fucose;OTN-5 and TN-5 could not detect glucuronic acid;OTN-6 could not detect glucuronic acid,galacturonic acid and fucose.Experimental conclusion:In this paper,the comprehensive evaluation method and response surface method were used to optimize the extraction process of Achyranthes bidentata polysaccharides,which can further save materials,save time and improve production efficiency;the crude polysaccharides were gradually purified,and the chemical structure of Achyranthes bidentata polysaccharides after wine processing was preliminarily determined by UV,IR,HPLC and other means Identification,inspection,extract,content determination and other items of Achyranthes bidentata in Chinese Pharmacopoeia（2020 Edition）confirmed that the quality of Achyranthes bidentata and wine Achyranthes bidentata pieces was qualified.At the same time,the content of polysaccharide,uronic acid,protein and other items were increased,and HPLC and infrared fingerprint were established to improve the quality evaluation system of Achyranthes bidentata and its processed wine Achyranthes bidentata. |
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