Comparative Study On The Basic Structure And Immunocompetence Of The Polysaccharide In Polygonatum Sibiricum Before And After Processing With Wine

Processing is the traditional pharmaceutical method of Traditional Chinese medicines(TCMs),and steaming with wine is a key technology which can increase the immunocompetent of TCMs.Polysaccharides are one of immunocompetent components of TCMs.Because of the complex chemical structure and lack of effective qualitative and quantitative analysis methods of polysaccharides in TCMs,the research usually focuses on the changes in basic structure and content of monosaccharides or polysaccharides during the processing.In addition,the relative molecular mass(Mw)and its distribution,monosaccharide composition,functional group structure and glycosidic bond type of polysaccharides were reported to be closely related to their immunocompetent.Polygonatum sibiricum(PS)is the dried rhizomes of Polygonatum coll.et He msl.,P.sibiricum Red.or P.cyrtonema Hua.Processed Polygonatum sibiricum(PPS)can eliminate the irritation of PS and enhance the effect of nourishing.Polygonatum sibiricum Polysaccharides(PSP)are the main active components of PS,which have better immunomodulatory activity.Pharmacodynamics studies showed that,PS and PPS all can improve the non-specific immune function of mice,and PS has the better pharmacological effect.At present,most PPS studies focus on optimizing processing technology and extraction of polysaccharides and their content.But it is not clear which molecular weight fractions of PPS are related to immunocompetence.To study the change of content and basic structure of PSP before and after processing and to clarify the effective polysaccharide fractions is the basis for Synergy.According to the transformation of the basic structure of PS,the quality standard of formula granules of PS and PPS was established.In this experiment,PS and PPS were used as the subject,and the polysaccharides were obtained by the optimized method.MW and its distribution,monosaccharide composition,functional group,and glycosidic bond type of PS before and after processing were analyzed by HPGPC,HPLC,.FT-IR.Mice peritoneal macrophage model and immunosuppressive mouse model were used to evaluate the immunocompetence of PS and its different fractions before and after processing.The main findings of the study are as follows:1.Study on the extraction,divorce,and purification of polysaccharides in PS and PPS.First,20 batches of 3 varieties of PS were collected from the main producing areas.According to the previous research results,PS and PPS were prepared.The content of polysaccharides was determined by the anthrone-sulfuric acid method to determine the difference in PS between different producing areas and varieties.Secondly,MW and its distribution and the polysaccharide yield were taken as the index to compare the effects of ultrasonic assistant extraction and hot water extraction.The optimum extraction,separation,and purification process of polysaccharides were investigated.The results showed that:(1)There was no significant difference in polysaccharide content of PS and PPS in the same batch(2)The best extraction method of PS and PPS is hot water extraction,the best extraction process parameter is as follow:ratio of water to raw material 1:10(g/mL),extraction 1h,extraction 2 times,the best separation process parameter is as follow:Concentration ratio 1:2(g 生药/m L),processing concentration is 90%;optimum purification process parameter is as follow:the ratio of Sevage reagent and crude polysaccharide solution 1:3.3 times of deproteinization.2.Comparison of the basic structure of PSP before and after processing20 batches of purified PS and PPS were as the research object.Firstly,the HPGPC was used to compare and analyze the MW and distribution of PSP before and after processing.Secondly,compare the result of PMP-HPLC,HPLC-ELSD,HPLC-RID to select the suitable method for the determination of monosaccharides of PSP and Processed Polygonatum sibiricum polysaccharides(PPSP);At the same time.a monosaccharide composition fingerprint was established to compare and analyze the monosaccharide composition of polysaccharides from different areas and varieties of PS and PPS.Third,FT-IR was used to determine the functional group structure of polysaccharides.Finally,NMR was used to determine the type of glycosidic bond and the linkage model of the polysaccharide.The results showed that:(1)After processing,the Mw and distribution of PSP all changed,and it was different in 3 varieties of PS;The area of MW>100 kDa fractions increase after processing;There are three new chromatographic peaks in processed P.cyrtonema Hua and the area of MW<10 kDa reduced(2)PMP-HPLC method is not suitable for the detection of fructose-containing polysaccharides;The sensitivity of the HPLC-RID method is too low;HPLC-ELSD method can detect more monosaccharide components.(3)The similarity of the monosaccharide composition of PSP is>0.8 when the similarity of polysaccharide of PPSP drops to 0.4,The main monosaccharide components between PSP and PPS are glucose,mannose,fructose and arabinose;PSP and PPSP are neutral heteropolysaccharides.monosaccharide composition of polysaccharide has changed due to processing.(4)Fructose is the main component of PSP;The monosaccharide content of PSP is greatly different;The ratio of glucose,mannose and arabinose is about 1:1:1;The ratio of fructose is greatly differently,which indicates that fructose content is an important factor for the difference in the molar ratio of monosaccharides.(5)FT-IR:The infrared spectra of PSP and PPSP are basically the same,indicating that the structures of PSP are highly similar before and after processing.There may be differences in the number of C-OH and C-O-C structures,and absorption of mannose and arabinose is lower.(6)PSP and PPSP are both linked by β(2→1)glycosidic bonds.3.Comparative study on the immunological activity of PSP and its different fractions before and after processingFirstly,compare the separation effect of dialysis method and ultrafiltration method and the different polysaccharide fractions of PPS were prepared.Secondly,normal and lipopolysaccharide(LPS)-induced mouse peritoneal macrophages were used as the research object.To explore the effects of polysaccharides and their different fractions on the immunocompetence of macrophages of PS before and after processing.To establish an immunosuppressive mouse model and compare the immunity of polysaccharides and different fractions of PS before and after the processing.The results showed that:(1)The dialysis membrane method can better divide the polysaccharides from PSP into two polysaccharide fractions with MW>50 kDa and MW<50 kDa compared with ultrafiltration.(2)Under normal physiological conditions,only the polysaccharide fraction of PSP with MW<50 kDa has a significant effect on the secretion of NO(P<0.05);The polysaccharide fractions of PSP and PPSP have no significant effect on the secretion of IL-1β;When macrophages were induced by LPS,PSP and its different fractions before and after processing can inhibit the secretion of TNF-α,IL-1β and NO,which indicate that PSP mainly enhances the immune regulation of the body by inhibiting the occurrence of inflammation.The effect on inhibiting excessive inflammation of PS was enhanced after processing.The effect of the MW<50 kDa fraction of the polysaccharides from PPS is stronger than that of the MW>50 kDa fraction.MW<50 kDa fraction is the main active component of PSP.(3)Compared with the model group,PSP and its different fractions before and after processing can improve the atrophy of mouse spleen and thymus induced by cyclophosphamide and promote the phagocytic ability of mononuclear macrophages(P<0.05 or P<0.01).Compared with the model group,the content of SOD was increased after the intervention of PSP and its different fractions before and after processing while the content of MDA was reduced(P<0.05).Results showed that both PSP and PPSP could enhance the immune function of the body by increasing the antioxidant enzyme activity of immunosuppressed mice and reducing the content of MDA in the serum.PPSP and its different fractions have better effects.4.Quality Control of Polygonatum Formula GranulesDextrin and starch were the research objects to investigate the optimal reaction conditions of enzymatic removal excipients.According to the<<Technical Requirements for Quality Control and Standard Formulation of Chinese Medicine Formula Granules(Draft for Comments>>,the PS and PPS formula granules are prepared according to the maximum amount of excipients(excipients:extract powder=1:1).With the MW and distribution of polysaccharides as indicators,the quality standard of formula granules is established by HPGPC method.The results showed that(1)high-temperature α-amylase and glucoamylase had no effect on the Mw and distribution of PSP and PPSP.The Mw and distribution of dextrin produced by different manufacturers are relatively consistent,while the soluble starch is quite different.A single enzyme cannot completely hydrolyze soluble starch and dextrin and the synergistic effect of the two enzymes is better.The optimal amount of high-temperature α-amylase(40 U/mL)and glucoamylase(50 U/mL)are 1:1.5(g/mL)andl:2(g/mL),respectively.(2)The quality control method of PS and PPS was established by HPGPC method.The precision,stability and repeatability of HPGPC method all met the requirements,which indicates that the method is stable and reliable.After enzymatic treatment of dextrin and soluble starch,the part with MW>10 kDa is all converted into oligosaccharides,which will not interfere with the determination of this part of the formulated granules.In part with Mw>10 kDa,the peak areas of polysaccharides from PPS formula granules are larger than those of PS formula.Therefore,The formula granules of PS and PPS can be distinguished.ConclusionThe processing has increased the number and peak area of some chromatographic peaks in the range of MW>100 kDa,and some PSP will produce an oligosaccharide peak in the range of MW<10 kDa after processed.The main monosaccharide components of PSP are not changed before and after processing,but the content of arabinose,mannose,and fructose decreased while the content of glucose increased.The number of functional groups of PSP was different after Processed,which indicates that the basic structure of Polysaccharides changed before and after processing.After processing,the immunomodulatory activity of PSP was enhanced.The Mw<50 kDa fraction is the active fragments.The quality standard of formula granules Based on the HPGPC method can successfully distinguish the formula granules of PSP and PPSP.