Structural Characterization And In Vitro Antioxidant Activities Of Polysaccharides From Both Roots And Stems Of Two Species Of Radix Codonopsis

Polysaccharides,as one of the central active ingredients of radix Codonopsis,have assorted biological activities,including immune regulation and antioxidant activity.Due to the variety of radix Codonopsis and their wide sources,most studies only focus on the polysaccharides of a certain category and a part.At present,the studies on the polysaccharides of Codonopsis pilosula mainly focus on the roots,while the stems,as an important part of C.pilosula,have rich chemical composition and physiological activities,but few related studies.In our previous studies,the polysaccharides of C.pilosula Nannf.var.modesta(Nannf.)L.T.Shen have been well studied.Thus,the main aim of this study is to compare the structure,antioxidant and prebiotic activities,and the structure-function relationship of the purified fractions from both roots and stems of two species of Radix Codonopsis,including C.pilosula(Franch.)Nannf.and C.tangshen Oliv.,which are stated in the Pharmacopoeia of the People’s Republic of China(2015).It will be isolated under same extraction and purification conditions.1 Extraction and fractionation of neutral and acidic polysaccharides from both roots and stems of two species of Radix CodonopsisAfter hot-water extraction,80% ethanol was added and obtained the crude polysaccharide from roots of C.pilosula and C.tangshen,and stems of C.pilosula and C.tangshen,and denominated as CPP,CTP,CPSP and CTSP.The yield was 20.3%,22.8%,4.6% and 8.5%,respectively.The crude polysaccharides were purified by anion-exchange chromatography with DEAE-Sepharose column to fractionate the neutral polysaccharides,CPPN(obtaind form CPP)and CTPN(obtaind form CTP),and acidic polysaccharides,CPP-1(obtaind form CPP),CTP-1(obtaind form CTP),CPSP-1(obtaind form CPSP)and CTSP-1(obtaind form CTSP),while the yield were 44.6%,51.4%,31.3 %,30.3%,56.4%and 31.6%.Then,they were purified further with size exclusion chromatography and it was found that all above acidic polysaccharides contained only one fraction.The results showed that different regions and parts of C.pilosula is an important factor to affect the yield of both crude polysaccharides and purified one.The yield of crude polysaccharides and neutral one in C.tangshen and roots are higher than that in C.pilosula and stems,while the yield of acidic fractions in C.pilosula and stems are more superior.Since the content of neutral fractions derived from stems are extremely low and have no use value,it would not subject to further studies.2 Characterization of inulin-type fructans from two species of Radix Codonopsis and their antioxidant activitiesThe structural characterization of inulin-type fructans was detected using the gas chromatography(GC),Urea-HCl paper chromatography(Urea-HCl),gas chromatography-mass spectrometry(GC-MS),the size-exclusion chromatography-multiangle laser scattering(SEC-MALLS)and nuclear magnetic resonance(NMR),including monosaccharide compositions,glycosidic linkage,molecular weight(Mw)and degree of polymerization(DP).An in vitro oxidative model was used in intestinal porcine epithelial cells(IPEC-J2)to further study the antioxidant effects of CPPN and CTPN.The cell viability,levels of malondialdehyde(MDA),total antioxidant capacity(T-AOC),and the activities of lactate dehydrogenase(LDH),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)and catalase(CAT)were investigated.The results of characterization showed that the CPPN and CTPN consist of a large amount of fructose(Fru)and a small amount of glucose(Glc),which is based on β-(2-1)linked inulin-type fructans with non-reducing terminal Glcp,with the Mw of 2.3 k Da and4.0 k Da,and with the DP of 19.6 and 25.2.The results of antioxidative research showed that both CPPN and CTPN can significantly enhance cell viability(P<0.05),T-AOC and the activities of antioxidase(P<0.05),reducing the levels of MDA and LDH(P<0.05).While CTPN,the inulin with higher DP,showed better antioxidant activity.3 Characterization of acidic polysaccharides from both roots and stems of two species of Radix Codonopsis and their antioxidant activitiesThe structural characterization of acidic polysaccharides was detected using the GC,Urea-HCl,GC-MS,SEC-MALLS and NMR,including monosaccharide compositions,glycosidic linkage and Mw.An in vitro oxidative model was used in IPEC-J2 cells to further study the antioxidant effects of CPP-1,CTP-1,CPSP-1 and CTSP-1.The cell viability,levels of reactive oxygen species(ROS)and MDA,T-AOC,and the activities of LDH,GSH-Px,SOD and CAT were investigated,and the molecular mechanism was explored.The results of structure elucidation indicated that all of CPP-1,CTP-1,CPSP-1 and CTSP-1 are pectic polysaccharides with long homogalacturonan regions(HG)and rhamnogalacturonan I(RG-I)regions.The major side chains of roots polysaccharides CPP-1 and CTP-1 are both arabinogalactan type II(AG-II),and with partly type I(AG-I).But only CPP-1 has the methyl esterified galacturonic acid(Gal Ap A6Me)units.The stems polysaccharides CPSP-1 and CTSP-1 both contain some Gal Ap A6 Me units.Side chains for CTSP-1 are AG-I and AG-II,while CPSP-1 only has AG-II.The Mw of CPP-1,CTP-1,CPSP-1 and CTSP-1 are 21.0,29.5,13.1 and 23.0 k Da,respectively.The results of antioxidative research showed that all above polysaccharides can significantly enhance cell viability(P<0.05),T-AOC and the activities of antioxidase(P<0.05),reducing the levels of MDA,LDH and ROS(P<0.05).All of these fractions can also significantly up-regulate the expression of antioxidant enzyme genes,including GPXs,SOD1 and CAT,and that of tight junction protein ZO-1 with a dose-dependent trend(P<0.05).The roots fractions CPP-1and CTP-1 can up-regulate the expression of Nuelear Faetor-E2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase 1(NQO1)and oxidoreductase 1(HO-1)(P<0.05).While the stems fractions CPSP-1 and CTSP-1 can not up-regulate the expression of Nrf2、NQO1 and HO-1(P>0.05).In general,the neutral and acidic fractions from both roots and stems of C.pilosula and C.tangshen have different extraction and purification yields,while the diverse of regions and parts of radix Codonopsis is the momentous factor to impact the structure and activity of polysaccharides.Multiple structural inspections have shown that the neutral fractions of C.pilosula(CPPN)and C.tangshen(CTPN)are inulin-type fructans with DP of 19.6 and 25.2,respectively.The acidic fractions from both roots and stems of C.pilosula and C.tangshen are pectic one with different main chain lengths and the amount of side chains,while the number of branch chains and Mw are related to the source and part.Among them,inulins have a better antioxidant activity,and these activities were found to be related to the DP.Both the polysaccharides from roots and stems have antioxidant activity,but only that from roots has activation of the Nrf2 signaling pathway at the transcription level.These differences may be related to the structures,but the specific one that activating Nrf2 cannot be determined for the time being,and the specific molecular mechanism is still to be studied.Finally,it also showed the different regions and parts of polysaccharides are the important factors affecting structure,activity and structure-function relationship.In addition,these results also indicated that both roots and stems of C.pilosula and C.tangshen contain highly active polysaccharides.The stems are rich in yield but less used,so it is worthy to be developed and utilized.However,the active mechanism and structure-activity relationship still need further study.