Structural Analysis And Anti-aging Activity Of Polysaccharides From Panax Notoginseng

Panax notoginseng(Burk.)F.H.Chen is a famous traditional Chinese medicine,and polysaccharide is one of main active components.It has been reported that water-extracted polysaccharides from Panax notoginseng were mainly composed of Glc,Gal and Ara,having many biological functions such as anti-oxidation,anti-aging,anti-tumor,regulating immunity and neuroprotection.Due to the interference of starch granules in cell wall of plants,polysaccharides from Panax notoginseng cannot be completely extracted by hot water,which leads to the omission of total water-soluable polysaccharides from Panax notoginseng.To make best use of active ingredient in Panax notoginseng resources,Panax notoginseng roots were firstly extracted by hot water and then continuously extracted by ?-Amylase to get water-soluble polysaccharides as entirely as possible in this study.Further,the structures of these homogeneous polysaccharide fractions were studied by a combination of chemical,biological and instrumental methods.Meanwhile,the anti-aging activity was explored on C.elegans model.The specific research results are summarized as:1.Water extraction of polysaccharides from Panax notoginsengFirstly,polysaccharide named as PNP was extracted by Panax notoginseng roots by hot water.The yield of the polysaccharide was 4.7% and the monosaccharide composition of it was Glc(62.1%),Gal(17.5%),Gal A(9.9%),Ara(6.1%)and Rha(2.5%).PNP was separated and purified by anion-exchange chromatography and gel-filtration chromatography into one neutral polysaccharide(PNPN)and six homogeneous acidic polysaccharides(PNPA-1A,PNPA-2A,PNPA-3A,PNPA-1B,PNPA-2B and PNPA-3B).Enzymatic hydrolysis,methylation,FT-IR and NMR methods were used to analyze the structure of homogeneous fractions.The results revealed that:1)The neutral polysaccharide fraction(PNPN)was a starch-like glucan,having more than 80% Glc.PNPN had a molecular weight of 191 k Da,and the branching degree of its backbone was about 6%.2)PNPA-1A and PNPA-2A were AG type pectin,containing over 80% Ara and Gal.The Ara and Gal residues were assigned to be ?-D-1,4-Galp,?-L-1,5-Araf and ?-L-1,3,5-Araf.According to the monosaccharide composition result,PNPA-3A had over 25% Rha and Gal A,and the ratio of Rha to Gal A in PNPA-3A was close to 1,being RG-I type pectin.PNPA-3A was composed of disaccharides [-2-?-L-Rhap-1?4-?-D-Galp A-1-] repeating units as backbone,with a degree of branching 67%.The side chains of PNPA-3A included linear or branched(1?5)-?-L-arabinan,(1?4)-?-D-galactan,AG-I and AG-II.PNPA-1B,PNPA-2B and PNPA-3B were HG type pectin,with a molecular weight of 3 k Da,6 k Da and 18 k Da,respectively.The degrees of methylation(DM)of the three HG rich fractions were below 12%,indicating that low esterified pectin was the main component in Panax notoginseng.2.?-Amylase-assisted extraction of polysaccharides from Panax notoginsengThe residues of Panax notoginseng after hot-water extraction were further proceeded in ?-Amylase-assisted extraction to obtain the total polysaccharide(PNPE).The yield of PNPE was 6.5%,which was higher than that of PNP(4.7%),indicating that there were still a lot of polysaccharides in Panax notoginseng after hot water extraction.PNPE mainly contained Glc(76.1%),Gal(10.2%),Gal A(6.0%),Ara(6.0%)and Rha(1.3%).PNPE was purified by anion-exchange and gel-filtration chromatography,and one neutral polysaccharide(PNPEN)and six homogeneous acidic polysaccharide fractions(PNPEA-1A,PNPEA-2A,PNPEA-3A,PNPEA-1B,PNPEA-2B and PNPEA-3B)were prepared.Enzymatic hydrolysis,methylation,FT-IR and NMR methods were combined to analyze the structure of homogeneous fractions.The results showed that:1)PNPEN was a glucan with low molecular weight(2 k Da),which contained more than 80% Glc.It inferred that PNPEN was produced from starch-like glucan after ?-Amylase extraction.2)PNPEA-1A and PNPEA-2A were both AG type pectin with molecular weights of 170 k Da and 140 k Da,respectively.The contents of Ara and Gal were more than 80%,and were assigned to be ?-D-1,4-Galp,?-L-1,5-Araf and ?-L-1,3,5-Araf.The results showed that the structural characteristics of AG type pectin extracted by the two methods were same.AG type pectin prepared by ?-Amylase-assisted extraction had higher molecular weight than that from hot-water extraction.PNPEA-3A and PNPEA-1B were RG-I type pectin.According to the monosaccharide composition result,both PNPEA-3A and PNPEA-1B had over 25% Rha and Gal A,and the ratios of Rha to Gal A were close to 1.Both PNPEA-3A and PNPEA-1B were composed of disaccharides [-2-?-L-Rhap-1?4-?-D-Galp A-1-] repeating units as backbone,with a degree of branching 43% and 26%,respectively.The side chains of PNPEA-3A and PNPEA-1B mainly included linear or branched(1?5)-?-L-arabinan,(1?4)-?-D-galactan or AG-I,containing less AG-II.PNPEA-2B and PNPEA-3B were HG type pectins with molecular weight of 14 k Da and 42 k Da,respectively.The degrees of methylation(DM)of the three HG rich fractions were below 12%,being low esterified pectins.Except little higher molecular weights,HG type pectins prepared by ?-Amylase-assisted extraction had similar structures with that from from hot-water extraction.3.Anti-aging activity of polysaccharides from Panax notoginsengC.elegans model was used for evaluating the anti-aging activity of Panax notoginseng polysaccharides.The results showed that PNP and PNPE extended the mean life span of N2 nematodes by 7.8%(P = 0.0002)and 16.0%(P <0.0001),respectively,indicating that PNPE had a more significant effect on the life span of N2 nematodes than PNP.Polysaccharide fractions(PNPN,PNPEN,PNPA and PNPEA)purified by anion-exchange chromatography were further analyzed.Both PNPEN and PNPN were ?-1,4-glucan,and the molecular weight of PNPEN(2 k Da)was significantly lower than that of PNPN(191 k Da).PNPEN could significantly prolong the life of wild-type C.elegans,while there was no significant difference between PNPN(P = 0.2357)and the control group,indicating that the lower molecular weight may have better anti-aging activity.The effects of acidic polysaccharide PNPA and PNPEA on the lifespan of N2 nematodes were similar,and the average lifespan of N2 nematodes was increased by 13.7% and 15.6%(P < 0.0001).Four homogeneous pectic polysaccharide(PNPA-2A,PNPA-2B,PNPEA-1A and PNPEA-2B)were main components of pectin in Panax notoginseng,using for seeking bioactive pectic domains.All of them could significantly extended the mean life span of N2 nematodes by about 15.0%.The results indicated that AG type pectin and HG type pectin were bioactive pectic domains in Panax notoginseng,exerting anti-aging activity.In summary,water-soluable polysaccharides in Panax notoginseng were sequential and fully extracted by hot water and ?-Amylase-assisted extraction,the yield was 2 times more than single hot water extraction.The types and structural features of polysaccharides in Panax notoginseng have been analyzed and concluded.Meanwhile,the anti-aging activity of representative polysaccharides was studied,and the bioactive structural domains have been indentified.These results enriched the structural information of carbohydrates in Panax notoginseng,laid the foundation for study the structure-activity relationship,and provided information for full development of Panax notoginseng resources.