Studies On The Physicochemical Properties And Activities Of Polysaccharides Sequentially Extracted From Polygonatum Cyrtonema Hua

Polygonatum cyrtonema Hua is a perennial herb,which can be used as medicine and food.Polysaccharides are one of their main active ingredients,which have many functions such as improving immunity,anti-oxidation,delaying senility,anti-inflammation and anti-tumor.In this study,four kinds of P.cyrtonema Hua polysaccharides were sequentially extracted by hot buffer,chelating agent,dilute alkaline and concentrated alkaline,and named as HBSS,CHSS,DASS and CASS,respectively.The physicochemical properties such as composition and structure,thermodynamic properties,rheological properties,emulsifying properties and physiological activities such as antioxidant,antibacterial properties,anti-inflammatory,anticoagulant and inhibition of cancer were studied,which may provide a theoretical basis for their direct application in the development of functional foods,medicine and cosmetics.The main results are as follows:（1）Composition and structural analysis of P.cyrtonema Hua Polysaccharides.Four kinds of polysaccharides（HBSS,CHSS,DASS and CASS）were obtained by sequentially extracted from P.cyrtonema Hua.The content of sugar for HBSS,CHSS,DASS and CASS were 78.39%,71.86%,69.99%,73.73%,respectively.The content of uronic acid for HBSS,CHSS,DASS and CASS were 7.02%,17.73%,28.34%,8.68%,respectively.Four polysaccharides had the characteristic absorption peaks of saccharide,and they were the acid pyranose which were connected by theβ-glycosidic bond.Furthermore,CHSS and DASS had the ether bond of the sugar ring;whereas,HBSS hadα-glycosidic bond.The molecular weights of HBSS,CHSS,DASS and CASS were12718,9112,7658 and 607 kDa,respectively.Four polysaccharides were heteropolysaccharides which consisted of rhamnose,arabinose,xylose,glucose and galactose with the lowest content of mannose.HBSS had the largest mannose content（54.55%）;while,CHSS had the highest arabinose content（36.97%）.On the other hand,DASS possessed the highest galactose（54.91%）,CASS was reported with the highest glucose content（26.1%）,followed by xylose（24.05%）and galactose（20.06%）.These results confirmed that the composition and structure of sequentially extracted four polysaccharides from P.cyrtonema Hua.were different.（2）Comparison of Polygonatum cyrtonema Hua polysaccharides on thermodynamic,rheological and emulsifying properties.The enthalpy values of four kinds of polysaccharides were greater than that of xanthan gum,which had good thermal stability.Among all the fractions,HBSS had the best thermal stability.Rheological studies of four kinds of polysaccharides showed thinning behavior,which were a typical non-Newtonian fluid property.The apparent viscosity of polysaccharides was expressed as CHSS>HBSS>DASS>CASS.Concentration,temperature and salt ions have obvious influence on the apparent viscosity of polysaccharides in a dose-dependent manner.The apparent viscosity decreased with increasing system temperature and heat treatment,but increased with cold treatment.The apparent viscosity decreased under the conditions as acid（pH4.0）and alkaline（pH10.0）;whereas,the effect of alkaline condition was stronger than acidic pH.The increasing concentration of salt ions has influenced the apparent viscosity to a greater extent.On the other hand,the apparent viscosity was decreased by addition of Ca²⁺.The apparent viscosity of HBSS was increased by addition of Na⁺,and the apparent viscosity of CHSS increased by addition of 0.1 mol/L Na⁺,but decreased for others by adding Na+.Oscillatory dynamic tests indicated that polysaccharides were viscoelastic materials,and the linear viscoelastic region was observed as 1-4%.HBSS,DASS,CASS（0.1%）showed higher viscosity at low frequency,and showed elastic behavior at high frequency.At other concentrations,G’>G’’in the test frequency range was observed,which showed that due to elastic behavior,P.cyrtonema Hua polysaccharides solutions could form gel network.The emulsifying activity and emulsifying stability of P.cyrtonema Hua polysaccharides for the peanut oil were increased with the increase of polysaccharides concentration,which showed obvious concentration dependence.The emulsifying activity of CHSS was the highest,and HBSS had the strongest emulsifying stability.Whereas,pH and NaCl had no significant influence on emulsifying activity and emulsifying stability of four polysaccharides.（3）Study on the physiological activities of P.cyrtonema Hua polysaccharides.The physiological activities of four kinds of polysaccharides had some differences in a concentration dependent manner.DASS showed the best antioxidant potential;while,HBSS was reported with the weakest antioxidant ability.Four kinds of polysaccharides could inhibit the secretion of TNF-αin mouse spleen lymphocytes,which showed a certain inhibitory effect on inflammation.CASS had the best anti-inflammatory effect;while,weak effects were observed in case of HBSS and CHSS.HBSS,DASS could promote the secretion of INF-γin mouse spleen lymphocytes and improve cellular immunity with the strongest effect of HBSS.CHSS,CASS were not significantly different from control which confirmed no immunity activity.Four kinds of polysaccharides had a certain antibacterial properties,especially against Gram-positive bacteria.The antibacterial ability of four test organisms was in following order:Bacillus.subtilis>Staphylococcus.aureus>Escherichia.coli>Salmonella.typhimurium.The antibacterial ability of four polysaccharides was in order:DASS>CHSS>CASS>HBSS.Among all the fractions,DASS showed the strongest antibacterial properties.Four kinds of polysaccharides had anticoagulant activities;which could prolong activated partial thromboplastin time（APTT）and thrombin time（TT）.It had the greatest influence on TT,but no effect on prothrombin time（PT）,which indicated that polysaccharides showed anticoagulant activity through the intrinsic coagulation pathway and common coagulation pathways,but not by exogenous coagulation pathway.The effect of CASS was the remarkable,followed by HBSS;while,the effects of DASS and CHSS were non-significant.（4）Inhibiting cancer and its mechanism.Four kinds of polysaccharides displayed inhibitory effects on proliferation,toxicity and inducing apoptosis for（Hela cells）cervical cancer in a concentration dependence manner.The inhibitory effect on Hela cell proliferation was expressed as CASS>HBSS>CHSS>DASS,with the maximum inhibition rate of 74.453±3.399%.Hela cell cytotoxicity was in the following order:CASS>CHSS>HBSS>DASS,with the maximum cell death rate of 82.472±3.488%.The activity of the Hela cell Caspase-3 was induced by CASS>HBSS>DASS>CHSS,and the maximum multiple was 2.954±0.103.The above results indicated that cancer inhibiting activities were different for four polysaccharides.Among all the tested fractions,CASS had the maximum inhibitory effect on Hela cells.CASS induced Hela cell cycle block at the G2/M phase by enhancing levels of mRNA expression of the genes of CyclinD1,p21,p53 and Wee1,and reducing the expression levels of mRNA of the genes of Survivin,CHK2,Wee1,CyclinB1 and cdk-1in cell cycle.CASS could effectively induce Hela cell apoptosis in a dose-dependent manner due to regulation of the expression of related genes in different apoptotic pathways.CASS enhanced mRNA expression levels of the of DR3,DR5,FasL,FADD,PARP,TNF-α,TNF-R1,TRDAA,caspases-8 and caspases-10 in the death receptor pathway;while,reduced mRNA expression levels of antiapoptotic proteins genes of Bcl-2 and Bcl-xL genes were observed.The mRNA expression levels of apoptosis protein genes of Bak,Cytc,Puma,caspases-3,caspases-7 and caspases-9 gene in the mitochondria pathway were up regulated which led to Hela cell apoptosis.