Molecular Modification And Biological Activities Of Ziziphus Jujuba Fruit Polysaccharide

Polysaccharide is the most important bioactive component in jujube, the fruit of Zizyphusjujuba Mill. Due to its high potential for pharmaceutical use, many studies had been done toinvestigate the extraction, purification, structure and biological activities of Ziziphus jujubafruit polysaccharide (ZJP). Molecular modification of ZJP favors to improve its biologicalactivities and extend its application range. It was also helpful for the research on therelationship between structure and bioactivities of ZJP, so as to other polysaccharides. In thisdissertation, the ZJP was extracted, purified, degraded and chemically modified, and thebiological activities, including antioxidant activity, hypopoglycemic effect and inhibitoryeffect to hyaluronidase were studied for both the native and modified ZJP. It was proved thatthe ZJP could inhibit the activities of α-amylase, α-glucosidease, hyaluronidase andnon-enzymatic glycation, and molecular modification could alter the biological activitiessignificantly. Furthermore, the change of advanced structure of ZJP was thought to be morerelated to the alteration of its biological activities. The results detailed as follows:Preparation of Ziziphus jujuba fruit polysaccharide (ZJP)The optimum conditions for extraction of polysaccharide from Ziziphus jujuba fruit withhot water were:1:10for the ratio of material to water (w/v),80℃, extracting two times, andone hour for each time. By XAD-761resin adsorption, the combined extraction ofpolyphenols and polysaccharides from Ziziphus jujuba fruit was achieved with less effect onthe polysaccharide extraction.The protein in ZJP extract could be removed efficiently by Sevag method. And the colorcould be removed by macroporous adsorption resin with little loss of polysaccharide, whileactive carbon usually caused higher loss of polysaccharide due to its high adsorption topolysaccharide with poor decoloration effect to ZJP extract.By DE52-cellulose column chromatography, one neutral polysaccharide fraction namedas ZJP-0and three acidic polysaccharide fractions named as ZJP-1, ZJP-2and ZJP-3, wereobtained. The molecular weights of four fractions were about1000kDa. Among of them, ZJP-2was the major constituent. The monosacchaaride composition analysis by HPLC showedthat ZJP was a heteropolysaccharide which was composed of galacouronic acid, rhamnose, arabinose, galactose, xylose and glucose etc. Results of periodate oxidation indicated that themain link type of ZJP was (1→4) linkage.Molecular modification of Ziziphus jujuba fruit polysaccharide (ZJP)Ultrasonic treatment could cause the degradation of ZJP. Lower temperature, higherultrasonication power, lower concentration of ZJP and frequency conversion treatment washelpful for the ultrasonic degradation. Under the experimental conditions, Ultrasonictreatment only decreased the molecular weight of ZJP to a certain degree and the finalmolecular weight of ZJP was still above300kDa. Hydrochloric acid could degrade ZJPefficiently even at lower concentration under room temperature. Degradation of ZJP was moreviolent by increasing the concentration of hydrochloric acid and the molecular weight of ZJPreduced sharply. The degradation product of ZJP treated with a certain concentration ofhydrochloric acid was also relative homogeneous polysaccharide. Hydrogen peroxide couldalso degrade ZJP violently, but the molecular weight distribution of degradation product wastoo wide with a large number of low molecular sugars.By chlorosu1fonic acid-pyridine method, the ZJP could be sulfated successfully. Theoptimum conditions for sulfation of ZJP were:1:3of volume ratio of chlorosu1fonic acid topyridine,70℃of reaction temperature and2.5h of reaction time. Under these conditions, thedegree of substitution of sulfated ZJP was up to1.39.Carboxymethylated derivatives of ZJP were obtained by using sodium hydroxide-momochloroacetic acid reaction system. The degree of substitution of carboxymethylated ZJPincreased with higher concentration of NaOH in reaction system, but the yield of productsdecreased. There was a significant negative correlation between the degree of substitution andthe yield. Increasing the ratio of carboxy-methylation reagent to ZJP was helpful for theincrease of the degree of substitution of carboxymethyl.Acetylation of ZJP was achieved by using both acetic anhydride method and glacialacetic acid method respectively. By glacial acetic acid method, the degree of substitution ofacetylated ZJP was lower, so this method was not suitable for preparing acetylated ZJP withhigher degree of substitution. High temperature and low pH were unfavorable for the acetylsubstitution of ZJP by acetic anhydride method, and reaction at room temperature andconstant pH of9.5±0.5were in favor of the acetyl substitution.Sulfation, carboxymethylation and acetylation of ZJP could significantly improve thedistribution statue and solubility of ZJP in water.Biological activity of Ziziphus jujuba fruit polysaccharide (ZJP) and its molecularmodification productsAntioxidant activity The antioxidant activity of ZJP and the fractions separated by DE52-cellulose columnchromatography was evaluated by using free radical scavenging test in various in vitro system.And ZJP and the fractions separated by DE52-cellulose column chromatography weredemonstrated to exhibit strong scavenging capacities towards superoxide anion radicalsgenerated by illuminating riboflavin, hydroxyl radical generated by Fenton reaction andDPPH with a dose-dependent pattern. Under the experiment conditions, the maximumscavenging percentage of ZJP to superoxide anion radical, hydroxyl radical and DPPH was upto99.01%,49.46%, and36.89%respectively. As compared with the native ZJP, the freeradical scavenging activities of fractions separated by DE52-cellulose columnchromatography decreased. Among of the fractions, acidic polysaccharide (ZJP-1, ZJP-2andZJP-3) showed stronger activity than the neutral polysaccharide (ZJP-0), which suggested thatgalacouronic acid may play an important role in free radical scavenging activity for ZJP.Properly degradation by ultrasonication or hydrochloric acid treatment could improve thefree radical scavenging capacity of ZJP, but the activity would decrease when the molecularweight was lower than230kDa. Ultrasonication treatment would iuflence the antioxidantactivity of ZJP by dual mechanisms. The molecular size and conformation of ZJP, whichcould be changed by ultrasonication, would both have impact on the antioxidant activity.Under the experiment conditions, the maximum scavenging percentage of ZJP to superoxideanion radical, hydroxyl radical and DPPH was increased by3.16,2.00and0.16timesrespectively by ultrasonication treatment, while increased by4.78,1.46and0.22times whendegraded with hydrochloric acid.The free radical scavenging effects of chemical modified ZJP derivatives were related tothe radical testing system, and the configuration might play an important role in the freeradical scavenging activity of ZJP. Appropriate sulfation could obviously improve thescavenging activity of ZJP to superoxide anion radical, and the IC50of sulfated ZJP withmoderate degree of substitution (DS) of0.79and1.03decreased by half compared with thatof the native ZJP. But there was less effect on hydroxyl radical and DPPH scavenging effect.Carboxymethylation could significantly improve the scavenging activity of ZJP to hydroxylradical, but was not favorable for superoxide anion radical and DPPH scavenging activity.The scavenging effect of five acetylation products of ZJP to hydroxyl radical were improvedsignificantly, but acetylation would lead to the decrease of the scavenging effect to superoxideanion radical. The acetylation also changed the kinetics of scavenging effect of ZJP to DPPH.Therefore, moderate degree of sulfation was the most effective method to improve thesuperoxide anion radical scavenging effect of ZJP, and acetylation and carboxymethylationwere important means to improve the hydroxyl radical scavenging effect of ZJP. Hypoglycemic effectThe hypoglycemic effect of ZJP was evaluated by determination of the inhibitory effectagainst α-amylase and α-glucosidease in vitro. Results showed that ZJP could inhibitα-amylase and α-glucosidease effectively with an obvious dose-dependent manner. Under theexperiment conditions, the maximum inhibitory percentage against α-amylase andα-glycosidase was53.53%and62.65%with IC50of11.39mg·mL-1and16.61mg·mL-1respectively. Thus, ZJP might reduce postprandial hyperglycemia by affecting the wholeprocess from enzymatic hydrolysis of starch to the production of glucose and prolonging therelease and absorption of monosaccharide.As compared to the native ZJP, the inhibitory effect on α-amylase of fractions separatedby DE52-cellulose column chromatography, ZJP-1, ZJP-2and ZJP-3, were improvedsignificantly except ZJP-0with a slight decrease, and the inhibitory effects on α-glucosideaseof the four fractions were all improved to different degree. Among of the fractions, ZJP-1showed the strongest inhibitory activity on α-amylase and α-glucosidease,which suggestedthat ZJP-1may play an important role in the hypoglycemic activity of ZJP.The inhibitory effect against α-amylase of ZJP was decreased, but the inhibitory effectagainst against α-glucosidease was improved after ultrasonication treatment. Furthermore, theinhibitory effect against α-glucosidease increased with prolonging of treatment duration.When degraded with hydrochloric acid, the inhibitory effects of ZJP against α-amylase andα-glucosidease were improved at lower concentration of hydrochloric acid, but excessivedegradation led to lower activity. ZJP fractions with molecular weight from300to600kDashowed stronger inhibitory activity against α-glucosidease, but the activity was weaker whenthe molecular weight was lower than290kDa. Maintaining certain length of chain and spatialconformation of ZJP was crucial to perform its activity. Under the experiment conditions, themaximum inhibitory percentage of ZJP against α-glucosidease could be increased by2.09and1.56times respectively when treated with ultrasonication and hydrochloric acid, and themaximum inhibitory percentage of ZJP againstα-amylase was also improved by38.25%whendegraded with hydrochloric acid.The inhibitory effects against α-amylase of ZJP substituted with different groups alldecreased as compared to that of the native ZJP, but the inhibitory effects againstα-glucosidease were improved. Under the experiment conditions, the inhibitory percentage ofZJP modificated with sulfation, carboxymethylation and acetylation against α-glucosideasewas increased by1.87,5.08and3.97times respectively. Carboxymethylation was one of themost efficient methods to improve the inhibitory effect of ZJP against α-glucosidease.Inhibitory effect on non-enzyme glycation ZJP and the fractions separated by DE52-cellulose column chromatography weredemonstrated to inhibit the formation of Amadori products and AGEs, the intermediates andadvanced glycation endproducts of non-enzyme glycation (NEG), which suggested that ZJPmay improve the diabetic complications such as microangiopathy caused by NEG and AGEs.As compared to the native ZJP, the inhibitory effect on Amadori products formation of ZJP-0and ZJP-3increased, while that of ZJP-1and ZJP-2decreased. Furthermore, only ZJP-3showed stronger inhibitory effect on AGEs formation than that of the native ZJP, whichsuggested that ZJP-3may be a good inhibitor against non-enzyme glycation.The inhibitory effects of ZJP on non-enzyme glycation were improved with degradationby both ultrasonication and hydrochloric acid, but there was no obvious correlation betweenthe inhibitory effect and degradation degree. Under the experiment conditions, the maximuminhibitory percentage on Amadori products and AGEs formation of ZJP degraded withultrasonication was increased by108.93%and29.41%respectively, and that of ZJP degradedwith hydrochloric acid was improved by95.55%and29.65%respectively.Chemical modification affected the inhibitory effect of ZJP on non-enzyme glycationgreatly. As compared to the native ZJP, the sulfated ZJP with lower degree of substitutionshowed a higher inhibitory effect on Amadori products and AGEs formation, while thesulfated ZJP with higher degree of substitution showed a much lower inhibitory effect.Carboxymethylation ZJP with higher degree of substitution strongly inhibited the non-enzymeglycation with a good correlation between activity and degree of substitution. But there wasno relationship between activity and degree of substitution of aceylated ZJP. Among of thethree modification methods of different substitution groups, carboxymethylation showed thestrongest influence on inhibitory activity on non-enzyme glycation. Under the experimentconditions, the inhibitory percentage on Amadori products and AGEs formation of CM-ZJP-7was up to89.5%and86.3%respectively. Thus, carboxymethylation could be used as animportant means to improve the inhibitory effect of ZJP on non-enzyme glycation.Inhibitory activity on hyaluronidaseExcessive degradation of hyaluronic acid could lead to joint diseases, allergy and othertype of inflammatory response and closely related to initiation and development of cancer.The ZJP was demonstrated to exhibit strong inhibitory effect on hyaluronidase with a strongdose-dependent pattern. Therefore, ZJP could be used as preventive and therapeutic agent forthe diseases caused by excessive degradation of hyaluronic acid due to its prevention ofdegradation of hyaluronic acid and maintaining the function of hyaluronic acid. Under theexperiment conditions, the maximum inhibitory percentage was up to93.55%with an IC₅₀of0.095mg·mL^-1. All of ZJP and the fractions separated by DE52-cellulose column chromatographyshowed inhibitory effect on hyaluronidase. Among of the fractions, the inhibitory effect ofZJP-0and ZJP-1increased slightly, but that of ZJP-2and ZJP-3decreased, which suggestedthat ZJP-0and ZJP-1may play an important role in inhibitory activity on hyaluronidase.ZJP of molecular weight from600to660kDa by degradation with ultrasonicationshowed the strongest inhibitory effect on hyaluronidase, but descended when the molecularweight was lower than600kDa. There was little effect on the inhibitory effect onhyaluronidase when degraded with lower concentration of hydrochloric acid, but theinhibitory activity decreased sharply when the hydrochloric acid concentration was above0.4mol/L, which could cause the molecular weight of ZJP decreased to less than200kD. Thisindicated that higher molecular weight of ZJP was important to maintain its strongerinhibitory effect on hyaluronidase, the length of chain was crucial for the inhibitory activity.Ultrasonication degradation to a moderate extent was helpful to improve the inhibitory effectof ZJP on hyaluronidase.Chemical modification of ZJP with different substitution groups affected the inhibitoryeffect on hyaluronidase differently. As compared to the native ZJP, the inhibitory effect ofsulfated ZJP was improved with a good correlation between degree of substitution andinhibitory activity. While acetylation of ZJP caused the decreasing of inhibitory activity, andthere was no relationship between inhibitory activity and degree of substitution. Thecarboxymethylated ZJP with lower degree of substitution showed a lower inhibitory effect onhyaluronidase, while the carboxymethylated ZJP with higher degree of substitution showed ahigher inhibitory effect. Under the experiment conditions, the inhibitory percentage onsulfation and carboxymethylation could increase the inhibitory percentage of ZJP onhyaluronidase by45.80%and39.76%respectively, which indicated that sulfation andcarboxymethylation with higher degree of substitution could be used as important means toimproved the inhibitory activity of ZJP on hyaluronidase.In conclusion, the biological activities of ZJP were influenced by various mechanisms.The effect of molecular modification on biological activity was also related to the differentmechanism involved in each biological activity. By molecular modification, the solubility ofZJP was improved, thus the activity could be performed better in water soluble test system.Furthermore, with the change of spatial conformation due to molecular modification of ZJP,more or less active groups were revealed. Otherwise, the active configuration of ZJP wasformed or destroyed. All of these changes in structure or physical-chemical characteristicscould lead to the alteration of biological activities. The difference existed in the mechanismand active group or configuration characteristics according to different biological activity would be responsible for the different effect of the same modification. The change ofconfiguration caused by molecular modification seemed to be more important thanphysical-chemical characteristics for ZJP. Maintaining certain length of chain and spatialconformation of ZJP was crucial to perform its activity.