红枣多糖的分子修饰与生物活性研究
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摘要
多糖是红枣中最重要的活性成分之一，具有较高的药用价值。对红枣多糖进行分子修饰和改性有助于提高和改善其生物活性，拓宽应用范围，同时也为进一步探索红枣多糖构效关系和丰富多糖构效关系理论积累基础资料。为此，本论文在对红枣多糖进行提取和分离纯化的基础上，将分子修饰技术应用到红枣多糖研究中，通过超声处理、盐酸降解和硫酸酯化、羧甲基化及乙酰化修饰，进一步对其进行分子修饰和改性，并采用体外试验法研究了红枣多糖及其分子修饰产物的抗氧化活性、降血糖作用以及对透明质酸酶的抑制作用，证实红枣多糖对α-淀粉酶、α-葡萄糖苷酶及透明质酸酶和非酶糖化反应具有抑制作用，分子修饰可显著改变红枣多糖的生物活性，且这种改变更多的与分子修饰导致的红枣多糖高级结构的变化有关。具体结果如下：
一、红枣多糖制备
1、热水浸提法提取红枣多糖的适宜条件为：料液比1:10、温度80℃、时间1h、提取2次。采用XAD-761树脂吸附法可有效分离红枣多糖提取液中的多酚物质，而且不影响红枣多糖的提取分离效果，达到了联合提取红枣多酚、多糖的目的。
2、Sevag法可有效脱除红枣多糖中的蛋白质，活性炭脱色造成的多糖损失率较高。大孔吸附树脂对红枣多糖具有较好的脱色效果，而且可以重复使用，是红枣多糖的较佳脱色方法。
3、采用DE52纤维素柱层析法可将红枣多糖分级纯化为1个中性多糖组分和3个酸性多糖组分，平均分子量均在1000kDa左右，其中酸性多糖组分ZJP-2为红枣多糖中的主要组分。通过HPLC分析和高碘酸氧化研究红枣多糖的单糖组成和连接键型，结果表明，红枣多糖是由半乳糖醛酸、鼠李糖、阿拉伯糖、半乳糖、木糖、葡萄糖等组成的杂多糖，主要连接键型为1→4糖苷键连接。
二、红枣多糖的分子修饰
1、超声处理可引起红枣多糖的降解。较低的温度、较高的超声功率、较低的多糖溶液浓度和变频处理有利于多糖的超声降解。在试验条件下，超声处理仅使红枣多糖分子量得到一定程度的降低，超声降解后红枣多糖的平均分子量仍在300kDa以上。盐酸降解可大幅降低红枣多糖的分子量，并随盐酸浓度的增加而增强。在一定盐酸浓度条件下得到的红枣多糖降解产物仍为相对均一的多糖。过氧化氢可强烈降解红枣多糖，但其分子量分布很宽，并且存在大量的小分子糖。
2、采用氯磺酸-吡啶法可成功对红枣多糖进行硫酸酯化修饰，其最佳工艺条件为：氯磺酸与吡啶的体积比1:3，反应温度60℃，反应时间2.5h，在此条件下制备得到的红枣多糖硫酸酯的硫酸基取代度可以达到1.39。
3、采用氢氧化钠-氯乙酸反应体系对红枣多糖进行羧甲基化修饰，产物的羧甲基取代度随着反应体系中NaOH浓度的增加而提高，但得率呈下降趋势，呈明显负相关关系。在使反应液的pH保持在最适条件下的同时，适当增大羧甲基化试剂与红枣多糖的比例，有利于羧甲基取代度的增加。在试验条件下，制备得到的羧甲基化红枣多糖的羧甲基取代度最高可达到0.22。
4、用冰醋酸法和乙酸酐法均可对红枣多糖进行乙酰化修饰。用冰醋酸法制备的乙酰化红枣多糖的取代度较低，因此不适用于高乙酰基取代度的乙酰化修饰。高温和低pH条件不利于乙酸酐法对红枣多糖的乙酰化修饰，在室温条件下保持反应液的pH为9.5±0.5，可以较好地实现红枣多糖的乙酰化修饰。在试验条件下，制备得到的乙酰化红枣多糖的乙酰基取代度最高可达到0.29。
5、红枣多糖的硫酸酯化、羧甲基化和乙酰化修饰均可明显改善其在水溶液中的分布状态，提高其溶解性。
三、红枣多糖及其分子修饰产物的生物活性研究
1、抗氧化活性
红枣多糖及其经DE52纤维素柱层析分离纯化得到的各个级分对光照核黄素体系产生的超氧阴离子自由基、Fenton反应产生的羟自由基及DPPH自由基均具有较好的清除作用，并呈现一定的量效关系。在试验条件下，红枣多糖对超氧阴离子自由基、羟自由基及DPPH自由基的清除率分别可达99.01%、49.46%和36.89%。与原红枣多糖（ZJP）相比，经DE52纤维素柱层析分离纯化得到的各个级分的自由基清除活性均有一定程度的降低，酸性多糖组分ZJP-1、ZJP-2、ZJP-3较中性多糖ZJP-0表现出更强的自由基清除活性，说明红枣多糖中半乳糖醛酸对于其自由基清除活性具有重要作用。
适度超声降解或者盐酸降解均可提高红枣多糖对不同体系产生的自由基的清除能力，但分子量降低至230kDa以下时其自由基清除活性大幅降低。超声处理对红枣多糖抗氧化活性的影响存在双重效应，既可通过调整分子大小影响红枣多糖抗氧化活性，又可通过声场作用造成多糖构象改变从而影响红枣多糖抗氧化活性。在试验条件下，超声处理最高可使红枣多糖对超氧阴离子自由基、羟自由基及DPPH自由基的清除率分别提高3.16、2.00、0.16倍，而盐酸降解则可使其分别提高4.78、1.46、0.22倍。
不同取代基修饰对红枣多糖自由基清除活性产生的效应与自由基测试体系有关，红枣多糖清除自由基的效果与其构象密切相关。中等取代度的硫酸酯化红枣多糖S-ZJP-2（DS=0.79）、S-ZJP-3（DS=1.03）对超氧阴离子自由基的半抑制剂量（IC50）可较原红枣多糖（ZJP）降低一半以上，但对羟自由基和DPPH自由基清除活性的影响较小；羧甲基化修饰可显著提高红枣多糖的羟自由基清除活性，但不利于其超氧阴离子自由基和DPPH自由基清除活性的发挥；乙酰化修饰可大幅提高红枣多糖的羟自由基清除活性，在试验条件下，五种乙酰化红枣多糖对羟自由基的清除效果均较原多糖（ZJP）提高一倍以上，但乙酰化修饰导致红枣多糖超氧阴离子自由基活性的降低，并改变其清除DPPH自由基的动力学。中等硫酸酯化修饰是提高红枣多糖超氧阴离子自由基清除活性的最有效方法，而乙酰化修饰和羧甲基化修饰则是提高其羟自由基清除活性的重要手段。
2、降血糖作用
红枣多糖对-淀粉酶和-葡萄糖苷酶活性均具有抑制作用，其抑制效果随着多糖浓度的增加而增大，具有明显的量效关系。在试验条件下，红枣多糖对α-淀粉酶和-葡萄糖苷酶活性的最高抑制率分别为53.35%和62.65%，IC50分别为11.39mg·mL-1和16.61mg·mL-1。说明红枣多糖可能对淀粉酶促水解直至生成葡萄糖的整个过程的不同阶段产生影响，从而可有效延缓单糖的释放和吸收，抑制餐后高血糖。
与原红枣多糖（ZJP）相比，除了中性多糖组分ZJP-0的抑制活性略有降低外，经DE52纤维素柱层析分离纯化得到的其它三种酸性多糖组分ZJP-1、ZJP-2、ZJP-3对-淀粉酶的抑制活性均有大幅提高，而四种级分对-葡萄糖苷酶的抑制作用和原多糖相比均有不同程度的提高。其中尤以ZJP-1对-淀粉酶和-葡萄糖苷酶的抑制作用最强，在同浓度条件下可分别较原红枣多糖（ZJP）提高0.63倍和1.67倍，说明ZJP-1在红枣多糖的-淀粉酶和-葡萄糖苷酶抑制活性中发挥主要作用。
红枣多糖经超声降解处理后其对-淀粉酶的抑制活性减弱，而对-葡萄糖苷酶活性的抑制作用得到增强，并且抑制效果随着超声处理时间的延长而提高。低浓度的盐酸降解可提高红枣多糖对-淀粉酶和-葡萄糖苷酶活性的抑制作用，但过度降解则会造成抑制活性的降低。红枣多糖分子量在300~600kDa之间表现出较强的-葡萄糖苷酶抑制活性，但分子量降低至290kDa以下时其-葡萄糖苷酶抑制活性大幅降低。维持一定的链长和空间构象是发挥其活性的关键。在试验条件下，超声处理和盐酸降解最高可使红枣多糖对-葡萄糖苷酶活性的抑制率分别提高2.09倍和1.56倍，而盐酸降解还可使其对-淀粉酶活性的抑制率提高38.25%。
不同取代基修饰均降低了红枣多糖对-淀粉酶的抑制活性，但可增强其对-葡萄糖苷酶的抑制活性。在试验条件下，硫酸酯化、羧甲基化和乙酰化修饰最高可使红枣多糖对-葡萄糖苷酶活性的抑制率分别提高1.87、5.08和3.97倍。羧甲基化修饰是提高红枣多糖-葡萄糖苷酶抑制活性的最有效方法之一。
3、对非酶糖化反应的抑制作用
红枣多糖及其经DE52纤维素分离纯化得到的不同级分对BSA-Glu体系非酶糖化反应中间产物Amadori和终产物AGEs的形成均具有一定的抑制作用，说明红枣多糖可能对由非酶糖化反应以及由此形成的糖化终产物引起的微血管病变等糖尿病并发症具有改善作用。与原红枣多糖（ZJP）相比，ZJP-0和ZJP-3对Amadori产物形成的抑制作用增强，而ZJP-1抑制作用略有降低，ZJP-2的抑制作用最弱。而从AGEs形成的情况来看，则只有ZJP-3的抑制作用高于原多糖，其它组分均较低，说明ZJP-3是一种较好的非酶糖化反应的抑制剂。
超声降解和盐酸降解都可增强红枣多糖对非酶糖化反应的抑制作用，但抑制效果与降解程度没有明显的相关性。在试验条件下，超声降解最高可使红枣多糖对Amadori产物和AGEs形成的抑制率分别提高108.93%和29.41%，而盐酸降解最高可使其分别提高95.55%和29.65%。
取代基修饰可对红枣多糖非酶糖化反应抑制活性产生较大影响。与未修饰的红枣多糖相比，低取代度的硫酸酯化红枣多糖对Amadori产物和AGEs的形成的抑制作用均得到提高，而高取代度的硫酸酯化红枣多糖的抑制活性却大幅下降；而较高取代度的羧甲基化修饰可以显著增强红枣多糖对非酶糖化反应的抑制作用，并且抑制活性的增强与羧甲基取代度之间有较好的相关关系；各乙酰化红枣多糖的取代度与其对非酶糖化反应的抑制效果没有相关性。在试验的三种取代基修饰中，以羧甲基化修饰对红枣多糖非酶糖化反应抑制活性的影响最大，在试验条件下羧甲基取代度最高的CM-ZJP-7对BSA-Glu非酶糖化反应体系中Amadori产物和AGEs的形成的抑制率分别可达到89.5%和86.3%。因此，羧甲基化修饰可作为提高红枣多糖非酶糖化反应抑制效果的重要手段之一。
4、透明质酸酶抑制活性
透明质酸的过度降解可导致关节疾病和过敏及其它类型的炎症反应，而且与肿瘤的发生、发展密切相关。本文研究结果表明，红枣多糖对透明质酸酶具有较强的抑制作用，而且随着红枣多糖浓度的增加而增强，呈现出较好的量效关系。因此，红枣多糖可以阻止体内透明质酸的分解，有助于维持透明质酸的功能，对于由于透明质酸过度降解造成的各种疾病可能会具有一定的防治作用。在试验条件下，其对透明质酸酶活性的抑制率最高可达93.55%，IC50为0.095mg·mL~(-1)。
红枣多糖经DE52纤维素柱层析分离纯化得到的各个级分对透明质酸酶均有不同程度的抑制作用，其中ZJP-0和ZJP-1对透明质酸酶的抑制活性较原多糖略有提高，而ZJP-2和ZJP-3则较低，说明ZJP-0和ZJP-1在红枣多糖的透明质酸酶抑制活性中发挥主要作用。
红枣多糖超声降解至平均分子量在600-660kDa时透明质酸酶抑制活性最强，降至600kDa以下时即呈下降趋势。低浓度盐酸降解对红枣多糖透明质酸酶的影响不大，但当盐酸浓度增大至0.4mol/L以上即红枣多糖平均分子量被降低到200kDa以下时，其对透明质酸酶的抑制活性急剧降低。这说明较高分子量对于维持红枣多糖较强的透明质酸酶抑制活性十分重要，保持一定的链长是发挥红枣多糖对透明质酸酶抑制活性的关键。适度超声降解有助于提高红枣多糖的透明质酸酶抑制活性。
不同取代基修饰对红枣多糖的透明质酸酶抑制活性产生的效应有较大差别。红枣多糖经不同程度硫酸酯化修饰后对透明质酸酶活性的抑制作用均有不同程度的增强，并且其抑制活性随硫酸基取代度的增加而增强；而红枣多糖的乙酰化修饰则使其对透明质酸酶的抑制活性大大降低，且与乙酰基取代度之间没有相关性；低取代度羧甲基化修饰使红枣多糖对透明质酸酶的抑制活性降低，但较高的羧甲基取代会使红枣多糖的透明质酸酶抑制活性有所提高。在试验条件下，硫酸酯化和羧甲基化修饰最高可使红枣多糖对透明质酸酶活性的抑制率分别提高45.80%和39.76%。硫酸酯化修饰和高取代度羧甲基化修饰可作为提高红枣多糖对透明质酸酶抑制活性的重要手段。
综上所述，分子修饰可通过多种机制影响红枣多糖的生物活性，且与各种活性的不同机理有关。通过分子修饰，一方面可以改善红枣多糖的溶解性，从而可使其在水溶性测试系统中发挥更好的活性。同时，通过分子修饰，还可以改变红枣多糖的空间构象，使更多/少的活性基团裸露出来，或形成/破坏红枣多糖的活性构型，造成红枣多糖活性的改变。不同生物活性产生的机理和要求的活性基团及构象特征不同，所以分子修饰产生的效果也存在很大差异。分子修饰导致的红枣多糖构象变化对于其生物活性的影响较理化性质改变更为重要，维持一定的链长和空间构象是发挥其活性的关键。
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 products
Antioxidant 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 effect
The 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 hyaluronidase
Excessive 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 IC_(50)of0.095mg·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.
一、红枣多糖制备
1、热水浸提法提取红枣多糖的适宜条件为：料液比1:10、温度80℃、时间1h、提取2次。采用XAD-761树脂吸附法可有效分离红枣多糖提取液中的多酚物质，而且不影响红枣多糖的提取分离效果，达到了联合提取红枣多酚、多糖的目的。
2、Sevag法可有效脱除红枣多糖中的蛋白质，活性炭脱色造成的多糖损失率较高。大孔吸附树脂对红枣多糖具有较好的脱色效果，而且可以重复使用，是红枣多糖的较佳脱色方法。
3、采用DE52纤维素柱层析法可将红枣多糖分级纯化为1个中性多糖组分和3个酸性多糖组分，平均分子量均在1000kDa左右，其中酸性多糖组分ZJP-2为红枣多糖中的主要组分。通过HPLC分析和高碘酸氧化研究红枣多糖的单糖组成和连接键型，结果表明，红枣多糖是由半乳糖醛酸、鼠李糖、阿拉伯糖、半乳糖、木糖、葡萄糖等组成的杂多糖，主要连接键型为1→4糖苷键连接。
二、红枣多糖的分子修饰
1、超声处理可引起红枣多糖的降解。较低的温度、较高的超声功率、较低的多糖溶液浓度和变频处理有利于多糖的超声降解。在试验条件下，超声处理仅使红枣多糖分子量得到一定程度的降低，超声降解后红枣多糖的平均分子量仍在300kDa以上。盐酸降解可大幅降低红枣多糖的分子量，并随盐酸浓度的增加而增强。在一定盐酸浓度条件下得到的红枣多糖降解产物仍为相对均一的多糖。过氧化氢可强烈降解红枣多糖，但其分子量分布很宽，并且存在大量的小分子糖。
2、采用氯磺酸-吡啶法可成功对红枣多糖进行硫酸酯化修饰，其最佳工艺条件为：氯磺酸与吡啶的体积比1:3，反应温度60℃，反应时间2.5h，在此条件下制备得到的红枣多糖硫酸酯的硫酸基取代度可以达到1.39。
3、采用氢氧化钠-氯乙酸反应体系对红枣多糖进行羧甲基化修饰，产物的羧甲基取代度随着反应体系中NaOH浓度的增加而提高，但得率呈下降趋势，呈明显负相关关系。在使反应液的pH保持在最适条件下的同时，适当增大羧甲基化试剂与红枣多糖的比例，有利于羧甲基取代度的增加。在试验条件下，制备得到的羧甲基化红枣多糖的羧甲基取代度最高可达到0.22。
4、用冰醋酸法和乙酸酐法均可对红枣多糖进行乙酰化修饰。用冰醋酸法制备的乙酰化红枣多糖的取代度较低，因此不适用于高乙酰基取代度的乙酰化修饰。高温和低pH条件不利于乙酸酐法对红枣多糖的乙酰化修饰，在室温条件下保持反应液的pH为9.5±0.5，可以较好地实现红枣多糖的乙酰化修饰。在试验条件下，制备得到的乙酰化红枣多糖的乙酰基取代度最高可达到0.29。
5、红枣多糖的硫酸酯化、羧甲基化和乙酰化修饰均可明显改善其在水溶液中的分布状态，提高其溶解性。
三、红枣多糖及其分子修饰产物的生物活性研究
1、抗氧化活性
红枣多糖及其经DE52纤维素柱层析分离纯化得到的各个级分对光照核黄素体系产生的超氧阴离子自由基、Fenton反应产生的羟自由基及DPPH自由基均具有较好的清除作用，并呈现一定的量效关系。在试验条件下，红枣多糖对超氧阴离子自由基、羟自由基及DPPH自由基的清除率分别可达99.01%、49.46%和36.89%。与原红枣多糖（ZJP）相比，经DE52纤维素柱层析分离纯化得到的各个级分的自由基清除活性均有一定程度的降低，酸性多糖组分ZJP-1、ZJP-2、ZJP-3较中性多糖ZJP-0表现出更强的自由基清除活性，说明红枣多糖中半乳糖醛酸对于其自由基清除活性具有重要作用。
适度超声降解或者盐酸降解均可提高红枣多糖对不同体系产生的自由基的清除能力，但分子量降低至230kDa以下时其自由基清除活性大幅降低。超声处理对红枣多糖抗氧化活性的影响存在双重效应，既可通过调整分子大小影响红枣多糖抗氧化活性，又可通过声场作用造成多糖构象改变从而影响红枣多糖抗氧化活性。在试验条件下，超声处理最高可使红枣多糖对超氧阴离子自由基、羟自由基及DPPH自由基的清除率分别提高3.16、2.00、0.16倍，而盐酸降解则可使其分别提高4.78、1.46、0.22倍。
不同取代基修饰对红枣多糖自由基清除活性产生的效应与自由基测试体系有关，红枣多糖清除自由基的效果与其构象密切相关。中等取代度的硫酸酯化红枣多糖S-ZJP-2（DS=0.79）、S-ZJP-3（DS=1.03）对超氧阴离子自由基的半抑制剂量（IC50）可较原红枣多糖（ZJP）降低一半以上，但对羟自由基和DPPH自由基清除活性的影响较小；羧甲基化修饰可显著提高红枣多糖的羟自由基清除活性，但不利于其超氧阴离子自由基和DPPH自由基清除活性的发挥；乙酰化修饰可大幅提高红枣多糖的羟自由基清除活性，在试验条件下，五种乙酰化红枣多糖对羟自由基的清除效果均较原多糖（ZJP）提高一倍以上，但乙酰化修饰导致红枣多糖超氧阴离子自由基活性的降低，并改变其清除DPPH自由基的动力学。中等硫酸酯化修饰是提高红枣多糖超氧阴离子自由基清除活性的最有效方法，而乙酰化修饰和羧甲基化修饰则是提高其羟自由基清除活性的重要手段。
2、降血糖作用
红枣多糖对-淀粉酶和-葡萄糖苷酶活性均具有抑制作用，其抑制效果随着多糖浓度的增加而增大，具有明显的量效关系。在试验条件下，红枣多糖对α-淀粉酶和-葡萄糖苷酶活性的最高抑制率分别为53.35%和62.65%，IC50分别为11.39mg·mL-1和16.61mg·mL-1。说明红枣多糖可能对淀粉酶促水解直至生成葡萄糖的整个过程的不同阶段产生影响，从而可有效延缓单糖的释放和吸收，抑制餐后高血糖。
与原红枣多糖（ZJP）相比，除了中性多糖组分ZJP-0的抑制活性略有降低外，经DE52纤维素柱层析分离纯化得到的其它三种酸性多糖组分ZJP-1、ZJP-2、ZJP-3对-淀粉酶的抑制活性均有大幅提高，而四种级分对-葡萄糖苷酶的抑制作用和原多糖相比均有不同程度的提高。其中尤以ZJP-1对-淀粉酶和-葡萄糖苷酶的抑制作用最强，在同浓度条件下可分别较原红枣多糖（ZJP）提高0.63倍和1.67倍，说明ZJP-1在红枣多糖的-淀粉酶和-葡萄糖苷酶抑制活性中发挥主要作用。
红枣多糖经超声降解处理后其对-淀粉酶的抑制活性减弱，而对-葡萄糖苷酶活性的抑制作用得到增强，并且抑制效果随着超声处理时间的延长而提高。低浓度的盐酸降解可提高红枣多糖对-淀粉酶和-葡萄糖苷酶活性的抑制作用，但过度降解则会造成抑制活性的降低。红枣多糖分子量在300~600kDa之间表现出较强的-葡萄糖苷酶抑制活性，但分子量降低至290kDa以下时其-葡萄糖苷酶抑制活性大幅降低。维持一定的链长和空间构象是发挥其活性的关键。在试验条件下，超声处理和盐酸降解最高可使红枣多糖对-葡萄糖苷酶活性的抑制率分别提高2.09倍和1.56倍，而盐酸降解还可使其对-淀粉酶活性的抑制率提高38.25%。
不同取代基修饰均降低了红枣多糖对-淀粉酶的抑制活性，但可增强其对-葡萄糖苷酶的抑制活性。在试验条件下，硫酸酯化、羧甲基化和乙酰化修饰最高可使红枣多糖对-葡萄糖苷酶活性的抑制率分别提高1.87、5.08和3.97倍。羧甲基化修饰是提高红枣多糖-葡萄糖苷酶抑制活性的最有效方法之一。
3、对非酶糖化反应的抑制作用
红枣多糖及其经DE52纤维素分离纯化得到的不同级分对BSA-Glu体系非酶糖化反应中间产物Amadori和终产物AGEs的形成均具有一定的抑制作用，说明红枣多糖可能对由非酶糖化反应以及由此形成的糖化终产物引起的微血管病变等糖尿病并发症具有改善作用。与原红枣多糖（ZJP）相比，ZJP-0和ZJP-3对Amadori产物形成的抑制作用增强，而ZJP-1抑制作用略有降低，ZJP-2的抑制作用最弱。而从AGEs形成的情况来看，则只有ZJP-3的抑制作用高于原多糖，其它组分均较低，说明ZJP-3是一种较好的非酶糖化反应的抑制剂。
超声降解和盐酸降解都可增强红枣多糖对非酶糖化反应的抑制作用，但抑制效果与降解程度没有明显的相关性。在试验条件下，超声降解最高可使红枣多糖对Amadori产物和AGEs形成的抑制率分别提高108.93%和29.41%，而盐酸降解最高可使其分别提高95.55%和29.65%。
取代基修饰可对红枣多糖非酶糖化反应抑制活性产生较大影响。与未修饰的红枣多糖相比，低取代度的硫酸酯化红枣多糖对Amadori产物和AGEs的形成的抑制作用均得到提高，而高取代度的硫酸酯化红枣多糖的抑制活性却大幅下降；而较高取代度的羧甲基化修饰可以显著增强红枣多糖对非酶糖化反应的抑制作用，并且抑制活性的增强与羧甲基取代度之间有较好的相关关系；各乙酰化红枣多糖的取代度与其对非酶糖化反应的抑制效果没有相关性。在试验的三种取代基修饰中，以羧甲基化修饰对红枣多糖非酶糖化反应抑制活性的影响最大，在试验条件下羧甲基取代度最高的CM-ZJP-7对BSA-Glu非酶糖化反应体系中Amadori产物和AGEs的形成的抑制率分别可达到89.5%和86.3%。因此，羧甲基化修饰可作为提高红枣多糖非酶糖化反应抑制效果的重要手段之一。
4、透明质酸酶抑制活性
透明质酸的过度降解可导致关节疾病和过敏及其它类型的炎症反应，而且与肿瘤的发生、发展密切相关。本文研究结果表明，红枣多糖对透明质酸酶具有较强的抑制作用，而且随着红枣多糖浓度的增加而增强，呈现出较好的量效关系。因此，红枣多糖可以阻止体内透明质酸的分解，有助于维持透明质酸的功能，对于由于透明质酸过度降解造成的各种疾病可能会具有一定的防治作用。在试验条件下，其对透明质酸酶活性的抑制率最高可达93.55%，IC50为0.095mg·mL~(-1)。
红枣多糖经DE52纤维素柱层析分离纯化得到的各个级分对透明质酸酶均有不同程度的抑制作用，其中ZJP-0和ZJP-1对透明质酸酶的抑制活性较原多糖略有提高，而ZJP-2和ZJP-3则较低，说明ZJP-0和ZJP-1在红枣多糖的透明质酸酶抑制活性中发挥主要作用。
红枣多糖超声降解至平均分子量在600-660kDa时透明质酸酶抑制活性最强，降至600kDa以下时即呈下降趋势。低浓度盐酸降解对红枣多糖透明质酸酶的影响不大，但当盐酸浓度增大至0.4mol/L以上即红枣多糖平均分子量被降低到200kDa以下时，其对透明质酸酶的抑制活性急剧降低。这说明较高分子量对于维持红枣多糖较强的透明质酸酶抑制活性十分重要，保持一定的链长是发挥红枣多糖对透明质酸酶抑制活性的关键。适度超声降解有助于提高红枣多糖的透明质酸酶抑制活性。
不同取代基修饰对红枣多糖的透明质酸酶抑制活性产生的效应有较大差别。红枣多糖经不同程度硫酸酯化修饰后对透明质酸酶活性的抑制作用均有不同程度的增强，并且其抑制活性随硫酸基取代度的增加而增强；而红枣多糖的乙酰化修饰则使其对透明质酸酶的抑制活性大大降低，且与乙酰基取代度之间没有相关性；低取代度羧甲基化修饰使红枣多糖对透明质酸酶的抑制活性降低，但较高的羧甲基取代会使红枣多糖的透明质酸酶抑制活性有所提高。在试验条件下，硫酸酯化和羧甲基化修饰最高可使红枣多糖对透明质酸酶活性的抑制率分别提高45.80%和39.76%。硫酸酯化修饰和高取代度羧甲基化修饰可作为提高红枣多糖对透明质酸酶抑制活性的重要手段。
综上所述，分子修饰可通过多种机制影响红枣多糖的生物活性，且与各种活性的不同机理有关。通过分子修饰，一方面可以改善红枣多糖的溶解性，从而可使其在水溶性测试系统中发挥更好的活性。同时，通过分子修饰，还可以改变红枣多糖的空间构象，使更多/少的活性基团裸露出来，或形成/破坏红枣多糖的活性构型，造成红枣多糖活性的改变。不同生物活性产生的机理和要求的活性基团及构象特征不同，所以分子修饰产生的效果也存在很大差异。分子修饰导致的红枣多糖构象变化对于其生物活性的影响较理化性质改变更为重要，维持一定的链长和空间构象是发挥其活性的关键。
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 products
Antioxidant 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 effect
The 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 hyaluronidase
Excessive 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 IC_(50)of0.095mg·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.
引文
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