柑橘果胶多糖可控性降解和寡糖片段的分离分析及其活性研究
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摘要
果胶多糖是一类结构复杂的高分子化合物,广泛用于食品和医药领域。由果胶多糖降解产生的果胶寡糖,尤其是半乳糖醛酸寡糖,是重要的生物信息分子,具有多种生物活性。本研究以柑橘果胶多糖为原料,对其理化性质、降解条件进行了研究,并对半乳糖醛酸寡糖的分离纯化、分析检测和生物活性进行了较为系统的研究,结果如下：
1.理化性质的测定采用GC法测定了柑橘果胶多糖的单糖组成,结果显示柑橘果胶主要由GalA、Gal、Rha、Ara、Glc和Xyl六种单糖组成,各组分的相对摩尔比为42.8：7.0：2.4：1.2：1.0：0.3,其中Ga1A占总糖含量的78.2%；硫酸-苯酚法测得柑橘果胶中总糖含量为80.5%；间羟基联苯法测得其糖醛酸含量为71.6%；用HPLC法对其甲酯化度和乙酰化度同时测定,结果分别为78.5%和28.5%。以上实验结果显示,柑橘果胶是富含半乳糖醛酸的高酯化度的酸性杂多糖。
2.降解条件的优化通过对多种降解方法的比较试验,优化确定了果胶多糖的两种降解途径：(1)果胶多糖的酸法-酶法联合降解。先将果胶多糖用0.5 mol/L三氟乙酸于80℃水浴中水解5 h,离心,取沉淀部分,继续用0.05 mg/mL的pectinase于40℃下反应60 min,可得DP 1-10的半乳糖醛酸寡糖；(2)果胶酸的酶法降解。以果胶酸为底物,用0.05 mol/L的pectinase于40℃水浴中酶解60 min,可得DP1-5的半乳糖醛酸寡糖。两种方法各有优劣,可满足不同的实际需要。后者虽然得到的寡糖片段少,但相对含量更高,适用于低聚合度(DP1-5)半乳糖醛酸寡糖的大量制备；前者降解释放的寡糖片段较多,可用于更高聚合度(DP>5)半乳糖醛酸寡糖的制备。
药理实验证实半乳糖醛酸五糖具有一定的抗肿瘤活性,本研究应用苯胺稳定同位素标记对还原性寡糖链进行质谱相对定量的分析方法,对影响活性半乳糖醛酸五糖产率的因素进行了优化,确定的制备条件为：以果胶酸为底物,用0.05 mg/mL果胶酶在40℃反应90min。在该条件下,活性半乳糖醛酸五糖的得率最高,可达20%。
3.分析方法的建立(1)建立了果胶寡糖的TLC分析方法寡糖的分析检测是糖类物质研究的难点之一本研究对果胶中性寡糖和酸性寡糖的TLC分析条件进行了优化,并在此基础上建立了薄层扫描方法,可以对多糖的降解过程、寡糖的分离纯化过程进行更为精确的半定量分析。色谱条件为：Sillca gel-100 UV254 TLC薄层分析板；酸性寡糖采用正丁醇-甲酸-水(4：7：1)体系上行展开；中性寡糖采用正丁醇-乙酸-水(3：3：2)体系上行展开；两种寡糖均采用地衣酚-硫酸甲醇溶液显色；检测波长为λmax=490 nm,单波长反射式线性扫描,狭缝0.04×4mm。
(2)建立了AEC柱前衍生HPLC方法,用于多糖或寡糖类物质单糖组成的快速、准确分析。该方法克服了传统GC法样品前处理步骤繁琐、操作复杂、不适合快速检测等缺点,使样品前处理步骤大大简化(衍生反应一次即可完成)、样品损失少,测定结果更接近样品的真实组成,效果满意,可作为糖类物质组成分析的常规手段。
4.分离纯化方法的建立
(1)建立了半乳糖醛酸寡糖的分离纯化方法。以果胶酸为底物酶法降解,所得半乳糖醛酸寡糖混合物过强阴离子交换树脂Dowex-1×4-400(HCOO型),用甲酸钱溶液梯度洗脱,混合组分在交换柱上获得良好的分离,最终得到五个单一聚合度的半乳糖醛酸寡糖：GalA.(GalA)2、(GalA)3、(GalA)4和(GalA)5, DP2-5的收率分别为3.5%、12.7%、8.0%、8.2%。
(2)建立了果胶中性寡糖的分离纯化方法。果胶多糖经TFA部分酸水解,水解液用阴离子交换树脂Dowex-1×4-400 (HCOO型)除去酸性糖,再经硅胶柱,正丁醇-乙酸-水混合溶剂梯度洗脱分离,得聚合度DP 1-5的果胶中性寡糖单体。根据糖组成分析和ESI-MS结果推测,该系列中性寡糖可能以六碳聚糖(Hex)。(n=2-10)的形式存在。
(3)初步建立了鼠李半乳糖醛酸聚糖RG-I的制备方法。果胶多糖经控制性酸水解(HCl+TFA),所得产物主要由GalA、Rha和Gal组成,GalA为最主要成分,占总糖含量的80%,Rha约占13.8%,Rha/GalA比值为0.17,结合ESI-MS分析数据,确定该水解产物为半乳糖醛酸聚糖HG和鼠李半乳糖醛酸聚糖RG-I的混合物,进一步色谱分离,即可得到鼠李半乳糖醛酸聚糖RG-I
5.生物活性试验试验证实,半乳糖醛酸寡糖的生物活性与其聚合度大小密切相关,聚合度高的寡糖表现出强的生物活性,且具有一定的量效相关性。抑菌试验、体外抗肿瘤试验及体外抗氧化试验结果显示,只有DP 6-10的半乳糖醛酸寡糖才表现出一定的抑菌、抗肿瘤及清除自由基的活性。
Pectin is a family of complex polysaccharides present in all plant primary cell walls and widely used in the food industry as a gelling agent and in the health care industry as a immunomodulator. The results of many studies on pectin suggested that pectic oligosaccharides, especially oligogalacturonic acids (oligo-GalAs), possessed many biological activities which were different from that of pectic polysaccharide. In this study, the physico-chemical properties and degradation of Citrus pectic polysaccharides and the isolation, purification, structure characterization and antioxidant activity of oligosaccharides were investigated. The results were summarized as follows:
1. The citrus pectic polysaccharide (CPPS) consists of galacturonic acid, galactose, rhamnose, arabinose, glucose and xylose with the molar ratios of 42.8:7.0:2.4:1.2:1.0:0.3 by gas chromatography assay. The content of total sugar was assayed to be 80.5% by phenol-sulphuric acid method and the content of uronic acid was 71.6% by m-hydroxydiphenyl method. With high-performance liquid chromatography assay, degrees of methylation and acetylation were assayed to be 78.5% and 28.5% in citrus pectic polysaccharide, respectively. Therefore, CPPS is a kind of acidic heteropolysaccharide with high degree of esterification.
2. The degradation is an ideal approach to manufacture oligosaccharides from polysaccharide. Firstly, different pectin hydrolysis procedures were carried out and the optimal parameter values were achieved. A high productivity of oligo-GalAs was obtained under emzymatic hydrolysis condition using pectic acid as the substrate, so this method was suitable for preparation of oligo-GalAs with DP 1-5. Secondly, combined chemical and enzymatic hydrolysis (0.5 mol/L trifluoroacetic acid at 80℃for 5 h and 0.05 mg/mL pectinase at 40℃for 1 h) could offer more oligosaccharide fragments, so this method could be employed to prepare the oligo-GalAs with DP>5.Thirdly, based on aniline stable isotopic labeling and relative quantification in ESI-MS analysis, the optimization of the preparation of active pentogalacturonic acid was investigated. The yield of pentogalacturonic acid reached 20% under the optimized condition.
3.For a long time, the detection of oligosaccharides is one of the difficulties in the study on saccharide. The two analytical methods for pectic oligosaccharides were established in this study. Firstly, thin layer chromatography (TLC) was applied to the analysis of acidic and neutral oligosaccharides and the conditions were optimized. The results showed that it is an effective and convenient method for the analysis of saccharides. The separation of neutral oligosaccharides was improved by multiple development with n-butanol-acetic acid-water (3:3:2) and acidic oligosaccharides with n-butanol-formic acid-water (4:7:1) on TLC silica gel-100 UV254 plates. On that basis, a TLC scanning method was developed for qualitative analysis of oligosaccharides accurately. Secondly, the simultaneous determination of aldoses and uronic acids was available by high-performance liquid chromatography. The procedure involves acid hydrolysis followed by derivatization with 3-amino-9-ethyl-carbazole, which was first employed for aldoses and uronic acids derivatization. The usefulness of this method was seen in the ability to analyze citrus pectin. Compared to the GC method. The described method was suitable for routine analysis of pectin or other polysaccharides containing uronic acids.
4. Firstly, an efficient and inexpensive method for large-scale preparation of oligo-GalAs (DP 1-5) from pectic acid was described. The mixture of oligo-GalA was obtained after pectic acid was digested with an available pectinase, and this mixture was effectively isolated by the anion exchange resin (Dowex-1×4-400, HCOO-) to obtain pure oligo-GalA of DP 1-5 with the yielding rate of 3.5%,12.7%,8.0% and 8.2% for DP 2-5, respectively. Secondly, another efficient method for large-scale preparation of the neutral pectic oligosaccharides was described. The mixture of pectic oligosaccharides was obtained after pectin was hydrolysed by trifluoroacetic acid. Then the mixture was effectively isolated by a combination of anion exchange resin (Dowex-1×4-400, HCOO-) and silica gel column chromatography to obtain pure neutral pectic oligosaccharides of DP 1-5, which were deduced to be probably present as (Hex)n (n=2-10) based on the analysis of GC and ESI-MS assay. Thirdly, a preparation method of rhamnogalacturonans I (RG-I) fragment was investigated. Pectin was hydrolysed by hydrochloric acid followed by trifluoroacetic acid. The hydrolysate was composed of galacturonic acid, galactose and rhamnose with the molar ratio of Rha to GalA as 0.17, which indicated the presence of RG-Ⅰfragment in the hydrolysates.
5.The results of the biological activity experiments for oligo-GalA with different degree of polymerization (DP) showed that higher DP of oligo-GalA had stronger biological activities. Only oligo-GalA with DP more than 5 showed apparent inhibitory effect on the putrefying bacteria in food, antitumor effect on human hepatoma cell line SMMC7721 and scavenging effect on DPPH free radicals.
1.理化性质的测定采用GC法测定了柑橘果胶多糖的单糖组成,结果显示柑橘果胶主要由GalA、Gal、Rha、Ara、Glc和Xyl六种单糖组成,各组分的相对摩尔比为42.8：7.0：2.4：1.2：1.0：0.3,其中Ga1A占总糖含量的78.2%；硫酸-苯酚法测得柑橘果胶中总糖含量为80.5%；间羟基联苯法测得其糖醛酸含量为71.6%；用HPLC法对其甲酯化度和乙酰化度同时测定,结果分别为78.5%和28.5%。以上实验结果显示,柑橘果胶是富含半乳糖醛酸的高酯化度的酸性杂多糖。
2.降解条件的优化通过对多种降解方法的比较试验,优化确定了果胶多糖的两种降解途径：(1)果胶多糖的酸法-酶法联合降解。先将果胶多糖用0.5 mol/L三氟乙酸于80℃水浴中水解5 h,离心,取沉淀部分,继续用0.05 mg/mL的pectinase于40℃下反应60 min,可得DP 1-10的半乳糖醛酸寡糖；(2)果胶酸的酶法降解。以果胶酸为底物,用0.05 mol/L的pectinase于40℃水浴中酶解60 min,可得DP1-5的半乳糖醛酸寡糖。两种方法各有优劣,可满足不同的实际需要。后者虽然得到的寡糖片段少,但相对含量更高,适用于低聚合度(DP1-5)半乳糖醛酸寡糖的大量制备；前者降解释放的寡糖片段较多,可用于更高聚合度(DP>5)半乳糖醛酸寡糖的制备。
药理实验证实半乳糖醛酸五糖具有一定的抗肿瘤活性,本研究应用苯胺稳定同位素标记对还原性寡糖链进行质谱相对定量的分析方法,对影响活性半乳糖醛酸五糖产率的因素进行了优化,确定的制备条件为：以果胶酸为底物,用0.05 mg/mL果胶酶在40℃反应90min。在该条件下,活性半乳糖醛酸五糖的得率最高,可达20%。
3.分析方法的建立(1)建立了果胶寡糖的TLC分析方法寡糖的分析检测是糖类物质研究的难点之一本研究对果胶中性寡糖和酸性寡糖的TLC分析条件进行了优化,并在此基础上建立了薄层扫描方法,可以对多糖的降解过程、寡糖的分离纯化过程进行更为精确的半定量分析。色谱条件为：Sillca gel-100 UV254 TLC薄层分析板；酸性寡糖采用正丁醇-甲酸-水(4：7：1)体系上行展开；中性寡糖采用正丁醇-乙酸-水(3：3：2)体系上行展开；两种寡糖均采用地衣酚-硫酸甲醇溶液显色；检测波长为λmax=490 nm,单波长反射式线性扫描,狭缝0.04×4mm。
(2)建立了AEC柱前衍生HPLC方法,用于多糖或寡糖类物质单糖组成的快速、准确分析。该方法克服了传统GC法样品前处理步骤繁琐、操作复杂、不适合快速检测等缺点,使样品前处理步骤大大简化(衍生反应一次即可完成)、样品损失少,测定结果更接近样品的真实组成,效果满意,可作为糖类物质组成分析的常规手段。
4.分离纯化方法的建立
(1)建立了半乳糖醛酸寡糖的分离纯化方法。以果胶酸为底物酶法降解,所得半乳糖醛酸寡糖混合物过强阴离子交换树脂Dowex-1×4-400(HCOO型),用甲酸钱溶液梯度洗脱,混合组分在交换柱上获得良好的分离,最终得到五个单一聚合度的半乳糖醛酸寡糖：GalA.(GalA)2、(GalA)3、(GalA)4和(GalA)5, DP2-5的收率分别为3.5%、12.7%、8.0%、8.2%。
(2)建立了果胶中性寡糖的分离纯化方法。果胶多糖经TFA部分酸水解,水解液用阴离子交换树脂Dowex-1×4-400 (HCOO型)除去酸性糖,再经硅胶柱,正丁醇-乙酸-水混合溶剂梯度洗脱分离,得聚合度DP 1-5的果胶中性寡糖单体。根据糖组成分析和ESI-MS结果推测,该系列中性寡糖可能以六碳聚糖(Hex)。(n=2-10)的形式存在。
(3)初步建立了鼠李半乳糖醛酸聚糖RG-I的制备方法。果胶多糖经控制性酸水解(HCl+TFA),所得产物主要由GalA、Rha和Gal组成,GalA为最主要成分,占总糖含量的80%,Rha约占13.8%,Rha/GalA比值为0.17,结合ESI-MS分析数据,确定该水解产物为半乳糖醛酸聚糖HG和鼠李半乳糖醛酸聚糖RG-I的混合物,进一步色谱分离,即可得到鼠李半乳糖醛酸聚糖RG-I
5.生物活性试验试验证实,半乳糖醛酸寡糖的生物活性与其聚合度大小密切相关,聚合度高的寡糖表现出强的生物活性,且具有一定的量效相关性。抑菌试验、体外抗肿瘤试验及体外抗氧化试验结果显示,只有DP 6-10的半乳糖醛酸寡糖才表现出一定的抑菌、抗肿瘤及清除自由基的活性。
Pectin is a family of complex polysaccharides present in all plant primary cell walls and widely used in the food industry as a gelling agent and in the health care industry as a immunomodulator. The results of many studies on pectin suggested that pectic oligosaccharides, especially oligogalacturonic acids (oligo-GalAs), possessed many biological activities which were different from that of pectic polysaccharide. In this study, the physico-chemical properties and degradation of Citrus pectic polysaccharides and the isolation, purification, structure characterization and antioxidant activity of oligosaccharides were investigated. The results were summarized as follows:
1. The citrus pectic polysaccharide (CPPS) consists of galacturonic acid, galactose, rhamnose, arabinose, glucose and xylose with the molar ratios of 42.8:7.0:2.4:1.2:1.0:0.3 by gas chromatography assay. The content of total sugar was assayed to be 80.5% by phenol-sulphuric acid method and the content of uronic acid was 71.6% by m-hydroxydiphenyl method. With high-performance liquid chromatography assay, degrees of methylation and acetylation were assayed to be 78.5% and 28.5% in citrus pectic polysaccharide, respectively. Therefore, CPPS is a kind of acidic heteropolysaccharide with high degree of esterification.
2. The degradation is an ideal approach to manufacture oligosaccharides from polysaccharide. Firstly, different pectin hydrolysis procedures were carried out and the optimal parameter values were achieved. A high productivity of oligo-GalAs was obtained under emzymatic hydrolysis condition using pectic acid as the substrate, so this method was suitable for preparation of oligo-GalAs with DP 1-5. Secondly, combined chemical and enzymatic hydrolysis (0.5 mol/L trifluoroacetic acid at 80℃for 5 h and 0.05 mg/mL pectinase at 40℃for 1 h) could offer more oligosaccharide fragments, so this method could be employed to prepare the oligo-GalAs with DP>5.Thirdly, based on aniline stable isotopic labeling and relative quantification in ESI-MS analysis, the optimization of the preparation of active pentogalacturonic acid was investigated. The yield of pentogalacturonic acid reached 20% under the optimized condition.
3.For a long time, the detection of oligosaccharides is one of the difficulties in the study on saccharide. The two analytical methods for pectic oligosaccharides were established in this study. Firstly, thin layer chromatography (TLC) was applied to the analysis of acidic and neutral oligosaccharides and the conditions were optimized. The results showed that it is an effective and convenient method for the analysis of saccharides. The separation of neutral oligosaccharides was improved by multiple development with n-butanol-acetic acid-water (3:3:2) and acidic oligosaccharides with n-butanol-formic acid-water (4:7:1) on TLC silica gel-100 UV254 plates. On that basis, a TLC scanning method was developed for qualitative analysis of oligosaccharides accurately. Secondly, the simultaneous determination of aldoses and uronic acids was available by high-performance liquid chromatography. The procedure involves acid hydrolysis followed by derivatization with 3-amino-9-ethyl-carbazole, which was first employed for aldoses and uronic acids derivatization. The usefulness of this method was seen in the ability to analyze citrus pectin. Compared to the GC method. The described method was suitable for routine analysis of pectin or other polysaccharides containing uronic acids.
4. Firstly, an efficient and inexpensive method for large-scale preparation of oligo-GalAs (DP 1-5) from pectic acid was described. The mixture of oligo-GalA was obtained after pectic acid was digested with an available pectinase, and this mixture was effectively isolated by the anion exchange resin (Dowex-1×4-400, HCOO-) to obtain pure oligo-GalA of DP 1-5 with the yielding rate of 3.5%,12.7%,8.0% and 8.2% for DP 2-5, respectively. Secondly, another efficient method for large-scale preparation of the neutral pectic oligosaccharides was described. The mixture of pectic oligosaccharides was obtained after pectin was hydrolysed by trifluoroacetic acid. Then the mixture was effectively isolated by a combination of anion exchange resin (Dowex-1×4-400, HCOO-) and silica gel column chromatography to obtain pure neutral pectic oligosaccharides of DP 1-5, which were deduced to be probably present as (Hex)n (n=2-10) based on the analysis of GC and ESI-MS assay. Thirdly, a preparation method of rhamnogalacturonans I (RG-I) fragment was investigated. Pectin was hydrolysed by hydrochloric acid followed by trifluoroacetic acid. The hydrolysate was composed of galacturonic acid, galactose and rhamnose with the molar ratio of Rha to GalA as 0.17, which indicated the presence of RG-Ⅰfragment in the hydrolysates.
5.The results of the biological activity experiments for oligo-GalA with different degree of polymerization (DP) showed that higher DP of oligo-GalA had stronger biological activities. Only oligo-GalA with DP more than 5 showed apparent inhibitory effect on the putrefying bacteria in food, antitumor effect on human hepatoma cell line SMMC7721 and scavenging effect on DPPH free radicals.
引文
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