薤白多糖的制备、性质、结构及其生物活性研究
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摘要
目前有关多糖的研究已成为天然药物及保健品研发中的重要组成部分,国外大部分制药公司都有糖类药物研发项目。作为天然生物活性成分,研究已发现多糖具有抗氧化、抗肿瘤、抗炎及免疫调节等多种功能。薤白(Allium macrostemon Bunge)为葱属多年生草本植物,广泛分布于中国、日本和韩国等国的平原地区,多食用,其干燥鳞茎也可入药。截至目前,对薤白的研究国内外主要集中在其挥发油及单体化合物等方面,有关薤白多糖的研究还很不完善,尚未做较为深入系统的研究。鉴于此,为了对现代多糖类保健食品或糖类药物的开发奠定技术基础,为薤白及其方剂的进一步应用提供理论基础,同时在理论上、学术上及生产实践上为我国活性多糖研究与开发利用提供更多的参考资料,本文拟对薤白多糖从提取工艺优化、纯品制备、结构分析鉴定、活性筛选评价及结构和功能的构效关系等方面进行较为系统深入的研究,通过对这些问题的阐释,不仅有助于充分开发利用我国的葱属植物资源,拓宽其应用范围,而且对于其做为传统药物作用机制的揭示,也具有一定的科学价值。
本文首先以单因素提取实验为基础,结合响应面优化法,对薤白多糖的分离提取工艺进行了优化,得出实现薤白多糖高效提取的最佳工艺条件。在此条件下,采用分步醇沉技术对薤白多糖水浸提液进行分段沉降,得到薤白粗多糖。而后采用离子交换柱层析和凝胶柱层析相结合的方法,纯化分离得到均一多糖。通过对薤白多糖的总糖含量(苯酚-硫酸法)、蛋白质含量(考马斯亮蓝法)、糖醛酸含量(硫酸-间羟联苯法)、硫酸基含量(氯化钡-明胶比色法)的测定,初步了解其理化性质,然后应用GC、HPLC、IR、NMR、GC/MS等仪器及结合高碘酸氧化、Smith降解、甲基化等化学手段分析薤白多糖,探讨薤白多糖各纯化组分的分子量、单糖组成、结构特征。最后对薤白多糖采用体外抗氧化活性、抗肿瘤活性、抑菌活性、神经营养活性以及小鼠体内化学急性肝损伤等模型进行活性筛选,评价其活性,并探讨了薤白多糖活性与结构的初步关系。主要研究结果如下：
1、薤白多糖提取条件的响应面法优化研究
本研究采用响应曲面法,优化薤白多糖提取工艺条件。首先选择提取温度、水料比、提取时间及提取次数4个因素进行单因素实验,比较多糖得率,确定提取温度90℃、水料比12mL/g、提取时间90min及提取次数2次为中心实验点。在此基础上,采用Box-Behnken实验设计法,进行4因素3水平响应曲面法实验,同时进行统计分析,从而优化出薤白多糖最佳提取工艺条件为提取温度87℃,水料比12mL/g,提取时间100min以及提取次数3次,在此条件下,薤白多糖得率预测值为25.51%,实验值为25.49%,数据分析表明实验值与预测值间存在较好的一致性。此优化条件的得出为进一步开发和利用薤白多糖提供了基础实验数据和理论分析依据。
2、多糖提取、分离纯化、理化性质及结构分析
通过对薤白干燥鳞茎进行预处理后,经干燥、粉碎、热水提取、40%,60%和80%乙醇分步沉淀、离心,冻干,分别得到三种级分的薤白粗多糖AMP40、AMP60和AMP80,其提取率分别为3.2%、11.8%和9.8%。AMP40、AMP60和AMP80再经过阴离子交换色谱(DEAE-纤维素)和凝胶过滤色谱(Sephadex G-100)进一步分离纯化,得到四个薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)。分别采用苯酚-硫酸法、考马斯亮蓝法、硫酸-间羟联苯法、氯化钡-明胶法对薤白粗多糖(AMP40、AMP60及AMP80)和纯化组分(AMP40-1、AMP40-2、AMP60-1及AMP80-1)中总糖、蛋白质、糖醛酸、硫酸基含量进行了分析。结果表明,各多糖组分不含蛋白质,硫酸基含量普遍较低,但薤白粗多糖AMP40的糖醛酸含量达12.56%,远高于AMP60和AMP80, AMP40经纯化后,AMP40-2中糖醛酸的含量更是高达30.22%,远高于其它薤白多糖。
采用高效液相凝胶渗透色谱法对薤白多糖纯化组分AMP40-1. AMP40-2. AMP60-1及AMP80-1的均一性进行了鉴定,同时测定了其相对分子质量,分别为18.2kDa、105.0kDa、11.2kDa和8.1kDa,表明酸性多糖AMP40-2相比其它薤白多糖具有较高的相对分子质量。
通过气相色谱分析了各多糖的单糖组成,AMP40-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖=1.00：5.03；AMP40-2单糖组成及摩尔比为：鼠李糖：阿拉伯糖：葡萄糖：半乳糖=1.41：2.34：1.62：1.00；AMP60-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖：半乳糖=9.73:1.10:1.00; AMP80-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖=1.00：2.41。结果表明,AMP40-2目比其它多糖拥有更为复杂的单糖组成。
红外光谱分析结果表明：AMP40-1、AMP40-2、AMP60-1及AMP80-1四种多糖均主要为吡喃糖的糖苷键构型,且其中AMP40-2既有β-型糖苷键,又存在仅α-型糖苷键连接。在1741cm-1处出现强吸收峰进一步验证了AMP40-2中的高糖醛酸含量。紫外光谱分析显示薤白多糖在260-280nm处均未出现明显的吸收峰,说明薤白多糖不含核酸和蛋白质。
薤白多糖样品(AMP40-1、AMP40-2、AMP60-1及AMP80-1)通过高碘酸氧化、Smith降解、甲基化反应、FT-IR、GC/MS以及NMR技术对其糖苷键连接方式、糖链分支情况进行分析。结果表明：AMP40-1中各糖苷主要以β-吡喃糖形式存在,以1,2-葡萄糖残基和1,2-阿拉伯糖残基构成主链,同时以葡萄糖为非还原末端,含有1,2,6-葡萄糖分支的杂多糖；AMP40-2其主链主要由Glc(1→6)和Gal(1→6)连接组成,支链由Rha(1→2)构成,分支点位于Glc(1→6)的C-2处,末端残基为Glc、Rha及Ara,各糖苷主要以吡喃糖形式存在,且糖苷键构型存在α型和β型两种构型；AMP60-1主要由Glc(1→2)和Ara(1→5)构成主链,以Gal(1→6)和Glc(1→6)为支链,分支点位于Glc(1→2)的C-6处,末端残基为Ara,各糖苷主要以β-吡喃糖形式存在；AMP80-1王链主要由Glc(1→2)连接组成,支链由Glc(1→6)构成,分支点位于Glc(1→2的C-6处,以葡萄糖和阿拉伯糖为非还原末端,各糖苷主要以β-吡喃糖形式存在。同时刚果红实验结果表明薤白多糖AMP40-2具有三股螺旋结构。
3、薤白多糖体外抗氧化活性及其对小鼠急性肝损伤的保护作用研究
通过测定薤白多糖清除1,1-二苯基-2-苦肼基自由基(DPPH-)的能力、清除羟基自由基(OH·)的能力、清除超氧阴离子自由基(O2·能力、金属离子螯合能力和还原能力,分别评价了薤白粗多糖(AMP40、AMP60和AMP80)及其纯化组分(AMP40-1、 AMP40-2、AMP60-1和AMP80-1)的体外抗氧化活性,结果表明：AMP40-2在大多数抗氧化评价体系中比其它薤白多糖具有更强的抗氧化活性,对比其它薤白多糖表明其较强的抗氧化活性可能与其较高的分子量,较高的糖醛酸含量以及较为复杂的单糖组成有关。
采用CC14肝损伤模型评价了薤白多糖(AMP40、AMP60和AMP80)的体内抗氧化能力,结果发现,通过预先灌胃不同剂量的薤白多糖AMP40,可以抑制小鼠化学性急性肝损伤的发生使肝中毒的各项生化指标趋于正常值,表明薤白多糖AMP40具有一定的肝保护作用。与AMP60及AMP80相比,AMP40在所含多糖分子量大小及糖醛酸含量方面存在明显差异,这也说明多糖的肝保护作用可能与上述因素密切相关。
4、薤白多糖抑菌、抑瘤及神经营养活性研究
采用琼脂扩散法,对葱属植物薤白中薤白水煮部分(AMW)、薤白醇提部分(AWE)和薤白多糖部分(AMP)进行了抑茵活性测定,结果表明：葱属植物薤白具有一定的抗菌活性,其抑菌成分主要存在于醇提部分,可能为挥发油或其它小分子物质,而薤白多糖(水溶性大分子)部分的抗菌活性较弱。
采用MTT法对薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)在体外抑制人肺癌细胞A549和人胃癌细胞BGC-823(?)勺增殖作用进行了研究。结果显示,AMP40-1、AMP60-1和AMP80-1三种薤白多糖对人肺癌细胞A549的生长不具有抑制作用,而AMP40-2对人肺癌细胞A549的生长具有一定的抑制作用,且在实验范围内其抑制作用与浓度呈明显的浓度依赖效应。对人胃癌细胞BGC-823的增殖实验结果表明,薤白多糖均能不同程度的抑制BGC-823细胞的增殖,且抑制作用与多糖之间存在不同程度的正相关量效关系和时效关系。结果也进一步表明薤白多糖对癌细胞生长抑制作用的强弱与其多糖分子量的大小、糖醛酸含量的高低以及单糖组成等因素存在明显的对应关系。
通过大鼠肾上腺嗜铬细胞瘤细胞PC12培养体系,对薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)进行了神经营养活性研究。结果表明,薤白多糖AMP40-2对大鼠嗜铬细胞瘤PC12细胞具有一定的促分化作用,即AMP40-2具有一定的神经营养活性。
Polysaccharide has become an important part of the research and development of natural medicines and health products. Most of the foreign pharmaceutical companies engaged in the carbohydrate drug research. As naturally occurring biological constituents, these high molecular weight polymers are highly appreciated for their multipurpose therapeutic properties, such as antioxidant, antitumor, immune-modulating and anti-inflammatory activities. A. macrostemon Bunge, a perennial herb that propagates vegetative and grows wild on the plains of China, Korea and Japan, belongs to the botanic family of Alliaceae. The young leaves and bulbs of this plant are used as a vegetable. Furthermore, the dried bulbs of A. macrostemon Bunge are well known as the traditional Chinese medicine "Xiebai".
Up to now, research about A. macrostemon Bunge is mainly concentrated in the volatile oil and monomer compounds at home and abroad. The research in polysaccharide of A. macrostemon Bunge is far from perfect, which lack of an in-depth and a systematic study. In view of this, to establish the technological foundation for the development of modern multi-carbohydrate health food or sugar substances, to provide a theoretical basis for the further application of the A. macrostemon Bunge and its prescription, while to provide more references about research and development of polysaccharides in theory, academic and production practices were necessary. In this paper, we conducted a more systematic and in-depth research of polysaccharide from A. macrostemon Bunge about the optimization of the extraction process, the pure preparation methods, structural analysis and identification, activity screening evaluation and the structure and function relationship. Interpretation of these questions not only help to fully develop the use of our resources of Allium species, and to broaden its scope of application, but also to reveal its mechanism of action of traditional medicines will have a certain scientific value.
A four-factor and three-level Box-Behnken design was used to optimise the extraction parameters for polysaccharides from A. macrostemon Bunge. Then, a graded ethanol precipitation was used to fractionate the water-extractable polysaccharides, resulting in three polysaccharides fractions. And the crude polysaccharides were purified by anion-exchange and gel filtration chromatography; The purified fractions were characterized by using the phenol-sulfuric method, periodic acid oxidation, Smith degradation, methylation combined with high-performance liquid chromatography (HPLC), gas chromatography (GC), Fourier-infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS), etc. Finally, we investigated the antioxidant activities, the antitumor activities, antibacterial activity, neurotrophic activity, as well as hepatoprotective effects of polysaccharides from A. macrostemon Bunge, and described the initial relationship of the activity and structure of the polysaccharide. The main findings are as follows.
1. Extraction optimisation of polysaccharides from Allium macrostemon Bunge
Response surface methodology (RSM), based on a Box-Behnken design (BBD), was used to optimize the extraction conditions of polysaccharides (AMP). Four independent variables such as extraction temperature (℃), ratio of water to raw material (mL/g), extraction time (min) and extraction time were investigated. As a result, the optimal conditions for AMP extraction were the following:extraction temperature,87℃; ratio of water to raw material,12mL/g; extraction time,100min; and extraction times,3. Under these conditions, the experimental value was25.49±0.11%, which is well matched with value predicted by the model. The optimal conditions provide the basis of experimental data and theoretical analysis for the further development and utilization polysaccharides from A. macrostemon Bunge.
2. Isolation, purification, physico-chemical characterization and structural elucidation
The dried pretreated bulbs of A. macrostemon Bunge was extracted with water by use of the optimal extraction conditions determined as mentioned above. The extract was then centrifuged, and the supernatant was mixed with absolute ethanol to a final ethanol concentration of40%,60%,80%. The resulting precipitate was obtained from centrifugation and dried, affording AMP40, AMP60and AMP80. The recovery rates were3.2%,11.8%and9.83%, respectively. The crude AMP were purified by DEAE-Cellulose52chromatography and Sephadex G-100chromatography to afford4fractions, AMP40-1, AMP40-2, AMP60-1and AMP80-1. Based on the characterization results of AMP by the phenol-sulfuric acid method, the Bradford assay method, the hydroxydiphenyl assay, we found that AMP40-2was quite different from the others, especially AMP40-2had relative higher content of uronic acid (30.22%).
The molecular weights of AMP were determined by HPLC with size exclusion column. All of them showed a single and symmetrically sharp peak, indicated that they were homogeneous polysaccharide. According to the calibration curve of the elution times of standards, the molecular weights of AMP40-1, AMP40-2, AMP60-1and AMP80-1were estimated to be18.2kDa、105.0kDa、11.2kDa and8.1kDa, respectively.
Monosaccharide composition analysis was acted by using the GC method. GC analysis showed that AMP40-1was composed of arabinose and glucose in a molar ratio of1.00:5.03. AMP40-2was composed of rhamnose, arabinose, glucose and galactose in a molar ratio of1.41:2.34:1.62:1.00. AMP60-1was composed of arabinose, glucose and galactose in a molar ratio of9.73:1.10:1.00. AMP80-1was composed of arabinose and glucose in a molar ratio of1.00:2.41. Results indicated that monosaccharide composition of AMP40-2was more complicated than the others.
Infrared spectroscopy analysis indicated that the four samples showed typical peaks of polysaccharide. The characteristic absorption bands at833cm-1and894cm-1indicated that AMP40-2contained both a-glycosidic and β-glycosidic linkages, the band near1741cm-1was in coincidence with the fact that AMP40-2had relative higher uronic acid contents. UV analysis showed that the polysaccharides in the260-280nm no obvious absorption peak, indicate that it does not contain nucleic acids and proteins.
The structural characterizations of purified polysaccharides were studied by using periodic acid oxidation, Smith degradation, methylation combined with HPLC, GC, GC-MS, FTIR and NMR spectra. Results showed that sugar rings of AMP40-1, AMP40-2, AMP60-1and AMP80-1were pyranose rings. AMP40-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl and (1,2)-linked arabinosyl residues. The branch chains with non-reducing terminal glucose residues were attached to backbone chain by1→6glycosidic bonds; AMP40-2were connected by a-configuration and β-configuration glycosidic bonds. Backbone chains of AMP40-2were linked by (1,6)-linked D-glucosyl and (1,6)-linked galactosyl residues. The branch chains with non-reducing terminal glucose, rhamnose and arabinose residues were attached to backbone chain by (1,2)-linked rhamnosyl glycosidic bonds; It was concluded that AMP60-1contained a backbone of (1,6)-linked D-glucosyl and (1,5)-arabinosyl residues with small amounts of the non-reducing terminal arabinose residues, also contained a minor (1,2,6)-D-glucose residue; AMP80-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl residues. The branch chains with non-reducing terminal glucose and arabinose residues were attached to backbone chain by1→6glycosidic bonds. Congo red dye experiments results indicated that AMP40-2possessed the triple helical structure.
3. Antioxidant activities in vitro and hepatoprotective effects of polysaccharides from Allium macrostemon Bunge
The antioxidant activities in vitro of polysaccharides from A. macrostemon Bunge were evaluated by measuring2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical and hydroxyl radical (OH·) scavenging activity, superoxide radical scavenging activity (O2·), metal ion chelating activity and reducing power. During in vitro antioxidant assay, AMP40-2exhibited relative stronger scavenging activities than the others in most cases. The differences in antioxidant activities in vitro might be due to their differences in molecular weight, monosaccharide composition and uronic acid content.
The antioxidant activities in vivo of crude AMP were investigated of against CCl4-induced acute liver damage in Kuming mice. The results showed that pretreatments with AMP40could improve markedly the activities of those antioxidant enzymes and the levels of CCl4-treated mice. The antioxidative system tended to be normalized by the protective action of AMP40. Results showed that molecular weight, monosaccharide composition and the uronic acid content of polysaccharide affects its antioxidant property directly.
4. Antibacterial activity, the antitumor activity and neurotrophic activity of polysaccharides from Allium macrostemon Bunge
The antimicrobial effects of extracts (AMW, AME and AMP) were evaluated by agar diffusion method. The results showed that:A. macrostemon Bunge has some anti-bacterial activity, its antibacterial ingredients present in the ethanol extract, may be a volatile oil or other small molecules, while the anti-bacterial activity of polysaccharides (water-soluble macromolecules) was weak.
MTT assay was used to investigate the anti-proliferation activities against human gastric cancer cells (BGC-823) and human lung adenocarcinoma epithelial cells (A549) in vitro of AMP40-1、AMP40-2、AMP60-1and AMP80-1. The results showed that AMP40-1, AMP60-1and AMP80-1did not inhibit the growth of human lung cancer cell line A549, while AMP40-2inhibited proliferation of A549in a dose-dependent manner. For human gastric carcinoma cell BGC-823, AMP40-1、AMP40-2、AMP60-1and AMP80-1significantly inhibited proliferation in dose-dependent and time-dependent manner. The results demonstrate that the molecular weight, chemical property, monosaccharide composition and linkage type of polysaccharide play important roles in the antitumor activity of polysaccharide.
The neurotrophic activity of polysaccharides from A. macrostemon Bunge were investingated by PC12cell assay. The screening results showed that AMP40-2induced the differentiation of PC12cell and has obvious neurotrophic activity.
本文首先以单因素提取实验为基础,结合响应面优化法,对薤白多糖的分离提取工艺进行了优化,得出实现薤白多糖高效提取的最佳工艺条件。在此条件下,采用分步醇沉技术对薤白多糖水浸提液进行分段沉降,得到薤白粗多糖。而后采用离子交换柱层析和凝胶柱层析相结合的方法,纯化分离得到均一多糖。通过对薤白多糖的总糖含量(苯酚-硫酸法)、蛋白质含量(考马斯亮蓝法)、糖醛酸含量(硫酸-间羟联苯法)、硫酸基含量(氯化钡-明胶比色法)的测定,初步了解其理化性质,然后应用GC、HPLC、IR、NMR、GC/MS等仪器及结合高碘酸氧化、Smith降解、甲基化等化学手段分析薤白多糖,探讨薤白多糖各纯化组分的分子量、单糖组成、结构特征。最后对薤白多糖采用体外抗氧化活性、抗肿瘤活性、抑菌活性、神经营养活性以及小鼠体内化学急性肝损伤等模型进行活性筛选,评价其活性,并探讨了薤白多糖活性与结构的初步关系。主要研究结果如下：
1、薤白多糖提取条件的响应面法优化研究
本研究采用响应曲面法,优化薤白多糖提取工艺条件。首先选择提取温度、水料比、提取时间及提取次数4个因素进行单因素实验,比较多糖得率,确定提取温度90℃、水料比12mL/g、提取时间90min及提取次数2次为中心实验点。在此基础上,采用Box-Behnken实验设计法,进行4因素3水平响应曲面法实验,同时进行统计分析,从而优化出薤白多糖最佳提取工艺条件为提取温度87℃,水料比12mL/g,提取时间100min以及提取次数3次,在此条件下,薤白多糖得率预测值为25.51%,实验值为25.49%,数据分析表明实验值与预测值间存在较好的一致性。此优化条件的得出为进一步开发和利用薤白多糖提供了基础实验数据和理论分析依据。
2、多糖提取、分离纯化、理化性质及结构分析
通过对薤白干燥鳞茎进行预处理后,经干燥、粉碎、热水提取、40%,60%和80%乙醇分步沉淀、离心,冻干,分别得到三种级分的薤白粗多糖AMP40、AMP60和AMP80,其提取率分别为3.2%、11.8%和9.8%。AMP40、AMP60和AMP80再经过阴离子交换色谱(DEAE-纤维素)和凝胶过滤色谱(Sephadex G-100)进一步分离纯化,得到四个薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)。分别采用苯酚-硫酸法、考马斯亮蓝法、硫酸-间羟联苯法、氯化钡-明胶法对薤白粗多糖(AMP40、AMP60及AMP80)和纯化组分(AMP40-1、AMP40-2、AMP60-1及AMP80-1)中总糖、蛋白质、糖醛酸、硫酸基含量进行了分析。结果表明,各多糖组分不含蛋白质,硫酸基含量普遍较低,但薤白粗多糖AMP40的糖醛酸含量达12.56%,远高于AMP60和AMP80, AMP40经纯化后,AMP40-2中糖醛酸的含量更是高达30.22%,远高于其它薤白多糖。
采用高效液相凝胶渗透色谱法对薤白多糖纯化组分AMP40-1. AMP40-2. AMP60-1及AMP80-1的均一性进行了鉴定,同时测定了其相对分子质量,分别为18.2kDa、105.0kDa、11.2kDa和8.1kDa,表明酸性多糖AMP40-2相比其它薤白多糖具有较高的相对分子质量。
通过气相色谱分析了各多糖的单糖组成,AMP40-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖=1.00：5.03；AMP40-2单糖组成及摩尔比为：鼠李糖：阿拉伯糖：葡萄糖：半乳糖=1.41：2.34：1.62：1.00；AMP60-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖：半乳糖=9.73:1.10:1.00; AMP80-1单糖组成及摩尔比为：阿拉伯糖：葡萄糖=1.00：2.41。结果表明,AMP40-2目比其它多糖拥有更为复杂的单糖组成。
红外光谱分析结果表明：AMP40-1、AMP40-2、AMP60-1及AMP80-1四种多糖均主要为吡喃糖的糖苷键构型,且其中AMP40-2既有β-型糖苷键,又存在仅α-型糖苷键连接。在1741cm-1处出现强吸收峰进一步验证了AMP40-2中的高糖醛酸含量。紫外光谱分析显示薤白多糖在260-280nm处均未出现明显的吸收峰,说明薤白多糖不含核酸和蛋白质。
薤白多糖样品(AMP40-1、AMP40-2、AMP60-1及AMP80-1)通过高碘酸氧化、Smith降解、甲基化反应、FT-IR、GC/MS以及NMR技术对其糖苷键连接方式、糖链分支情况进行分析。结果表明：AMP40-1中各糖苷主要以β-吡喃糖形式存在,以1,2-葡萄糖残基和1,2-阿拉伯糖残基构成主链,同时以葡萄糖为非还原末端,含有1,2,6-葡萄糖分支的杂多糖；AMP40-2其主链主要由Glc(1→6)和Gal(1→6)连接组成,支链由Rha(1→2)构成,分支点位于Glc(1→6)的C-2处,末端残基为Glc、Rha及Ara,各糖苷主要以吡喃糖形式存在,且糖苷键构型存在α型和β型两种构型；AMP60-1主要由Glc(1→2)和Ara(1→5)构成主链,以Gal(1→6)和Glc(1→6)为支链,分支点位于Glc(1→2)的C-6处,末端残基为Ara,各糖苷主要以β-吡喃糖形式存在；AMP80-1王链主要由Glc(1→2)连接组成,支链由Glc(1→6)构成,分支点位于Glc(1→2的C-6处,以葡萄糖和阿拉伯糖为非还原末端,各糖苷主要以β-吡喃糖形式存在。同时刚果红实验结果表明薤白多糖AMP40-2具有三股螺旋结构。
3、薤白多糖体外抗氧化活性及其对小鼠急性肝损伤的保护作用研究
通过测定薤白多糖清除1,1-二苯基-2-苦肼基自由基(DPPH-)的能力、清除羟基自由基(OH·)的能力、清除超氧阴离子自由基(O2·能力、金属离子螯合能力和还原能力,分别评价了薤白粗多糖(AMP40、AMP60和AMP80)及其纯化组分(AMP40-1、 AMP40-2、AMP60-1和AMP80-1)的体外抗氧化活性,结果表明：AMP40-2在大多数抗氧化评价体系中比其它薤白多糖具有更强的抗氧化活性,对比其它薤白多糖表明其较强的抗氧化活性可能与其较高的分子量,较高的糖醛酸含量以及较为复杂的单糖组成有关。
采用CC14肝损伤模型评价了薤白多糖(AMP40、AMP60和AMP80)的体内抗氧化能力,结果发现,通过预先灌胃不同剂量的薤白多糖AMP40,可以抑制小鼠化学性急性肝损伤的发生使肝中毒的各项生化指标趋于正常值,表明薤白多糖AMP40具有一定的肝保护作用。与AMP60及AMP80相比,AMP40在所含多糖分子量大小及糖醛酸含量方面存在明显差异,这也说明多糖的肝保护作用可能与上述因素密切相关。
4、薤白多糖抑菌、抑瘤及神经营养活性研究
采用琼脂扩散法,对葱属植物薤白中薤白水煮部分(AMW)、薤白醇提部分(AWE)和薤白多糖部分(AMP)进行了抑茵活性测定,结果表明：葱属植物薤白具有一定的抗菌活性,其抑菌成分主要存在于醇提部分,可能为挥发油或其它小分子物质,而薤白多糖(水溶性大分子)部分的抗菌活性较弱。
采用MTT法对薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)在体外抑制人肺癌细胞A549和人胃癌细胞BGC-823(?)勺增殖作用进行了研究。结果显示,AMP40-1、AMP60-1和AMP80-1三种薤白多糖对人肺癌细胞A549的生长不具有抑制作用,而AMP40-2对人肺癌细胞A549的生长具有一定的抑制作用,且在实验范围内其抑制作用与浓度呈明显的浓度依赖效应。对人胃癌细胞BGC-823的增殖实验结果表明,薤白多糖均能不同程度的抑制BGC-823细胞的增殖,且抑制作用与多糖之间存在不同程度的正相关量效关系和时效关系。结果也进一步表明薤白多糖对癌细胞生长抑制作用的强弱与其多糖分子量的大小、糖醛酸含量的高低以及单糖组成等因素存在明显的对应关系。
通过大鼠肾上腺嗜铬细胞瘤细胞PC12培养体系,对薤白多糖纯化组分(AMP40-1、AMP40-2、AMP60-1和AMP80-1)进行了神经营养活性研究。结果表明,薤白多糖AMP40-2对大鼠嗜铬细胞瘤PC12细胞具有一定的促分化作用,即AMP40-2具有一定的神经营养活性。
Polysaccharide has become an important part of the research and development of natural medicines and health products. Most of the foreign pharmaceutical companies engaged in the carbohydrate drug research. As naturally occurring biological constituents, these high molecular weight polymers are highly appreciated for their multipurpose therapeutic properties, such as antioxidant, antitumor, immune-modulating and anti-inflammatory activities. A. macrostemon Bunge, a perennial herb that propagates vegetative and grows wild on the plains of China, Korea and Japan, belongs to the botanic family of Alliaceae. The young leaves and bulbs of this plant are used as a vegetable. Furthermore, the dried bulbs of A. macrostemon Bunge are well known as the traditional Chinese medicine "Xiebai".
Up to now, research about A. macrostemon Bunge is mainly concentrated in the volatile oil and monomer compounds at home and abroad. The research in polysaccharide of A. macrostemon Bunge is far from perfect, which lack of an in-depth and a systematic study. In view of this, to establish the technological foundation for the development of modern multi-carbohydrate health food or sugar substances, to provide a theoretical basis for the further application of the A. macrostemon Bunge and its prescription, while to provide more references about research and development of polysaccharides in theory, academic and production practices were necessary. In this paper, we conducted a more systematic and in-depth research of polysaccharide from A. macrostemon Bunge about the optimization of the extraction process, the pure preparation methods, structural analysis and identification, activity screening evaluation and the structure and function relationship. Interpretation of these questions not only help to fully develop the use of our resources of Allium species, and to broaden its scope of application, but also to reveal its mechanism of action of traditional medicines will have a certain scientific value.
A four-factor and three-level Box-Behnken design was used to optimise the extraction parameters for polysaccharides from A. macrostemon Bunge. Then, a graded ethanol precipitation was used to fractionate the water-extractable polysaccharides, resulting in three polysaccharides fractions. And the crude polysaccharides were purified by anion-exchange and gel filtration chromatography; The purified fractions were characterized by using the phenol-sulfuric method, periodic acid oxidation, Smith degradation, methylation combined with high-performance liquid chromatography (HPLC), gas chromatography (GC), Fourier-infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS), etc. Finally, we investigated the antioxidant activities, the antitumor activities, antibacterial activity, neurotrophic activity, as well as hepatoprotective effects of polysaccharides from A. macrostemon Bunge, and described the initial relationship of the activity and structure of the polysaccharide. The main findings are as follows.
1. Extraction optimisation of polysaccharides from Allium macrostemon Bunge
Response surface methodology (RSM), based on a Box-Behnken design (BBD), was used to optimize the extraction conditions of polysaccharides (AMP). Four independent variables such as extraction temperature (℃), ratio of water to raw material (mL/g), extraction time (min) and extraction time were investigated. As a result, the optimal conditions for AMP extraction were the following:extraction temperature,87℃; ratio of water to raw material,12mL/g; extraction time,100min; and extraction times,3. Under these conditions, the experimental value was25.49±0.11%, which is well matched with value predicted by the model. The optimal conditions provide the basis of experimental data and theoretical analysis for the further development and utilization polysaccharides from A. macrostemon Bunge.
2. Isolation, purification, physico-chemical characterization and structural elucidation
The dried pretreated bulbs of A. macrostemon Bunge was extracted with water by use of the optimal extraction conditions determined as mentioned above. The extract was then centrifuged, and the supernatant was mixed with absolute ethanol to a final ethanol concentration of40%,60%,80%. The resulting precipitate was obtained from centrifugation and dried, affording AMP40, AMP60and AMP80. The recovery rates were3.2%,11.8%and9.83%, respectively. The crude AMP were purified by DEAE-Cellulose52chromatography and Sephadex G-100chromatography to afford4fractions, AMP40-1, AMP40-2, AMP60-1and AMP80-1. Based on the characterization results of AMP by the phenol-sulfuric acid method, the Bradford assay method, the hydroxydiphenyl assay, we found that AMP40-2was quite different from the others, especially AMP40-2had relative higher content of uronic acid (30.22%).
The molecular weights of AMP were determined by HPLC with size exclusion column. All of them showed a single and symmetrically sharp peak, indicated that they were homogeneous polysaccharide. According to the calibration curve of the elution times of standards, the molecular weights of AMP40-1, AMP40-2, AMP60-1and AMP80-1were estimated to be18.2kDa、105.0kDa、11.2kDa and8.1kDa, respectively.
Monosaccharide composition analysis was acted by using the GC method. GC analysis showed that AMP40-1was composed of arabinose and glucose in a molar ratio of1.00:5.03. AMP40-2was composed of rhamnose, arabinose, glucose and galactose in a molar ratio of1.41:2.34:1.62:1.00. AMP60-1was composed of arabinose, glucose and galactose in a molar ratio of9.73:1.10:1.00. AMP80-1was composed of arabinose and glucose in a molar ratio of1.00:2.41. Results indicated that monosaccharide composition of AMP40-2was more complicated than the others.
Infrared spectroscopy analysis indicated that the four samples showed typical peaks of polysaccharide. The characteristic absorption bands at833cm-1and894cm-1indicated that AMP40-2contained both a-glycosidic and β-glycosidic linkages, the band near1741cm-1was in coincidence with the fact that AMP40-2had relative higher uronic acid contents. UV analysis showed that the polysaccharides in the260-280nm no obvious absorption peak, indicate that it does not contain nucleic acids and proteins.
The structural characterizations of purified polysaccharides were studied by using periodic acid oxidation, Smith degradation, methylation combined with HPLC, GC, GC-MS, FTIR and NMR spectra. Results showed that sugar rings of AMP40-1, AMP40-2, AMP60-1and AMP80-1were pyranose rings. AMP40-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl and (1,2)-linked arabinosyl residues. The branch chains with non-reducing terminal glucose residues were attached to backbone chain by1→6glycosidic bonds; AMP40-2were connected by a-configuration and β-configuration glycosidic bonds. Backbone chains of AMP40-2were linked by (1,6)-linked D-glucosyl and (1,6)-linked galactosyl residues. The branch chains with non-reducing terminal glucose, rhamnose and arabinose residues were attached to backbone chain by (1,2)-linked rhamnosyl glycosidic bonds; It was concluded that AMP60-1contained a backbone of (1,6)-linked D-glucosyl and (1,5)-arabinosyl residues with small amounts of the non-reducing terminal arabinose residues, also contained a minor (1,2,6)-D-glucose residue; AMP80-1were linked mainly by β-configuration glycosidic bond, and contained backbone chains of (1,2)-linked D-glucosyl residues. The branch chains with non-reducing terminal glucose and arabinose residues were attached to backbone chain by1→6glycosidic bonds. Congo red dye experiments results indicated that AMP40-2possessed the triple helical structure.
3. Antioxidant activities in vitro and hepatoprotective effects of polysaccharides from Allium macrostemon Bunge
The antioxidant activities in vitro of polysaccharides from A. macrostemon Bunge were evaluated by measuring2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical and hydroxyl radical (OH·) scavenging activity, superoxide radical scavenging activity (O2·), metal ion chelating activity and reducing power. During in vitro antioxidant assay, AMP40-2exhibited relative stronger scavenging activities than the others in most cases. The differences in antioxidant activities in vitro might be due to their differences in molecular weight, monosaccharide composition and uronic acid content.
The antioxidant activities in vivo of crude AMP were investigated of against CCl4-induced acute liver damage in Kuming mice. The results showed that pretreatments with AMP40could improve markedly the activities of those antioxidant enzymes and the levels of CCl4-treated mice. The antioxidative system tended to be normalized by the protective action of AMP40. Results showed that molecular weight, monosaccharide composition and the uronic acid content of polysaccharide affects its antioxidant property directly.
4. Antibacterial activity, the antitumor activity and neurotrophic activity of polysaccharides from Allium macrostemon Bunge
The antimicrobial effects of extracts (AMW, AME and AMP) were evaluated by agar diffusion method. The results showed that:A. macrostemon Bunge has some anti-bacterial activity, its antibacterial ingredients present in the ethanol extract, may be a volatile oil or other small molecules, while the anti-bacterial activity of polysaccharides (water-soluble macromolecules) was weak.
MTT assay was used to investigate the anti-proliferation activities against human gastric cancer cells (BGC-823) and human lung adenocarcinoma epithelial cells (A549) in vitro of AMP40-1、AMP40-2、AMP60-1and AMP80-1. The results showed that AMP40-1, AMP60-1and AMP80-1did not inhibit the growth of human lung cancer cell line A549, while AMP40-2inhibited proliferation of A549in a dose-dependent manner. For human gastric carcinoma cell BGC-823, AMP40-1、AMP40-2、AMP60-1and AMP80-1significantly inhibited proliferation in dose-dependent and time-dependent manner. The results demonstrate that the molecular weight, chemical property, monosaccharide composition and linkage type of polysaccharide play important roles in the antitumor activity of polysaccharide.
The neurotrophic activity of polysaccharides from A. macrostemon Bunge were investingated by PC12cell assay. The screening results showed that AMP40-2induced the differentiation of PC12cell and has obvious neurotrophic activity.
引文
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