苓桂术甘汤中多糖结构组成及其抗氧化活性考察

目的:研究苓桂术甘汤中多糖的结构,包括单糖组成以及官能团检测,并对其抗氧化能力进行评估,为体外生物测定法运用于苓桂术甘汤的质量评价提供基础。方法:利用高效凝胶色谱对全方多糖分子量进行研究;进一步采用气相色谱-质谱(GC-MS)柱前衍生化法及红外光谱扫描分析全方多糖结构组成;采用1,1-二苯基-2-三硝基苯肼(DPPH)及羟基自由基测定全方粗多糖及精多糖的抗氧化能力。结果:全方多糖由单一峰组成,且相对分子质量为3 689 Da,主要由阿拉伯糖、甘露糖、葡萄糖、半乳糖、果糖组成,摩尔比为6. 85∶1. 00∶109. 21∶1. 04∶21. 82,其中葡萄糖和果糖为主要组成成分;红外结果显示聚糖结构中存在吡喃糖及糖醛酸,并且糖苷键存在2种立体异构(α-糖苷键及β-糖苷键);抗氧化研究发现全方多糖有一定清除DPPH自由基及羟基自由基的能力,粗多糖的活性优于精多糖。进一步采用LC-Q-TOF-MS对粗多糖中的其他成分进行定性分析,提示这与甘草中五环三萜类成分的吸附有关。结论:苓桂术甘汤中多糖及五环三萜类物质均是苓桂术甘汤抗氧化作用的物质基础,利用体外生物测定法测定抗氧化活性可作为全面控制苓桂术甘汤质量的评价指标。     

亲水作用色谱-质谱法测定麻黄根多糖单糖的组成

目的:建立一种亲水相互作用色谱-质谱(HILIC-UPLC-MS/MS)检测分析方法,用于直接检测麻黄根多糖的单糖组成。方法:采用ACQUITY UPLC BEH Amide色谱柱(2.1 mm×100 mm,1.7μm),流动相A为乙腈-水(95∶5),B为乙腈-水(5∶95),柱温60℃,梯度洗脱(0~10 min,92%~70%A;10~10.1 min,70%~92%A;10.1~15 min,92%A),进样量2μL,流速0.2 m L·min~(-1);质谱采用电喷雾离子源,负离子模式下多反应离子检测。Turbo V离子源参数如下:毛细管电压-4.5 k V,源温度550℃;气帘气和碰撞气压力分别设为30 psi和10 psi;雾化气和气化气压力均为55 psi,碰撞电池入口电位和出口电位均为-10 V。结果:该方法在12 min内实现8种未衍生单糖成分的完全基线分离,精密度、稳定性和重复性的RSD均2.1%,各成分的回收率在99.3%~102.5%,RSD在1.5%~2.9%,应用上述建立的方法分别对5批麻黄根多糖进行单糖组成分析,结果显示,麻黄根中检测到5种单糖分别为L-鼠类糖,L-阿拉伯糖,D-甘露糖,D-半乳糖,D-半乳糖醛酸。结论:HILICUPLC-MS/MS方法灵敏度高、重复性好、快速、准确、样品前处理简单、无需衍生化,可用于植物多糖中单糖及寡糖的快速检测。     

分步醇沉对玛咖多糖单糖组成及抗氧化活性的影响

目的:分析分步醇沉所得玛咖多糖的单糖组成,并对各部分玛咖多糖的抗氧化能力进行研究。方法:采用水提醇沉法得到玛咖粗多糖,通过分步醇沉对玛咖粗多糖进行分离、纯化;采用气相色谱分析法对各玛咖多糖的单糖组成进行分析;最后采用三氯乙酸法和清除DPPH自由基的方法考察各部分玛咖多糖的抗氧化能力。结果:分步醇沉随乙醇浓度增加依次得到0~40%乙醇多糖部分,40%~50%乙醇多糖部分,50%~60%乙醇多糖部分,60%~70%乙醇多糖部分及70%~80%乙醇多糖部分,依次命名为MP1,MP2,MP3,MP4和MP5;其中收率最大的为MP1,纯度最高的为MP4,分别为0.856%和68.5%;经气相色谱技术分析发现不同体积分数乙醇所得玛咖多糖的单糖组成种类及比例不同;抗氧化能力试验结果表明各部分玛咖多糖的抗氧化能力存在显著性差异,其中抗氧化能力最强的为MP5,MP4抗氧化能力次之,抗氧化能力最弱的为MP1。结论:分步醇沉所得各部分玛咖多糖的抗氧化能力有较大差异,玛咖多糖的抗氧化活性与其单糖组成的种类、比例及其空间构象密切相关,分步醇沉对分离纯化玛咖多糖具有重要意义,为玛咖多糖进一步分离纯化、结构组成和药理研究提供可靠的参考依据。     

琵琶甲多糖的提取分离、结构表征及生物活性研究

目的 研究昆虫琵琶甲多糖的结构表征及生物活性。方法 采用水提醇沉和分级沉淀法制备得琵琶甲粗多糖组分BP50、BP75和BP90,并用葡聚糖凝胶G-75进一步分离纯化BP90得琵琶甲多糖组分BP90-Ⅱ。用苯酚-硫酸法、BCA法、HPLC、凝胶渗透色谱联用多角度激光光散射和示差检测器、红外光谱法分析表征多糖结构特征;测定各组分清除DPPH、ABTS和OH自由基的能力来评估抗氧化活性;此外,采用脂多糖诱导的BV-2小胶质细胞初步研究琵琶甲多糖的抗炎活性。结果 多糖组分BP90的抗氧化能力最强。BP90-Ⅱ的总糖、蛋白质含量分别为82.13%和11.28%。BP90-Ⅱ的红外光谱呈现多糖的典型特征吸收峰,重均分子质量为56 200 g·mol^–1。BP90-Ⅱ是由甘露糖、鼠李糖、氨基葡萄糖、葡萄糖醛酸、半乳糖醛酸、氨基半乳糖、葡萄糖、半乳糖、阿拉伯糖和岩藻糖10种单糖组成的杂多糖,其中葡萄糖含量最高,达74.85%。结果显示琵琶甲多糖BP90-Ⅱ对脂多糖诱导产生的NO有一定的抑制作用,当BP90-Ⅱ浓度为10 μg·mL^–1和100 μg·mL^–1时,NO抑制率分别为(14.86±1.63)%、(36.43±5.62)%。结论 琵琶甲多糖具有良好的抗氧化能力和一定的抗炎活性,具有一定的药用和食用开发价值。     

P-ERK、P-p38及iNOS、GLUT4在冠心病患者冬眠心肌中表达及意义

目的探讨冬眠心肌（HM）细胞内磷酸化ERK（P-ERK）、磷酸化p38（P-p38）、葡萄糖转运因子4（GLUT4）、诱导型一氧化氮合酶（iNOS）的变化和意义，探讨P-ERK、P-p38与GLUT4、iNOS的关系。方法选择行冠脉搭桥手术的冠心病患者10例，术前1周用多巴酚丁胺超声负荷试验结合多普勒组织成像确定HM及正常心肌（NM）的存在部位，术中根据检测结果取材，用免疫印迹法检测P-ERK、P-p38、iNOS、GLUT4的表达情况，分析HM与NM的P-ERK、P-p38、iNOS、GLUT4含量；分析四者之间的相关性。结果HM细胞内P-ERK、P-p38、GLUT4、iNOS水平较正常心肌高；P-ERK与GLUT4呈正相关（r=0.665，P〈0.05），P-p38与GLUT4、iNOS呈正相关（r=0.708、0.676，P〈0.05）。结论心肌缺血缺氧可触发ERK、p38活化，活化的ERK、p38促使心肌细胞增加GLUT4及iNOS表达，促进HM形成。     

悬钩子木多糖的分离纯化及免疫调节活性研究

目的 从悬钩子木中分离纯化多糖，初步分析其结构特征并进行免疫调节活性研究。方法 采用热水浸提法提取悬钩子木粗多糖（Rubus sachalinensis polysaccharides，RSP），再经Cellulose DE-52纤维素离子交换柱、Sephadex G-100凝胶柱色谱进行分离纯化；利用高效凝胶渗透色谱法（HPGPC）鉴定纯度并测定相对分子质量；采用气相色谱分析其单糖组成；采用红外光谱和核磁共振波谱对其结构进行初步分析；采用CCK-8法检测其对小鼠脾淋巴细胞增殖的影响；利用ELISA试剂盒测定其对白细胞介素-2（IL-2）、γ-干扰素（IFN-γ）和肿瘤坏死因子-α（TNF-α）释放能力的影响。结果 从悬钩子木中分离纯化得到RSP1-1和RSP1-2 2种多糖，相对分子质量分别为13 227（多分散指数1.15）和9 343（多分散指数1.08）；2种多糖均主要含阿拉伯糖、甘露糖、葡萄糖和半乳糖，其物质的量比分别是RSP1-1（9.5：7.0：10.3：18.6）和RSP1-2（5.7：11.1：10.3：14.2）；红外光谱及核磁波共振波谱对其结构分析证明RSP1-1可能主要含有α-1，3-Ara、β-1，4-Gal、β-1，3-Glc、β-1，3-Man片段，RSP1-2可能主要含有β-1，4-Gal、β-1，3-Glc、β-1，3-Man片段。质量浓度在5~200 μg/mL时，RSP1-1、RSP1-2和RSP2均可刺激小鼠脾淋巴细胞的增殖（P<0.05），并且明显促进淋巴细胞分泌IFN-γ和TNF-α（P<0.05），质量浓度为5 μg/mL时能促进淋巴细胞分泌IL-2（P<0.05）。结论 RSP1-1和RSP1-2均为首次从悬钩子木中分离得到的天然来源的均一多糖。红外光谱及核磁共振波谱进一步表征了其纯度和结构特征。RSP1-1、RSP1-2和RSP2均能不同程度促进脾淋巴细胞的增殖，促进淋巴细胞分泌IL-2、IFN-γ和TNF-α，呈现一定的免疫调节活性。     

Synthetic di-sulfated iduronic acid attenuates asthmatic response by blocking T-cell recruitment to inflammatory sites

Identification of carbohydrate sequences that determine affinity to specific chemokines is a critical step for strategies to interfere with chemokine-mediated leukocyte trafficking. Here, we first characterized the development of allergic asthma in Tie2-dependent and inducible Ext1-knockout (Tie2-Ext1^iKO) mice. We showed that heparan sulfate is essential for leukocyte recruitment in the peribronchial region and bronchoalveolar lavage fluid (BALF), and is crucial for induction of airway hyperresponsiveness. Our glycan microarray showed a unique affinity profile of chemokine CCL20 to substructures of heparin and heparin-like oligo/di/monosaccharides. Among them, we identified a synthetic and not naturally occurring monosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA), as a potential inhibitor for CCL20–heparan sulfate interaction. Mice injected with Di-S-IdoA via tail vain or nasal inhalation showed attenuated leukocyte recruitment into inflammatory sites and BALF. These results demonstrate a critical role of chemokine–heparan sulfate interaction in the asthma development and Di-S-IdoA as a potential drug for asthma treatment.Asthma is a common allergic disease characterized by chronic airway inflammation, mucus hypersecretion, and airway hyperreactivity to inhaled allergens (1). Despite the importance of T lymphocytes in adaptive immunity and host defense, their accumulation in airway in allergic asthma causes Th2-mediated pulmonary inflammation. The asthmatic inflammatory response is orchestrated by T-cell trafficking network among lung, blood circulation, secondary lymphoid organ, and peripheral tissue (2). Of note, the significant increase of T cells in the airway in asthma is mostly due to T-cell recruitment from regional lymph nodes rather than their proliferation at the inflamed site (3). Therefore, a therapeutic approach that shuts off the trafficking pathway of pathogenic T cells should significantly inhibit the Th2-mediated inflammation in allergic asthma.It is well known that the destination of T-cell trafficking pathway is tightly restricted by the profile of chemokines, lipid chemoattractants, and T-cell chemokine receptors. As a part of immune surveillance, naïve T cells and central memory T cells constantly access secondary lymphoid organs from blood circulation via specialized high endothelial venules (HEVs). The interaction between T cells and HEV cells includes in a stepwise manner (4, 5), L-selectin–dependent tethering and rolling, activation, firm arrest, and transendothelial migration. Besides 6-sulfo sialyl Lewis X as a L-selectin ligand, HEVs constitutively express chemokine CCL21 and CCL19 and attract T cells that express its cognate receptor CCR7 (5). In contrast to this homeostatic homing, circulating T cells interact with inflamed blood vessels in lung after asthmatic exposure to an inhaled allergen. Among numerous combinations of chemokines and their receptors, there is considerable evidence that CCL20 and its cognate receptor CCR6 may contribute to the pathogenesis of asthma (6). CCL20-CCR6 plays a key role in the recruitment of Th17 (7) cells and Th2 cells (8). Indeed, CCL20 is highly enriched on inflammatory epithelium (9) and CCR6 is expressed on memory T cells infiltrated in the lung during allergic inflammation (7). In addition, CCR6-deficient mice have decreased airway responsiveness, and reduced recruitment of eosinophils into lung (10, 11). These findings suggest that CCL20-CCR6 axis is a putative target for the treatment of asthma.Cumulative evidence in vivo and in vitro indicates that chemokines cannot be functionally active in HEVs and inflamed sites without their interaction with heparan sulfate (12). Heparan sulfate protects chemokines from proteolysis, immobilizes them on the endothelium surface and produces chemokine gradients in the vasculature. Heparan sulfate is composed of repeating disaccharide units of uronic acid [glucuronic acid (GlcA) or iduronic acid (IdoA)] and N-acetylglucosamine (GlcNAc) carbohydrates. Some of GlcA (or IdoA) carbohydrates are subsequently O-sulfated, and GlcNAc carbohydrates are partially modified with N-deacetylation and N-sulfation (13). Previous reports have indicated that the sulfation patterns in heparan sulfate are more restricted than expected (14), and the sulfation is associated with respiratory distress (15) and asthma (16). It is believed that there are specific interactions between heparan sulfate and chemokines (17). Nevertheless, the nontemplate nature of long carbohydrate chains (<25,000 disaccharide units) and conformational plasticity still make it difficult to identify the common sequences of heparan sulfate that display affinity to specific chemokines. In this regard, our previously established glycan microarray system (18, 19) is a powerful tool to define the selectivity in heparan sulfate–chemokine interactions.Recently, we established the exostoses-1 (Ext1) gene conditional knockout Tie2-Ext1^iKO mouse model, in which GlcA/IdoA-GlcNAc repeat of heparan sulfate can be abrogated in endothelial cells in a tetracycline-inducible manner (20). In this study, Tie2-Ext1^iKO mouse showed significant reduction of both leukocyte recruitment to lung tissues and of airway hyperresponsiveness in ovalbumin (OVA) asthma model. Moreover, glycan microarray analysis surprisingly identified that an unnatural and synthetic monosaccharide, 2,4-O-di-sulfated iduronic acid (Di-S-IdoA) has a high affinity to recombinant CCL20. Intravenous and inhalation challenges of Di-S-IdoA significantly inhibited the leukocyte infiltration in bronchoalveolar lavage fluid (BALF). Our finding that even a monosaccharide can attenuate airway inflammation suggests its potential use as an antiasthma therapy that can be administered by inhalation.     

高效液相色谱法建立黄芪多糖特征图谱研究

目的 探讨高效液相色谱法建立黄芪多糖特征图谱.方法 利用高效液相色谱法对PMP柱前衍生化的黄芪多糖进行糖组成分析.结果 黄芪多糖特定图谱由鼠李糖、葡萄糖、半乳糖、阿拉伯糖4种单糖4个共有峰组成.结论 高效液相色谱法建立的黄芪多糖特征图谱,其操作方法简便,具有良好的重复性及稳定性,可有效控制黄芪多糖的内在质量.     

板蓝根多糖的系统分离纯化与组成分析

目的建立板蓝根多糖的系统分离纯化方法,并对分离得到的均一多糖进行组成测定。方法以抗病毒活性较好的80%醇沉板蓝根粗多糖为研究对象,对其进行DEAE-Sepharose Fast Flow柱色谱、Sephacryl-200葡聚糖凝胶柱色谱以及高效凝胶色谱系统分离纯化。分别采用1-苯基-3-甲基-5-吡唑啉酮(PMP)和邻苯二甲醛(OPA)衍生化HPLC法对分离得到的板蓝根均一多糖进行单糖组成和氨基酸组成测定。结果利用系统分离纯化策略,从80%醇沉板蓝根粗多糖中分离得到2种均一板蓝根多糖(IRPS1A和IRPS1B)和2种均一板蓝根糖蛋白(IRPS2A和IRPS3A)。其中,IRPS1A与IRPS1B的单糖组成均为甘露糖、葡萄糖、半乳糖和阿拉伯糖;IRPS2A的单糖组成为半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖;IRPS3A的单糖组成为甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖。糖蛋白IRPS2A与IRPS3A均含有天门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、苏氨酸、精氨酸、丙氨酸、胱氨酸、缬氨酸、苯丙氨酸、异亮氨酸、亮氨酸、赖氨酸,共14种氨基酸残基。同时,IRPS2A还含有酪氨酸残基。结论该系统分离纯化的策略可应用于板蓝根均一多糖(糖蛋白)的获取,为板蓝根多糖的结构分析及药理活性研究奠定基础。     

基于多糖和黄酮类成分的铁皮石斛优良内生真菌筛选

为了揭示铁皮石斛内生真菌与功效成分的关系,该研究利用HPLC和紫外分光光度法测定21种铁皮石斛内生真菌菌丝及发酵液提取物中糖类和黄酮类成分。结果表明:21个供试菌株发酵产物乙酸乙酯提取物中均可检测到黄酮类成分,对比色谱保留时间和紫外光谱特征,发现DO49可产柚皮素,DO23,DO81,DO83可产芦丁;21个供试菌株发酵产物水提取物中均检测到多糖,但不同菌株单糖的组成存在差异,参照单糖种类以及甘露糖/葡萄糖的峰面积比,筛选与铁皮石斛多糖相关的优良菌株DO23,DO26,DO81,DO54,DO55,DO83。综合糖类和黄酮类两类成分,筛选出优良菌株DO23,DO81,DO83,既能产生与铁皮石斛相同的黄酮成分,又能产生单糖组分与铁皮石斛相近的多糖。