CN114790251A - 一种具有免疫活性的滇重楼多糖及其组合物和应用 - Google Patents
一种具有免疫活性的滇重楼多糖及其组合物和应用 Download PDFInfo
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Abstract
本发明提供一种具有免疫活性的滇重楼多糖及其组合物和应用,属于活性多糖技术领域。本发明的滇重楼多糖RP66‑19主链为α(1→4)连接的葡聚糖,侧链上葡萄糖以(1→6)连接于主链,单糖组成为葡萄糖,分子量为2380 Da。是由滇重楼根茎通过干燥粉碎、脱色脱脂、热水提取、分级醇沉、淀粉检测与处理、凝胶柱层析法和离子交换柱层析法分离纯化后获得。通过高效液相色谱法、质谱、气质联用色谱法、一维和二维核磁图谱、紫外和红外光谱法等方法对滇重楼多糖RP66‑19的结构进行分析确认。滇重楼多糖RP66‑19能促进小鼠单核巨噬细胞白血病细胞RAW264.7释放NO和细胞因子(IL‑10、IL‑6)的分泌,同时增强巨噬细胞的吞噬能力,且与浓度呈一定的相关性。滇重楼多糖RP66‑19及其组合物可作为开发免疫调节药物及功能性食品的重要来源。
Description
技术领域:
本发明属于活性多糖技术领域,具体涉及一种具有增强免疫活性的滇重楼多糖、其制备方法、以其为活性成分的药物组合物及其在制备治疗免疫缺陷类疾病的药物中的应用。
背景技术:
重楼是我国的传统中药,《中国药典(2020版)》规定,重楼为百合科植物云南重楼Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.或七叶一枝花Parispolyphylla Smith var.chinensis(Franch.)Hara的干燥根茎,具有清热解毒,消肿止痛,凉肝定惊等功效,常用于治疗咽喉肿痛,蛇虫咬伤,惊风抽搐,功能性子宫出血,神经炎,外科炎等。
多糖又称聚糖(polysaccharide),是一类以单糖为基本组成单元,由20个或20个以上单糖残基通过糖苷键连接在一起的天然大分子化合物,其分子量从几万到几千万。多糖作为生命体中三大生物分子之一,在植物体内广泛分布。植物多糖是植物体能量存储的一种形式,作为供能物质,也是植物体内起支撑作用的结构性物质,大量存在于植物的根、茎、叶、种子和果实等重要部位,例如淀粉、纤维素、果胶和其他功能性多糖等。现有的研究表明,植物多糖在免疫调节、抗病毒、抗癌、抗凝血、降脂降血糖、免疫调节、抗氧化等方面有良好的药理药效活性。天然植物来源的多糖是一类无细胞毒性且功能活性突出的极性大分子物质,因其无细胞毒性,多糖成为了药品、保健食品、化妆品等的研发热点。
目前国内外关于滇重楼中主要功效物质是皂苷类化合物。关于滇重楼多糖的研究报道较少,多糖作为无细胞毒性的天然植物来源化合物,应该有更深入的研究。因此,有必要进一步探讨滇重楼多糖的分离纯化、结构鉴定,并探讨其免疫活性作用,为滇重楼多糖的开发利用提供基础。
迄今为止,现有技术中未见有滇重楼多糖RP66-19的报道,也没有其具有免疫活性的报道。
发明内容:
本发明的目的在于提供一种具有增强免疫活性的滇重楼多糖RP66-19、其制备方法、以其为活性成分的药物组合物,及其在制备治疗免疫缺陷类疾病的药物中和/或在制备提高免疫功能的保健食品中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
一种具有免疫活性的滇重楼多糖RP66-19,其主链为α(1→4)连接的葡聚糖,侧链上葡萄糖以(1→6)连接于主链,重均分子量(Mw)为2380Da,数均分子量(Mn)为2771Da,分子量分散系数(Mw/Mn)为1.16,表明滇重楼多糖RP66-19的分子量分布范围可能较窄,纯度较高。根据现代分析手段,确定滇重楼多糖RP66-19的分子结构如下所示:
一种具有免疫活性的滇重楼多糖的制备方法,该方法包含以下步骤:将滇重楼根茎粉碎,乙醇脱色脱脂,热水浸提,得到多糖水提物;分级醇沉,利用α-淀粉酶除去淀粉后醇沉,经SephadexG100凝胶柱纯化,再经DEAE-52阴离子交换柱层析,依次用去离子水和NaCl溶液进行洗脱,得到滇重楼多糖,该多糖可以从滇重楼中提取、分离纯化得到滇重楼多糖RP66-19。
具体地,所述的具有免疫活性的滇重楼多糖RP66-19的制备方法包括步骤:
1)制备滇重楼粉末:将滇重楼根茎除泥和须根,干燥处理后,粉碎过筛,得到滇重楼根茎粉末。
2)脱色和脱脂:称取干燥的滇重楼根茎粉,按料液比1g:1-100mL加入乙醇,水浴温度40-100℃处理1-6h,浓缩后冻干。
3)热水提取:将上述提取物按原料液比1g:1-100mL加入去离子水,水浴温度40-100℃提取1-6h,1000-10000rpm离心5-30min,浓缩后冻干,得到粗多糖水提物。
4)分级醇沉:将步骤3)的粗多糖水提物依次用终浓度为10%-100%(v/v)的乙醇洗涤沉淀,干燥后得到醇沉粗多糖。
5)测定总糖含量:采用硫酸-苯酚法测定总糖含量,利用葡萄糖标准曲线计算总糖并获得根茎多糖的提取率。
6)淀粉的去除:滇重楼根茎粗多糖检测含有淀粉,利用α-淀粉酶的专一性酶解淀粉。
7)凝胶柱层析:将步骤6)得到的多糖用去离子水溶解,1000-10000rpm离心5-30min,注入到SephadexG100凝胶柱的顶端,用0.01-10M NaCl溶液洗脱。用苯酚-硫酸法对其多糖含量进行检测。合并属于同一洗脱峰的洗脱液,用100-5000Da的透析袋进行脱盐处理,透析后得粗多糖。
8)阴离子交换柱层析:粗多糖经DEAE-52cellulose进行洗脱,依次用去离子水及浓度为0.01-10mol/L的NaCl溶液进行洗脱,收集洗脱组分,得到滇重楼多糖RP66-19。
根据所述的滇重楼多糖的制备方法,其中,所属脱色和脱脂的条件为在40-100℃回流1-6h;所述热水浸提的条件为在40-100℃下热水浸提1-6h;所述的分级醇沉是指将粗多糖水提物依次用终浓度体积比为10%-100%的乙醇洗涤沉淀1-5次,干燥后得到醇沉粗多糖;所述的除淀粉操作为用α-淀粉酶的专一性酶解淀粉在40-100℃酶解1-6h后,1000-10000rmp离心5-30min后上清液分级醇沉;所述凝胶柱层析为选用SephadexG100凝胶用去离子水溶解,1000-10000rpm离心5-30min,上清液注入到SephadexG100凝胶柱的顶端用NaCl溶液洗脱,用100-5000Da的透析袋进行脱盐处理,透析后样品冻干并称重;所述经DEAE-52阴离子交换柱层析,依次用去离子水及浓度为0.1mol/L、0.3mol/L的NaCl溶液进行洗脱,每个梯度洗脱2个柱体积。
由所述方法制得的一种具有免疫活性的滇重楼多糖RP66-19。
所述的一种具有免疫活性的滇重楼多糖在制备治疗免疫缺陷类疾病的药物中的应用和/或在制备提高免疫功能的保健食品中的应用。
一种滇重楼多糖组合物,所述的组合物中含有活性成分滇重楼多糖RP66-19。
一种药物组合物,其含有活性成分滇重楼多糖RP66-19,和可药用载体。
所述的一种滇重楼多糖组合物或药物组合物在制备治疗免疫缺陷类疾病的药物中的应用和/或在制备提高免疫功能的保健食品中的应用。
小鼠单核巨噬细胞白血病细胞RAW264.7是机体先天和后天免疫的关键,其具有吞噬功能,能主动吞噬和清除外来病原体和伤口处坏死的组织和细胞,并释放多种活性物质,比如多种细胞因子,从而对外界不良侵害起到防御作用。外源物质作用于巨噬细胞,通过产生NO等分子杀伤靶细胞,同时产生多种生物活性物质发挥其免疫调节作用。
细胞实验证明,滇重楼多糖RP66-19可以促进小鼠单核巨噬细胞白血病细胞RAW264.7的NO释放、细胞因子(IL-10、IL-6)的分泌量,增强巨噬细胞的吞噬能力,从而提高机体的免疫能力。
所述的滇重楼多糖RP66-19能应用于免疫缺陷类相关疾病的辅助治疗和提高免疫功能的保健食品中。
本发明同时还提供一种药物组合物,所述的药物组合物包括上述滇重楼多糖RP66-19和药物载体或赋形剂。同时,还提供所述药物组合物在免疫治疗药物中的应用,包括但不限于癌症的免疫治疗。
本发明与现有技术相比,具有如下的优点和技术效果:
本发明的滇重楼多糖RP66-19为首次提出,填补了现有技术的空白。滇重楼多糖RP66-19主链为α(1→4)连接的葡聚糖,侧链上葡萄糖以(1→6)连接于主链,分子量为2380Da,分子量均一,具有良好的免疫活性,可用于免疫缺陷类疾病的辅助治疗,也可用于制备提高免疫功能的保健食品。为滇重楼植物资源的进一步开发利用提供理论基础和实践指导。
本发明的制备工艺简单合理,绿色环保,科学严谨,易于实现工业化生产。
此外,本发明的实例验证,滇重楼多糖RP66-19在12.5-200μg/mL的范围内,可显著促进RAW264.7巨噬细胞对NO的释放,对RAW264.7巨噬细胞吞噬能力有增强作用,对巨噬细胞分泌IL-6、IL-10有促进作用,具有刺激巨噬细胞分泌细胞因子的能力。
附图说明:
图1是滇重楼多糖RP66-19的结构图;
图2是滇重楼多糖RP66-19的分子量分布曲线图;
图3是滇重楼多糖RP66-19的标准单糖混合和RP66-19的PMP衍生物HPLC图;
图4是滇重楼多糖RP66-19的紫外光谱图;
图5是滇重楼多糖RP66-19的红外光谱图;
图6是滇重楼多糖RP66-19的1H-NMR谱图;
图7是滇重楼多糖RP66-19的13C-NMR谱图;
图8是滇重楼多糖RP66-19的1H-1H COSY谱图;
图9是滇重楼多糖RP66-19的1H-1H TCOSY谱图;
图10是滇重楼多糖RP66-19的1H-13C HSQC谱图;
图11是滇重楼多糖RP66-19的1H-13C HMBC谱图;
图12是滇重楼多糖RP66-19的1H-13C HSQC-TCOSY谱图;
图13是滇重楼多糖RP66-19的1H-1H ROESY谱图;
图14是滇重楼多糖RP66-19对巨噬细胞RAW264.7 NO释放的影响;
图15是滇重楼多糖RP66-19对巨噬细胞RAW264.7吞噬能力的影响;
图16是细胞因子(白介素IL-10、白介素IL-6)含量的标准曲线;
图17是滇重楼多糖RP66-19对巨噬细胞RAW264.7分泌IL-10的影响;
图18是滇重楼多糖RP66-19对巨噬细胞RAW264.7分泌IL-6的影响。
具体实施方式:
下面结合附图,用本发明的实施例来进一步说明本发明的实质性内容,但并不以此来限定本发明。
实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行,所用的仪器或试剂未注明生产厂商者,均为可通过市购获得的常规产品。
本发明所用的重楼品种为滇重楼,即云南重楼Paris polyphylla Smithvar.yunnanensis(Franch.)Hand.-Mazz.。本发明所用的滇重楼药材均来自于丽江云鑫绿色生物开发有限公司。
实施例1
一种滇重楼根茎多糖的提取分离:
1)制备滇重楼粉末:将滇重楼的根茎除泥和须根,干燥处理后,粉碎过20-80目筛,得到滇重楼根茎粉末。
2)脱色和脱脂:称取干燥的滇重楼根茎粉,按料液比1g:10mL加入95%乙醇,水浴温度80℃加热处理2h,提取结束后,趁热抽滤得到95%乙醇提取物,提取2次后合并提取物,浓缩后冻干。
3)热水提取:将上述提取物按原料液比1g:10mL加入去离子水,水浴温度80℃恒温提取4h,冷却至室温,4000rpm离心15min,收集上清液,提取2次后合并提取物,浓缩后冻干,得到粗多糖水提物。
4)分级醇沉:将步骤3)的粗多糖水提物依次用终浓度为40%(v/v)、60%(v/v)、80%(v/v)的乙醇洗涤沉淀3次,干燥后得到40%乙醇沉淀粗多糖PPRP40、60%乙醇沉淀粗多糖PPRP60、80%乙醇沉淀粗多糖PPRP80。
5)测定总糖含量:采用硫酸-苯酚法测定总糖含量,利用葡萄糖标准曲线计算总糖并获得根茎多糖的提取率。测得根茎粗多糖提取率为10.40%。
6)淀粉的去除:滇重楼根茎粗多糖PPRP60检测含有淀粉,利用α-淀粉酶的专一性酶解淀粉。称取一定质量的PPRP60样品,加去离子水溶解,至浓度大约为50mg/mL,此时pH值在6-7之间,酶活力较高,按体积的0.01%加入α-淀粉酶,70℃酶解2h,酶解后4000rpm离心15min,上清液进行分级醇沉,得40%醇沉RP64、60%醇沉RP66、80%醇沉RP68。
7)凝胶柱层析:选用SephadexG100凝胶。称取根茎粗多糖RP66-19约200mg,10mL去离子水溶解,4000rpm离心15min,上清液经0.45μm的滤膜过滤后缓慢均匀的注入到SephadexG100凝胶柱的顶端用0.1M NaCl溶液洗脱控制流速为0.1mL/min,每管收集2mL,用苯酚-硫酸法对其多糖含量进行检测以吸光值为纵坐标,管数为横坐标,绘制多糖组分的洗脱曲线合并属于同一洗脱峰的洗脱液,用500Da的透析袋进行脱盐处理,透析后样品冻干并称重。经过SephadexG100纯化后得到组分(RP66-19)进行液相分析,是单一对称峰。使用亚甲蓝检测未见明显颜色变化,初步判定为粗多糖。
8)阴离子交换柱层析:活化与装柱DEAE-52cellulose后,依次用去离子水及浓度为0.1mol/L-0.3mol/L的NaCl溶液进行洗脱,每个梯度洗脱2个柱体积。最终获得纯化滇重楼多糖RP66-19,保留时间在19min左右。
实施例2
对实施例1得到的滇重楼多糖纯品RP66-19进行结构解析,包括:
1)分子量测定:利用高效凝胶渗透色谱法HPGPC测定RP66-19的重均分子量为2380Da,具体分子量如图2所示。
2)单糖组成分析:利用PMP能使单糖在250nm处有紫外吸收的特点,借助高效液相色谱进行分析鉴定。由标准单糖组成的混合单糖和多糖RP66-19经TFA水解PMP衍生化后的液相图谱如图3所示,结果表明多糖RP66-19仅由葡萄糖组成,故该来源于根茎的多糖为葡聚糖。
3)甲基化分析:采用Needs法,称取多糖样品3~5mg,置于COD水解管中,加入碱性DMSO溶液1mL快速溶解多糖样品,后加入碘甲烷1mL,加塞密闭,在室温避光条件下60℃超声反应1h,加入2mL去离水终止反应,按体积1:1加氯仿萃取3次中和后的甲基化样品,减压干燥后,加入2M的TFA溶液2mL,充分溶解甲基化产物,于110℃酸水解4h。水解液用2M的NaOH溶液调pH10,加入15-25mg NaBD4,50℃水浴还原2h后,滴加冰乙酸终止反应,减压干燥。干燥样品中加入吡啶0.5mL和乙酸酐0.5mL,转移到试管中封口,100℃反应1h,加入1mL水破坏乙酸酐,再加入2mL二氯甲烷萃取3次,用水反向萃取3次,有机相减压旋蒸至200μL左右,进行GC-MS分析。滇重楼多糖RP66-19的甲基化结果见表1。
表1滇重楼多糖RP66-19甲基化产物的GC-MS信号归属
4)UV光谱分析:取RP66-19多糖加水溶解至1mg/mL,在190~800nm范围内检测样品的紫外吸收光谱。紫外光谱图由图4所示,RP66-19多糖杂质少,纯度较高。
5)红外光谱分析:取2mg滇重楼粗多糖RP66-19,40℃真空干燥24h后,溴化钾压片,红外光谱仪对样品进行4000~400cm-1扫描,记录光谱图。RP66-19的红外光谱见图5所示,可以看出,出现了多糖的特征吸收峰。在3392cm-1处的特征峰为葡萄糖糖环醇羟基O-H伸缩振动引起的;在2929cm-1处的峰值为葡萄糖糖环亚甲基C-H的伸缩振动引起的;1639cm-1为醛基峰;1412~1200cm-1为C-H的变角振动;1200~1000cm-1为糖环上C-O-C的伸缩振动;928cm-1为吡喃环末端次甲基的横摇振动;700~500cm-1糖环呼吸振动(伸缩振动)。
6)核磁共振(NMR)波谱分析:取样品约10mg,经3次D2O交换,D2O溶解至20mg/mL,以3-(三甲基硅基)氘代丙酸钠为内标,800MHz核磁仪检测1H-NMR/13C-NMR谱图和1H-1H COSY、1H-1H TCOSY、1H-13C HSQC、1H-13C HMBC、1H-13C HSQC-TCOSY、1H-1H ROESY图谱。滇重楼多糖RP66-19的1D/2D核磁结果如图6-13所示,根据图6-13的核磁图谱对各残基的各个C和H的化学位移值进行归属,1H、13C信号归属如表2所示。
表2滇重楼多糖RP66-19的1H和13C NMR的信号归属
由1H-13C HSQC谱图和1H-13C HMBC可以更清楚的看到糖环上C-H之间的相关信号,根据1H-NMR中端基氢的化学位移,结合二维谱图中1H-1H ROESY、1H-13C HSQC和1H-13C HMBC可以判断糖苷键为α(1→4),同时存在α(1→6),根据对RP66-19的1H/13C-NMR以及相关二维谱图分析,结合GC-MS结果,推断RP66-19是以α(1→4)糖苷键为主链,且有α(1→6)糖苷键葡萄糖侧链取代的的葡聚糖。
实施例3
取上述实施例制备得到的滇重楼多糖RP66-19,进行免疫调节活性筛选,具体步骤如下:
1)滇重楼多糖RP66-19对RAW264.7细胞释放NO的影响:取RAW264.7对数生长期的细胞,制成细胞密度为2×106cells/mL的细胞悬液,接种于96孔细胞培养板中,每孔80μL,实验组加入不同浓度多糖样品RP66-19,以LPS为阳性对照组,设空白组,在原培养条件下培养24h后收集细胞上清,作为实验组样本和对照组样本。按50μL/孔,在96孔板中加入不同浓度多糖样品和LPS对照组,室温下先后加入Griess I和Griess II各50μL,避光10min后振板,使用酶标仪于570nm下测吸光值,测定结果如图14所示。从图中可以看出,多糖RP66-19在浓度为12.5-200μg/mL时,对巨噬细胞分泌NO的含量具有促进作用。
2)滇重楼多糖RP66-19对RAW264.7细胞吞噬能力的影响:取RAW264.7对数生长期的细胞,制成细胞密度为2×106cells/mL的细胞悬液,接种于96孔细胞培养板中,每孔80μL,实验组加入不同浓度多糖样品RP66-19,以LPS为阳性对照组,设空白组,在原培养条件下培养24h后吸弃细胞上清,每孔加入0.075%中性红生理盐水溶液100μL,继续培养30min弃上清,每孔加入200μL预温的PBS,清洗细胞,重复操作3次加入200μL细胞溶解液,使用酶标仪于570nm下测吸光值,测定结果如图15所示。从图中可以看出,在浓度为12.5-800μg/mL时,多糖RP66-19可以提高细胞吞噬能力。
3)滇重楼多糖RP66-19对巨噬细胞分泌IL-6、IL-10的影响:取不同浓度多糖样品RP66-19,以LPS为阳性对照组,设空白组,细胞培养24h后收集细胞上清,作为实验组样本和对照组样本。参考试剂盒说明书进行检测,IL-10和IL-6含量检测操作如下:在酶标板中加入标准品稀释液和不同浓度样本,以及阳性对照LPS各100μL每孔加入50μL稀释1/100的检测抗体,封板,于室温下300转/min震荡孵育2h,待孵育结束后,弃掉液体并加入300μL洗液6次,完成洗涤后,在吸水纸上扣干,每孔加入100μL稀释1/100的辣根过氧化物酶标记的链霉亲和素,封板,室温300转/min震荡孵育45min,按上一步骤洗涤每孔加入显色底物TMB100μL,避光,室温静置孵育30min后加100μL终止液以终止反应在短时间内使用酶标仪进行双波长检测,校准后的OD值为OD450与OD570的差值。
根据OD值绘制细胞因子(白介素IL-10、白介素IL-6)含量的标准曲线(图16),并计算2个细胞因子的分泌量(表3),滇重楼多糖RP66-14DH对巨噬细胞RAW264.7分泌细胞因子IL-10和IL-6的影响分别如图17和图18所示。结果表明,滇重楼多糖RP66-19在浓度为12.5-200μg/mL时可以促进巨噬细胞分泌细胞因子IL-10、IL-6。
综上所述,通过滇重楼多糖RP66-19对巨噬细胞吞噬能力、释放NO能力以及对巨噬细胞细胞因子(包括IL-6、IL-10等)分泌的影响等一系列实验,发现滇重楼多糖RP66-19具有促进巨噬细胞释放NO的作用,提高巨噬细胞吞噬的能力,对于细胞因子分泌也有促进作用。结果表明滇重楼多糖RP66-19能够有效激活巨噬细胞的免疫应答,具有一定的免疫活性。
表3滇重楼多糖RP66-19对巨噬细胞RAW264.7分泌细胞因子的影响
制剂实施例
1.取滇重楼多糖RP66-19,按其与赋形剂重量比1:1的比例加入赋形剂,制粒压片。
2.取滇重楼多糖RP66-19,按其与赋形剂重量比1:2的比例加入赋形剂,制粒压片。
3.取滇重楼多糖RP66-19,按常规胶囊制剂方法制成胶囊。
4.取滇重楼多糖RP66-19100 mg,再取淀粉适量、玉米浆适量、硬脂酸镁适量,制成片剂。
5.胶囊剂:取滇重楼多糖RP66-19100 mg,淀粉适量,硬脂酸模适量,制备方法:将活性部位与助剂混合,过筛,在合适的容器中均匀混合,把得到的混合物装入硬明胶胶囊。
6.鼻喷雾剂:
制备方法:搅拌下于适当体积的重蒸榴水中每次加入一种成分,直至完全溶解,然后再加入另一种成分。加水至2mL后,将该溶液在无菌过滤器上过滤,装入瓶中并按照适当的剂量分隔。
7.滴丸:取滇重楼多糖RP66-19 1g,聚乙二醇6000g。制法:按上述处方量称取滇重楼多糖RP66-19,加入适量无水乙醇,微热溶解后,加入处方量的聚乙二醇熔融液中(60℃水浴保温),搅拌混合均匀,直至乙醇挥尽为止,静置于60℃水浴中保温30分钟,待气泡除尽,将除尽气泡的上述混匀熔融液转入贮液筒内,在80~85℃保温条件下,控制滴速,逐滴滴入冷凝液中,待冷凝完全,倾去冷凝液,收集滴丸,沥净和用滤纸除去丸上的冷凝液,放置硅胶干燥器中或自然干燥即可。
Claims (10)
2.权利要求1所述的一种具有免疫活性的滇重楼多糖的制备方法,其特征在于,该方法包含以下步骤:将滇重楼根茎粉碎,乙醇脱色脱脂,热水浸提,得到多糖水提物;分级醇沉,利用α-淀粉酶除去淀粉后醇沉,经SephadexG100凝胶柱纯化,再经DEAE-52阴离子交换柱层析,依次用去离子水和NaCl溶液进行洗脱,得到滇重楼多糖。
3.权利要求1所述的一种具有免疫活性的滇重楼多糖的制备方法,其特征在于,该方法包含以下步骤:
1)制备滇重楼粉末:将滇重楼根茎除泥和须根,干燥处理后,粉碎过20-80目筛,得到滇重楼根茎粉末;
2)脱色和脱脂:称取干燥的滇重楼根茎粉,按料液比1g:1-100mL加入乙醇,水浴温度40-100℃处理1-6h,浓缩后冻干;
3)热水提取:将上述提取物按原料液比1g:1-100mL加入去离子水,水浴温度40-100℃提取1-6h,1000-10000rpm离心5-30min,浓缩后冻干,得到粗多糖水提物;
4)分级醇沉:将步骤3)的粗多糖水提物依次用终浓度体积比为10%-100%的乙醇洗涤沉淀,干燥后得到醇沉粗多糖;
5)测定总糖含量:采用硫酸-苯酚法测定总糖含量,利用葡萄糖标准曲线计算总糖并获得根茎多糖的提取率;
6)淀粉的去除:滇重楼根茎粗多糖检测含有淀粉,利用α-淀粉酶的专一性酶解淀粉;
7)凝胶柱层析:将步骤6)得到的粗多糖用去离子水溶解,1000-10000rpm离心5-30min,注入到SephadexG100凝胶柱的顶端,用0.01-10M NaCl溶液洗脱,用苯酚-硫酸法对其多糖含量进行检测,合并属于同一洗脱峰的洗脱液,用100-5000Da的透析袋进行脱盐处理,透析后得粗多糖;
8)阴离子交换柱层析:粗多糖经DEAE-52cellulose进行洗脱,依次用去离子水及浓度为0.01-10mol/L的NaCl溶液进行洗脱,收集洗脱组分,得到滇重楼多糖RP66-19。
4.根据权利要求2或3所述的滇重楼多糖的制备方法,其特征在于:所属脱色和脱脂的条件为在40-100℃回流1-6h;所述热水浸提的条件为在40-100℃下热水浸提1-6h;所述的分级醇沉是指将粗多糖水提物依次用终浓度体积比为10%-100%的乙醇洗涤沉淀1-5次,干燥后得到醇沉粗多糖;所述的除淀粉操作为用α-淀粉酶的专一性酶解淀粉在40-100℃酶解1-6h后,1000-10000rmp离心5-30min后上清液分级醇沉;所述凝胶柱层析为选用SephadexG100凝胶用去离子水溶解,1000-10000rpm离心5-30min,上清液注入到SephadexG100凝胶柱的顶端用NaCl溶液洗脱,用100-5000Da的透析袋进行脱盐处理,透析后样品冻干并称重;所述经DEAE-52阴离子交换柱层析,依次用去离子水及浓度为0.1mol/L、0.3mol/L的NaCl溶液进行洗脱,每个梯度洗脱2个柱体积。
5.由权利要求2或3或4所述的滇重楼多糖的制备方法制得的一种具有免疫活性的滇重楼多糖RP66-19。
6.权利要求1或5所述的一种具有免疫活性的滇重楼多糖在制备治疗免疫缺陷类疾病的药物中的应用和/或在制备提高免疫功能的保健食品中的应用。
7.一种滇重楼多糖组合物,其特征在于所述的组合物中含有权利要求1或5所述的活性成分滇重楼多糖RP66-19。
8.一种药物组合物,其含有权利要求1或5所述的活性成分滇重楼多糖RP66-19,和可药用载体。
9.权利要求7或8所述的一种滇重楼多糖组合物或药物组合物在制备治疗免疫缺陷类疾病的药物中的应用和/或在制备提高免疫功能的保健食品中的应用。
10.权利要求8所述的药物组合物的制备方法,其特征在于将滇重楼根茎粉碎,乙醇脱色脱脂,热水浸提,得到多糖水提物;分级醇沉,利用α-淀粉酶除去淀粉后醇沉,经SephadexG100凝胶柱纯化,再经DEAE-52阴离子交换柱层析,依次用去离子水和NaCl溶液进行洗脱,得到滇重楼多糖将滇重楼根茎粉碎,乙醇脱色脱脂,热水浸提,得到多糖水提物;分级醇沉,利用α-淀粉酶除去淀粉后醇沉,经SephadexG100凝胶柱纯化,再经DEAE-52阴离子交换柱层析,依次用去离子水和NaCl溶液进行洗脱,得到滇重楼多糖,然后,再加入可药用载体。
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申世安: ""滇重楼多糖的分离纯化与结构鉴定及其生物活性研究"", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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