CN109400742B - 一种齿瓣石斛精制多糖及其制备方法和应用 - Google Patents
一种齿瓣石斛精制多糖及其制备方法和应用 Download PDFInfo
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- CN109400742B CN109400742B CN201811330972.1A CN201811330972A CN109400742B CN 109400742 B CN109400742 B CN 109400742B CN 201811330972 A CN201811330972 A CN 201811330972A CN 109400742 B CN109400742 B CN 109400742B
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Abstract
本发明公开了一种齿瓣石斛精制多糖及其制备方法和应用,齿瓣石斛精制多糖的制备方法包括:齿瓣石斛先用水提取,提取液浓缩后添加乙醇醇沉,沉淀用水溶解后脱蛋白,干燥得粗多糖,粗多糖经离子交换柱纯化得到半精制多糖,半精制多糖再经凝胶柱纯化后得到齿瓣石斛精制多糖。该齿瓣石斛精制多糖具有增强免疫,缓解体力疲劳的作用,安全有效,可用于制备增强免疫力和缓解体力疲劳的药物和功能性食品中,还可作为TLR4激动剂在制备添加到疫苗中的免疫佐剂,和TLR4相关的抗肿瘤药物的应用。
Description
技术领域
本发明涉及齿瓣石斛技术领域,具体涉及一种齿瓣石斛精制多糖及其制备方法和应用。
背景技术
TLR4是Toll样受体(TLR)家族中的关键跨膜模式识别受体,主要表达于免疫细胞,也表达于正常上皮细胞和癌细胞。TLR4通过病原体相关分子模式(PAMPs)对包括病毒、细菌、原生动物和真菌在内的各种入侵外源病原体作出反应。此外,它通过危险(或损伤)相关的分子模式(DAMPs)来识别内源性物质。这些内源性物质通常从炎症、氧化、损伤或坏死的细胞中释放,包括β-防御素、β-淀粉样蛋白、过氧化物酶、高迁移率族蛋白1(HMGB1)、热休克蛋白(HSPs)、透明质酸、硫酸肝素、P物质等。这些能力使TLR4成为先天免疫和适应性免疫反应的关键调节因子,并参与许多免疫和其他疾病。因此,TLR4已成为重要的药物靶标。TLR4拮抗剂正在开发用于治疗炎症性疾病,包括哮喘、动脉硬化、2型糖尿病、自身免疫和神经炎性疾病,而TLR4激动剂直接或作为疫苗中的免疫佐剂用于治疗癌症和传染性疾病(例如真菌和寄生虫)。
齿瓣石斛为兰科植物齿瓣石斛(Dendrobium devonianum Paxton.)的干燥茎,又名紫皮石斛、紫皮兰、香棍草、大黄草,收载于《云南省中药材标准》,具有益胃生津,滋阴清热等功效。同时齿瓣石斛收载于《云南省食品安全地方标准》,是目前唯一由行政主管部门发布了食品安全标准的石斛品种,对齿瓣石斛的研究与开发已越来越受到重视。
申请号为201510040088.4(授权公告号为CN104628798B)的中国专利申请公开了从紫皮石斛原料中同时制备花色苷及多糖的方法。所述方法包括以下步骤:1)制备水提液:将新鲜紫皮石斛茎条在水中提取,并进行固液分离,所得液体为水提液;2)浓缩水提液:将水提液蒸发,得到浓缩水提液;3)提取多糖:将乙醇加入浓缩水提液中,静置后进行固液分离,所得固体干燥即为多糖;4)提取花色苷:将步骤3)固液分离所得液体去除乙醇后,再将溶液干燥即得花色苷。所述方法能够简便地将紫皮石斛中的花色苷色素和有功能活性的多糖同时分别提取出,并且得到的花色苷色素和多糖纯度较高;提取过程中能耗较低;该方法既可以提高时效,也可以充分利用资源,具有较好的应用前景。
申请号为201610121973.X(申请公布号为CN106632708A)的中国发明专利申请公开了一种分离提纯齿瓣石斛均一多糖的方法,先用分步醇沉方法进行分离,然后经凝胶柱纯化,能够得到纯净的齿瓣石斛的均一多糖。另外,本发明还采用多光谱解析鉴定了齿瓣石斛均一多糖的骨架结构,能够对该多糖的一级结构准确鉴定,明确齿瓣石斛中的主要多糖为乙酰基葡甘露聚糖。
申请号为201710214362.4(申请公布号为CN107033253A)的中国发明专利申请公开了一种具有免疫促进和降血糖活性的紫皮石斛多糖及其制备方法,通过水提、醇沉、复溶、干燥或水提、超滤、干燥等步骤制备紫皮石斛多糖,其特征:碘-碘化钾试剂不显色,分子量为3×105-5×105Da,单糖组成主要为甘露糖、乙酰化甘露糖(Acetyl-Man)并含少量葡萄糖(Glc)且甘露糖和葡萄糖组成比大于20:1,糖苷键主要为β-1,4-Manp和β-1,4-Glcp。该紫皮石斛多糖具有免疫促进和降血糖作用,可用于治疗免疫低下和高血糖相关产品的开发。
发明内容
本发明的目的是提供了一种齿瓣石斛精制多糖及其制备方法和应用,该齿瓣石斛精制多糖具有增强免疫的作用,还有该多糖作为TLR4激动剂在制备添加到疫苗中的免疫佐剂,和TLR4相关的抗肿瘤药物的应用。
为达此目的,本发明采用以下技术方案:齿瓣石斛先用水提取,提取液浓缩后添加乙醇使含醇量达到约80%,醇沉,沉淀用水溶解后脱蛋白,干燥得粗多糖,粗多糖经离子交换柱纯化得到半精制多糖,半精制多糖再经凝胶柱纯化后得到齿瓣石斛精制多糖。
一种齿瓣石斛精制多糖的制备方法,包括以下步骤:
1)将齿瓣石斛原料前处理,使之呈粉状、颗粒状、片状或条状,得到前处理后的齿瓣石斛;
2)将前处理后的齿瓣石斛置于容器中,加水,提取,过滤出提取液;
3)将提取液减压浓缩成浓缩液;
4)在浓缩液中添加乙醇,使药液含醇量达到50%~90%,醇沉,得沉淀;
5)将沉淀用水溶解,形成沉淀溶解液,用氯仿-正丁醇的混合液萃取,取最上层液体减压浓缩,冷冻干燥,得齿瓣石斛粗多糖;
6)将齿瓣石斛粗多糖用水溶解,上样离子交换柱,用超纯水洗脱,收集洗脱液,苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,减压浓缩,透析,冷冻干燥,得齿瓣石斛半精制多糖;
7)将齿瓣石斛半精制多糖用水溶解,上样凝胶柱,用超纯水洗脱,收集洗脱液,苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,减压浓缩,透析,冷冻干燥,得齿瓣石斛精制多糖。
步骤1)中,将齿瓣石斛碎成粉,过20~50目筛,得到粉状齿瓣石斛;或将齿瓣石斛切成片,每片的厚度为2~5mm,得到片状齿瓣石斛,切成上述的厚度,有利于药物有效成分的提取。
步骤2)中,所述的前处理后的齿瓣石斛与水的重量比为1:10~100,进一步优选为1:20~40。
所述的提取的条件为:加热至95℃~105℃,保温提取2~4小时。通过上述条件的提取能将药物有效成分提取出来。
所述的提取的次数为2~4次,具体包括:先加水,提取,过滤出提取液,然后将滤渣再加同样水同样提取,如此往复,共分2~4次得到提取液,最后合并提取液。
步骤3)中,所述的减压浓缩的条件为:在50℃~70℃下减压浓缩,进一步优选,在60℃~70℃下减压浓缩。
步骤4)中,所述的药液含醇量是指乙醇的体积比上浓缩液加上乙醇的体积。
步骤5)中,氯仿-正丁醇的混合液中氯仿和正丁醇的体积比为3~5:1,进一步优选为4:1。
所述的氯仿-正丁醇(体积比4:1)的混合液用量与沉淀溶解液的体积比为1:4。所述的减压浓缩的条件为:在50℃~60℃下减压浓缩。
步骤6)、步骤7)中,所述的减压浓缩的条件为:在50℃~60℃下减压浓缩。所述的透析条件为:以流动的蒸馏水透析(透析膜截留分子量7000)24h。
制备得到的齿瓣石斛精制多糖经高效液相色谱法测定分子量,分子量为8×104-5×106;
制备得到的齿瓣石斛精制多糖经气相色谱法分析,其单糖组成为甘露糖和葡萄糖,葡萄糖与甘露糖的摩尔比为1:10-1:20。
制备得到的齿瓣石斛多糖经核磁共振分析,其结构:
其中,n为重复的结构单元数。
制备得到的齿瓣石斛多糖,可用于制备增强免疫作用和缓解体力疲劳的药物和功能性食品。所述的增强免疫作用和缓解体力疲劳的药物和功能性食品中齿瓣石斛精制多糖的重量百分含量为0.1%~99.5%。
所述的增强免疫作用和缓解体力疲劳的药物和功能性食品,有效成分为质量比1:1的齿瓣石斛精制多糖和人参皂苷Rg1;或者,有效成分为齿瓣石斛精制多糖、人参皂苷Rg1、***糖和半乳糖,质量比为1:1:0.4:0.2。
本发明的具体研究结果阐述如下:首先,通过研究,创造了一种离子交换柱结合凝胶柱的纯化齿瓣石斛多糖的方法,从齿瓣石斛中分离到一种新的齿瓣石斛精制多糖,其分子量为9.5×104Da,摩尔比为15.85:1,研究发现该齿瓣石斛精制多糖是TLR4激动剂,在体外和斑马鱼体内可直接刺激巨噬细胞的活化,表明该齿瓣石斛精制多糖可以作为TLR4激动剂,用于制备添加到疫苗中的免疫佐剂和TLR4相关的抗肿瘤药物,以及作为免疫功能的调节剂用于保健食品。
与现有技术相比,本发明具有如下优点:
本发明中,齿瓣石斛是具有益胃生津,滋阴清热功效的名贵中药,是目前唯一具备药食两用资质的石斛品种。本发明首次公开了通过采用离子交换柱结合凝胶柱纯化的方法制备齿瓣石斛多糖,因制备得到的齿瓣石斛多糖组成和结构明确,并且具有明显的增强免疫作用,适用于开发具有增强免疫作用的功能性食品和药品,具备广阔的应用前景。
附图说明
图1是本发明实施例1制备的齿瓣石斛精制多糖的气相色谱图;
图2是本发明实施例1制备的齿瓣石斛精制多糖的1H-NMR谱图(500MHz);
图3是本发明实施例1制备的齿瓣石斛精制多糖的13C-NMR谱图(125MHz);
图4是本发明实施例1制备的齿瓣石斛精制多糖作为TLR-4激动剂体外诱导巨噬细胞活化的实验结果图;
其中图4中a为齿瓣石斛精制多糖对TLR4的直接作用图,横坐标为精制多糖浓度,纵坐标为TLR4相对刺激强度;图4中b为齿瓣石斛精制多糖对分泌TNF-α的影响图,横坐标为精制多糖浓度,纵坐标为TNF-α的含量。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚,以下结合实施例对本发明作进一步说明,但这些实施例不得用于解释对本发明保护范围的限制。
实施例1
本实施例齿瓣石斛精制多糖,按照下述具体方式进行制备:
1)将齿瓣石斛原料粉碎,过40目筛网,得到粉状齿瓣石斛,备用;
2)将粉状的齿瓣石斛置于容器中,加齿瓣石斛重量40倍量的水,100℃~105℃保温提取3小时,过滤得滤液,滤渣再如此重复提取一次,过滤得滤液,合并两次提取液;
3)将提取液在60℃~70℃温度下减压浓缩成浓缩液;
4)在浓缩液中添加4倍量的无水乙醇使药液含醇量达到80%,醇沉,得沉淀;
5)将沉淀用水溶解,形成沉淀溶解液,用氯仿-正丁醇(体积比4:1)的混合液萃取,氯仿-正丁醇(体积比4:1)的混合液用量与沉淀溶解液的体积比为1:4,最上层液体加同样体积的氯仿-正丁醇混合液,重复上述操作6次,最后取最上层液体在50℃~60℃温度下减压浓缩,冷冻干燥,得齿瓣石斛粗多糖;
6)将齿瓣石斛粗多糖用水溶解,上样DEAE-Sepharose Fast Flow柱,用超纯水洗脱,自动收集器收集,蠕动泵流速为1mL/min,8min收集一管。苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,在50℃~60℃温度下减压浓缩,再以流动的蒸馏水透析(透析膜截留分子量7000)24h,冷冻干燥,得齿瓣石斛半精制多糖;
7)将齿瓣石斛半精制多糖用水溶解,上样Sephadex G-200柱,用超纯水洗脱,自动收集器收集,蠕动泵流速为1mL/min,11min收集一管。苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,在50℃~60℃温度下减压浓缩,再以流动的蒸馏水透析(透析膜截留分子量7000)24h,冷冻干燥,得齿瓣石斛精制多糖;
8)齿瓣石斛精制多糖经高效液相色谱法测定分子量,分子量为9.5×104。
9)制备得到的齿瓣石斛精制多糖经气相色谱法分析,气相色谱图如图1所示,其单糖组成为甘露糖和葡萄糖,葡萄糖与甘露糖的摩尔比为1:15.85;
10)齿瓣石斛精制多糖经核磁共振分析,进一步确认多糖的单糖成分主要为甘露糖,多糖中含有乙酰基的吸收峰。1H-NMR谱图(500MHz)见图2,13C-NMR谱图(125MHz)见图3,多糖的结构单元如下所示:
实施例2
本实施例齿瓣石斛精制多糖,按照下述具体方式进行制备:
1)将齿瓣石斛原料切片,得到厚度为2~5mm的片状齿瓣石斛;
2)将片状的齿瓣石斛置于容器中,加齿瓣石斛重量30倍量的水,100℃~105℃保温提取3小时,过滤得滤液,滤渣再如此重复提取一次,过滤得滤液,合并两次提取液;
3)将提取液在60℃~70℃温度下减压浓缩成浓缩液;
4)在浓缩液中添加4倍量的无水乙醇使药液含醇量达到80%,醇沉,得沉淀;
5)将沉淀用水溶解,用氯仿-正丁醇(体积比4:1)的混合液萃取,氯仿-正丁醇(体积比4:1)的混合液用量与沉淀溶解液的体积比为1:4,最上层液体加同样体积的氯仿-正丁醇混合液,重复上述操作6次,最后取最上层液体在50℃~60℃温度下减压浓缩,冷冻干燥,得齿瓣石斛粗多糖;
6)将齿瓣石斛粗多糖用水溶解,上样DEAE-Sepharose Fast Flow柱,用超纯水洗脱,自动收集器收集,蠕动泵流速为1mL/min,8min收集一管。苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,在50℃~60℃温度下减压浓缩,再以流动的蒸馏水透析(透析膜截留分子量7000)24h,冷冻干燥,得齿瓣石斛精制多糖。
对比例1
按照下述具体方式进行制备:
按照申请号为201610121973.X(申请公布号为CN106632708A)的中国发明专利申请说明书实施例1制备的齿瓣石斛均一多糖。
对比例2
按照下述具体方式进行制备:
按申请号为201710214362.4(申请公布号为CN107033253A)的中国发明专利申请说明书实施例1制备紫皮石斛多糖。
安全性试验
经动物实验证实齿瓣石斛精制多糖具有很高的安全性。
1实验材料
1.1样品:实施例1制备的齿瓣石斛精制多糖。
1.2实验动物:黑色素等位基因突变型Albino品系斑马鱼,以自然成对交配繁殖方式进行。共240尾,年龄为受精后2天(2dpf),饲养于28℃的养鱼用水中(水质:每1L反渗透水中加入200mg速溶海盐,电导率为480~510μS/cm;pH为6.9~7.2;硬度为53.7~71.6mg/LCaCO3),实验动物使用许可证号为:SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证的要求。
1.3主要试剂:长春瑞滨,无色溶液,批号为140501,4℃避光密闭保存;临用时用生理盐水稀释成浓度为0.05mg/mL的母液。
1.4主要仪器:解剖显微镜(SZX7,OLYMPUS,Japan);与显微镜相连的相机(VertA1,China);6孔板(Fisher Scientific,China);精密电子天平(CP214,OHAUS,America);显微注射仪(IM-300,Narishige);拉针仪(PC-10,Narishige,Japan)。
2实验方法
随机选取240尾受精后2天(2dpf)黑色素等位基因突变型Albino品系斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,静脉注射给予长春瑞滨0.25ng/尾剂量建立斑马鱼巨噬细胞减少症模型。模型斑马鱼分别水溶给予齿瓣石斛精制多糖125、250、500、1000、1500和2000μg/mL浓度,同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组,每孔(实验组)容量为3mL。在实验过程中,每天观察记录斑马鱼的死亡情况并移除死亡的斑马鱼。齿瓣石斛精制多糖处理3天后,统计各实验组的斑马鱼死亡数量和毒性情况,根据斑马鱼的死亡数量和毒性反应情况确定齿瓣石斛精制多糖对巨噬细胞减少症模型斑马鱼的最大耐受浓度(MTC)。
3实验结果
齿瓣石斛精制多糖在125、250、500、1000、1500和2000μg/mL浓度下,未引起斑马鱼死亡,且未见明显毒性反应,与正常对照组和模型对照组相似,可确定齿瓣石斛精制多糖对巨噬细胞减少症模型斑马鱼的MTC为2000μg/mL,表明齿瓣石斛精制多糖在该剂量范围内具有很高安全性。详见表1。
表1.齿瓣石斛精制多糖处理后“浓度-死亡率”实验结果(n=30)
药效试验
经动物实验证实齿瓣石斛精制多糖具有增强免疫的作用。
1.1样品:实施例1制备的齿瓣石斛精制多糖;对比例1制备的齿瓣石斛均一多糖、对比例2制备的紫皮石斛多糖。
1.2实验动物:黑色素等位基因突变型Albino品系斑马鱼,以自然成对交配繁殖方式进行。共150尾,年龄为受精后2天(2dpf),饲养于28℃的养鱼用水中(水质:每1L反渗透水中加入200mg速溶海盐,电导率为480~510μS/cm;pH为6.9~7.2;硬度为53.7~71.6mg/LCaCO3),实验动物使用许可证号为:SYXK(浙)2012-0171。饲养管理符合国际AAALAC认证的要求。
1.3主要试剂:长春瑞滨,无色溶液,批号为140501,购自江苏豪森药业股份有限公司,4℃避光密闭保存;临用时用生理盐水稀释成浓度为0.05mg/mL的母液。甲基纤维素(Sigma,China);中性红(Sigma,China)。
1.4主要仪器:解剖显微镜(SZX7,OLYMPUS,Japan);与显微镜相连的相机(VertA1,China);6孔板(Fisher Scientific,China);精密电子天平(CP214,OHAUS,America);显微注射仪(IM-300,Narishige);拉针仪(PC-10,Narishige,Japan)。
2实验方法
随机选取150尾受精后2天(2dpf)黑色素等位基因突变型Albino品系斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,静脉注射给予长春瑞滨0.25ng/尾剂量建立斑马鱼巨噬细胞减少症模型。模型斑马鱼分别水溶给予齿瓣石斛精制多糖222、667μg/mL浓度,对比例1组分别水溶给予齿瓣石斛均一多糖222、667μg/mL浓度,对比例2组分别水溶给予紫皮石斛多糖222、667μg/mL浓度,同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组,每孔(实验组)容量为3mL。斑马鱼处理2天后,加入2μg/mL中性红溶液对斑马鱼进行活体染色,染色结束后统计斑马鱼头部巨噬细胞数量(N),以统计学意义评价齿瓣石斛精制多糖对巨噬细胞形成的促进作用。齿瓣石斛精制多糖对斑马鱼巨噬细胞形成促进作用计算公式如下:
用方差分析和Dunnett’s T-检验进行统计学分析,p<0.05表明具有显著性差异。
3实验结果
模型对照组斑马鱼头部巨噬细胞数量(39.9个)与正常对照组(61.9个)比较p<0.001,提示斑马鱼巨噬细胞减少症模型构建成功;齿瓣石斛精制多糖222、667μg/mL浓度组斑马鱼头部巨噬细胞数量分别为49.6、54.5和46.6个,与模型对照组比较,222μg/mL浓度组p<0.01,667μg/mL浓度组p<0.001,其对斑马鱼巨噬细胞形成促进作用分别为24.3%、36.6%,显示齿瓣石斛精制多糖对巨噬细胞形成具有明显的促进作用,结果详见表2。
表2.齿瓣石斛精制多糖对巨噬细胞形成促进作用评价实验结果(n=10)
与模型对照组比较,**p<0.01,***p<0.001
由表2可见,相比于实施例1和实施例2,本发明齿瓣石斛精制多糖显示齿瓣石斛精制多糖对巨噬细胞形成具有明显的促进作用,具有明显的增强免疫作用。
同时,将本发明实施例1制备的齿瓣石斛精制多糖与人参皂苷Rg1,***糖,半乳糖复配,第一组是将齿瓣石斛精制多糖与人参皂苷Rg1按质量比1:1复配,形成有效成分,第二组是将齿瓣石斛精制多糖与人参皂苷Rg1、***糖,半乳糖按质量比1:1:0.4:0.2复配,形成有效成分,之后配成有效成分浓度222、667μg/mL浓度,进行重复试验,结果如表3所示。
表3(n=3)
由表3可知,采用复配后的有效成分,对巨噬细胞形成具有更加明显的促进作用,具有更加明显的增强免疫作用,效果显著。
药效试验
经实验证实实施例1制备的齿瓣石斛精制多糖是TLR4激动剂,能直接诱导巨噬细胞活化。
1实验方法
1.1细胞系与细胞培养
RAW264.7细胞(购自中国科学院上海生物科学研究所),在添加10%热灭活FBS、100U/mL青霉素和100μg/mL链霉素的DMEM培养基中培养。HEK-BLUETM-hTLR4细胞(美国路易斯维尔大学Thomas C.Mitchell教授馈赠),在添加10%热灭活FBS、100U/mL青霉素、100μg/mL链霉素和0.4%HEK-BlueTM选择抗生素混合物的DMEM培养基中培养。细胞在37℃、5%CO2培养箱中培养。
1.2RAW264.7细胞分泌细胞因子的测定
将RAW264.7细胞(5×105个)接种于24孔平底平板培养24h,然后加入DvP-1(齿瓣石斛精制多糖)、LPS、PMB(10μg/ml),最终体积为2mL。收集培养基上清液,用ELISA试剂测定细胞因子TNF-α的含量。
1.3TLR4活化测定
将HEK-BLUETM-HTLR4细胞(1×104个细胞)接种于96孔平板中。培养2h后,加入DvP-1、LPS(阳性对照),最终体积为200μl,继续培养24h,收集上清液,与定量的QUANTI-blueTM反应,在630nm处测定吸光度。
2实验结果
齿瓣石斛精制多糖是TLR4激动剂,并能直接诱导巨噬细胞活化。
我们用稳定表达TLR4的HEK293细胞系HEK-BLUETM-hTLR4细胞研究了齿瓣石斛精制多糖(DvP-1)对TLR4的直接作用,结果(图4a)表明与阳性对照组LPS相似,DvP-1在3.125~50μg/mL浓度之间能浓度依赖性方式显著刺激的TLR4活化(P<0.001)。进一步采用巨噬细胞RAW264.7研究发现,DvP-1还浓度依赖性显著刺激RAW264.7细胞分泌TNF-α。并且该作用没有受到多年菌素B(PMB)的影响。这些结果表明DvP-1是TLR4激动剂,能直接刺激巨噬细胞活化。
Claims (2)
1.一种齿瓣石斛精制多糖的制备方法,其特征在于,包括以下步骤:
1)将齿瓣石斛原料粉碎,过40目筛网,得到粉状的齿瓣石斛,备用;
2)将粉状的齿瓣石斛置于容器中,加齿瓣石斛重量40倍量的水,100℃~105℃保温提取3小时,过滤得滤液,滤渣再如此重复提取一次,过滤得滤液,合并两次提取液;
3)将提取液在60℃~70℃温度下减压浓缩成浓缩液;
4)在浓缩液中添加4倍量的无水乙醇使药液含醇量达到80%,醇沉,得沉淀;
所述的药液含醇量是指乙醇的体积比上浓缩液加上乙醇的体积;
5)将沉淀用水溶解,形成沉淀溶解液,用体积比4:1的氯仿-正丁醇的混合液萃取,体积比4:1的氯仿-正丁醇的混合液用量与沉淀溶解液的体积比为1:4,最上层液体加同样体积的氯仿-正丁醇混合液,重复上述操作6次,最后取最上层液体在50℃~60℃温度下减压浓缩,冷冻干燥,得齿瓣石斛粗多糖;
6)将齿瓣石斛粗多糖用水溶解,上样DEAE-Sepharose Fast Flow柱,用超纯水洗脱,自动收集器收集,蠕动泵流速为1mL/min,8min收集一管,苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,在50℃~60℃温度下减压浓缩,再以流动的蒸馏水透析24 h,透析膜截留分子量7000,冷冻干燥,得齿瓣石斛半精制多糖;
7)将齿瓣石斛半精制多糖用水溶解,上样Sephadex G-200柱,用超纯水洗脱,自动收集器收集,蠕动泵流速为1mL/min,11min收集一管,苯酚硫酸法测定吸光度,每个梯度洗至无糖被检出,根据吸收峰合并相同流份,在50℃~60℃温度下减压浓缩,再以流动的蒸馏水透析24 h,透析膜截留分子量7000,冷冻干燥,得齿瓣石斛精制多糖;
8)齿瓣石斛精制多糖的分子量为9.5×104,其单糖组成为甘露糖和葡萄糖,葡萄糖与甘露糖的摩尔比为1:15.85。
2.根据权利要求1所述的制备方法制备的齿瓣石斛精制多糖在制备功能性食品和增强免疫作用和缓解体力疲劳的药物中的应用,其特征在于,所述的功能性食品和增强免疫作用和缓解体力疲劳的药物,有效成分为质量比1:1的齿瓣石斛精制多糖和人参皂苷Rg1;或者,有效成分为齿瓣石斛精制多糖、人参皂苷Rg1、***糖和半乳糖,质量比为1:1:0.4:0.2。
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Effective date of registration: 20231229 Address after: No. 1 Yunshan Road, Longshan Town, Longling County, Baoshan City, Yunnan Province, 678000 Patentee after: Yunnan Pinhutang Biotechnology Co.,Ltd. Address before: 310013 No. 182 Tianmu Mountain Road, Zhejiang, Hangzhou Patentee before: ZHEJIANG ACADEMY OF MEDICAL SCIENCES |