CN111228317A - 一种桦褐孔菌提取物的制备方法及其应用 - Google Patents
一种桦褐孔菌提取物的制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种桦褐孔菌提取物的制备方法。包括浸泡、高压提取、分离、沉淀、纯化、干燥等步骤，本发明主要是通过以上几个步骤来完成的本发明公开的人参饮品制备方法与现有技术相比，不仅在提取过程中保护了桦褐孔菌中的成分，还提高了提取率，同时还明确了桦褐孔菌多糖抗糖尿病和抗肿瘤的活性成分。
Description
技术领域
本发明涉及一种天然产物的提取方法，特别是涉及一种从桦褐孔菌中提取化合物的方法。
背景技术
桦褐孔菌属于真菌门，担子菌亚门，层菌纲，非褐菌目，锈革孔菌科，褐卧孔菌属。目前桦褐孔菌的化学组成主要有羊毛脂烷型甾体类化合物、多糖类、多酚类、木质素类、黑色素、生物碱类、单宁化合物、叶酸衍生物以及芳香的香草酸、丁香酸和γ- 羟基苯甲酸等。目前，对桦褐孔菌的***研究已引起许多科学家的关注，且对桦褐孔菌的研究主要包括药理研究、成分分析和提取工艺优化等三个方面，而针对桦褐孔菌的药理研究主要集中在抗肿瘤作用及治疗糖尿病、心血管疾病、肝病方面。
由于桦褐孔菌体内的活性成分种类多，应用价值高，目前本领域研究并未充分利用和开发桦褐孔菌体活性成分的价值，也缺少相应的活性成分的提取制备方法。
发明内容
针对上述问题，本发明的一个目的在于提供一种桦褐孔菌提取物的制备方法，该制备方法步骤简单、易操作、能够实现桦褐孔菌提取物的工业化规模生产。为达到上述目的，本发明提供一种桦褐孔菌提取物的制备方法，该方法包括以下步骤：
步骤一、将桦褐孔菌原材料粉碎，加入60-80%乙醇浸泡2-4h，料液比为1：3-5对原材料进行处理；
步骤二、将上述经过浸泡处理的桦褐孔菌粉和浸泡液一起装袋，密封，常温下，在300-400Mpa下高压处理30-150s，反复高压处理3次，合并提取液，利用分子量为800-1000Da的膜过滤设备过滤，收集滤液，滤液减压回收乙醇，用体积为上述浓缩后桦褐孔菌供试液10倍体积的5-20%的乙醇水溶液洗涤，之后离心分离，收集沉淀物，将沉淀物冻干得到桦褐孔菌提取物A；
步骤三、将步骤二中提取后的残渣加入去离子水，料液比为1：5-8，常温下，在500-600Mpa下高压处理100-150s，反复高压处理3次，合并提取液，减压浓缩提取液，然后用3-5倍的无水乙醇沉淀，沉淀用无水乙醇反复洗涤3次；
步骤四、将步骤三中的沉淀用水溶解，料液比为1：5-10，经膜分离和葡聚糖凝胶纯化，得到3种成分均一、纯度高的桦褐孔菌多糖PP-1、PP-2、PP-3。
优选地，步骤二中膜过滤设备所使用的膜为无机膜陶瓷，流速为2-5L/h，步骤四中膜分离设备使用的膜为无机膜纤维滤膜，滤膜的分子量分别为50KDa、100KDa和200KDa。
优选地，步骤二中桦褐孔菌提取物A的得率为1.72%-2. 41%。
优选地，步骤二中桦褐孔菌提取物A的主要成分及含量分别为麦角甾醇35.56-36.71%，羊毛甾醇43.53%-45.33%，桦褐孔菌醇13.67%-16.25%，白桦脂酸3.81%-4.72%，栓菌酸0.67%-0.94%。
优选地，步骤四中葡聚糖凝胶的上样流速为3-5倍柱体积/h，每个梯度的洗量为3-4倍柱体积。
优选地，步骤三中沉淀（粗多糖）的得率为17.44%-19.29%，沉淀中多糖的含量为65.33%-74.79%。
优选地，步骤四中桦褐孔菌多糖PP-1、PP-2、PP-3的收率分别为26.83%-27.92%，42,61%-44.73%，12.74%-15.51%。
优选地，步骤四中桦褐孔菌多糖PP-1、PP-2、PP-3的含量分别为89.94%-91.15%，95.55%-97.73%，94.96%-95.39%。
优选地，步骤四中桦褐孔菌多糖PP-1、PP-2、PP-3的分子量分别为31.3KDa、65.4KDa、179 KDa。
与现有技术相比，本发明的积极效果在于：
1.本发明采用超高压结合膜分离的技术制备桦褐孔菌提取物A，并利用高效液相明确了桦褐孔菌提取物A中成分，提取物A收率达到1.72%-2. 41%。
2.通过优化各制备步骤中的工艺参数，使得制备得到的桦褐孔菌提取物中的多糖的含量达到65.33%-74.79%，且得率达到17.44%-19.29％，高于采用常规热回流实验方法的得率（常规得率为12.76%）。
3.本发明通过膜分离和葡聚糖凝胶技术将桦褐孔菌多糖分离出三种成分均一、分子量明确、纯度高的多糖。
4.本发明通过活性试验发现桦褐孔菌降血糖和抗肿瘤的主要有效活性成分是分子量为65.4KDa的PP-2多糖。
5.本发明提供的桦褐孔菌提取物的制备方法的工艺简单、易操作，实现了桦褐孔菌提取物的工业化规模生产，并有利于降低生产成本。
附图说明
图1桦褐孔菌提取物A中麦角甾醇HPLC色谱图
图2桦褐孔菌提取物A中羊毛甾醇HPLC色谱图
图3桦褐孔菌提取物A中桦褐孔菌醇HPLC色谱图
图4桦褐孔菌提取物A中白桦脂酸HPLC色谱图
图5桦褐孔菌提取物A中栓菌酸HPLC色谱图
具体实施方式：
本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
实施例1
步骤一、将5kg桦褐孔菌原材料粉碎，加入80%乙醇浸泡4h，料液比为1：5对原材料进行处理；
步骤二、将上述经过浸泡处理的桦褐孔菌粉和浸泡液一起装袋，密封，常温下，在300Mpa下高压处理60s，反复高压处理3次，合并提取液，利用分子量为1000Da的膜过滤设备过滤，收集滤液，滤液减压回收乙醇，用体积为上述浓缩后桦褐孔菌供试液10倍体积的5%的乙醇水溶液洗涤，之后离心分离，收集沉淀物，将沉淀物冻干得到桦褐孔菌提取物A107.5g，得率为2.15%，提取物A的主要成分及含量分别为麦角甾醇36.33%，羊毛甾醇44.57%，桦褐孔菌醇16.25%，白桦脂酸3.95%，栓菌酸0.94%；
步骤三、将步骤二中提取后的残渣加入去离子水，料液比为1：8，常温下，在500Mpa下高压处理100s，反复高压处理3次，合并提取液，减压浓缩提取液，然后用5倍的无水乙醇沉淀，沉淀用无水乙醇反复洗涤3次，冻干得到沉淀864.5g，得率为17.29%，沉淀中多糖的含量为74.79%。；
步骤四、将步骤三中的沉淀864.5g用水溶解，料液比为1：10，经滤膜的分子量分别为50KDa、100KDa和200KDa膜分离设备和葡聚糖凝胶纯化，葡聚糖凝胶的上样流速为5倍柱体积/h，每个梯度的洗量为4倍柱体积。得到3种成分均一、纯度高的桦褐孔菌多糖PP-1、PP-2、PP-3，收率分别为27.92%，43.55%，14.92%，纯度分别为91.15%，95.63%，95.39%，其分子量分别为31.3KDa、65.4KDa、179 KDa。
比较例：桦褐孔菌多糖常规提取
采用常规实验方法制备桦褐孔菌提取物，具体步骤如下：1)取经干燥的桦褐孔菌原材料5kg(采用与实施例1相同的桦褐孔菌的子实体作为原材料，于1L蒸馏水中浸泡2小时，之后进行加热回流提取1.5小时，收集提取液，对提取液进行过滤，连续回流提取3次，滤液合并，将浓缩液冷却至室温，加入浓缩液5倍的95％的乙醇，置于4℃下静置12小时，收集沉淀物，将沉淀物在干燥至恒重，得到638g桦褐孔菌提取物，计算得到桦褐孔菌提取物的得率为12.76％，多糖含量为58.48%。
实施例2桦褐孔菌多糖对糖尿病小鼠血糖的影响
以实施例1和对比例中得到的桦褐孔菌多糖给药机分为6组，分别为模型组、二甲双胍组（阳性组）、pp-1组、pp-2组、pp-3组、对比例组。空白组和模型组小鼠每天灌胃0.9%的生理盐水，阳性组灌胃250mg/kg，桦褐孔菌多糖给药组的剂量均为20 mg/kg，每天一次，连续灌胃4周，给药体积均为0.01mL/g。
桦褐孔菌多糖对糖尿病小鼠血糖的影响（mmol/L）
与空白组相比##P<0.01；与模型组相比*P<0.05,**P<0.01
桦褐孔菌多糖对糖尿病小鼠血清中SCr、BUN、INS影响
与空白组相比##P<0.01；与模型组相比*P<0.05,**P<0.01
实施例3桦褐孔菌多糖对肿瘤小鼠的影响
以实施例1和比较例中得到的桦褐孔菌多糖给药机分为6组进行抗肿瘤活性试验。
Claims (4)
1.一种桦褐孔菌提取物的制备方法，其特征在于，包括以下步骤：
步骤一、将桦褐孔菌原材料粉碎，加入60-80%乙醇浸泡2-4h，料液比为1：3-5对原材料进行处理；
步骤二、将上述经过浸泡处理的桦褐孔菌粉和浸泡液一起装袋，密封，常温下，在300-400Mpa下高压处理30-150s，反复高压处理3次，合并提取液，利用分子量为800-1000Da的膜过滤设备过滤，收集滤液，滤液减压回收乙醇，用体积为上述浓缩后桦褐孔菌供试液10倍体积的5-20%的乙醇水溶液洗涤，之后离心分离，收集沉淀物，将沉淀物冻干得到桦褐孔菌提取物A；
步骤三、将步骤二中提取后的残渣加入去离子水，料液比为1：5-8，常温下，在500-600Mpa下高压处理100-150s，反复高压处理3次，合并提取液，减压浓缩提取液，然后用3-5倍的无水乙醇沉淀，沉淀用无水乙醇反复洗涤3次；
步骤四、将步骤三中的沉淀用水溶解，料液比为1：5-10，经膜分离和葡聚糖凝胶纯化，冻干得到3种成分均一、纯度高的桦褐孔菌多糖PP-1、PP-2、PP-3。
2.根据权利要求1所述的制备方法，其特征在于，步骤二中膜过滤设备所使用的膜为无机膜陶瓷，流速为2-5L/h，步骤四中膜分离设备使用的膜为无机膜纤维滤膜，滤膜的分子量分别为50KDa、100KDa和200KDa。
3.根据权利要求1所述的制备方法，其特征在于，步骤四中葡聚糖凝胶的上样流速为3-5倍柱体积/h，每个梯度的洗量为3-4倍柱体积。
4.根据权利要求1所述的制备方法，其特征在于，桦褐孔菌提取物主要用于制备降血糖和抗肿瘤的保健品、功能食品、药品。
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