CN108047349B - 一种卷边桩菇多糖提取物及其制备方法和医用用途 - Google Patents
一种卷边桩菇多糖提取物及其制备方法和医用用途 Download PDFInfo
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Abstract
本发明提供一种卷边桩菇多糖提取物及其制备方法和医用用途，从天然菌物卷边桩菇中获得具有新颖结构和多种生物活性的多糖PIP2‑1；并在该多糖制备过程中创造性的增加了两次80%乙醇沉淀步骤改进了卷边桩菇粗多糖的溶解性和纯度，同时公开了该多糖具有抗氧化、免疫调节和保护神经细胞等功效，可用于制备治疗抗氧化、免疫调节和保护神经细胞的药物，为卷边桩菇后续的开发和利用提供研究基础，具有重要的经济价值和市场价值。
Description
技术领域
本发明涉及一种卷边桩菇多糖提取物，具有抗氧化，免疫调节，保护神经细胞作用，本发明进一步提供了其制备方法，属于天然药物开发领域。
背景技术
随着生活水平的提高，人们逐渐增强了保健意识。另一方面，由于饮食不健康，工作压力大，环境污染，这些因素会使免疫力下降以及自由基增多而导致的氧化损伤等健康问题，随着年龄增长这些因素也是导致老年痴呆的原因之一。因此，开发具有抗氧化，免疫调节和保护神经细胞作用的药物或功能食品成为当务之急。
大型真菌是一种重要的天然生物资源，包括灵芝，虫草等在内的大型真菌由于其药效显著，副作用少，已成为药物开发的重要资源。卷边桩菇（Paxillus involutus），所属担子菌纲，伞菌目，广泛分布于北半球。“长白山植物药志”记录追风散寒，舒筋活络。主治手足麻木，筋络不舒，腰腿疼痛”。而目前，对卷边桩菇抗氧化，免疫调节和保护神经细胞等功能的开发未见报道。因此，对其进行药用价值的开发和利用具有重要意义。
发明内容
本发明涉及一种卷边桩菇多糖提取物，具有抗氧化，免疫调节，保护神经细胞作用，本发明进一步提供了其制备方法，
本发明所述的一种卷边桩菇多糖提取物，其特征在于由卷边桩菇通过乙醇提取得到。
本发明所述的卷边桩菇多糖提取物，其特征在于：卷边桩菇多糖提取物含量为82.96%，蛋白含量为3.1%，分子量为32 kDa；
该多糖由葡萄糖，甘露糖，半乳糖，岩藻糖组成，比例分别为62.2%, 2.8%，25.4%和9.6%；连接方式为T-岩藻糖、1,3-甘露糖、T-葡萄糖、1,4-葡萄糖、1,6葡萄糖、T-半乳糖、1,6-半乳糖、1,4,6-半乳糖及1,2,6-半乳糖。
本发明所述的卷边桩菇多糖提取物的制备方法，包括如下步骤：
1）将卷边桩菇烘干，粉碎过200目筛，将卷边桩菇菌粉在79℃，液料比为43.1 mL/g条件下提取3 h；提取液浓缩至原浓度1/5~1/4，加入无水乙醇至终浓度80%，沉淀，5000r/min离心去上清，沉淀烘干后，加入去离子水溶解，再加入终浓度为80%的乙醇沉淀，去上清后，用冷冻干机冷冻干燥备用；
2）将冻干的多糖样品加入去离子水溶解，通过3次sevag试剂(正丁醇：氯仿=1:4，v/v)去蛋白，再通过3500 Da截留量透析袋透析去除小分子，寡糖后浓缩并再通过80%乙醇沉淀，5000r/min离心去上清，沉淀通过冷冻干燥机冷冻干燥获得多糖卷边桩菇初级纯化多糖PIP；
3）将PIP用去离子水溶解，进行DEAE Cellulose-52(5.0×30 cm)柱层析，NaCl浓度由0~1 mol/L梯度洗脱，洗脱速度为1 mL/min，每管收集5 mL，通过苯酚-硫酸法测定多糖含量将含有多糖部分收集，分别获得多糖组分PIP1和PIP2，进一步将PIP2进行Sephadex-G100 (2.6×60 cm)层析，获得卷边桩菇多糖提取物PIP2-1。
本发明所述的卷边桩菇多糖提取物在制备免疫调节药物中的用途。
本发明所述的卷边桩菇多糖提取物在制备抗氧化药物中用途。
本发明所述的卷边桩菇多糖提取物在制备保护神经细胞药物中用途。
本发明所述的一种药物组合物，其特征在于含有卷边桩菇多糖提取物提取物和任选地药学上可接受的辅料。
本发明所述的药物组合物，其特征在于为片剂、胶囊剂、滴丸剂、颗粒剂、散剂、微丸、溶液剂、糖浆剂、乳剂或注射剂。
本发明的积极效果在于：首次从天然菌物卷边桩菇中获得具有新颖结构和多种生物活性的多糖PIP2-1；并在该多糖制备过程中创造性的增加了两次80%乙醇沉淀步骤改进了卷边桩菇粗多糖的溶解性和纯度，同时公开了该多糖具有抗氧化、免疫调节和保护神经细胞等功效，可用于制备治疗抗氧化、免疫调节和保护神经细胞的药物，为卷边桩菇后续的开发和利用提供研究基础，具有重要的经济价值和市场价值。
附图说明
图1为本发明卷边桩菇多糖提取物洗脱曲线（A）DEAE Cellulose-52分析，（B）Sephadex-G 100层析；
图2 为本发明PIP2-1单糖组成分析；
图3为本发明PIP2-1体外抗氧化活性；
图4 为本发明PIP2-1对巨噬细胞分泌IL-6和TNFα的影响；（*: 与对照组比p<0.05；**: 与对照组比p<0.01）；
图5 为本发明PIP2-1对L-谷氨酸所致的神经细胞PC12 (A)和HT22 (B)损伤的影响（##：与对照组比，p<0.01; *: 与模型组比p<0.05；**: 与模型组比p<0.01）。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不限制发明范围。此外应理解，在阅读了本发明的内容之后，本领域技术人员可以对发明作各种改动或修改，这些等价形式同样落于本申请所附后权利要求书限定的范围。
实施例1：
卷边桩菇多糖提取物提取物的制备
1、将吉林松原地区采集的卷边桩菇烘干，粉碎过200目筛，将卷边桩菇菌粉在79℃，液料比为43.1 mL/g条件下提取3 h。提取液浓缩至原浓度1/5~1/4，加入无水乙醇至终浓度80%，沉淀，5000r/min离心去上清，沉淀烘干后，加入去离子水溶解，在加入终浓度为80%的乙醇沉淀，去上清后，沉淀用冻干机冷冻干燥备用。
2、将冻干多的多糖样品加入去离子水溶解，通过3次sevag试剂(正丁醇：氯仿=1:4，v/v)去3 蛋白，再通过透析液出去小分子，寡糖后浓缩并通过80%乙醇沉淀，5000r/min离心去上清，沉淀通过冷冻干燥机冷冻干燥获得多糖卷边桩菇初级纯化多糖PIP
3、将PIP用去离子水溶解，进行DEAE Cellulose-52(5.0×30 cm)柱层析，NaCl浓度由0~1 mol/L梯度洗脱，洗脱速度为1 mL/min，每管收集5 mL，通过苯酚-硫酸法测定多糖含量将含有多糖部分收集，分别获得多糖组分PIP1和PIP2（图 1A），进一步将PIP2进行Sephadex-G 100 (2.6×60 cm)层析，获得PIP2-1（图 1B）。
实施例2：
卷边桩菇多糖提取物PIP2-1单糖组成分析
取2 mg多糖样品通过含有1 M HCl的甲醇水解，水解产物通过2M TFA水解后，水解产物通过1-苯基-3-甲基-5-吡唑啉酮（PMP）衍生化后，产物进行Compass C18 column (250× 4.6 mm) 连接岛津HPLC分析洗脱产物在UV245 nm出检测，结果如图2所示。结果表明，PIP2-1主要由由葡萄糖，甘露糖，半乳糖，岩藻糖组成，比例分别为62.2%,，2.8%，25.4%和9.6%。
实施例3：
卷边桩菇多糖提取物PIP2-1的甲基化分析
5 mg 多糖在氮气条件下溶解在含有NaOH的无水DMSO后，样品用CH3I甲基化，全甲基化产物进一步用2.5 M 三氟乙酸121℃下水解1.5h。衍生化产物在安捷伦GC7890A-MS5975C GC-MS系统上，用 HP-5 ms quartz capillary 柱分析，结果如表1所示，结果表明PIP2-1主要的连接方式为T-岩藻糖, 1,3-甘露糖，T-葡萄糖, 1,4-葡萄糖，1,6葡萄糖，T-半乳糖，1,6-半乳糖，1,4,6-半乳糖，1,2,6-半乳糖。
表1 PIP2-1甲基化分析结果
| Methylation sugar | Retention time (min) | Linkage type | Molar ratio (%) |
| 2,3,4-Me<sub>3</sub>-Fuc | 14.89 | T-Fuc | 8.8 |
| 2,4,6-Me<sub>3</sub>-Man | 19.03 | 1,3-Man | 2.0 |
| 2,3,4,6-Me<sub>4</sub>-Glc | 17.15 | T-Glc | 21.4 |
| 2,3,6-Me<sub>3</sub>- Glc | 19.33 | 1,4-Glc | 32.2 |
| 2,3,4-Me<sub>3</sub>-Glc | 19.60 | 1,6-Glc | 4.6 |
| 2,3,4,6-Me<sub>4</sub>-Gal | 17.61 | T-Gal | 2.9 |
| 2,3,4-Me<sub>3</sub>-Gal | 20.26 | 1,6-Gal | 14.8 |
| 2,3-Me<sub>2</sub>-Gal | 21.33 | 1,4,6-Gal | 6.1 |
| 3,4-Me<sub>2</sub>-Gal | 21.92 | 1,2,6-Gal | 7.1 |
通过以下试验证明本发明卷边桩菇多糖提取物的医用用途：
试验例1：
卷边桩菇多糖提取物PIP2-1具有清除羟自由基，ABTS, DPPH和超氧阴离子作用
分别对卷边桩菇多糖提取物的清除羟自由基，ABTS, DPPH和超氧阴离子能力进行测定，以其说明其抗氧化活性，采用VC作为阳性对照；
清除羟自由基活性实验：取1 mL 0.02M的磷酸盐缓冲液（pH=7）, 0.8mL 0.15 mM亚甲蓝溶液，0.4 mL 0.01M Fe(Ⅱ)-EDTA溶液，0.2 mL 7.5 mM H2O2溶液，加入卷边桩菇多糖提取物溶液（0.2, 0.6, 1.0, 1.2, 1.6, 2.0 mg/mL）0.5 mL，用90%乙醇溶液稀释至10mL,混匀反应2 h, 测定660 nm处吸光度，A0为初始亚甲蓝溶液的吸光度，A1为加入Fenton试剂后的吸光度，A2为加入Fenton试剂及卷边桩菇多糖提取物溶液后的吸光度。按以下公式计算卷边桩菇多糖提取物对羟自由基的清除能力：
清除率（%）=[1-(A0-A2)/ (A0-A1)]×100%
清除ABTS活性实验：7 mM的ABTS溶液100 mL与2.45 mM的过硫酸钾溶液溶液混合，避光16 h后作为ABTS反应液使用。取1mL ABTS反应液加入100 mL无水乙醇，并用无水乙醇逐级稀释该溶液至OD734为0.7±0.02。将3.9 mL的ABTS反应液分别加入100 μL卷边桩菇多糖提取物溶液（0.2, 0.6, 1.0, 1.2, 1.6, 2.0 mg/mL），或乙醇溶液，避光30 min, 分别测定其吸光度A样品及A空白, 按以下公式计算卷边桩菇多糖提取物对ABTS的清除能力：
清除率（%）=[(A空白-A样品)/ A空白)]×100%
清除DPPH活性方法：0.75 mL的0.1 mM DPPH与1.5 mL不同浓度卷边桩菇多糖提取物（0.2, 0.6, 1.0, 1.2, 1.6, 2.0 mg/mL）溶液混匀，避光30 min，测定无水乙醇在517nm处吸光度A1; 0.75 mL无水乙醇与1.5mL卷边桩菇溶液混匀，避光30 min后，测定517nm处吸光度A2；0.75 mL的0. 1mM DPPH与1.5 mL无水乙醇混匀，避光30 min后，测定517nm处吸光度A0。按以下公式计算卷边桩菇多糖提取物对DPPH自由基的清除能力：清除率（%）=[1-(A1-A2)/A0)]×100%
清除超氧阴离子活性方法：取4 mL Tris-HCl溶液（pH=8.2）, 加入卷边桩菇多糖提取物溶液（0.2, 0.6, 1.0, 1.2, 1.6, 2.0 mg/mL）0.5 mL，采用乙醇定容至10 mL, 48℃反应40 min, 加入48 ℃预热后的3 mM 邻苯三酚 0.3 mL,立刻混匀，测定其319.5 nm处吸光度，空白样品的吸光值为A0,加入卷边桩菇多糖提取物后样品的吸光值为A1。按以下公式计算卷边桩菇多糖提取物对超氧阴离子的清除能力：
清除率（%）=[1-A1/A0]×100%
以上四种抗氧化实验结果如图3所示，卷边桩菇多糖提取物PIP2-1对·OH，ABTS,DPPH清除率的IC50值分别为0.1mg/mL，0.92mg/mL和0.38 mg/mL而对超氧阴离子清除能力与阳性药物VC接近显著高于其他同类多糖。
试验例2：
卷边桩菇多糖提取物提取物PIP2-1激活巨噬细胞促进其分泌TNFα和IL-6
取对数生长的小鼠巨噬细胞RAW264.7,采用含有10% FBS和1% biomyc-3的DMEM培养基稀释至1*105个/mL,每孔接种100μL，于37 ℃，10% CO2细胞培养箱中过夜培养。每孔分别加入卷边桩菇多糖提取物PIP2-1（50，100， 200，400μg/mL，由基础培养基配制）,阳性对照孔加入1μg/mL LPS，阴性对照孔加入基础培养基，每孔3个平行。药物处理24h，分别取上清。采用ELISA试剂盒测定上清中细胞因子(IL-6,TNFα)水平。结果如图4所示，卷边桩菇多糖提取物较对照组能够显著刺激小鼠巨噬细胞分泌IL-6和TNFα，结果说明卷边桩菇多糖提取物具有显著的免疫调节活性。
试验例3：
卷边桩菇多糖提取物PIP2-1对L-谷氨酸所致的神经细胞损伤具有保护作用
采用MTT方法，分别取对数生长期的PC12和HT22细胞，用完全培养基将其分别稀释成5*105个/mL的细胞悬液，每孔接种100μL, 于37 ℃，10% CO2细胞培养箱中过夜培养。移去培养基，每孔分别加入100μL不同浓度（10-200 μg/mL）的卷边桩菇多糖提取物PIP2-1（采用基础培养基稀释）处理3h，，再用终浓度为25 μM的L-谷氨酸处理24 h，同时以不加谷氨酸和多糖组为对照组，以只加谷氨酸不加多糖预处理为模型组，每孔加入10μL MTT（5 mg/mL），37 ℃培养箱孵育4 h。弃上清后每孔加入150μL DMSO,均匀震荡，读取540 nm波长吸光度。结果如图5所示，PIP2-1对L-谷氨酸所致的神经细胞PC12和HT22均有显著的保护作用。
Claims (4)
1. 一种卷边桩菇多糖提取物PIP2-1，其特征在于由卷边桩菇通过乙醇提取得到；卷边桩菇多糖含量为82.96%，蛋白含量为3.1%，分子量为32 kDa；
包含：葡萄糖，甘露糖，半乳糖，岩藻糖组成，比例分别为62.2%、2.8%、25.4%和9.6%；
连接方式为T-岩藻糖、1,3-甘露糖、T-葡萄糖、1,4-葡萄糖、1,6葡萄糖、T-半乳糖、1,6-半乳糖、1,4,6-半乳糖及1,2,6-半乳糖；
上述的卷边桩菇多糖提取物PIP2-1的制备方法，包括如下步骤：
1）将卷边桩菇烘干，粉碎过200目筛，将卷边桩菇菌粉在79℃，液料比为43.1 mL/g条件下提取3 h；提取液浓缩至原浓体积1/5~1/4，加入无水乙醇至终浓度80%，沉淀，5000r/min离心去上清液，沉淀烘干后，加入去离子水溶解，加入终浓度为80%的乙醇沉淀，去上清液后，用冷冻干燥机冷冻干燥备用；
2）将冻干的多糖样品加入去离子水溶解，通过3次sevag试剂，去蛋白，该试剂组成为正丁醇：氯仿=1:4，v/v；再通过3500 Da截留量透析袋透析去除小分子，寡糖后浓缩并再通过80%乙醇沉淀，5000r/min离心去上清，沉淀通过冷冻干燥机冷冻干燥获得卷边桩菇初级纯化多糖PIP；
3）将PIP用去离子水溶解，进行DEAE Cellulose-52柱层析，层析柱型号为5.0×30 cm；NaCl浓度由0~1 mol/L梯度洗脱，洗脱速度为1 mL/min，每管收集5 mL，通过苯酚-硫酸法测定多糖含量将含有多糖部分收集，分别获得多糖组分PIP1和PIP2，进一步将PIP2进行Sephadex-G 100柱层析，层析柱型号为2.6×60 cm，获得卷边桩菇多糖提取物PIP2-1。
2.如权利要求1所述的卷边桩菇多糖提取物PIP2-1在制备免疫调节药物中的用途。
3.如权利要求1所述的卷边桩菇多糖提取物PIP2-1在制备抗氧化药物中用途。
4.如权利要求1所述的卷边桩菇多糖提取物PIP2-1在制备保护神经细胞药物中用途。
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