CN105504085B - 蛹虫草葡聚糖及其制备方法和应用 - Google Patents
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Abstract
本发明为一种蛹虫草子实体葡聚糖及其制备方法和用途。本发明涉及的制备方法主要包括以下步骤:烘干的蛹虫草子实体、粉碎、乙醇脱脂、热水超声提取、提取液经浓缩、醇沉、脱蛋白、柱层析、透析、冷冻干燥得蛹虫草子实体葡聚糖TY517。本发明蛹虫草多糖具有降脂功效,在25mg/kg即可显著降低高脂诱导的实验动物模型的胆固醇、甘油三酯和低密度脂蛋白胆固醇水平,提升高密度脂蛋白胆固醇水平。
Description
技术领域
本发明涉及一种生物活性物质及其制备方法和用途,具体是指从蛹虫草子实体中提取的一种葡聚糖及其制备方法和应用。
背景技术
蛹虫草(Cordyceps militaris L. Link)又名北虫草、北冬虫夏草,其与冬虫夏草属于同属真菌,不仅具有与冬虫夏草相近的生物活性,且易于人工培养,成为冬虫夏草极具潜力的替代品。
多糖在自然界中含量极其丰富,其具有显著的免疫调节、抗肿瘤、降血糖、降脂等生物学活性,已引起国内外医学界的高度重视,成为近年来研究和应用的热点。蛹虫草多糖是蛹虫草含量较高的生物活性物质之一,且具有类似于冬虫夏草的生物学活性,例如:增强免疫、抗肿瘤、抗病毒、抗衰老、降血糖、降脂等多种功能。因此,从蛹虫草中制备多糖,并确定其生物学活性,可以提升蛹虫草的临床转化应用价值和经济价值,对蛹虫草的综合利用具有重要意义。
蛹虫草多糖的单糖组成、相对分子质量、糖苷键连接方式可对其生物活性产生重要影响。现在普遍采用水提醇沉法来提取蛹虫草多糖,水提醇沉法获得多糖的方法虽然工艺流程简单,但获得的多糖相对分子质量范围广,组成成分复杂,既不利于多糖构-效关系的阐述,也不利于产品质量控制和后续临床转化应用。
高血脂是诱发心脑血管疾病的主要因素之一。当体内胆固醇过高,特别是低密度脂蛋白水平(LDL-C)以及甘油三酯增高时,氧化的LDL则可被巨噬细胞吞噬,进而沉积在动脉内皮下,引起内皮下变性,进而导致血小板在动脉内壁聚集,若同时伴有动脉壁损伤或胆固醇转运障碍,则易在动脉内膜形成脂斑,最后,血管壁隆起、向官腔内突起形成粥样斑块。
2013年至今,先后有国内外研究者报道了蛹虫草提取液单独或联合用药的降脂效果,但蛹虫草子实体多糖中具有降脂功能的活性成分及其确切结构和制备方法未见报道。
发明内容
本发明的目的是提供了一种蛹虫草子实体葡聚糖及其制备方法和在降脂方面的应用。为解决上述问题,本发明知道了如下技术方案:
蛹虫草子实体葡聚糖的制备方法,包含有如下步骤:
(1)制备蛹虫草子实体粗多糖:
采集蛹虫草子实体,阴干并粉碎,过筛;
向粉碎的蛹虫草子实体中加水,经冻融后超声处理,过滤得破壁蛹虫草子实体;
在破壁后的蛹虫草子实体加入95%的乙醇回流浸提脱脂,过滤得固形物;向固形物中再加入蒸馏水,加热超声浸提,抽滤除去残渣得上清液;
将上清液减压浓缩后加入95%的乙醇,置于0~4℃过夜,离心,收集沉淀。收集的沉淀经真空冷冻干燥,即得蛹虫草子实体粗多糖;
(2) 除蛋白质:将制备得到的蛹虫草子实体粗多糖,溶于蒸馏水中,加入适量sevage试剂,剧烈震荡,然后3000-5000×g离心得上清液,重复多次,上清液合并;
(3)透析:将上述脱蛋白的多糖溶液移入透析袋中,密封后先在自来水中透析,再在蒸馏水中透析,每4-8小时换一次水;之后溶液经浓缩、醇沉、离心、干燥得蛹虫草子实体多糖;
(4) 蛹虫草子实体多糖的纯化:蛹虫草子实体多糖半纯品再经阴离子交换柱和凝胶渗透色谱柱分离纯化,即可分离得到纯的蛹虫草子实体葡聚糖命名为TY517,该多糖的结构式是:
α-D-Glcp (1[→6)-β-D-Glcp (1]13→6)β-D-Glcp
其中:Glcp为吡喃型葡萄糖。
蛹虫草子实体葡聚糖在制备降脂药物上的应用。
本发明的有益效果是:
本发明首次公开由蛹虫草子实体获得结构明确的葡聚糖制备方法及该葡聚糖在降脂方面的应用,为蛹虫草多糖的构效关系研究提供了详实的实施案例。在本发明的实施例中,蛹虫草子实体葡聚糖可显著下调apoE(-/-)小鼠、豚鼠和Wistar大鼠血浆TC、TG和LDL-C水平,并上调HDL-C,能;在制备方面,本发明的实施方法简单易于大规模生产,为该发明的转化奠定了基础。
附图说明
图1所示为蛹虫草子实体葡聚糖TY517的1H(A)和DEPT(B)核磁共振图谱;
图2所示为蛹虫草子实体葡聚糖TY517的1H-1H COSY图谱;
图3所示为蛹虫草子实体葡聚糖TY517的HSQC核磁共振图谱;
图4所示为蛹虫草子实体葡聚糖TY517的HMBC核磁共振图谱;
图5所示为蛹虫草子实体葡聚糖TY517的NEOSY核磁共振图谱。
具体实施方式
实施例1:
提取制备本发明的蛹虫草子实体葡聚糖TY517并验证其降脂活性时,采取了下述个步骤:
1) 制备蛹虫草子实体粗多糖:采集蛹虫草子实体,阴干后用植物粉碎机粉碎,过100目筛。向粉碎的蛹虫草子实体中加入10倍量的水,搅拌均匀,置于超低温冰箱中-80℃冷冻12小时。迅速投入80℃水浴锅中解冻,水浴2小时。然后500W超声处理30分钟,过滤得固形物为破壁蛹虫草子实体。破壁后的蛹虫草子实体中加入95%乙醇回流2小时,料液比为1:5(W/V),浸提脱脂及可溶性分子,过滤得固形物。向固形物中加入蒸馏水,料液比为1:10 (W/V),500W超声加热,温度控制在85℃,提取时间为3小时,抽滤除去残渣得上清液。重复提取1次。多次的上清液合并,减压浓缩得浓缩液。向浓缩液中加入4倍体积95%的乙醇,置于4℃过夜,3000×g,离心15 分钟,收集沉淀。收集的沉淀经真空冷冻干燥即得蛹虫草子实体粗多糖。
2) 除蛋白质:将制备得到的蛹虫草子实体粗多糖,溶于料液比为1:5 (W/V)的蒸馏水中,加入体积比为料液比为3:1 (V/V)的sevage试剂。剧烈震荡,然后5000×g 离心得上清液。重复4次,上清液合并。
3)透析除盐、寡糖、单糖等:将脱蛋白的多糖溶液移入透析袋中,密封后先在自来水中透析,再在蒸馏水中透析,每4小时换一次水。之后溶液经浓缩、醇沉、离心、干燥得蛹虫草子实体多糖。
4) 蛹虫草子实体多糖的纯化:蛹虫草子实体多糖先经DEAE-52纤维素阴离子交换柱,采用去离子水和0.2 mol/L的氯化钠洗脱,收集0.2mol/L氯化钠的洗脱液,经浓缩、透析、冷冻干燥得蛹虫草子实体葡聚糖半成品。上述蛹虫草子实体葡聚糖半成品再经Sephadex G-200葡聚糖凝胶柱层析,即可分离得到蛹虫草子实体葡聚糖成品TY517,经NMR分析(图谱见附图),确定其结构。
分离得到的蛹虫草子实体葡聚糖TY517的核磁共振波谱(NMR)数据(见表1):
连接方式 | H1/C1 | H2/C2 | H3/C3 | H4/C4 | H5/C5 | H6/C6 |
α-D-Glcp(1→ | 4.88/98.12 | 3.45/71.25 | −/72.3 | −/68.56 | −/72.81 | 3.70,3.62/63.22 |
→6)β-D-Glcp(1→ | 4.38/103.28 | 3.19/73.62 | 3.36/75.87 | 3.48/70.24 | 3.62/75.18 | 4.07,3.78/69.13 |
→6)β-D-Glcp(1→ | 4.38/103.28 | 3.19/73.62 | 3.36/75.87 | 3.33/70.24 | 3.72/75.18 | 4.07,3.72/69.13 |
→6)β-D-Glcp | 4.78/94.06 | 3.30/73.76 | 3.38/76.28 | 3.54/70.67 | 3.7474.86 | 3.96,3.78/69.13 |
5)蛹虫草子实体葡聚糖TY517的降脂应用:SPF级载脂蛋白E敲除(apoE-/-)小鼠,雄性,体重20±2 g。在实验条件下给予普通饲料适应性喂养1周,将实验动物随机分为4组:空白组12只、模型组12只、TY517低剂量组12只、TY517中剂量组12只和TY517高剂量组12只。除空白组外,均给予高脂饲料(配方:胆固醇1.25%、胆汁盐0.5%、猪油10%、蛋黄粉10%、基础饲料78.25%);空白组给予普通饲料。喂养1个月后,各组在饲料不变的情况下,按照0.2 mL/kg灌胃给予相应药物,空白组和模型组给予同体积的生理盐水,每天1次,连续给药2个月。各组的受试药物及剂量如下:
空白组:0.2 mL/kg生理盐水;
模型组:0.2 mL/kg生理盐水;
TY517低剂量组:12.5 mg/kg蛹虫草子实体葡聚糖;
TY517中剂量组:25 mg/kg蛹虫草子实体葡聚糖;
TY517高剂量组:50 mg/kg蛹虫草子实体葡聚糖。
实验期间,各组均无动物死亡。于末次给药后禁食12h,采用肝素抗凝取血管眼眶静脉采血0.8-1.0 mL,戊巴比妥钠麻醉处死,分离血浆,-80 ℃下保存备用。所得血清样本检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白水平(LDL-C)和高密度脂蛋白水平(HDL-C)。
实验结果见表2蛹虫草子实体葡聚糖TY517对apoE(-/-)小鼠血浆TC、TG、LDL-C和HDL-C的影响,实验结果显示,模型组相对于空白对照组TC、TG和LDL-C显著上升,而HDL-C显著下降;给药组均可下调TC、TG和LDL-C,并上调HDL-C。TY517中、高剂量组效果明显,相对于模型组具有统计学意义(P<0.05或P<0.01)。实验结果表明:蛹虫草子实体葡聚糖TY517在25 mg/kg即可起到显著降低高脂诱导的apoE-/-小鼠血浆脂质的效果。
表2
组别 | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | HDL-C (mg/dl) |
空白组 | 457.88 ± 47.83 | 148.93 ± 38.35 | 119.44 ± 12.16 | 98.95 ±17.60 |
模型组 | 875.67 ± 79.03 ## | 198.63 ± 30.24 # | 272.17 ± 17.08 ## | 75.27 ± 8.22 # |
TY517低剂量 | 709.75 ± 75.62* | 178.08 ± 23.95 | 259.06 ± 42.14 | 82.63 ± 25.05 |
TY517中剂量 | 657.24 ± 81.34 ** | 167.26 ± 15.38* | 209.12 ± 37.09* | 88.15 ± 12.37* |
TY517高剂量 | 563.04 ± 86.17 ** | 159.68 ± 14.92** | 188.46 ± 27.35** | 96.27 ± 10.26 ** |
与空白组相比,#P<0.05;##P<0.01;
与模型组相比,*P<0.05;**P<0.01;
实施例2:
提取制备本发明的蛹虫草子实体葡聚糖TY517并验证其降脂活性时,采取了下述个步骤:
1) 制备蛹虫草子实体粗多糖:采集蛹虫草子实体,阴干后用植物粉碎机粉碎,过200目筛。向粉碎的蛹虫草子实体中加入8倍量的水,搅拌均匀,置于超低温冰箱中-40℃冷冻24小时。迅速投入90℃水浴锅中解冻,水浴1小时。然后200W超声处理1小时,过滤得固形物为破壁蛹虫草子实体。破壁后的蛹虫草子实体中加入95%乙醇回流3小时,料液比为1:4(W/V),浸提脱脂及可溶性分子,过滤得固形物。向固形物中加入蒸馏水,料液比为1:15 (W/V),400W超声加热,温度控制在90℃,提取时间为2小时,抽滤除去残渣得上清液。重复提取2次。多次的上清液合并,减压浓缩得浓缩液。向浓缩液中加入3倍体积95%的乙醇,置于4℃过夜,4000×g,离心10 分钟,收集沉淀。收集的沉淀经真空冷冻干燥即得蛹虫草子实体粗多糖。
2) 除蛋白质:将制备得到的蛹虫草子实体粗多糖,溶于料液比为1:4 (W/V)的蒸馏水中,加入体积比为料液比为4:1 (V/V)的sevage试剂。剧烈震荡,然后3000×g 离心得上清液。重复6次,上清液合并。
3)透析除盐、寡糖、单糖等:将脱蛋白的多糖溶液移入透析袋中,密封后先在自来水中透析,再在蒸馏水中透析,每8小时换一次水。之后溶液经浓缩、醇沉、离心、干燥得蛹虫草子实体多糖。
4) 蛹虫草子实体多糖的纯化:蛹虫草子实体多糖经Q-Sepharose Fast Flow阴离子交换柱,采用去离子水和0.2 mol/L的氯化钠洗脱,收集0.2mol/L氯化钠的洗脱液,经浓缩、透析、冷冻干燥得蛹虫草子实体葡聚糖半成品。上述蛹虫草子实体葡聚糖半成品再经Sephacryl-S200HR凝胶柱层析,即可分离得到蛹虫草子实体葡聚糖成品TY517,经NMR分析(图谱见附图),确定其结构。
所分离得到的蛹虫草子实体葡聚糖的波谱数据(见表1):
5)蛹虫草子实体葡聚糖TY517的降脂应用:SPF级豚鼠,雄性,体重20±2 g。在实验条件下给予普通饲料适应性喂养1周,将实验动物随机分为4组:空白组12只、模型组12只、TY517低剂量组12只、TY517中剂量组12只和TY517高剂量组12只。除空白组外,均给予高脂饲料(配方:胆固醇1.25%、胆汁盐0.5%、猪油10%、蛋黄粉10%、基础饲料78.25%);空白组给予普通饲料。喂养1个月后,各组在饲料不变的情况下,按照0.2 mL/kg灌胃给予相应药物,空白组和模型组给予同体积的生理盐水,每天1次,连续给药2个月。各组的受试药物及剂量如下:
空白组:0.2 mL/kg生理盐水;
模型组:0.2 mL/kg生理盐水;
TY517低剂量组:10 mg/kg蛹虫草子实体葡聚糖;
TY517中剂量组:20 mg/kg蛹虫草子实体葡聚糖;
TY517高剂量组:40 mg/kg蛹虫草子实体葡聚糖。
实验期间,各组均无动物死亡。于末次给药后禁食12h,采用肝素抗凝取血管眼眶静脉采血1.2-2.0 mL,戊巴比妥钠麻醉处死,分离血浆,-80 ℃下保存备用。所得血清样本检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白水平(LDL-C)和高密度脂蛋白水平(HDL-C)。
实验结果见表3蛹虫草子实体葡聚糖TY517对豚鼠血浆TC、TG、LDL-C和HDL-C的影响,实验结果显示,模型组相对于空白对照组TC、TG和LDL-C显著上升,而HDL-C显著下降;给药组均可下调TC、TG和LDL-C,并上调HDL-C。TY517中、高剂量组效果明显,相对于模型组具有统计学意义(P<0.05或P<0.01)。实验结果表明:蛹虫草子实体葡聚糖TY517在20 mg/kg即可起到显著降低高脂诱导的豚鼠血浆脂质的效果。
表3
组别 | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | HDL-C (mg/dl) |
空白组 | 115.34 ± 35.46 | 98.17 ± 25.18 | 79.25 ± 20.94 | 28.54 ±17.85 |
模型组 | 457.60 ± 69.27 ## | 358.64 ± 71.84 # | 170.33 ± 65.38 ## | 76.37 ± 10.34 # |
TY517低剂量 | 385.25 ± 77.51 | 318.55 ± 63.50 | 155.27 ± 32.59 | 65.28 ± 15.43 |
TY517中剂量 | 358.47 ± 59.96** | 260.26 ± 35.61** | 129.02 ± 47.51* | 58.35 ± 22.27** |
TY517高剂量 | 323.74 ± 66.58** | 252.90 ± 44.25** | 108.36 ± 39.75** | 56.57 ± 14.06** |
与空白组相比,#P<0.05;##P<0.01;
与模型组相比,*P<0.05;**P<0.01;
实施例3
提取制备本发明的蛹虫草子实体葡聚糖TY517,采取了下述步骤:
1) 制备蛹虫草子实体粗多糖:采集蛹虫草子实体,阴干后用植物粉碎机粉碎,过150目筛。向粉碎的蛹虫草子实体中加入9倍量的水,搅拌均匀,置于超低温冰箱中-40℃冷冻18小时。迅速投入85℃水浴锅中解冻,水浴1.5小时。然后350W超声处理45分钟,过滤得固形物为破壁蛹虫草子实体。破壁后的蛹虫草子实体中加入95%乙醇回流2.5小时,料液比为1:4.5 (W/V),浸提脱脂及可溶性分子,过滤得固形物。向固形物中加入蒸馏水,料液比为1:13 (W/V),450W超声加热,温度控制在90℃,提取时间为2.5小时,抽滤除去残渣得上清液。重复提取2次。多次的上清液合并,减压浓缩得浓缩液。向浓缩液中加入3.5倍体积95%的乙醇,置于4℃过夜,3500×g,离心12 分钟,收集沉淀。收集的沉淀经真空冷冻干燥即得蛹虫草子实体粗多糖。
2) 除蛋白质:将制备得到的蛹虫草子实体粗多糖,溶于料液比为1:4 (W/V)的蒸馏水中,加入体积比为料液比为4:1 (V/V)的sevage试剂。剧烈震荡,然后4000×g 离心得上清液。重复5次,上清液合并。
3)透析除盐、寡糖、单糖等:将上述脱蛋白的多糖溶液移入透析袋中,密封后先在自来水中透析,再在蒸馏水中透析,每6小时换一次水。之后溶液经浓缩、醇沉、离心、干燥得蛹虫草子实体多糖。
4) 蛹虫草子实体多糖的纯化:蛹虫草子实体多糖经Q-Sepharose Fast Flow阴离子交换柱,采用去离子水和0.2 mol/L的氯化钠洗脱,收集0.2mol/L氯化钠的洗脱液,经浓缩、透析、冷冻干燥得蛹虫草子实体葡聚糖半成品。上述蛹虫草子实体葡聚糖半成品再经Sephacryl-S200HR凝胶柱层析,即可分离得到蛹虫草子实体葡聚糖成品TY517,经NMR分析(图谱见附图),确定其结构。
所分离得到的蛹虫草子实体葡聚糖的波谱数据(见表1):
5)蛹虫草子实体葡聚糖TY517的降脂应用:SPF级Wistar大鼠,雄性,体重80±5 g。在实验条件下给予普通饲料适应性喂养1周,将实验动物随机分为5组:空白组8只、模型组8只、TY517低剂量组8只、TY517中剂量组8只和TY517高剂量组8只。除空白组外,均给予高脂饲料(配方:胆固醇1.25%、胆汁盐0.5%、猪油10%、蛋黄粉10%、基础饲料78.25%);空白组给予普通饲料。喂养1个月后,各组在饲料不变的情况下,按照0.2 mL/kg灌胃给予相应药物,空白组和模型组给予同体积的生理盐水,每天1次,连续给药2个月。各组的受试药物及剂量如下:
空白组:0.2 mL/kg生理盐水;
模型组:0.2 mL/kg生理盐水;
TY517低剂量组:10 mg/kg蛹虫草子实体葡聚糖;
TY517中剂量组:20 mg/kg蛹虫草子实体葡聚糖;
TY517高剂量组:40 mg/kg蛹虫草子实体葡聚糖。
实验期间,各组均无动物死亡。于末次给药后禁食12h,采用肝素抗凝取血管眼眶静脉采血1.2-2.0 mL,戊巴比妥钠麻醉处死,分离血浆,-80 ℃下保存备用。所得血清样本检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白水平(LDL-C)和高密度脂蛋白水平(HDL-C)。
实验期间,各组均无动物死亡。于末次给药后禁食12h,采用肝素抗凝取血管眼眶静脉采血1.2-2.0 mL,戊巴比妥钠麻醉处死,分离血浆,-80 ℃下保存备用。所得血清样本检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白水平(LDL-C)和高密度脂蛋白水平(HDL-C)。
如表4蛹虫草子实体葡聚糖TY517对Wistar大鼠血浆TC、TG、LDL-C和HDL-C的影响所示,实验结果显示,模型组相对于空白对照组TC、TG和LDL-C显著上升,而HDL-C显著下降;给药组均可下调TC、TG和LDL-C,并上调HDL-C。TY517中、高剂量组效果明显,相对于模型组具有统计学意义(P<0.05或P<0.01)。实验结果表明:蛹虫草子实体葡聚糖TY517在20 mg/kg即可起到显著降低高脂诱导的Wistar大鼠血浆脂质的效果。
表4
组别 | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | HDL-C (mg/dl) |
空白组 | 151.38± 30.20 | 80.52 ± 10.39 | 60.08 ± 10.78 | 121.94 ±15..14 |
模型组 | 250.54 ± 30.63 ## | 100.75 ±11.77 ## | 148.64 ± 18.40 ## | 82.76 ± 7.46 # |
TY517低剂量 | 228.06 ± 20.28 | 90.11 ±8.41 | 130.10 ± 15.30 | 85.56 ± 8.42 |
TY517中剂量 | 210.17± 14.80** | 82.40 ±4.65** | 107.62 ± 17.34* | 90.27 ± 11.19 |
TY517高剂量 | 189.32 ± 17.23** | 77.83 ± 19.40** | 90.37± 12.71** | 100.94 ± 10.88* |
与空白组相比,#P<0.05;##P<0.01; 与模型组相比,*P<0.05;**P<0.01。
当然,上述说明并非是对本发明的限制,本发明也并不仅限于上述举例,本技术领域的技术人员在本发明的实质范围内所做出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (2)
1.一种蛹虫草子实体葡聚糖,其特征在于,该多糖的结构式是:
α-D-Glcp (1[→6)-β-D-Glcp (1]13→6)β-D-Glcp
其中:Glcp为吡喃型葡萄糖;该多糖简称TY517。
2.权利要求1所述的蛹虫草子实体葡聚糖的制备方法,其特征在于,包含有如下步骤:
(1)制备蛹虫草子实体粗多糖:
采集蛹虫草子实体,阴干并粉碎,过筛;
向粉碎的蛹虫草子实体中加水,经冻融后超声处理,过滤得破壁蛹虫草子实体;
在破壁后的蛹虫草子实体加入95%的乙醇回流浸提脱脂,过滤得固形物;向固形物中再加入蒸馏水,加热超声浸提,抽滤除去残渣得上清液;
将上清液减压浓缩后加入95%的乙醇,置于0~4℃过夜,离心,收集沉淀;
收集的沉淀经真空冷冻干燥,即得蛹虫草子实体粗多糖;
(2) 除蛋白质:将制备得到的蛹虫草子实体粗多糖,溶于蒸馏水中,加入适量sevage试剂,剧烈震荡,然后3000-5000×g离心得上清液,重复多次,上清液合并;
(3)透析:将上述脱蛋白的多糖溶液移入透析袋中,密封后先在自来水中透析,再在蒸馏水中透析,每4-8小时换一次水;之后溶液经浓缩、醇沉、离心、干燥得蛹虫草子实体多糖;
(4) 蛹虫草子实体多糖的纯化:蛹虫草子实体多糖先经DEAE-52纤维素阴离子交换柱或Q-Sepharose Fast Flow阴离子交换柱,采用去离子水和0.2 mol/L的氯化钠洗脱,收集0.2mol/L氯化钠的洗脱液,经浓缩、透析、冷冻干燥得蛹虫草子实体葡聚糖半成品, 上述蛹虫草子实体葡聚糖半成品再经Sephadex G-200葡聚糖凝胶柱或Sephacryl-S200HR凝胶柱层析,即可分离得到蛹虫草子实体葡聚糖成品TY517,经NMR分析,确定其结构。
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