CN114230646A - 一种抗肿瘤灰树花糖蛋白及其制备方法和应用 - Google Patents
一种抗肿瘤灰树花糖蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了一种抗肿瘤灰树花糖蛋白及其制备方法和应用。所述抗肿瘤灰树花糖蛋白的蛋白质含量为54.0±3.0%,多糖含量为20.3±2.0%,重均分子量为17.5kDa,单糖组成及摩尔比为岩藻糖:盐酸氨基葡萄糖:半乳糖:葡萄糖:甘露糖:半乳糖醛酸:葡萄糖醛酸=2.4:0.8:3.9:72.3:7.7:3.2:9.6;氨基酸组成为:丙氨酸、甘氨酸、苏氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、天门冬氨酸、苯丙氨酸、酪氨酸和谷氨酸。本发明灰树花糖蛋白经体内验证了增强肿瘤机体的免疫应答,抑制乳腺癌体内生长的作用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抗肿瘤灰树花糖蛋白及其制备方法和应用。
背景技术
已知癌症源于自体细胞的基因突变,具有强的遗传不稳定性和异质性。经过近几十年的发展,放、化疗及手术取得巨大进步,但在绝大多数情况不能完全控制肿瘤生长,且易导致耐药性和更难治疗的复发。肿瘤学和免疫学的深入发展和理解,研究发现肿瘤的形成是机体形成免疫逃逸的结果。由于免疫抑制检查点抑制剂PD-1、PD-L1以及CTLA-4抗体在癌症晚期治疗中的突出效果,2018年诺贝尔生理学或医学奖授予肿瘤免疫治疗,这也使肿瘤免疫治疗成为癌症治疗第四大疗法。免疫治疗基于癌细胞免疫逃逸的原理,采用修复和重塑肿瘤微环境,激活免疫细胞,尤其是T细胞识别、清除癌细胞,且实现持续性控制肿瘤生长。目前,肿瘤免疫治疗已形成了多种形式,包括靶向抗体、细胞因子、CAR-T、癌症疫苗、免疫检查点抑制剂及免疫佐剂。但目前疗效最好和广泛的抗PD-1/PD-L1抗体疗法临床有效率总体上也仅有20%-30%,且存在免疫相关不良反应。因此,基于抑制免疫逃逸恢复机体免疫识别和清除肿瘤细胞的策略,克服已有药物局限,开发新的免疫治疗药物成为热门研究和必须。
食药用菌是我国传统药物的重要组成部分,具有调节免疫力和抑制肿瘤生长的功能。灰树花(Grifola frondosa)是东亚地区传统药食同源真菌,具有抗肿瘤、调节免疫力、降血脂等生物活性。迄今为止,灰树花活性成分的研究主要集中在多糖,已报道40多种,绝大部分具有抑制肿瘤生长的作用。近年,灰树花糖蛋白和糖肽的活性及功能的报道逐渐增多。Hirokazu K等首次报道灰树花子糖蛋白GFL,含糖量3.3%,分子量30-52kDa,体外对Hela细胞有细胞毒作用(Hirokazu Kawagishi等,1990)。Gu CQ等报道了蛋白GFAHP(分子量为29.5kDa)具有抑制病毒HSV-1的活性(Gu CQ,2007)。2013年,Cui FJ等从灰树花菌丝体获得糖蛋白GFG-3a,含糖量为6.20%,分子量为88.01kDa,体外对S180和Bel-7402细胞具有抑制作用(Cui Fengjie等,2013)。Tsao YW等从灰树花子实体获得GFPr,是不含葡聚糖的蛋白,由两个分子量为41kDa的蛋白亚基组成。GFPr刺激从正常小鼠中获得脾细胞和NK细胞产生IFN-γ及髓系细胞分化成熟,且增强TH1反应;体内移植瘤实验显示抑制肿瘤的生长。专利US 7214778 B2公开了糖蛋白(Glyco-protein,20kDa)的抗肿瘤活性,专利CN103509091A公开了一种灰树花菌丝体抗肿瘤糖蛋白及制备方法。以上发表论文和公开专利,在提取糖蛋白的过程用到强酸碱及脱蛋白等工艺,提取的活性成分不同,并且在抗肿瘤的作用主要是在较高浓度下体外抑制肿瘤细胞株增殖,不能反映在机体内是否有效,且未能验证其抗肿瘤作用的关键成分组成。这在一定程度上影响了该类产品进一步开发应用。
发明内容
针对以上现有技术存在的缺点和不足之处,本发明的首要目的在于提供一种抗肿瘤灰树花糖蛋白。本发明的抗肿瘤灰树花糖蛋白具有确定分子量、氨基酸及单糖组成,经体内实验证明了该糖蛋白增强肿瘤机体的免疫应答,抑制乳腺癌的体内生长的作用。
本发明的另一目的在于提供上述抗肿瘤灰树花糖蛋白的制备方法。本发明制备方法以灰树花子实体为原料,采用提取、分离及纯化与体内活性评价结合的方法,简便安全地分离纯化出了一种高纯度的灰树花糖蛋白。
本发明的再一目的在于提供上述抗肿瘤灰树花糖蛋白在制备抑制肿瘤生长、增强机体免疫应答和/或治疗和预防肿瘤疾病药物或健康功能性食品中的应用。
本发明目的通过以下技术方案实现:
一种抗肿瘤灰树花糖蛋白,其蛋白质含量为54.0±3.0%,多糖含量为20.3±2.0%,重均分子量为17.5kDa,单糖组成及摩尔比为岩藻糖(Fuc):盐酸氨基葡萄糖(GlcN):半乳糖(Gal):葡萄糖(Glc):甘露糖(Man):半乳糖醛酸(GalA):葡萄糖醛酸(GlcUA)=2.4:0.8:3.9:72.3:7.7:3.2:9.6;氨基酸组成为:丙氨酸Ala、甘氨酸Gly、苏氨酸Thr、缬氨酸Val、亮氨酸Leu、异亮氨酸Lys、脯氨酸Pro、天门冬氨酸Asp、苯丙氨酸Phe、酪氨酸Tyr和谷氨酸Glu。
上述抗肿瘤灰树花糖蛋白的制备方法,包括如下制备步骤:
(1)将灰树花子实体经粉碎后以10~40倍纯水在70~110℃温度下提取0.5~10h,过滤收集滤液,真空浓缩至原液体积50%~10%,得到粗提取浓缩液;
(2)向步骤(1)的粗提取浓缩液中加入1~5倍体积的C1~C3的醇溶剂(甲醇、乙醇、丙醇)充分混匀,在4℃条件下静置过夜,离心,冷乙醇洗涤沉淀,得到沉淀物;
(3)用65~70℃纯水溶解步骤(2)的沉淀,在48~52℃条件下真空浓缩将乙醇蒸发,过滤除去滤渣,得滤液,将滤液经真空冷冻干燥,获得灰树花水溶性提取物GFI;
(4)用纯水、0.09~0.15mol/L、0.28~0.32mol/L、0.5~0.55mol/L梯度浓度的NaCl溶剂通过阴离子凝胶(DEAE-Sepharose Fast Flow)交换柱层析将步骤(3)获得的GFI进行级分,收集0.28~0.32mol/LNaCl溶剂洗脱组分,浓缩,透析脱盐,再浓缩,冷冻干燥,得到抗肿瘤灰树花糖蛋白(GFI-3)。
进一步地,步骤(1)中所述粉碎是指粉碎至40~100目。
进一步地,步骤(1)中所述纯水加入量优选为15~25倍原材料质量。
进一步地,步骤(1)中所述提取温度优选为80~100℃,提取时间优选为1~4h。
进一步地,步骤(1)中提取后的残渣再次以15~25倍体积的纯水溶剂提取1~4h,过滤后合并滤液。
通过两步提取和将每次提取后获得滤液合并可提高提取效率,但提取次数不局限于此。根据实验发现,在每步中检测提取效率总提取量的约80%至90%是通过两次提取得到的,从而说明两步提取与多于三次提取的多次提取相比具有显著的经济效益。另外,在本发明的提取方法中,如果用来制备灰树花提取物水溶剂的量太少,会导致提取物的溶解性降低导致提取效率下降。如果用来制备灰树花的溶剂的量太多,用于接下来纯化的醇的量会增加,而引起经济问题和处理问题。
进一步地,步骤(1)中所述真空浓缩优选至原液体积30%~10%。
进一步地,步骤(2)中所述C1~C3的醇溶剂加入量优选为3~4倍体积。
使用低级醇溶剂获得醇不溶物步骤是为了去除粗提取物中不必要的杂质如色素、果胶、脂肪酸及小分子脂溶性物质等。因此如果使用低级醇的量过少或过多,不能有效的去除杂质或者造成糖蛋白的损失。因此,优选在上述范围内调整低级醇的量。
进一步地,步骤(2)中所述C1~C3的醇溶剂优选无水乙醇。
进一步地,步骤(2)中所述离心是指在离心力8228g离心15min。
进一步地,步骤(3)中所述过滤采用0.8μm滤膜过滤。
进一步地,步骤(4)中所述透析脱盐是指采用截留分子量为3000Da的透析袋透析脱盐。
上述抗肿瘤灰树花糖蛋白在制备抑制肿瘤生长、增强机体免疫应答和/或治疗和预防肿瘤疾病药物或健康功能性食品中的应用。
与现有技术相比,本发明的有益效果是:
(1)本发明以灰树花子实体为原料,采用提取、分离及纯化与体内活性评价结合的方法,简便安全地分离纯化出了一种高纯度的灰树花糖蛋白组成,并确定分子量、氨基酸、单糖组成,且经体内实验证明了该糖蛋白增强肿瘤机体的免疫应答,抑制乳腺癌的体内生长的作用。
(2)本发明的抗肿瘤灰树花糖蛋白具有如下应用活性:1.口服给药方式显著地抑制肿瘤生长;2.注射给药方式极显著地抑制肿瘤生长;3.提升外周血总T淋巴细胞和CD8+T细胞的数量;4.抑制肿瘤组织免疫抑制检查点PD-L1和PD-1,激活CD8+T细胞增殖和活性。
附图说明
图1为实施例中GFI经阴离子凝胶层析柱分离组分在OD280nm的色谱分析图。
图2为实施例中所得抗肿瘤灰树花糖蛋白GFI-3的凝胶色谱图。
图3为实施例中GFI-3抑制小鼠肿瘤生长结果图。
图4为实施例中GFI-3对肿瘤组织T淋巴细胞及免疫抑制因子表达的作用结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
本实施例的一种抗肿瘤灰树花糖蛋白的制备方法,包括如下制备步骤:
(1)将灰树花子实体(广东粤微食用菌技术公司购买)60℃烘干至恒重,超微粉,过100目,取样品200g用4.0L纯水浸泡3小时,在100℃下加热,回流提取两次,每次持续2小时,合并滤液,将滤液在48~52℃下真空浓缩至原液的十分之一,离心,去除不溶物,得到粗提浓缩液。
(2)将步骤(1)的粗提取浓缩液与无水乙醇按照1:4的比例混合,4℃过夜,离心(离心力8228g,4℃,15min),得到沉淀物。将沉淀物经预冷无水乙醇洗涤两次,离心,去除上清,再次获得沉淀物。
(3)用65~70℃纯水溶解步骤(2)的沉淀物,在48~52℃条件下真空浓缩将乙醇蒸发,过0.8μm滤膜,除去滤渣,得滤液。将滤液经真空冷冻干燥,获得灰树花水溶性提取物GFI,共计28.1±3.01g。干燥保存,备用。
(4)用纯水、0.09~0.15mol/L、0.28~0.32mol/L、0.5~0.55mol/L梯度浓度的NaCl溶剂通过阴离子凝胶(DEAE-Sepharose Fast Flow)交换柱层析将步骤(3)获得的GFI进行级分,在洗脱过程中,OD280nm在线检测四个峰,分别是GFI-1、GFI-2、GFI-3、GFI-4(如图1所示),GFI-1为纯水洗脱部分,GFI-2为0.09~0.15mol/LNaCl洗脱部分,GFI-3为0.28~0.32mol/LNaCl洗脱部分,GFI-4为0.5~0.55mol/LNaCl洗脱部分。依次收集四个峰,将收集液浓缩、截留分子量3000透析袋透析脱盐,再浓缩,冷冻,获得冻干粉。计算四个组分得率,分析四个组分在GFI中的含量比例为:GFI-1:GFI-2:GFI-3:GFI-4=10.4±1.8:5.2±1.52:3.4±0.92:1±0.76。
其中GFI-3为本发明保护的一种抗肿瘤灰树花糖蛋白,经后续性能检测为抗肿瘤的关键活性组分。对本实施例所得灰树花糖蛋白GFI-3进行理化分析,用物理化学分析例如HPGPC、紫外分光光度计、离子测谱仪、GC-MS等进行实验。通过BCA方法检测GFI-3蛋白含量为54.0±3.0%,苯酚-硫酸法测的多糖20.3±2.0%。通过利用岛津LC-10A高效液相色谱仪配置RI-502示差检测器检测GFI-3的纯度和分子量。使用色谱柱为BRT105-104-102(8×300mm),柱温保持在40℃。样品浓度为5mg/mL,进样量为20μL。对于用作标准物质的不同分子量的多糖,其中分子量从5000~667800Da葡聚糖购买自sigma公司,Dextranstandards1152为源叶生物。流动相:0.05mol/LNaCl溶液,流速:0.6mL/min。利用岛津GPC软件计算GFI-3分子量。结果如图2所示,结果显示GFI-3为均一组分的糖蛋白,经分析计算其重均分子量为17.5kDa。
利用酸水解降解GFI-3多糖,经离子色谱仪检测,与单糖标准品对照,分析出GFI-3的单糖组成及比例。GFI-3单糖组成及比例为:岩藻糖:盐酸氨基葡萄糖:半乳糖:葡萄糖:甘露糖:半乳糖醛酸:葡萄糖醛酸=0.024:0.008:0.039:0.723:0.077:0.032:0.096,葡萄糖占绝对比重。
利用酸水解降解GFI-3蛋白质,经GC-MS检测和分析。GFI-3含有11中氨基酸,包括丙氨酸Ala、甘氨酸Gly、苏氨酸Thr、缬氨酸Val、亮氨酸Leu、异亮氨酸Lys、脯氨酸Pro、天门冬氨酸Asp、苯丙氨酸Phe、酪氨酸Tyr和谷氨酸Glu。
对本实施例所得抗肿瘤灰树花水溶性提取物GFI及糖蛋白GFI、GFI-1、GFI-2、GFI-3、GFI-4进行应用性能检测:
(1)口服灰树花提取物和组分激活机体免疫和抑制机体肿瘤生长的检测:
为了测定本实施例中灰树花提取物及级分组分增强肿瘤机体免疫应答和抑制肿瘤生长的效应,采用鼠源乳腺癌移植瘤小鼠模型进行激活淋巴细胞和抑瘤效应检测。
1实验方法
1.1小鼠乳腺癌细胞4T1准备
从液氮储存罐中取出4T1细胞,进行复苏、活化和培养,培养基为含1%青霉素、1%链霉素和10%胎牛血清(FBS)的DMEM培养基,培养条件为37℃、浓度5%的CO2细胞培养箱,培养至对数生长期。
1.2肿瘤动物模型构建和实验
小鼠为BALB/C雌性小鼠(6-8周龄,体重20±2g)来自广东省实验动物中心,适应性喂养一周,准备实验。将上述对数生长期的4T1细胞经消化、计数,调整到细胞密度为5×105cells/mL,植入小鼠皮下,并随机分配为模型对照组、测试组(GFI、GFI-1、GFI-2、GFI-3、GFI-4),每组小鼠10只,同时设置正常对照组。造模第二天,采用口服灌胃的方式对各个小组给药,测试组剂量为100mg/kg/day,模型组给予同等体积的生理盐水,连续给药4周。末次给药后,第二天牺牲小鼠,采用肝素钠小管取血、肿瘤等。按照下述实验目的进行保存。
1.3流式细胞仪检测外周血淋巴细胞
将上述取样血液在48小时内经流式细胞仪检测,获得外周血不同淋巴细胞的数量和比例。实验中涉及抗体包括:ANTI-MO CD4 PE,ANTI-MO CD8A 53-6.7FITC,ANTI-MO CD19APC,Anti-Mouse CD3 PerCP-CyTM5.5和Anti-Mouse CD45.2 APC-CyTM7。
简述实验步骤如下:
1)抗体的准备,按照抗体说明书,使用0.5%BSA缓冲液稀释(CD45+CD3+CD19+CD4+CD8)抗体,同时准备相应的单抗稀释液。
2)将60μL抗体混合液与等体积外周血液在流式管中混合,涡旋,避光,4℃孵育30min。
3)在上述流式管中加入2mL红细胞裂解液(ACK)裂解液,涡旋,避光室温孵育10min。
4)在4℃,离心力400g,离心5min,丢弃上清液。
5)加入1mLPBS缓冲液,涡旋,4℃,离心力400g,离心5min,丢弃上清液。(本步骤重复操作两次)。
6)加入300μLPBS,涡旋,避光4℃保藏。5h内上机完成流式检测。
1.4计算和分析
使用Prism 6.07和spss21.0等计算和分析上述数据。
2实验结果
2.1 GFI和各组分对肿瘤生长的效应
如表1所示,实验结果显示:所得GFI和GFI-3呈现出显著地抑制小鼠体内肿瘤生长。比较GFI级分获得的四个组分,发现在灌胃的条件下GFI-3抑瘤活性最强,是灰树花提取物主要的活性物质。因此,后续实验的样品为GFI和GFI-3。
表1.与模型组相比各给药组小鼠肿瘤体积的变化
注:给药组与模型组相比,*表示p<0.05(差异显著);**表示p<0.01(差异极显著)。
2.2 GFI和GFI-3对机体外周血淋巴细胞的效应
大量研究报告表明:癌症患者免疫力会降低,淋巴细胞增殖和活化会受到抑制。细胞毒性T细胞(CD8+)作为主要的效应免疫细胞,在肿瘤机体被抑制,因此增强和激活CD8+T细胞是肿瘤免疫治疗的关键。
因此,本实施例采用流式细胞仪分析外周血中不同淋巴细胞的含量,结果显示:与正常小鼠相比,肿瘤模型组小鼠总T细胞(CD3+)显著减少,且杀伤性T细胞(CD8+)与辅助性T细胞(CD4+)降低;与对照组相比,GFI和GFI-3促进外周血CD3+T细胞增多,杀CD8+数量提升,且CD8+/CD4+的比例上调(表2)。T细胞亚群直接反应机体细胞的免疫状态,本实验表明GFI和GFI-3提升了肿瘤小鼠机体的免疫功能。
表2.GFI和GFI-3对外周血免疫细胞的作用
注:给药组与模型组相比,*表示p<0.05(差异显著);**表示p<0.01(差异极显著)。
(2)腹腔注射GFI-3激活肿瘤组织CD8+T细胞及抑制肿瘤活性检测:
给药方式对药物的效应具有影响,因此本实施例在上述口服给药的基础上,采用腹腔注射给药的方式检测上述糖蛋白GFI-3激活和增强机体免疫,进而抑制肿瘤生长的活性。
1实验方法
1.1 4T1细胞准备
实验操作同(1)中1.1。
1.2肿瘤动物模型构建和实验
小鼠品系、来源、造模同上述(1)中1.2。将造模动物分为模型对照组、GFI-3组,设置紫杉醇阳性对照组,同时设置正常组。
造模第二天,按照剂量100mg/kg/每只小鼠腹腔注射给予GFI-3组小鼠,隔天给药,模型组给予同体积的生理盐水。周期为4周。
末次给药后,第二天牺牲小鼠,采用肝素钠小管取血、肿瘤等。按照下述实验目的进行保存。
1.3流式细胞仪检测外周血CD8+T淋巴细胞
实验操作同(1)中1.3。
1.4免疫组织化学检测肿瘤组织CD8+T淋巴细胞
1)组织切片的制备:取4.1.2中所获得的肿瘤组织,经10%中性***固定过夜,再经石蜡包埋、切片和贴片,制备得到肿瘤组织石蜡切片。
2)免疫组织化学染色:肿瘤组织免疫细胞杀伤性T细胞CD8+、辅助性T细胞CD4+和肿瘤细胞表面抑制分子PD-L1表达免疫组织化学染色。具体实现步骤如下:
A.切片常规二甲苯脱蜡,梯度酒精脱水:将石蜡切片在65~68℃烘箱中烤片2h,将石蜡溶解,然后将石蜡切片在二甲苯Ⅰ(浸泡时间15min)、二甲苯Ⅱ(浸泡时间15min)、二甲苯Ⅲ(浸泡时间15min)进行脱蜡。将脱蜡的切片经100%酒精Ⅰ浸泡5min、100%酒精Ⅱ浸泡5min、90%酒精Ⅰ浸泡5min、90%酒精Ⅱ浸泡5min、80%酒精浸泡5min、75%酒精浸泡5min脱水,之后用纯水清洗3次,每次5min。
B.灭活内源性抗氧化物酶:将切片放入用甲醇新鲜配置的0.3%过氧化氢中,室温放置15min,之后使用1×PBS缓冲液清洗3次,每次5min。
C.抗原修复:将切片放入0.01mol/L柠檬酸盐缓冲液中(保证液面可以将切片完全覆盖),在微波炉中调至中高火加热至沸腾8min,保温7分钟,然后调制中火加热三次,每次6min,之后保温6min,拿出待其降至室温后,将修复后的切片放入PBS缓冲液中清洗3次,每次5min。
D.血清封闭:37℃下用10%山羊血清封闭液将切片孵育30min,倾去。
E.一抗孵育:在切片滴加一滴一抗,覆盖样品,放置湿盒中并于37℃恒温箱中孵育1h,4℃过夜。第二天,1×PBS缓冲液清洗3次,每次5min。
F.二抗孵育:在切片上滴加二抗(山羊抗兔IgG H&L(HRP)),室温孵育1h,1×PBS缓冲液清洗3次,每次5min。
G.DAB显色:配置新鲜DAB显色液。在显微镜下滴加DAB显色液至切片,避光显色,此过程中显微镜下控制显色程度,观察到显色适度,立即用自来水清洗10min,终止显色。
H.复染:将切片置入苏木精染液中染色5min,用纯水冲洗(切勿将流水对着组织冲洗,以防脱片);放入苏木精透化液染盒中10s进行分化,冲洗,再放入苏木精返蓝液中10s。
I.常规脱水,透明,封片:将切片依次放入75%酒精(浸泡时间5min)、85%酒精(浸泡时间5min)、无水乙醇Ⅰ(浸泡时间5min)、无水乙醇Ⅱ(浸泡时间5min)、二甲苯Ⅰ(浸泡时间5min)中脱水至呈透明,再将切片从二甲苯Ⅰ中拿出来稍晾干,最后使用中性树胶封片。
2结果
2.1腹腔注射GFI-3抑制小鼠肿瘤生长的效应
为了测定GFI-3在腹腔注射给药的抑制肿瘤活性,按照上述实验进行测定的结果如下表3示出,并在图3中示出图例。实验结果显示,在腹腔注射给药的条件下,GFI-3极显著地抑制了小鼠乳腺癌的生长,与对照组相比,GFI-3抑制肿瘤生长的抑制率达到64%(p<0.01)。
表3.GFI-3腹腔注射给药对小鼠肿瘤生长的作用
注:给药组与模型组相比,*表示p<0.05(差异显著);**表示p<0.01(差异极显著)。
2.2流式分析外周血T淋巴细胞
按照上述实验方法测定腹腔注射给药肿瘤小鼠外周血T淋巴细胞测定,其结果在下表4示出。
表4.GFI-3对外周血T淋巴细胞的作用
注:#是表示模型组与正常组相比,*表示给药组与模型组相比,#和*表示p<0.05(差异显著);##和**表示p<0.01(差异极显著)。
2.3免疫组化检测肿瘤组织T淋巴细胞
按照上述实验方法测定肿瘤组织T淋巴细胞表达的测定,其结果如图4所示,模型组肿瘤组织可检测到CD4+和CD8+表达,标识肿瘤组织存在T淋巴细胞浸润。与模型对照组,GFI-3给药组肿瘤组织,CD8+表达量明显升高。同时,检测到GFI-3组肿瘤组织的免疫抑制因子PD-1和PD-L1表达下降。因此,糖蛋白GFI-3激活和增强肿瘤组织杀伤性T细胞(CD8+)免疫应答。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种抗肿瘤灰树花糖蛋白,其特征在于,所述抗肿瘤灰树花糖蛋白的蛋白质含量为54.0±3.0%,多糖含量为20.3±2.0%,重均分子量为17.5kDa,单糖组成及摩尔比为岩藻糖:盐酸氨基葡萄糖:半乳糖:葡萄糖:甘露糖:半乳糖醛酸:葡萄糖醛酸=2.4:0.8:3.9:72.3:7.7:3.2:9.6;氨基酸组成为:丙氨酸、甘氨酸、苏氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、天门冬氨酸、苯丙氨酸、酪氨酸和谷氨酸。
2.权利要求1所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,包括如下制备步骤:
(1)将灰树花子实体经粉碎后以10~40倍纯水在70~110℃温度下提取0.5~10h,过滤收集滤液,真空浓缩至原液体积50%~10%,得到粗提取浓缩液;
(2)向步骤(1)的粗提取浓缩液中加入1~5倍体积的C1~C3的醇溶剂充分混匀,在4℃条件下静置过夜,离心,冷乙醇洗涤沉淀,得到沉淀物;
(3)用65~70℃纯水溶解步骤(2)的沉淀,在48~52℃条件下真空浓缩将乙醇蒸发,过滤除去滤渣,得滤液,将滤液经真空冷冻干燥,获得灰树花水溶性提取物GFI;
(4)用纯水、0.09~0.15mol/L、0.28~0.32mol/L、0.5~0.55mol/L梯度浓度的NaCl溶剂通过阴离子凝胶交换柱层析将步骤(3)获得的GFI进行级分,收集0.28~0.32mol/LNaCl溶剂洗脱组分,浓缩,透析脱盐,再浓缩,冷冻干燥,得到抗肿瘤灰树花糖蛋白GFI-3。
3.根据权利要求2所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(1)中所述粉碎是指粉碎至40~100目。
4.根据权利要求2所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(1)中所述纯水加入量为15~25倍原材料质量;所述提取温度为80~100℃,提取时间为1~4h。
5.根据权利要求4所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(1)中提取后的残渣再次以15~25倍体积的纯水溶剂提取1~4h,过滤后合并滤液。
6.根据权利要求5所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(1)中所述真空浓缩至原液体积30%~10%。
7.根据权利要求2所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(2)中所述C1~C3的醇溶剂加入量为3~4倍体积,所述C1~C3的醇溶剂为无水乙醇。
8.根据权利要求7所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(2)中所述离心是指在离心力8228g离心15min;步骤(3)中所述过滤采用0.8μm滤膜过滤。
9.根据权利要求2所述的一种抗肿瘤灰树花糖蛋白的制备方法,其特征在于,步骤(4)中所述透析脱盐是指采用截留分子量为3000Da的透析袋透析脱盐。
10.权利要求1所述的一种抗肿瘤灰树花糖蛋白在制备抑制肿瘤生长、增强机体免疫应答和/或治疗和预防肿瘤疾病药物或健康功能性食品中的应用。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5854404A (en) * | 1996-03-08 | 1998-12-29 | Yukiguni Maitake Co., Ltd. | Antitumor substance extracted from grifola |
CN102408494A (zh) * | 2011-11-30 | 2012-04-11 | 杭州众芝康菇生物技术有限公司 | 一种灰树花多糖zzk组分及其制备方法 |
CN103509091A (zh) * | 2013-10-10 | 2014-01-15 | 江苏大学 | 一种灰树花菌丝体抗肿瘤糖蛋白及制备方法 |
CN103724445A (zh) * | 2013-12-25 | 2014-04-16 | 广东省微生物研究所 | 灰树花多糖f2的制备方法及其降血糖功能 |
WO2019137349A1 (zh) * | 2018-01-15 | 2019-07-18 | 中国科学院上海药物研究所 | 一种红花多糖、其制备方法及在抗肿瘤药物中的应用 |
-
2021
- 2021-12-07 CN CN202111486770.8A patent/CN114230646B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5854404A (en) * | 1996-03-08 | 1998-12-29 | Yukiguni Maitake Co., Ltd. | Antitumor substance extracted from grifola |
CN102408494A (zh) * | 2011-11-30 | 2012-04-11 | 杭州众芝康菇生物技术有限公司 | 一种灰树花多糖zzk组分及其制备方法 |
CN103509091A (zh) * | 2013-10-10 | 2014-01-15 | 江苏大学 | 一种灰树花菌丝体抗肿瘤糖蛋白及制备方法 |
CN103724445A (zh) * | 2013-12-25 | 2014-04-16 | 广东省微生物研究所 | 灰树花多糖f2的制备方法及其降血糖功能 |
WO2019137349A1 (zh) * | 2018-01-15 | 2019-07-18 | 中国科学院上海药物研究所 | 一种红花多糖、其制备方法及在抗肿瘤药物中的应用 |
Non-Patent Citations (1)
Title |
---|
崔凤杰等: "灰树花菌丝体抗肿瘤糖肽GFPS1b结构的研究", 食品与生物技术学报, vol. 25, no. 5, pages 66 - 71 * |
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