CN111410700B - 一种蝉花多糖及其分离纯化方法 - Google Patents
一种蝉花多糖及其分离纯化方法 Download PDFInfo
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- CN111410700B CN111410700B CN202010386370.9A CN202010386370A CN111410700B CN 111410700 B CN111410700 B CN 111410700B CN 202010386370 A CN202010386370 A CN 202010386370A CN 111410700 B CN111410700 B CN 111410700B
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- polysaccharide
- cordyceps sobolifera
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Abstract
本发明涉及一种蝉花多糖及其制备方法,所述蝉花多糖从蝉花中分离纯化得到,对胰腺癌、宫颈癌、白血病、胶质瘤等恶性肿瘤有明显的抑制作用。
Description
本申请是申请日为2016年8月23日、申请号为201610713106.5、发明名称为“一种蝉花多糖及其分离纯化方法”的发明专利申请的分案申请。
技术领域
本发明涉及一种蝉花多糖及其分离纯化方法,属于食品药品领域。
背景技术
蝉花(Isaria cicadae Miquel),是一种名贵中药材,是寄生在蝉幼虫上的一种虫草菌。属于真菌界(Fungi)的子囊菌门(Ascomycota)、盘菌亚门(Pezizomycotina)、粪壳菌纲(Sordariomycetes)、肉座菌目 (Hypocreales)、虫草科(Cordycipitaceae)、棒束孢属(Isaria)。
蝉花具有悠久的药用历史,最早可考证的记载是公元五世纪的南北朝时期雷斅在《雷公炮炙论》中记载了蝉花的加工方法:“凡使,要白花全者,收得后于屋下东南角悬干,去土后,用浆水煮一日至夜,焙干碾细用之”。后来,隋、唐甄权的《药性论》、宋代苏颂的《图经本草》、明代李时珍的《本草纲目》均有记载。一千多年年来,蝉花一直用于强身健体,治病防疾。
现代医学研究表明蝉花具有免疫调节、抗肿瘤、改善肾功能等药理作用。中国台湾辅仁大学的Kuo等人研究证实蝉花提取的麦角甾醇过氧化物能激活并促进人T淋巴细胞的增殖。程俊文等研究表明蝉花发酵菌丝多糖在12.5至100μg/ml的浓度范围内能明显刺激NO的产生,显示其免疫调节活性。Kim等研究发现蝉花发酵菌丝多糖可以通过TLR4信号通道激活巨噬细胞和诱导树突细胞的成熟。Wang等人研究证实蝉花水提物可以通过抑制MHCC97H的G2/M从而减缓人肝癌的增殖。暨南大学Wang Jing 等人研究表明蝉花中的环缩酚酸肽对HepG2具有细胞毒活性,且其半抑制浓度比阿霉素敏感25倍。朱戎等人通过活体研究证明蝉花提取物可抑制肾组织的纤维化,从而改善肾功能;Weng等人通过研究证明蝉花中含有人类单核细胞的生长调节物质。
发明内容
本发明的目的是提供一种蝉花多糖。
本发明的第二个目的在于提供所述蝉花多糖的制备方法。
本发明的第三个目的是提供该蝉花多糖在制备治疗肿瘤药物中的应用。
本发明的第四个目的是提供一种药物组合物。
本发明的目的是通过如下技术方案实现的:
蝉花多糖B-I-1,其分子量为100~120KDa,重复单元的结构如式1所示:
蝉花多糖B-I-2,其分子量为30~50KDa,重复单元的结构如式2所示:
蝉花多糖B-II-1,其分子量为110~130KDa,重复单元的结构如式3所示:
蝉花多糖B-II-2,其分子量为40~60KDa,重复单元的结构如式4所示:
本发明4种蝉花多糖成分均为单一的多糖成分。
上述蝉花多糖按如下方法制备:
步骤1,蝉花孢梗束以水提醇沉法提取,收集沉淀作为提取物;
步骤2,对步骤1的提取物进行去蛋白处理;
步骤3,将步骤2去除蛋白的提取物用乙醇沉淀,得蝉花粗多糖;
步骤4,将步骤3的蝉花粗多糖依次经过DEAE纤维素柱层析和Sephadex G凝胶柱层析纯化,得蝉花多糖。
优选的,步骤1所述醇沉浓度为60%-80%;进一步优选醇沉浓度为 65%-75%;最优选醇沉浓度为70%。
优选的,步骤1得到的沉淀进行纯化,纯化方法为依次用无水乙醇、丙酮、乙醚进行洗涤。
优选的,步骤2所述去蛋白方法为:将步骤1的沉淀溶解,调节pH近中性,分别加入胰蛋白酶和Sevag试剂,离心,去除沉淀(即蛋白质)。进一步优选的,上述去蛋白操作重复4~6次;更进一步优选为所述去蛋白操作重复5次。
本发明所述水提醇沉法系指在水提浓缩液中,加入乙醇使达到规定的含醇量(即醇沉浓度),某些成分在醇溶液中溶解度降低析出沉淀,使水提液得以精制的方法。
本发明还提供了所述蝉花多糖在制备治疗肿瘤的药物或保健品中的应用。
所述肿瘤包括包括白血病、恶性淋巴瘤、多发性骨髓瘤、胃肠道间质瘤、结肠癌、直肠癌、乳腺癌、肝癌、胃癌、卵巢癌、子宫癌、宫颈癌、阴道癌、肺癌、肾癌、前列腺癌、膀胱癌、胰腺癌、脑癌、黑色素瘤等。
本发明还提供了一种药物组合物,其包含上述蝉花多糖和药学上可接受的载体。所述药物组合物包括但不限于口服剂型、胃肠外给药剂型、外用剂型和直肠给药剂型。在一些实施方式中,所述药物组合物可以是口服的片剂、胶囊剂、丸剂、散剂、缓释制剂、溶液剂和悬浮液,用于胃肠外注射的无菌溶液、悬浮液或乳液,用于外用的软膏或乳膏,或者用于直肠给药的栓剂。在一些实施方式中,所述药物组合物和至少一种治疗剂分别以独立的剂型组合成组合产品,如药剂盒。
本发明采用凝胶柱层析法测定了各多糖成分的分子量,通过乙酰化法和气相-质谱联用鉴定了各多糖成分的单糖组成和比例,结合红外色谱、核磁共振等波谱学方法最终确定了各多糖成分的结构。
本发明分离得到的四种蝉花多糖均具有良好的抗肿瘤的效果,对恶性肿瘤有一定的抑制作用。
附图说明
图1 DEAE-52纤维素离子交换柱层析的洗脱曲线
图2 Sephadex G-150凝胶柱层析洗脱曲线
图3多糖纯度验证
图4单糖对照品和4个多糖组分水解后单糖GC谱(1-L-鼠李糖2-D-阿拉伯糖3-D-木糖4-D-甘露糖5-D-葡萄糖6-D-半乳糖)
图5 B-I-1的红外光谱图
图6 B-I-2的红外光谱图
图7 B-II-1的红外光谱图
图8 B-II-2的红外光谱图
图9 B-I-1 1H NMR谱图
图10 B-I-1 13C NMR谱图
图11 B-I-1 COSY-NMR谱图
图12 B-I-1 HSQC-NMR谱图
图13 B-I-1 HMBC-NMR谱图
图14 B-I-2 1H NMR谱图
图15 B-I-2 13C NMR谱图
图16 B-I-2 COSY-NMR谱图
图17 B-I-2 HSQC-NMR谱图
图18 B-I-2 HMBC-NMR谱图
图19 B-II-1 1H NMR谱图
图20 B-II-1 13C NMR谱图
图21 B-II-1 COSY-NMR谱图
图22 B-II-1 HSQC-NMR谱图
图23 B-II-1 HMBC-NMR谱图
图24 B-II-2 1H NMR谱图
图25 B-II-2 13C NMR谱图
图26 B-II-2 COSY-NMR谱图
图27 B-II-2 HSQC-NMR谱图
图28 B-II-2 HMBC-NMR谱图
图29 B-I-1抑制人胰腺癌细胞Wright-giemsa染色结果
A、对照;B、50μg/mL;C、100μg/mL;D、250μg/mL;E、500μg/mL; F、1mg/mL
图30 B-I-1抑制人胰腺癌细胞透射电镜观察结果
A、给药前;B、给药后
图31 B-I-2抑制人宫颈癌细胞Wright-giemsa染色结果
A、对照;B、50μg/mL;C、100μg/mL;D、250μg/mL;E、500μg/mL; F、1mg/mL
图32 B-I-2抑制人宫颈癌细胞透射电镜观察结果
A、给药前;B、给药后
图33 B-II-2抑制人胶质瘤Wright-giemsa染色结果
A、对照;B、50μg/mL;C、100μg/mL;D、250μg/mL;E、500μg/mL; F、1mg/mL
具体实施方式
实施例1多糖的分离纯化
1、多糖的提取
精密称取干燥的蝉花孢梗束粉末100g,按料液比1∶20加入蒸馏水, 90℃浸提2h。滤过,收集清液,滤渣按上述方法重复浸提1次并滤过,弃渣,合并2次清液,浓缩至提取液的1/4。缓慢地加入95%乙醇至乙醇的终浓度为70%,放入5℃冰箱中静置24h后4000r/min离心15min,收集沉淀;将沉淀物依次用无水乙醇、丙酮、乙醚洗涤。沉淀复溶于蒸馏水中,调节pH 值到7,加入一定量的胰蛋白酶,在65℃水中保温1.5h后加入一定量Sevag 试剂,振荡30min,以4000r/min离心10min除去沉淀,反复操作5次,弃去有机层后将上清液用3倍体积95%乙醇沉淀,沉淀依次用无水乙醇、丙酮、乙醚洗涤,干燥后即得蝉花孢梗束粗多糖。
2、DEAE-52纤维素层析
称取0.3g去蛋白的粗多糖,溶解于10ml蒸馏水中,10000rpm离心 15min,取上清液过DEAE-52离子交换柱(2.6cm×40cm),用0到1mol/mL NaCl溶液连续梯度洗脱,流速1mL/min,用苯酚-硫酸法检测多糖分布,收集含糖峰部分,浓缩,流水中透析过夜,蒸馏水透析48h,冷冻干燥,得蝉花孢梗束的初步纯化多糖组分。
孢梗束粗多糖经DEAE-52纤维素柱层析分离得到2个多糖组分,洗脱曲线图见图1。收集这两个多糖峰,并将前一个多糖组分命名为B-I,后一个多糖组分命名为B-II。
3、Sephadex G-150凝胶色谱层析
将透析后的多糖组分浓缩成10mL,上Sephadex G-150凝胶柱(2.6cm×40 cm),蒸馏水洗脱,流速0.45mL/min,苯酚-硫酸法检测糖分布,收集含糖主峰,冷冻干燥备用。
B-I经过Sephadex G-150凝胶层析柱的再次纯化得到两个组分,命名为B-I-1、B-I-2。B-II经过Sephadex G-150凝胶层析柱分离也得到两个组分,命名为B-II-1、B-II-2。洗脱曲线图见图2。
实施例2多糖组分纯度的验证
取部分冻干多糖纯品,过Sephadex G-150凝胶柱,2.0×20cm,蒸馏水洗脱,流速0.5mL/min,每管收集4mL,每隔1管取1管,用苯酚-硫酸法检测多糖分布。
将通过两次柱层析纯化得到的4个多糖组分(B-I-1、B-I-2、B-II-1、B-II-2) 分别再通过Sephadex G-150凝胶层析柱,对其纯度进行检验,得到4个单一的峰。表明通过柱层析分离纯化,蝉花孢梗束粗多糖分离出4个多糖纯品,见图3。
实施例3多糖的结构鉴定
1、分子量测定
采用凝胶柱层析法测定,将SephadexG-150层析柱,用蒸馏水平衡24h。用已知分子质量的标准葡聚糖Dextran T10、T40、T70、T500和T2000分别过Sephadex G-150层析柱,上柱样量为2.5mL(5mg/mL),用蒸馏水洗脱,流速为1mL/min,流出液按每管3mL体积分布部收集。用苯酚-硫酸法检测多糖分布,分别求得洗脱体积Ve,以T2000的洗脱体积为Vo,以Ve/Vo为纵标准,lgM为横坐标,得到分子质量测定标准曲线。Ve/Vo=-1.5214×lgMw +8.5202,R2=0.9998。分别将多糖样品过SephadexG-150层析柱,按与 Dextran标准品层析相同的条件操作,分别得到其相应洗脱液体积。根据标准曲线计算得各多糖组分的分子量。将B-I-1、B-I-2、B-II-1和B-II-2相应的洗脱体积代入公式,根据标准曲线分别计算得他们的分子分别为 108.70KDa、43.75KDa、114.40KDa和49.46KDa。
2、单糖组成测定
2.1标准单糖样品的乙酰化
精密称取等摩尔(2mol/L)的半乳糖、木糖、鼠李糖、葡萄糖、甘露糖和阿拉伯糖,分别溶于3mL蒸馏水中,加入20-30mg硼氢化钠,于室温下,间歇振荡,还原3h,然后用冰乙酸中和过量的硼氢化钠,至溶液不再产生气泡为止,pH应在4-5之间,加入3mL甲醇,减压浓缩蒸干,重复4-5次,以除去反应副产硼酸及水分,然后置于真空干燥器中过夜。次日,110℃烘箱中加热15min,充分去残留的水分后,加入4mL乙酸酐,100℃反应lh,冷却,然后加入3mL甲苯,减压浓缩蒸干,重复4-5次,以除去多余的乙酸酐。将乙酰化的产物用3mL用氯仿溶解后转移至分液漏斗中,加入少量的蒸馏水充分震荡后,除去上层水溶液,如此重复4次。氯仿层以适量的无水硫酸钠干燥,定容到10mL待GC-MS分析。
2.2多糖样品的乙酰化处理
取多糖样品5mg,加入4mL 2mol/L三氟乙酸(TFA),封管,110℃水解4h,水解液旋转蒸发至干,加入3mL甲醇,蒸干,重复4-5次,以完全除去三氟乙酸,然后按2.1进行衍生化。
2.3色谱条件
配备HP-5MS石英毛细管柱,30m×0.25mm×0.25μm。程序升温:柱初温80℃,保持1min,以5℃/min至200℃,再以2℃/min至215℃,最后以20℃/min至270℃。氦气作载气,进样口温度250℃,分流比1:50,柱流速为1mL/min。EI(70eV),倍增器电压350V,灯丝电流250μA,接口温度200℃,离子源温度250℃,质量数扫描范围40-450amu,扫描速率 2.5scan/sec。单糖对照品和4个多糖水解后单糖的GC谱见图4。
由图4可见,多糖B-I-1主要由阿拉伯糖、木糖和葡萄糖组成,三者的物质的量之比为1∶2.36∶33.14;多糖B-I-2由甘露糖、葡萄糖和半乳糖组成,三者的无质量之比为1.25∶1.28∶1;多糖B-II-1由甘露糖、葡萄糖和半乳糖组成,三者的物质的量之比为1∶16.08∶1.95;多糖B-II-2也是由甘露糖、葡萄糖和半乳糖组成,三者的物质的量之比为2.43∶1∶2.61。
3、红外光谱分析
蝉花多糖B-I-1,B-I-2,B-II-1,B-II-2的红外光谱分别见图5-8。
图5是蝉花孢梗束多糖B-I-1的红外光谱图,由图5可知,B-I-1在 3393cm-1出现一吸收强度大的宽峰,这是糖分子内或分子间氢键O-H伸缩振动引起的吸收,也包括有-NH:中N-H的伸缩振动,提示多糖中含有 -OH、-NH:或-NH基团。在2928cm-1左右的吸收是次甲基(-CH2-)中的C-H 键伸缩振动的吸收峰。1450-1220cm-1处的吸收峰是C-H键的变角振动,它和C-H键伸缩振动构成了糖环的特征吸收。1646cm-1处的吸收峰是羰基的C=O的伸缩振动,或形成了结晶水,1107cm-1,1080cm-1,1024cm-1处的吸收峰是一级醇羟基的变角振动,1151cm-1的吸收为吡喃糖环醚键 (C-O-C)伸缩振动,761cm-1是吡喃糖环醚键(C-O-C)对称环伸缩振动,在 848cm-1有吸收峰是α型葡萄糖端基差向异构的C-H变角振动。761cm-1、848cm-1的吸收是α-D葡聚糖的特征吸收峰。934cm-1的吸收峰可能是呋喃糖环的对称伸缩振动。故B-I-1中既具有吡喃糖又具有呋喃糖。
图6是蝉花孢梗束多糖B-I-2的红外光谱图,由图6可知,B-I-2在3382 cm-1出现一吸收强度大的宽峰,这是糖分子内或分子间氢键O-H伸缩振动引起的吸收,也包括有-NH:中N-H的伸缩振动,提示多糖中含有-OH、-NH: 或-NH基团。在2934cm-1左右的吸收是次甲基(-CH2-)中的C-H键伸缩振动的吸收峰。1450-1220cm-1处的吸收峰是C-H键的变角振动,它和C-H键伸缩振动构成了糖环的特征吸收。1647cm-1处的吸收峰是羰基的C=O的伸缩振动,或形成了结晶水,1112cm-1,1031cm-1处的吸收峰是一级醇羟基的变角振动,在812cm-1有吸收峰可能是呋喃糖端基差向异构的C-H变角振动,可也能可能含有α-D吡喃半乳糖。
图7是蝉花孢梗束多糖B-II-1的红外光谱图,由图7可知,B-II-1在 3393cm-1出现一吸收强度大的宽峰,这是糖分子内或分子间氢键O-H伸缩振动引起的吸收,也包括有-NH:中N-H的伸缩振动,提示多糖中含有 -OH、-NH:或-NH基团。在2927cm-1左右的吸收是次甲基(-CH2-)中的C-H 键伸缩振动的吸收峰。1450-1220cm-1处的吸收峰是C-H键的变角振动,它和C-H键伸缩振动构成了糖环的特征吸收。1647cm-1处的吸收峰是羰基的C=O的伸缩振动,或形成了结晶水,1079cm-1,1027cm-1处的吸收峰是一级醇羟基的变角振动,1151cm-1的吸收为吡喃糖环醚键(C-O-C)伸缩振动,1540cm-1是N-H的变角振动,证明有蛋白质存在,761cm-1是吡喃糖环醚键(C-O-C)对称环伸缩振动,在850cm-1有吸收峰是α型葡萄糖端基差向异构的C-H变角振动。761cm-1、850cm-1的吸收峰是α-D葡聚糖的特征吸收峰。
图8是蝉花孢梗束多糖B-II-2的红外光谱图,由图8可知,B-II-2在3374 cm-1出现一吸收强度大的宽峰,这是糖分子内或分子间氢键O-H伸缩振动引起的吸收,也包括有-NH:中N-H的伸缩振动,提示多糖中含有-OH、-NH: 或-NH基团。在2936cm-1左右的吸收是次甲基(-CH2-)中的C-H键伸缩振动的吸收峰。1450-1220cm-1处的吸收峰是C-H键的变角振动,它和C-H键伸缩振动构成了糖环的特征吸收。1652cm-1处的吸收峰是羰基的C=O的伸缩振动,或形成了结晶水。1054cm-1处的吸收峰是一级醇羟基的变角振动,1541 cm-1是N-H的变角振动,证明有蛋白质存在,在813cm-1有吸收峰可能是呋喃糖端基差向异构的C-H变角振动,也可能含有α-D吡喃半乳糖。
4、核磁共振分析
4.1蝉花多糖B-I-1的核磁共振分析
1H NMR谱图显示(图9),异头碳上氢主要化学位移δ为5.34,5.17, 4.92ppm,而δ为5.34ppm的信号最强,因此B-I-1主要以α型糖苷键构型存在。
13C NMR谱图显示(图10),异头碳区域的的化学位移值小于102ppm,可以判断多糖组分B-I-1的糖残基为吡喃环型,糖苷键的连接方式为α型,这与1H NMR的推测相一致。
COSY、HSQC和HMBC色谱图见图11-13。
B-I-1各图谱的数据分析见表1。
表1 B-I-1各图谱数据分析
综上所述,通过对B-I-1 1H-NMR、13C-NMR谱分析,并利用同核二维相关谱COSY、异核多量子二维相干相关谱HSQC、异核多键二维相关谱 HMBC将B-I-1的H和C进行了全归属,从而可确定其化学分子结构式。综合对该多糖单糖组成、红外光谱以及核磁共振测定及分析结果(表11),确定B-I-1的重复单元结构为:→4)-α-D-Glc(1→4)-α-D-Glc(1→即:
4.2蝉花多糖B-I-2的核磁共振分析
1H NMR谱图显示(图14),异头碳上氢主要化学位移δ为5.34,5.14, 5.10,4.92ppm。因此B-I-2有既有β糖苷键,又有α糖苷键。
13C NMR谱图显示(图15),异头碳区域的的化学位移值一部分小于 102ppm,一部分大于102ppm,可以判断多糖组分B-I-2糖苷键的连接方式有α和β型。
COSY、HSQC和HMBC色谱图见图16-18。
B-I-2各图谱的数据分析见表2。
表2 B-I-2各图谱数据分析
综合所述,通过对B-I-21H-NMR、13C-NMR谱分析,并利用同核二维相关谱COSY、异核多量子二维相干相关谱HSQC、异核多键二维相关谱 HMBC将B-I-2的H和C进行了全归属,从而可确定其化学分子结构式。综合对该多糖单糖组成、红外光谱以及核磁共振测定及分析结果(表2),确定B-I-2的重复单元结构为:
→1)-α-D-Glc(4→1)-α-Glc(3→1)-α-D-Gal(4→1)-α -D-Gal(3→1)-α-D-Glc(4→的主链,支链连接第1和第4单糖,α-D-Glc(3→4)-α-D -Gal(1→3)-β-D-man(1→4)-α-D-Gal(1→3)-α-Glc(1→3)-β-D-man(1→4)-α-D-Ga l即:
4.3蝉花多糖B-II-1的核磁共振分析
1H NMR谱图显示(图19),异头碳上氢主要化学位移δ为5.34,5.16, 5.10,4.92ppm,因此B-II-1主要是α糖苷键。
13C NMR谱图显示(图20),异头碳区域的的化学位移值小于102ppm,可以判断多糖组分B-II-1糖苷键的连接方式主要为α型,这与1H NMR的推测相一致。
COSY、HSQC和HMBC色谱图见图21-23。
B-II-1各图谱的数据分析见表3。
表3 B-II-1各图谱数据分析
通过对B-II-11H-NMR、13C-NMR谱分析,并利用同核二维相关谱 COSY、异核多量子二维相干相关谱HSQC、异核多键二维相关谱HMBC将 B-II-1的H和C进行了全归属,从而可初步确定其化学分子结构式。综合对该多糖单糖组成、红外光谱以及核磁共振测定及分析结果(表3),确定 B-II-1的重复单元结构为:→1)-α-D-Glc(4→1)-α-D-Glc(4→1)-α-D-Glc(4→ 1)-α-D-Glc(4→1)-α-D-Glc(4→1)-α-D-Glc(4→1)-α-D-Glc(4→1)-α-D-Glc(4→的主链,第4个Glc的3号位有取代α-D-Glc(3→4)-α-D-Gal即:
4.4蝉花多糖B-II-2的核磁共振分析
1H NMR谱图显示(图24),异头碳上氢主要化学位移δ为5.34,5.18, 5.15,5.10,5.08ppm。因此B-II-2主要是α糖苷键。
13C NMR谱图显示(图25),异头碳区域的的化学位移值一部分小于 102ppm,一部分大于102ppm,可以判断多糖组分B-II-2糖苷键的既有α型又有β型。
COSY、HSQC和HMBC色谱图见图26-28。
B-II-2各图谱的数据分析见表4。
表4 B-II-2各图谱数据分析
通过对B-II-21H-NMR、13C-NMR谱分析,并利用同核二维相关谱 COSY、异核多量子二维相干相关谱HSQC、异核多键二维相关谱HMBC将 B-II-2的H和C进行了全归属,从而可初步确定其化学分子结构式。综合对该多糖单糖组成、红外光谱以及核磁共振测定及分析结果(表4),确定 B-II-2的重复单元结构为:
→1)-α-D-Gal(4→1)-α-D-Gal(3→1)-α-D-man(3→1)-α-D-man(3→的主链,支链在第2单糖Gal,α-D-Gal(4→1)-α-D-Glc即:
抗肿瘤效果实验
以下实施例4-7中所用实验方法均为本领域常规方法:
所述“MTT”法检测参考文献《苦荞麦槲皮素对人胃癌细胞SGC-7901 增殖及细胞周期的影响》(中国细胞生物学学报,2013,35(5):615–621);
所述“瑞氏吉姆萨染色法”参考文献《吉姆萨染色法对大脑组织凋亡细胞的染色》(解剖学杂志,2005,28(03):362-363);
所述“对细胞周期和对细胞凋亡的影响”参考文献《莪术油对胃癌细胞SGC-7901增殖和凋亡的影响》(齐齐哈尔大学学报,2012, 28(4):33-37)和《苦荞麦槲皮素对人胃癌细胞SGC-7901增殖及细胞周期的影响》(中国细胞生物学学报2013,35(5):615–621)。
实施例4蝉花多糖B-I-1抗肿瘤活性研究
1、四甲基偶氮唑盐比色法(MTT)检测结果
结果见表5。
表5 B-I-1对人胰腺瘤SW1990抑制率
多糖用量μg/mL | 对照 | 50 | 100 | 250 | 500 | 1000 |
人胰腺瘤抑制率% | 0 | 2.87 | 66.29 | 63.77 | 69.18 | 77.05 |
2、瑞氏吉姆萨(Wright-giemsa)染色结果
结果见图29。
上述结果表明:蝉花多糖B-I-1对人胰腺癌SW1990的细胞增殖有一定的抑制作用。
3、蝉花多糖B-I-1对人胰腺癌SW1990细胞周期的影响
结果见表6
表6蝉花多糖B-I-1对人胰腺癌SW1990细胞周期的影响
流式细胞仪(PI单染)检测结果表明:蝉花多糖B-I-1可使人胰腺癌 SW1990细胞周期阻滞在G2期。
4、蝉花多糖B-I-1对人胰腺癌SW1990细胞凋亡的影响
结果见表7
表7蝉花多糖B-I-1对人胰腺癌SW1990细胞凋亡的影响
对照组作用人胰腺癌SW1990细胞48小时后,凋亡率为4.93%、坏死率为0.32%;蝉花多糖B-I-1作用人胰腺癌SW1990细胞48小时后,凋亡率为43.17%、坏死率为7.2%;显著高于对照组。
5、透射电镜观察结果
见图30。
图A为给药前的胰腺癌细胞,细胞核大,核形不规则,有切迹,核质比值大,核仁明显,细胞器丰富。
图B为给予蝉花多糖B-I-1后胰腺癌细胞,胞核变小,核浆比值减小,核仁减小,异染色质增多,沿核膜边集,呈早期凋亡形态。
实施例5蝉花多糖B-I-2抗肿瘤活性研究
1、四甲基偶氮唑盐比色法(MTT)检测结果
结果见表8。
表8 B-I-2对人宫颈癌Hela细胞的抑制率
多糖用量μg/mL | 对照 | 50 | 100 | 250 | 500 | 1000 |
人宫颈癌抑制率% | 0 | 26.26 | 42.10 | 44.68 | 44.29 | 61.78 |
2、瑞氏吉姆萨(Wright-giemsa)染色结果
结果见图31。
上述结果表明:蝉花多糖B-I-2对人宫颈癌Hela细胞的增殖有一定的抑制作用。
3、蝉花多糖B-I-2对对人宫颈癌Hela细胞的细胞周期的影响
结果见表9。
表9蝉花多糖B-I-2对对人宫颈癌Hela细胞的细胞周期的影响
流式细胞仪(PI单染)检测结果表明:蝉花多糖B-I-2可使人宫颈癌 Hela细胞周期阻滞在G2期。
4、蝉花多糖B-I-2对人宫颈癌Hela细胞凋亡的影响
结果见表10
表10蝉花多糖B-I-2对人宫颈癌Hela细胞凋亡的影响
对照组作用人宫颈癌Hela细胞48小时后,凋亡率为1.17%、坏死率为0.18%;蝉花多糖B-I-2作用人宫颈癌Hela细胞48小时后,凋亡率为 86.75、坏死率为3.78%,显著高于对照组。
5、透射电镜观察结果
见图32。
图A为给药前的宫颈癌细胞,核型不整,核膜有切迹,核浆比值大,双核仁,细胞表面有微绒毛。
图B为给予蝉花多糖B-I-2宫颈癌细胞后,核膜旁异染色质边集,胞质内线粒体肿胀,内质网扩张,细胞膜破损,呈坏死性凋亡。
实施例6蝉花多糖B-II-1抗肿瘤活性研究
1、四甲基偶氮唑盐比色法(MTT)检测结果
结果见表11。
表11 B-II-1对人白血病细胞K562抑制率
上述结果表明:蝉花多糖B-II-1对体外培养人白血病K562细胞的增殖有一定的抑制作用。
2、蝉花多糖B-II-1对人人白血病K562细胞周期的影响
结果见表12
表12蝉花多糖B-II-1对人人白血病K562细胞周期的影响
流式细胞仪(PI单染)检测结果表明:蝉花多糖B-II-1对人白血病细胞细胞周期阻滞在G1期。
3、蝉花多糖B-II-1对人白血病细胞K562细胞凋亡的影响
结果见表13
表13蝉花多糖B-II-1对人白血病细胞K562细胞凋亡的影响
对照组作用人白血病K562细胞48小时后,凋亡率为3.36%、坏死率为0.11%;蝉花多糖B-II-1作用人白血病K562细胞48小时后,凋亡率为52.59%、坏死率为11.51%;显著高于对照组。
实施例7蝉花多糖B-II-2抗肿瘤活性研究
1、四甲基偶氮唑盐比色法(MTT)检测结果
结果见表14。
表14 B-II-2对人胶质瘤LN229抑制率
2、瑞氏吉姆萨(Wright-giemsa)染色结果
结果见图33
上述结果表明:蝉花多糖B-II-2对人胶质瘤LN229细胞有一定的抑制作用。
Claims (13)
5.如权利要求1~4任一项所述的蝉花多糖的制备方法,其特征在于,所述制备方法包括如下步骤:
步骤1,蝉花孢梗束以水提醇沉法提取,收集沉淀作为提取物;
步骤2,对步骤1的提取物进行去蛋白处理;
步骤3,将步骤2去除蛋白的提取物用乙醇沉淀,得蝉花粗多糖;
步骤4,将步骤3的蝉花粗多糖依次经过DEAE纤维素柱层析和Sephadex G凝胶柱层析纯化,得蝉花多糖。
6.如权利要求5所述的制备方法,其特征在于,步骤1所述醇沉浓度为60%-80%。
7.如权利要求6所述的制备方法,其特征在于,所述醇沉浓度为65%-75%。
8.如权利要求7所述的制备方法,其特征在于,所述醇沉浓度为70%。
9.如权利要求5所述的制备方法,其特征在于,步骤1得到的沉淀进行纯化,纯化方法为依次用无水乙醇、丙酮、乙醚进行洗涤。
10.如权利要求5所述的制备方法,其特征在于,步骤2所述去蛋白方法为:将步骤1的沉淀溶解,调节pH近中性,分别加入胰蛋白酶和Sevag试剂,离心,去除沉淀。
11.如权利要求10所述的制备方法,其特征在于,所述去蛋白操作重复4~6次。
12.如权利要求11所述的制备方法,其特征在于,所述去蛋白操作重复5次。
13.一种药物组合物,其特征在于,所述组合物包括权利要求1~4任一项所述的蝉花多糖和药学上可接受的载体。
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