CN114805626B - 一种抗癌活性多糖及其制备和在制备抗癌药物中的应用 - Google Patents
一种抗癌活性多糖及其制备和在制备抗癌药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及医药技术领域,具体涉及抗癌药物。
技术背景
癌症被认为是仅次于心血管疾病(CVD)的全球第二大死亡原因。在2020年的统计数据中,约有180万人死于肺癌,这也是所有癌症中比例最高的(18%),其次是结肠癌(9.4%)和肝癌(8.3%)。癌症包括了很多不同的疾病,但是有一组共同的特点,即癌细胞不受控制的增殖、异常分化和侵袭迁移能力,这些都是恶性肿瘤最基本的生物学特性。目前各种癌症的常规治疗方法主要是外科手术、化学药物治疗、免疫治疗、内分泌法治疗、中医中药治疗、热源治疗、放射治疗等。然而目前最常见的疗法如化疗,在治疗过程中不可避免地会损害患者的健康,例如患者会出现骨髓抑制、刺激胃肠道粘膜而诱发一系列胃肠道症状、脱发、肝肾功能损害、心脏毒性以及神经毒性等。同时由于治疗费用昂贵,给患者及其家属带来很大的经济负担。因此急需发现一些特异性强、生物相容性好以及来源广泛的活性物质用于抗癌药物的研究。
发明内容
本发明第一目的在于,提供一种全新结构的抗癌活性多糖,旨在提供一种具备优异抗癌活性并对正常细胞具有良好促增殖的抗癌活性多糖。
本发明第二目的在于,提供所述的抗癌活性多糖的制备方法。
本发明第三目的在于,提供所述的抗癌活性多糖在制备抗癌药物中的应用。
本发明第四目的在于,提供包含所述抗癌活性多糖的抗癌药物。
一种抗癌活性多糖,为具有式1所示的结构的多糖:
本发明提供了一种全新式1结构的多糖,且研究表明,所述新结构的多糖具有良好的癌细胞和正常组织细胞的药理选择性,能够选择性地抑制癌细胞,且对正常细胞具有低毒性甚至具有一定的促增殖修复效果。
本发明中,所述的抗癌活性多糖,通过岩藻糖、半乳糖、葡萄糖、甘露糖以及葡萄糖醛酸单糖之间的糖苷键形成多糖。优选地,所述的抗癌活性多糖的分子量为13800~13900Da。
本发明还提供了所述的抗癌活性多糖的制备方法,步骤包括:
步骤(1):将云芝子实体粉末经脱脂、水提后浓缩,随后经醇沉、脱蛋白、脱盐处理,得到粗多糖;
步骤(2):将粗多糖预先经纤维素柱洗脱处理,洗脱过程包括依次进行的第一段洗脱和第二段洗脱;收集第二段洗脱液;其中,第一段洗脱的洗脱剂为水,第二段洗脱的洗脱剂为0.3~0.4mol/L的盐溶液;
将第二段的洗脱液经脱盐、浓缩处理后再经葡聚糖凝胶柱纯化处理即得所述的抗癌活性多糖。
本发明中,通过所述的制备方法,能够获得所述的具有优异癌细胞和正常细胞选择性,具有优异抗癌活性且对正常细胞有促增殖作用的全新结构的多糖。
本发明中,脱脂过程中,所采用的溶剂为75%~85%的乙醇水溶液;脱脂过程的温度为回流,优选为70℃~85℃。脱脂阶段,原料和溶剂的重量比例例如为1:15~25。
本发明中,脱脂处理后进行固液分离,获得的脱脂物进行后续的水提取;
优选地,水提取过程的水和原料的重量比为10~30:1,进一步可以为15~20:1。
优选地,水提取过程的温度为80℃~85℃;
优选地,水提取处理后进行固液分离,获得水提取液;
优选地,水提液浓缩后添加醇溶剂,进行醇沉处理;优选地,所述的浓缩比例为10~20%;
优选地,所述的醇溶剂为乙醇;
优选地,醇沉过程中,添加的醇为浓缩液的体积的3~5倍。
醇沉阶段的温度小于或等于20℃,进一步可以为2~10℃。
本发明中,可以采用常规的Sevage法进行脱蛋白处理。例如,将醇沉产物用水溶解,并采用中性蛋白酶和Sevage试剂进行脱蛋白处理,随后离心得到上清液,再进行透析、干燥处理,获得所述的粗多糖。
透析阶段使用的透析袋的截留分子量例如为3000~4000Da。
本发明中,所述的纤维素柱采用DEAE-52纤维素离子交换柱;
优选地,所述的盐溶液中的溶质为氯化钠、氯化钾中的至少一种;
优选地,第二段洗脱的盐溶液的浓度为0.34~0.36mol/L。
优选地,所述的葡聚糖凝胶柱为G-100葡聚糖凝胶柱;
优选地,葡聚糖凝胶柱纯化阶段的洗脱剂为水。
本发明所述的水均优选指去离子水。
本发明中,所述的透析、浓缩等工艺均可以采用现有的手段实现。
本发明还提供了一种所述的抗癌活性多糖在制备抗癌药物中的应用。
优选的应用,所述的抗癌药物为抗结肠癌、肺癌、肝癌中的至少一种癌症药物。本发明研究发现,所述的全新的多糖对结肠癌、肺癌、肝癌癌细胞以及对应的正常细胞具有更优的选择性,可以获得更优的抗癌活性,不仅如此,还能够获得更优的正常细胞促增殖修复效果。
作为优选,所述的抗癌药物为抗肺癌的药物。本发明研究发现,所述的全新的式1多糖,对肺癌细胞具有更优的抑制作用,且对正常细胞没有明显的毒副作用。
本发明还提供了一种抗癌药物,包含药学有效量的抗癌活性多糖。
本发明中,可以采用现有的制药辅料、手段以及原理,将本发明所述的活性成分制备成需要的制剂类型。
例如,本发明所述的抗癌药物,还包含其他抗癌活性成分;
优选地,还包含其他药学上可接受的辅料;
优选地,具有药学上可接受的药学有效的任意剂型。
优选地,所述的抗癌药物为抗肺癌药物。
技术效果:
本发明提供了一种全新结构的多糖,且所述新结构的多糖具有良好的癌细胞和正常组织细胞的药理选择性,能够选择性地抑制癌细胞,且对正常细胞具有一定的促增殖修复效果。
附图说明
图1为实施例一制备的粗多糖纤维素柱洗脱曲线图;
图2为实施例一制备的粗多糖葡聚糖柱洗脱曲线图;
图3为实施例一制备的粗多糖孵育后HCT116、HepG2及A549细胞存活率图;
图4为实施例一制备的粗多糖孵育后NCM460、L02及16HBE细胞存活率图
图5为实施例一制备的YZP-1a(式1多糖)孵育后HCT116、HepG2及A549细胞存活率图;
图6为实施例一制备的YZP-1a孵育后NCM460、L02及16HBE细胞存活率图;
图7为实施例一制备的YZP-1a孵育后,经AO/EB染色HCT116细胞和NCM460细胞的荧光图(高内涵拍摄,40x);
图8为实施例一制备的YZP-1a孵育后,经AO/EB染色的HepG2细胞和L02细胞荧光图(高内涵拍摄,40x);
图9为实施例一制备的YZP-1a孵育后,经AO/EB染色的A549细胞和16HBE细胞荧光图(高内涵拍摄,40x);
图10为实施例一制备的YZP-1a孵育后HCT116、HepG2、A549及L02细胞划痕图;
图11为实施例一制备的YZP-1a的红外光谱图;
图12为实施例一制备的YZP-1a甲基化后GC-MS色谱图;
图13为实施例一制备的YZP-1a一维核磁共振图(a)YZP-1a的1H核磁谱图(b)YZP-1a的13C核磁谱图(c)YZP-1a的DEPT135核磁谱图;
图14为实施例一制备的YZP-1a的HSQC核磁谱图;
图15为实施例一制备的YZP-1a的COSY核磁谱图;
图16为实施例一制备的YZP-1a的HMBC核磁谱图;
图17为实施例一制备的YZP-1a的NOESY核磁谱图;
具体实施方式
以下实施例旨在说明本发明而不是对本发明的进一步限定。
实施例一
(一)粗多糖的制备
1.1取烘干的云芝子实体(湖南中药谷集团研究院有限公司提供)20g于粉碎机得到原料粉末。将粉末置入圆底烧瓶内,加入75%乙醇-水溶液400mL,70℃下将圆底烧瓶于油浴锅内搅拌、冷凝回流2h。反应后,抽滤,收集滤饼于真空干燥箱内烘干。
1.2取滤饼15g于圆底烧瓶,加入300mL超纯水,80℃下将圆底烧瓶于油浴锅内搅拌2h。反应后,抽滤,收集滤液,留滤渣重复提取两次,合并滤液。
1.3取步骤1.2滤液旋蒸浓缩液体至原体积的1/10,将浓缩液缓慢倒入4倍体积的无水乙醇,置于4℃下醇沉过夜。
1.4离心步骤1.3液体,取沉淀加100mL超纯水于圆底烧瓶复溶,加入25mL Sevage试剂,剧烈搅拌40min后,离心取最上层清液,重复3-4次,将清液放入3500Da透析袋内透析48h,旋蒸浓缩透析袋内液体冻干得到粗多糖。
(二)层析柱洗脱
2.1装填DEAE-52纤维素离子交换柱,将500mg粗多糖(步骤(一)制得)溶于20mL超纯水中,湿法上样。依次用超纯水、0.35、0.70、1.0mol/L的氯化钠溶液梯度洗脱。洗脱液采用苯酚硫酸法进行监测,在紫外分光光度计490nm下隔管检测多糖含量,绘制洗脱曲线图(洗脱曲线如图1所示),收集主要组分,旋蒸浓缩后调pH至7-9室温透析48h除盐,真空冷冻干燥。其中,0.35mol/L氯化钠溶液的洗脱峰为组分YZP-1。
2.2装填Sephadex G-100葡聚糖凝胶色谱柱,将100mg YZP-1溶于5mL超纯水中湿法上样,之后用超纯水洗脱,洗脱液采用苯酚硫酸法进行监测,在紫外分光光度计490nm下隔管检测多糖含量,绘制洗脱曲线(洗脱曲线如图2所示),收集主要组分,旋蒸浓缩后真空冷冻干燥得到YZP-1a(即本发明中的式1多糖)。
采用实施例一获得的YZP-1a进行以下药效研究和结构分析,具体为:
实施例二
YZP-1a以及粗多糖(实施例一步骤(一)制备)对人结肠癌细胞、人肝癌细胞、人非小细胞肺癌细胞、人结肠上皮细胞、人正常肝组织细胞及人支气管上皮细胞的药效试验,包括对细胞活力、细胞凋亡和细胞迁移的影响,具体步骤如下:
使用细胞计数试剂盒(CCK-8)评估粗多糖与YZP-1a对人结肠癌细胞HCT116、人肝癌细胞HepG2、人非小细胞肺癌细胞A549、人结肠上皮细胞NCM460、人正常肝组织细胞L02及人支气管上皮细胞16HBE细胞活力的影响。将上述细胞分别接种到5x103细胞/孔的96孔板中,并在完全培养基中培养24小时。每个孔用PBS轻轻清洗两次,然后将100μL新鲜培养基添加到每个孔中,其中包含不同浓度的YZP-1a(0、100、200、300、400、500μg/mL)。温育24小时后,以无细胞的完全培养基的孔作为阴性对照,将CCK-8(10μL)添加至每个孔。并再温育1小时。使用酶标仪测定每个孔在450nm处溶液的吸光度OD,计算细胞存活率。
细胞存活率=(OD实验组-OD阴性对照组)/(OD空白组-OD阴性对照组),
其中OD实验组、OD空白组组和OD阴性对照组对分别代表加药实验组的平均吸光度、未加药实验组的平均吸光度和阴性对照组的平均吸光度。结果详见图3至图6;
由结果可以看出,经YZP-1a孵育24h后,在给药浓度500μg/mL时,粗多糖对HCT116细胞、HepG2细胞以及A549细胞的存活率分别是62.03%、62.50%和58.62%,YZP-1a对HCT116细胞、HepG2细胞以及A549细胞的存活率分别是56.35%、53.62%和45.39%。粗多糖与YZP-1a对三种癌细胞均有一定的抑制增殖作用,但YZP-1a相较于粗多糖对癌细胞的抗增殖作用有明显提升。与此同时,YZP-1a孵育24h后,在给药浓度500μg/mL时,粗多糖对NCM460细胞、L02细胞和16HBE细胞的存活率分别为104.64%、96.55%和92.94%,YZP-1a对NCM460细胞、L02细胞和16HBE细胞的存活率分别为105.93%、112.02%和108.48%,这表明粗多糖与YZP-1a对正常细胞均没有明显的细胞毒性,且YZP-1a体现出促进正常细胞增殖的活性。
使用吖啶橙/溴化乙锭(AO/EB)荧光染色法检测YZP-1a对HCT116细胞、HepG2细胞、A549细胞、NCM460细胞、L02细胞以及16HBE细胞在细胞凋亡方面的影响。AO/EB染色试剂盒用于分析细胞存活状态,并区分正常细胞与凋亡细胞和坏死细胞。具体步骤为,将细胞以5×103个细胞/孔接种到96孔板中,并培养24小时。用PBS轻轻清洗两次后,将包含不同浓度的粗多糖与YZP-1a(0、250、500μg/mL)的新鲜培养基(100μL)添加到每个孔中。将板孵育24小时后,使用配有试剂盒的1×缓冲液(90μL)替换每个孔中的培养基。之后,向每个孔中添加10μLAO/EB(1:1),然后在黑暗中于37℃细胞培养箱孵育放置5分钟,之后,吸取染料,用PBS轻轻清洗一两次后,每孔加入100μLPBS。染色细胞的荧光图像通过Operetta高内涵成像***拍摄,详见图7、图8及图9。
由结果可以看出,随着YZP-1a的给药浓度增加,HCT116细胞、HepG2细胞、A549细胞EB通道的荧光颜色明显逐渐增强,说明YZP-1a对上述癌细胞有促进凋亡的效果。与此同时,NCM460细胞、L02细胞和16HBE细胞AO/EB通道的荧光颜色无明显变化,说明YZP-1a对正常细胞没有明显的造成细胞损伤。
肿瘤细胞在体外仍具有迁移的能力,本发明借鉴体外细胞致伤愈合实验模型,利用细胞划痕法测定了肿瘤细胞的运动特性。具体步骤为,将细胞密度为5~10×105个/mL的HCT116细胞、HepG2细胞、A549细胞和L02细胞铺于6孔板(每孔1000μL),加入相应细胞的完全培养液,培养16~24h,使形成单层细胞。用200μL移液枪枪头在单层细胞上呈“一”字划痕,用PBS清洗3次,加入含不同浓度的YZP-1a(0、250、500μg/mL)的新鲜培养基,孵育0、12、24、48h,并于倒置荧光显微镜下观察和成像,详见图10。
由结果可以看出,随着YZP-1a的给药浓度增加,HCT116细胞、HepG2细胞、A549细胞在划痕后,恢复的能力逐渐减弱,说明YZP-1a在一定程度上可以抑制上述细胞的迁移能力。与此同时,随着YZP-1a的给药浓度增加,L02细胞在划痕后,恢复的能力无明显变化,说明YZP-1a对正常细胞的迁移能力基本没有影响。
实施例三
对实施例一获得的YZP-1a进行结构表征,包括分子量测定、单糖组成分析、红外光谱分析、甲基化分析及核磁共振分析。
(一)分子量测定
称取2mgYZP-1a多糖样品,充分溶解于1mL超纯水中制成样品溶液。采用T系列标准葡聚糖制作标准曲线。色谱柱采用TSK-GEL G4000PW(7.8mm×300mm);上样量为20μL;流动相为超纯水;流速为0.8mL/min,测得详见表1。
表1 YZP-1a分子量
(二)单糖组成分析
使用离子测谱仪测定单糖组成,分析结果详见表2,测试步骤如下:
4.2.1标准溶液的配制
取16种单糖标准品(岩藻糖、鼠李糖、***糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸、氨基半乳糖盐酸盐、盐酸氨基葡萄糖、N-乙酰-D氨基葡萄糖、古罗糖醛酸、甘露糖醛酸)配成约10mg/ml标准溶液。
4.2.2样品准备
精密称量10mg样品置于安瓿瓶中,加入3M TFA 10ml,120℃水解3h。准确吸取酸水解溶液转移至管中氮吹吹干,加入5ml水涡旋混匀,吸取100μL加入900μL去离子水,12000rpm离心5min。取上清进IC分析。
4.2.3色谱方法
色谱柱:DionexCarbopacTMPA20(3*150);流动相:A:H2O;B:15mM NaOH C:15mMNaOH以及100mM NaOAC;流速:0.3ml/min;进样量:5μL;柱温:30℃;检测器:电化学检测器。
表2 YZP-1a单糖组成
(三)红外光谱分析
红外光谱法在多糖结构分析中应用广泛,可用于初步判别多糖的一些特征官能团
充分干燥的YZP-1a(1mg)和KBr粉末(50mg)混合压片。用傅立叶变换红外光谱仪,在400~4000cm-1扫描范围,对其进行光谱扫描。
YZP-1a的红外光谱图如图11所示,出现在3423cm-1附近的吸收带属于-OH的伸缩振动,出现在2930cm-1附近的吸收带属于C-H伸缩振动。在1635cm-1附近范围内的吸收带和在1404cm-1附近范围内的吸收带可以归属于羰基C=O或C=C的伸缩振动。这些红外吸收都是多糖基本的特殊吸收带。而出现在800-1200cm-1附近范围内的吸收带对每种多糖来说各不相同,呈现出特异性。1151cm-1、1078cm-1和1045cm-1附近的吸收带表明云芝多糖结构内存在吡喃糖单糖环。此外,在912cm-1和827cm-1范围内的吸收带可以分别归属于β-糖苷键和α-糖苷键的吸收。
(四)甲基化分析与核磁共振分析
将多糖甲基化、水解、乙酰化后,经GC-MS测定并与标准质谱图库进行比对。
称量YZP-1a样品10mg置于玻璃反应瓶中,加入1mL无水DMSO,快速加入甲基化试剂A液(无水碱溶液),封闭,在超声作用下溶解,再加入甲基化试剂B液(碘甲烷溶液)。在磁力搅拌水浴30℃反应60min反应。最后将2mL超纯水加入到上述混合物中终止甲基化反应。
取甲基化后的多糖,加入1ml的2M三氟乙酸(TFA)水解90min,旋转蒸发仪蒸干。残基加入2ml双蒸水,60mg硼氢化钠还原8小时,加入冰醋酸中和,旋蒸,101℃烘箱烘干,然后加入1ml乙酸酐乙酰化100℃反应1h,冷却。然后加入3mL甲苯,减压浓缩蒸干,重复4-5次,以除去多余的醋酐。
将乙酰化后的产物用3mLCH2Cl2溶解后转移至分液漏斗,加入少量蒸馏水充分震荡后,除去上层水溶液,如此重复4次。CH2Cl2层以适量的无水硫酸钠干燥,定容10mL,放入液相小瓶。分析采用Shimadzu GCMS-QP 2010气相色谱-质谱联用仪测定乙酰化产物样品。
GC-MS条件:RXI-5SIL MS色谱柱30m*0.25mm*0.25um;程序升温条件为:起始温度120℃,以3℃/min升温至250℃/min;保持5min;进样口温度为250℃,检测器温度为250℃/min,载气为氦气,流速为1mL/min。
多糖甲基化后经GC-MS色谱分析,色谱图见图12,详细的糖苷键类型以及比例见表3所示。主要的糖苷键类型包括Glcp-(1→,→3)-Galp-(1→,→4)-Galp-(1→,→6)-Glcp-(1→,→4,6)-Glcp-(1→,→3,6)-Galp-(1→。除此之外一些较低含量的糖苷键类型包括Fucp-(1→,→3)-Fucp-(1→,Manp-(1→,→6)-Galp-(1→,→2,6)-Glcp-(1→。由甲基化分析结果可知,YZP-1a的基本骨架主要由葡萄糖和半乳糖的糖苷键连接所构成,与单糖组成实验结果相对应。
表3 YZP-1a的PMAAs结果
(五)核磁共振分析
将YZP-1a(60mg)溶解在D2O中,YZP-1a的1HNMR、13C NMR、DEPT135、HSQC、COSY、HMBC和NOESY谱图由Bruker光谱仪(600MHz,Rheinstetten Germany)测量,温度为25℃,详见图13-17。
1H NMR谱图(图13a)在4.4-5.9ppm为异头氢区域,其他区域谱峰重叠不易辨认,需要从COSY谱图(图15)和NOESY谱图(图17)补充其他位置H的化学位移。13C NMR谱图(图13b)在90-112ppm为异头碳区域,其他区域谱峰重叠,需要将H的化学位移结合HSQC谱图(图14)获得C2-C6的化学位移,其中C6的信息可对照DEPT135谱图(图13c)中的倒峰和HSQC谱图分析。再根据各个残基C/H的化学位移结合HMBC谱图(图16)找到各残基之间的连接信息,NOESY谱图可作为补充。详细数据见表4和表5。
表4 YZP-1a各残基的1H和13C NMR信号(ppm)
表5 YZP-1a中各糖残基之间的H1/C1相关联信息
通过所述的结构鉴定,可以确定所述的YZP-1a为具有式1重复结构的新型多糖。
Claims (11)
1.一种抗癌活性多糖,其特征在于,所述的抗癌活性多糖的制备步骤包括:
步骤(1):采用75%~85%的乙醇水溶液在70 ℃~85 ℃的温度下对云芝子实体粉末进行脱脂处理,随后固液分离获得脱脂物;将获得的脱脂物进行水提处理,得到水提液;将水提液浓缩得水提缩液,随后添加醇溶剂,进行醇沉处理得到醇沉产物;将醇沉产物用水溶解,并采用中性蛋白酶和Sevage试剂进行脱蛋白处理,随后离心得到上清液,再进行透析、干燥处理,获得粗多糖;
水提取过程的水和原料的重量比为10~30:1;水提取过程的温度为80 ℃~85 ℃;
醇沉过程中,添加的醇为浓缩液体积的3~5倍;
步骤(2):将粗多糖预先经纤维素柱洗脱处理,洗脱过程包括依次进行的第一段洗脱和第二段洗脱;收集第二段洗脱液;其中,第一段洗脱的洗脱剂为水,第二段洗脱的洗脱剂为0.35 mol/L的氯化钠溶液;
将第二段的洗脱液经脱盐、浓缩处理后再采用葡聚糖凝胶柱并以水为洗脱剂进行纯化处理,得所述的抗癌活性多糖;
所述的纤维素柱采用DEAE-52纤维素离子交换柱;
所述的葡聚糖凝胶柱为G-100葡聚糖凝胶柱。
2.如权利要求1所述的抗癌活性多糖,其特征在于,通过岩藻糖、半乳糖、葡萄糖、甘露糖以及葡萄糖醛酸单糖之间的糖苷键形成多糖。
3.如权利要求1所述的抗癌活性多糖,其特征在于,所述的抗癌活性多糖的分子量为13800~13900 Da。
4.如权利要求1所述的抗癌活性多糖,其特征在于,水提取处理后进行固液分离,获得水提取液;水提液浓缩后添加醇溶剂,进行醇沉处理;其中,所述的浓缩比例为10~20%。
5.如权利要求1所述的抗癌活性多糖,其特征在于,所述的醇溶剂为乙醇。
6.一种权利要求1~5任一项所述的抗癌活性多糖在制备抗癌药物中的应用。
7.如权利要求6所述的应用,其特征在于,所述的抗癌药物为抗结肠癌、肺癌、肝癌中的至少一种癌症药物。
8.一种抗癌药物,其特征在于,包含药学有效量的权利要求1~5任一项所述的抗癌活性多糖。
9.如权利要求8所述的抗癌药物,其特征在于,还包含其他抗癌活性成分。
10.如权利要求9所述的抗癌药物,其特征在于,还包含其他药学上可接受的辅料。
11.如权利要求9所述的抗癌药物,其特征在于,具有药学上可接受的药学有效的任意剂型。
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