CN113912664A - 土贝母苷甲的制备方法及应用 - Google Patents
土贝母苷甲的制备方法及应用 Download PDFInfo
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- CN113912664A CN113912664A CN202111337266.1A CN202111337266A CN113912664A CN 113912664 A CN113912664 A CN 113912664A CN 202111337266 A CN202111337266 A CN 202111337266A CN 113912664 A CN113912664 A CN 113912664A
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- tubeimoside
- ethanol
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Abstract
本发明公开了一种土贝母苷甲的制备方法及应用,所述应用具体指包含该皂苷的疫苗,以及该疫苗在用于预防或治疗传染性疾病中的应用。该方法制备土贝母苷甲,是以土贝母为原料,以乙醇提取,提取液回收乙醇,浓缩,上大孔吸附树脂柱去杂,再经柱色谱或HPLC色谱分离,得到土贝母苷甲。制备方法工艺简单、操作简便,产品收率和纯度高,避免使用氯仿、乙腈、甲醇等第一、二类有机溶剂,无有害溶剂残留,安全性好、生产成本低。制备的产品可以作为疫苗的免疫佐剂,发挥理想的免疫效果。
Description
技术领域
本发明涉及一种土贝母苷甲的制备方法及应用。
背景技术
土贝母是葫芦科植物土贝母Bolbostemma paniculatum(Maxim.)Franquet)的干燥块茎,是一味传统中药,最早记载于清代《本草纲目拾遗》。土贝母性味苦,微寒,归肺、脾经,具有解毒,散结,消肿之功效,主治用于乳痈,瘪痂,痰核。土贝母含皂苷类、黄酮类、蒽醌类、甾体类以及其他杂环类化合物。其中土贝母苷甲(Tubeimoside I)是土贝母的主要活性成分之一,其分子式为C63H98O29,分子量为1319.43。
现代药理研究表明:土贝母苷甲可通过多个分子靶点和不同信号通路,抑制肿瘤细胞生长和增殖,诱导肿瘤细胞分化、凋亡及周期阻滞,调节免疫应答,以及抑制血管生成、黏附、侵袭和转移,对一系列人肿瘤如胃癌、肝癌、宫颈癌、肺癌、卵巢癌、胶质瘤、食道癌、非小细胞肺癌、乳癌、结直肠癌、前列腺癌、红白血病和胶质母细胞瘤具有良好的治疗作用。同时,申请人研究发现:土贝母苷甲对猪口蹄疫灭活疫苗、猪瘟活疫苗等商品兽用疫苗具有显著的佐剂作用。
土贝母药材中土贝母苷甲含量较高。魏有良等研究结果表明:土贝母中土贝母苷甲含量在1.4%~1.9%[魏有良,杨小梅,徐长根,高海,孙文基.HPLC法测定土贝母中土贝母苷甲含量[J].西北药学杂志,2000;3:107-108]。黄瑾等报道不同产地土贝母中土贝母苷甲含量在1.4%~1.5%[黄瑾,张杰,顾正兵.高效液相色谱法同时测定土贝母中土贝母苷甲、乙和丙的含量[J].中国药业,2016,25(01):58-60.5]。2020版《中国药典》规定土贝母按干燥品计算,含土贝母昔甲(C63H98O29)不得少于1.0%[国家药典委员会.中华人民共和国药典,2020年版(一部),北京:中国医药科技出版社,2020:17]。因此,制备高纯度的土贝母苷甲单体化合物,作为抗肿瘤药物和疫苗佐剂开发具有良好的前景。
目前,土贝母苷甲的分离制备工艺已有发明专利申请[CN201110029127.2和CN201110026200.0]。申请的发明专利1(CN201110029127.2)涉及使用氯仿、二氯甲烷、乙腈、甲醇等第一、二类有机溶剂,易导致有机溶剂残留,对人体和动物有危害;同时,需经聚酰胺、Sephadex LH-20色谱和氧化铝等多种柱色谱分离纯化,步骤繁琐。申请的发明专利2(CN201110026200.0)同样需用二氯甲烷、乙腈、甲醇等第一、二类有机溶剂,存在溶剂残留的风险,且该发明需用硅藻土、活性炭等脱色,步骤繁琐,吸附损失严重,增加了生产成本。本发明涉及一种避免使用氯仿、乙腈、甲醇等危害较大的第一、二类有机溶剂,只经大孔吸附树脂去杂和反相硅胶分离,适合工业化生产纯度高、安全性好的土贝母苷甲的制备方法。同时,本发明所涉及方法制备的土贝母苷甲显示出比现有技术中已知的铝胶佐剂不仅能诱导机体对口蹄疫灭活疫苗和猪瘟活疫苗等商品兽用疫苗产生更强体液免疫反应,而且诱生机体产生细胞免疫反应以及Th1型和Th2型免疫应答,可以作为多种兽用疫苗的免疫佐剂,发挥理想的免疫效果。
发明内容
本发明的第一个目的是提供一种避免使用第一、二类有机溶剂、适用于工业化生产高纯度土贝母苷甲的制备方法。
本发明提供的土贝母苷甲是从中药土贝母中分离制备的,制备方法包括以下步骤:
a.土贝母药材粉碎,以乙醇提取。
b.提取液减压回收乙醇、浓缩,得到醇提取物。
c.醇提取物上大孔吸附树脂柱,以水和乙醇混合液进行梯度洗脱。
d.硅胶薄层色谱检查,具有相同斑点的洗脱液合并,浓缩,干燥,得土贝母苷甲粗品。
e.土贝母苷甲粗品经柱色谱或HPLC,以洗脱剂洗脱,得到土贝母苷甲。
上述步骤a采用加热回流、超声提取、微波提取、渗漉中的一种,提取1-3次。
上述所述步骤b中醇提取物的密度为1.05-1.35g/mL。
上述步骤c采用的大孔吸附树脂为D101、ADS21、DS401或ZTC-1大孔吸附树脂;洗脱剂为乙醇和水的混合物。
上述述步骤e柱色谱和HPLC所用的填料为Rp-18和Rp-8;洗脱剂为乙醇和水的混合物,乙醇浓度为5%-90%;流速为1-100mL/min。
本发明的第二个目的是提供土贝母苷甲对疫苗的佐剂作用,尤其对猪口蹄疫灭活疫苗、猪瘟活疫苗等商品疫苗具有显著的佐剂作用。
本发明的第三个目的是提供土贝母苷甲在制备疫苗中的应用。
疫苗中土贝母苷甲的剂量可以根据抗原种类、所需的抗体水平、接种对象的特异性及所需的免疫程序等各种因素进行确定。本发明的兽用疫苗中土贝母苷甲的量可为0.01μg~1g/单剂量,优选为0.1~100μg/单剂量。可以一次或多次施用。
本发明的优点
与现有技术已知的土贝母苷甲制备方法相比,本发明涉及的土贝母苷甲制备方法,工艺简单,操作简便,生产成本低,适于工业化生产;避免使用第一、二类有机溶剂,对环境和机体无危害,产品无有害溶剂残留,安全性好。因此,本发明所涉及的土贝母苷甲制备方法是一种简便、经济、适合大规模生产的制备工艺。
同时,本发明所制备的土贝母苷甲显示出比现有技术中已知的铝胶佐剂不仅能诱导机体对口蹄疫灭活疫苗和猪瘟活疫苗等商品疫苗产生更强的体液免疫反应,而且可以诱生机体的细胞免疫反应以及同时产生Th1型和Th2型免疫应答,可以作为兽用疫苗的免疫佐剂,发挥理想的免疫效果。
附图说明
图1为从土贝母中分离纯化得到的土贝母苷甲的HPLC图。
图2为土贝母苷甲的HR-ESI-MS图。
图3为土贝母苷甲的1H-NMR图。
图4为土贝母苷甲的13C-NMR)图。
图5为土贝母苷甲对猪口蹄疫疫苗免疫小鼠血清中特异性抗体效价的影响。FMDV146S:口蹄疫病毒粒子(FMDV 146S);Alum:铝胶;TBM I:土贝母苷甲;aP<0.05,bP<0.01和cP<0.001vs FMDV 146S对照组。
图6为土贝母苷甲对猪瘟活疫苗免疫小鼠刺激原和抗原刺激脾细胞增殖反应的影响。CSFV:猪瘟活疫苗(脾源);CSFV E2:猪瘟病毒E2蛋白;Quil A:皂苷佐剂阳性药物;TBMI:土贝母苷甲;bP<0.01和cP<0.001vs CSFV对照组。
具体实施方式
以下通过实例进一步说明本发明,而非限制其范围。
以下百分比浓度均指质量百分比浓度。
实施例1:土贝母苷甲制备
药材土贝母粗粉1kg,以70%乙醇回流提取3次,每次1.5小时,提取液滤过,合并滤液,减压回收乙醇,浓缩得醇提取物。醇提取物经D101大孔吸附树脂柱,用水和乙醇混合液进行梯度洗脱,用薄层色谱检查洗脱液,合并具有相同斑点的流份,浓缩至干,得到土贝母苷甲粗品。称取土贝母苷甲粗品2.0g,加50%乙醇5mL溶解,0.22μm微孔滤膜过滤,滤液注入制备型高效液相色谱仪,流动相为无水乙醇-水(43:57),流速为8mL/min,收集土贝母苷甲对应色谱峰流出液,减压回收溶剂至干,得透明状土贝母苷甲晶1.02g。
实施例2:土贝母苷甲纯度检测
采用高效液相色谱法(HPLC)对制备所得土贝母苷甲的纯度进行检测。色谱条件:色谱柱,Dikmond C18(4.6mm×250mm,粒径5μm);流动相为乙腈-水,乙腈比例变化如下:0-15min,30%→35%,15-30min,35%→50%,30-35min,50%→30%;柱温:35℃;流速,1.0mL/min;DAD检测器;进样量,10μL。采用面积归一法计算,上述制备所得土贝母苷甲的纯度为98.3%(图1)。
实施例3:土贝母苷甲的高分辨质谱分析
采用OrbitrapElite质谱仪(Thermo Scientific,Bremen,Germany)检测制备得到的土贝母苷甲的分子量。土贝母苷甲的高分辨电喷雾质谱(HR-ESI-MS)谱图见图2。HR-ESI-MS的准分子离子峰m/z 1317.6167[M-H]-(计算值:1318.6174),分子式:C63H98O29。
实施例4:土贝母苷甲的核磁共振分析
采用Angilent DD2-600型核磁共振仪(TMS作内标,DMSO-D6作溶剂),检测制备得到的土贝母苷甲的氢谱(1H-NMR)和碳谱(13C-NMR)。土贝母苷甲的核磁共振氢谱(1H-NMR)和碳谱(13C NMR)见图3、4。芳香区δ5.37ppm(1H,J=6.1Hz)为H-24双键上的特征信号。此外,δ5.17ppm(1H,d,J=6.4Hz)为葡萄糖基的端基氢信号。13C-NMR数据提示结构中含有5个糖残基和3个酯基,其中δC 95.21和δC178.01表明苷元C28羧基上连有阿拉伯糖糖残基。1H-NMR信号:δH1.98(3H,s),3.04和3.47(各1H,ABq,J=15.8Hz),3.06和3.30(各1H,ABq,J=15.6Hz);13C-NMR信号:δC 173.05、173.16、69.89、50.23、49.91和26.58ppm。这些NMR数据表明:化合物结构中存在3-甲基-3羟基戊二酸残基结构。13C NMR数据(DMSO-D6):44.0(C-1)、69.2(C-2)、85.0(C-3)、43.6(C-4)、48.2(C-5)、20.0(C-6)、34.9(C-7)、41.2(C-8)、48.6(C-9)、39.3(C-10)、20.5(C-11)、125.3(C-12)、146.5(C-13)、42.1(C-14)、28.2(C-15)、20.2(C-16)、49.0(C-17)、42.0(C-18)、46.5(C-19)、30.8(C-20)、34.4(C-21)、32.6(C-22)、64.2(C-23)、15.5(C-24)、17.8(C-25)、17.8(C-26)、26.5(C-27)、178.0(C-28)、33.4(C-29)、20.5(C-30);Glucose 105.2(C-1)、81.9(C-2)、80.4(C-3)、73.9(C-4)、79.2(C-5)、64.1(C-6);Arabinose 105.5(C-1)、76.2(C-2)、73.2(C-3)、73.3(C-4)、64.7(C-5);Arabinose95.2(C-1)、75.2(C-2)、71.8(C-3)、67.6(C-4)、66.3(C-5);Rhamnose100.8(C-1)、73.3(C-2)、79.7(C-3)、76.2(C-4)、68.7(C-5)、19.8(C-6);Xylose 108.1(C-1)、75.1(C-2)、79.8(C-3)、72.6(C-4)、67.6(C-5);Acyl moiety 173.1(C-1)、50.2(C-2)、69.9(C-3)、49.9(C-4)、173.2(C-5)、26.6(3-Me)。
根据上述HR-ESI-MS和NMR检测数据,本发明所获的样品被鉴定为土贝母苷甲。
土贝母苷甲(I)的化学结构如下。
实施例5:土贝母苷甲的溶血性
以真空采血管从兔耳静脉采集血液,加生理盐水,混匀,1500r/m离心10min,洗涤3次,收集红细胞以生理盐水稀释制成0.5%红细胞悬液,备用。取Quil A和土贝母苷甲,分别用生理盐水溶解,并作倍比稀释制成浓度为1000、500、250、125、62.5、31.25、15.63、7.81μg/ml的稀释液。于96孔微量板中,分别加入0.5%兔红细胞悬液100μl及不同浓度的皂苷稀释液100μl,混匀,每个浓度重复3孔。另设生理盐水及蒸馏水分别作为最小和最大的溶血性对照。37℃培养箱放置30min,1500r/m离心10min。各孔取上清液100μl,以酶标仪于波长405nm处测定OD值。重复3次,计算引起50%溶血的浓度(HD50)。结果显示:土贝母苷甲对0.5%兔红细胞的HD50值分别为5.38±0.06,而Quil A在相同条件下测得的HD50值为4.60±0.02μg/ml。说明土贝母苷甲的溶血性均显著低于Quil A (P<0.01)。
实施例6:土贝母苷甲的毒性
将6周龄雌性ICR小鼠分为3组,每组5只。土贝母苷甲以单剂量1.0mg和Quil A(150和200μg)分别皮下注射小鼠,给药后每天观察,称重,监测一周。生理盐水处理的动物作为对照。结果显示:土贝母苷甲组小鼠无一死亡,注射后体重无显著变化。而注射Quil A的小鼠出现局部肿胀、脱发和体重减轻,且72小时内分别死亡2和3只小鼠。
实施例7:土贝母苷甲对口蹄疫灭活疫苗的佐剂作用
实验方法:清洁级ICR小鼠随机分组,每组5只。水对照组:每只注射生理盐水0.2ml;口蹄疫病毒颗粒146S(FMDV 146S)对照组:每只注射含7.5μg/ml FMDV 146S的生理盐水0.2ml;氢氧化铝对照组:每只注射含氢氧化铝200μg和FMDV146S(1.5μg)的生理盐水0.2ml;土贝母苷甲实验组:每只注射含土贝母苷甲(50、100或200μg)和FMDV146S(1.5μg)的生理盐水0.2ml。各组皮下注射免疫2次,第一次免疫和第二次免疫间隔14天。二免后14天,采血,分离血清,进行抗原特异性抗体效价检测。
结果与分析:图5为土贝母苷甲对FMDV 146S免疫小鼠血清中特异性抗体效价的影响。土贝母苷甲能显著或极显著提高FMDV 146S免疫小鼠血清中特异性IgG、IgG1、IgG2a和IgG2b抗体滴度,且200μg剂量免疫小鼠血清中特异性IgG1、IgG2a和IgG2b抗体效价显著高于氢氧化铝(Alum)对照组。然而,Alum对FMDV 146S免疫小鼠血清中特异IgG2a和IgG2b抗体滴度无显著影响。这些结果说明土贝母苷甲既能诱导Th1型免疫应答反应,也能引发Th2型免疫应答反应,且对抗体应答的佐剂活性显著优于铝胶佐剂。
实施例8:土贝母苷甲对猪瘟活疫苗的佐剂作用
实验方法:清洁级ICR小鼠随机分组,每组6只。对照组:每只注射生理盐水0.2ml;猪瘟活疫苗对照组:每只注射含猪瘟活疫苗(兔源,CSFV)0.1头份的生理盐水0.2ml;氢氧化铝对照组:每只注射含氢氧化铝200μg和CSFV 0.1头份的生理盐水0.2ml;土贝母苷甲实验组:每只注射含土贝母苷甲(50、100或200μg)和CSFV 0.1头份的生理盐水0.2ml。各组皮下注射免疫2次,第一次免疫和第二次免疫间隔14天。二免后14天,将小鼠断颈处死,无菌条件下取脾,制备脾细胞悬液,采用MTT法检测脾细胞增殖反应。
结果与分析:图6为土贝母苷甲对CSFV免疫小鼠脾细胞增殖反应的影响。土贝母苷甲能显著或极显著促进Con A、LPS和猪瘟病毒E2蛋白(CSFV E2)诱导的免疫小鼠脾细胞增殖反应。说明土贝母苷甲能显著增强小鼠T、B淋巴细胞对CSFV的免疫应答反应。
综上所述,与现有技术已知的土贝母苷甲制备方法相比,本发明涉及的土贝母苷甲制备方法,工艺简单,操作简便,产品得率和纯度高,生产成本低,适于工业化生产;避免使用第一、二类有机溶剂,对环境和机体无危害,产品无有害溶剂残留,安全性好。因此,本发明所涉及的土贝母苷甲制备方法是一种简便、经济、适合大规模生产的制备工艺。本发明所涉及方法制备的土贝母苷甲显示出比现有技术中已知的铝胶佐剂能诱导机体对口蹄疫灭活疫苗和猪瘟活疫苗等商品兽用疫苗不仅能产生更强体液免疫反应,而且诱生机体的细胞免疫反应以及同时产生Th1型和Th2型免疫应答,可以作为多种兽用疫苗的免疫佐剂,发挥理想的免疫效果。
Claims (9)
1.一种土贝母苷甲的制备方法,其特征在于,包括以下步骤:
a.土贝母药材粉碎,以乙醇提取;
b.提取液减压回收乙醇、浓缩,得到醇提取物;
c.醇提取物上大孔吸附树脂柱,以水和乙醇混合液进行梯度洗脱;
d.硅胶薄层色谱检查,具有相同斑点的洗脱液合并,浓缩,干燥,得土贝母苷甲粗品;
e.土贝母苷甲粗品经柱色谱或HPLC,以洗脱剂洗脱,得到土贝母苷甲。
2.根据权利要求1所述的土贝母苷甲的制备方法,其特征在于,所述步骤a采用加热回流提取、超声提取、微波提取、渗漉提取中的一种,提取1-3次。
3.根据权利要求1所述的土贝母苷甲的制备方法,其特征在于,所述步骤b中醇提取物的密度为1.05-1.35g/mL。
4.根据权利要求1所述的土贝母苷甲的制备方法,其特征在于,所述步骤c采用的大孔吸附树脂为D101、ADS21、DS401或ZTC-1大孔吸附树脂;乙醇和水的混合物中乙醇浓度为5%-90%。
5.根据权利要求1所述的土贝母苷甲的制备方法,其特征在于,所述步骤e柱色谱和HPLC所用的填料为Rp-18和Rp-8;洗脱剂为乙醇和水的混合物,乙醇浓度为5%-90%;流速为1-100mL/min。
6.如权利要求1-5任一项所述方法制得的土贝母苷甲的应用,其特征在于,所述土贝母苷甲用于疫苗的制备。
7.如权利要求6所述的应用,其特征在于,所述土贝母苷甲用于制备兽用疫苗的佐剂。
8.如权利要求7所述的应用,其特征在于,所述的疫苗为猪口蹄疫灭活疫苗或猪瘟活疫苗。
9.如权利要求8所述的应用,其特征在于,所述土贝母苷甲的量为0.01μg~1g/单剂量。
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