CN114028555B - 具有免疫佐剂作用的土贝母皂苷及其制备方法和用途 - Google Patents
具有免疫佐剂作用的土贝母皂苷及其制备方法和用途 Download PDFInfo
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- CN114028555B CN114028555B CN202111506358.8A CN202111506358A CN114028555B CN 114028555 B CN114028555 B CN 114028555B CN 202111506358 A CN202111506358 A CN 202111506358A CN 114028555 B CN114028555 B CN 114028555B
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Abstract
本发明涉及具有免疫佐剂作用的土贝母皂苷及其制备方法和作为疫苗佐剂的应用。该皂苷是从中药土贝母中制备的土贝母苷乙、土贝母苷丙或包含有这2种皂苷化合物的土贝母总皂苷，具有免疫佐剂作用，可诱生机体对抗原同时产生Th1型和Th2型免疫应答，显示出比现有技术中已知的铝胶佐剂产生更强的细胞免疫和体液免疫反应，可以作为疫苗的佐剂。该方法制备土贝母皂苷工艺简单、操作简便，产品收率和纯度高，避免使用第一、二类有机溶剂，无有害溶剂残留，安全性好、生产成本低。
Description
技术领域
本发明涉及具有免疫佐剂作用的土贝母皂苷及其制备方法和应用。
背景技术
疫苗接种是控制和预防传染病最经济、有效的措施[O’Hagan DT,Lodaya RN,Lofano G.The continued advance of vaccine adjuvants–‘we can work it out’.Semin.Immunol.2020,50,101426]。由于SARS-CoV-2等复杂病原体的流行，疫苗的开发面临着越来越大的挑战。佐剂是传统疫苗和新型疫苗必不可少的组成成分，不仅影响机体对疫苗免疫应答的强度，而且能针对特定病原体诱导最有效的免疫应答类型[HarandiAM.Systems analysis of human vaccine adjuvants.Semin.Immunol.2018,39,30–34；Rappuoli R,Hanon E.Sustainable vaccine development:a vaccine manufacturer’sperspective.Curr.Opin.Immunol.2018,53,111–118]。目前研究报道的免疫佐剂种类甚多，但由于存在毒副作用或安全隐患等不可避免的缺陷多难以实际应用[Del Giudice G,Rappuoli R,Didierlaurent AM.Correlates of adjuvanticity:A review on adjuvantsin licensed vaccines.Semin.Immunol.2018,39,14–21；Shi S,Zhu H,Xia X,Liang Z,MaX,Sun B.Vaccine adjuvants:Understanding the structure and mechanism ofadjuvanticity.Vaccine 2019,37,3167–3178]。目前，铝胶、MF59、AS01、AS03和AS04是为数不多经欧盟或美国FDA批准应用于临床的疫苗佐剂。铝胶仍是疫苗生产中应用最广的佐剂，其存在一些问题：⑴主要通过诱导Th2型免疫应答产生抗体起保护作用，不能诱生Th1型免疫应答和细胞介导的免疫反应，只适用于以抗体为主要保护性免疫的疫苗；⑵对人免疫缺陷病毒、丙型肝炎病毒、单纯疱疹病毒、流感病毒、血吸虫病、百日咳和伤寒等多种抗原无佐剂作用；⑶促进IgE抗体产生，机体容易产生过敏反应；⑷局部反应，形成肉芽肿，甚至发生局部无菌性脓肿；⑸含铝胶的疫苗冷冻后，铝胶变性；⑹影响机体神经系统。随着新型疫苗应用的增多，对佐剂的需求越来越大，开发新型、高效、安全的佐剂对于增强疫苗的保护作用具有重要意义。
皂甙是一种天然糖苷化合物，被广泛研究用作疫苗的佐剂[Lacaille-Dubois MA,Wagner H.New perspectives for natural triterpene glycosides as potentialadjuvants.Phytomedicine 2017,37,49-57.]。其中研究最多的是南美皂树(Quillajasaponaria Molina)树皮中的总皂苷--Quil A及其活性成分QS-21[Lacaille-DuboisMA.Updated insights into the mechanism of action and clinical profile of theimmunoadjuvant QS-21:A review.Phytomedicine 2019,60,152905]。由于具有对外源性抗原既能刺激Th1免疫应答，又能诱导细胞毒性T淋巴细胞反应的独特特性，Quil A和QS-21广泛用于治疗性癌症疫苗[Cebon JS,Gore M,Thompson JF,et al.Results of arandomized,double-blind phase II clinical trial of NY-ESO-1vaccine withISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-riskresected melanoma.J.Immunother.Cancer 2020,8,e0004108]以及细胞内病原体(如HIV、结核病和疟疾)疫苗的临床试验[Van Der Meeren O,Hatherill M,Nduba V,et al.Phase2b controlled trial of M72/AS01E vaccine to prevent tuberculosis.NewEngl.J.Med.2018,379,1621–1634]。然而，QS-21具有严重的缺陷，①溶血性强、毒副作用大；②分子结构中均存在酰基，易水解，其去酰化水解产物失去诱导Th1型免疫应答及产生特异性CTL的能力，只能引发Th2型免疫应答；③南美皂树资源匮乏，含量低，分子量大，化学结构复杂，分离制备困难[Wang PF.Natural and synthetic saponins as vaccineadjuvants.Vaccines 2021,9,222]。事实上，QS-21不是单一分子，而是两种异构体的混合物[Wang P,Devalankar DA,Dai Q,Zhang P,Michalek SM.Synthesis and evaluation ofQS-21-based immunoadjuvants with a terminal-functionalized side chainincorporated in the west wing trisaccharide.J.Org.Chem.2016,81,9560-9566]。有必要进一步寻找高效、安全、稳定的皂苷佐剂[Pifferi C,Fuentes R,Fernández-TejadaA.Natural and synthetic carbohydrate-based vaccine adjuvants and theirmechanisms of action.Nat.Rev.Chem.2021,5,197–216]。
土贝母是葫芦科植物土贝母Bolbostemma paniculatum(Maxim.)Franquet的干燥块茎。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。临床上用于治疗急性乳腺炎、肿瘤等[Islam MS,et al.The potential role oftubeimosides in cancer prevention and treatment.Eur.J.Med.Chem.2018,162,109–121.]。
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[Zeng YL,etal.Rapid characterization of components in Bolbostemma paniculatum by UPLC/LTQ-Orbitrap MSn analysis and multivariate statistical analysis forherb.Molecules 2018,23,1155]。其中土贝母皂苷(Tubeimosides,TBMs)是土贝母的主要活性成分之一，具有抗肿瘤[Yang JB,et al.Tubeimoside-1induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cellsin vitro.Acta Pharmacol.Sin.2016,37,950–962；Wang K,Zhu X,Chen Y,Yin Y,MaT.Tubeimoside V sensitizes human triple negative breast cancer MDA-MB-231cells to anoikis via regulating caveolin-1-related signaling pathways.Arch.Biochem.Biophys.2018,646,10–15；Feng XS,Ma GY,Shi HL,Wang YW,Chao X.Anintegrative serum pharmacology-based approach to study the anti-tumoractivity of B.paniculatum aqueous bulb extract on the human hepatocellularcarcinoma cell line BEL-7404.Front.Pharmacol.2020,11,1261]、抗炎[He D,etal.Tubeimoside I protects dopaminergic neurons against inflammation-mediateddamage in lipopolysaccharide(LPS)-evoked model of parkinson’s disease inrats.Int.J.Mol.Sci.2018,19,2242；Liu Z,et al.Inhibitory effects of tubeimosideI on synoviocytes and collagen-induced arthritis in rats.J.Cell.Physiol.2018,233,8740–8753]和抗病毒[Ju X,et al.A novel cell culture system modeling theSARS-CoV-2life cycle.PLoS Pathog.2021,17,e1009439]作用。
发明内容
本发明的首要目的是提供一类具有免疫佐剂作用的皂苷。
本发明具有免疫佐剂作用的皂苷包括从中药土贝母中制备的土贝母苷乙、土贝母苷丙、以及包含有这2种皂苷化合物的土贝母总皂苷。
本发明的第二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乙、土贝母苷丙、以及土贝母总皂苷的制备方法。
本发明提供的土贝母苷乙、土贝母苷丙、以及包含有这2种皂苷化合物的土贝母总皂苷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，回收乙醇，浓缩，干燥，即得土贝母总皂苷。
e.土贝母总皂苷经柱色谱或HPLC，以洗脱剂洗脱，得到土贝母苷乙和土贝母苷丙。
上述所述步骤a采用加热回流、超声提取、微波提取、渗漉中的任一种；乙醇浓度5％-95％。
上述所述步骤b中醇提取物的密度为1.02-1.35g/mL。
上述所述步骤c采用的大孔树脂为D101、ADS21、DS401或ZTC-1大孔吸附树脂；乙醇和水的混合物中的乙醇浓度10％-90％。
上述所述步骤e柱色谱和HPLC所用的填料为Rp-18或Rp-8；洗脱剂为乙醇和水的混合物，乙醇浓度为5％-90％。
本发明的第三个目的是提供所述的土贝母苷乙、土贝母苷丙、以及土贝母总皂苷作为佐剂在制备疫苗中的应用。
本发明土贝母苷乙、土贝母苷丙或土贝母总皂苷在疫苗中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，0.0 1μg～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
本发明涉及的土贝母苷乙、土贝母苷丙、以及包含有这2种皂苷化合物的土贝母总皂苷)具有显著的佐剂活性，可诱导机体对抗原同时产生Th1型和Th2型免疫应答，显示出比现有技术中已知的铝胶佐剂产生更强的细胞免疫和体液免疫反应。可以作为疫苗的免疫佐剂，发挥理想的免疫效果。
本发明涉及的土贝母苷乙、土贝母苷丙、以及土贝母总皂苷的制备方法，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乙、土贝母苷丙、以及土贝母总皂苷的制备方法是一种简便、经济、适合大规模生产的制备工艺。
附图说明
图1为土贝母总皂苷的HPLC图(1.土贝母苷乙，2.土贝母苷丙)。
图2为土贝母苷乙的HR-ESI-MS图。
图3为土贝母苷乙的1H-NMR图。
图4为土贝母苷乙的13C-NMR图。
图5为土贝母苷丙的HR-ESI-MS图。
图6为土贝母苷丙的1H-NMR图。
图7为土贝母苷丙的13C-NMR图。
图8为土贝母总皂苷(BPS)对卵清蛋白(OVA)免疫小鼠血清中抗原特异性IgG、IgG1、IgG2a和IgG2b抗体效价的影响。aP<0.05、bP<0.01和cP<0.001vs OVA对照组。
图9为土贝母总皂苷(BPS)对卵清蛋白(OVA)免疫小鼠脾细胞增殖的影响。aP<0.05、bP<0.01和cP<0.001 vs OVA对照组。
图10是土贝母总皂苷(BPS)对OVA免疫小鼠自然杀伤细胞活性的影响。aP<0.05、bP<0.01和cP<0.001 vs OVA对照组。
图11为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)免疫小鼠血清中特异性抗体效价的影响。aP<0.05、bP<0.01和cP<0.001 vs OVA对照组。
图12为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)免疫小鼠脾细胞增殖反应的影响。aP<0.05、bP<0.01和cP<0.001 vs OVA对照组。
图13为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)免疫小鼠自然杀伤(NK)细胞活性的影响。
图14为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)免疫小鼠抗原特异性刺激脾细胞分泌细胞因子能力的影响。
图15为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)免疫小鼠抗原特异性刺激脾细胞Th1和Th2型细胞因子mRNA表达的影响。
图16为土贝母苷乙(TBM II)和土贝母苷丙(TBM III)对卵清蛋白(OVA)致敏小鼠迟发型超敏反应(DTH)的影响。
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：土贝母总皂苷制备
药材土贝母粗粉1kg，以70％乙醇回流提取3次，每次1.5小时，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、10％-95％乙醇混合液进行梯度洗脱。洗脱液用薄层色谱检查，具有相同斑点的洗脱液合并，减压回收乙醇、浓缩、冷冻干燥，得到灰白色土贝母总苷26.24g。土贝母总皂苷的HPLC色谱图见图1。
实施例2土贝母苷乙制备
称取土贝母总皂苷1.0g，加50％乙醇5mL溶解，0.22μm微孔滤膜滤过，滤液注入制备型高效液相色谱仪，流动相为无水乙醇-水(40∶60)，流速为10mL/min，收集土贝母苷乙对应色谱峰流出液，减压回收溶剂，冷冻干燥，得透明状土贝母苷乙(I)98.9mg。
采用OrbitrapElite质谱仪(Thermo Scientific,Bremen,Germany)检测制备得到的土贝母苷乙的分子量。土贝母苷乙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 1333.6135[M-H]-(计算值：1334.6143)，分子式：C63H98O30。
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乙的氢谱(1H-NMR)和碳谱(13C-NMR)。土贝母苷乙的1H-和13C-NMR见图3、4和表1。
表1土贝母苷乙的1C-and 13C-NMR
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乙。
土贝母苷乙(I)的化学结构如下。
实施例3土贝母苷丙制备
称取土贝母总皂苷1.0g，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(40:60)，流速为10mL/min，收集土贝母苷丙对应色谱峰流出液，减压回收溶剂，冷冻干燥，得透明状土贝母苷甲晶190.4mg。
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。土贝母苷丙的高分辨电喷雾质谱(HR-ESI-MS)谱图见图5。HR-ESI-MS的准分子离子峰m/z 1363.6233[M-H]-(计算值：1364.6249)，分子式：C64H100O31。
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丙的氢谱(1H-NMR)和碳谱(13C-NMR)。土贝母苷丙的1H-NMR和13C-NMR见图6、7和表2。
表2土贝母苷丙的1C-and 13C-NMR
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丙。
土贝母苷丙(II)的化学结构如下。
实施例4土贝母苷乙和土贝母苷丙的溶血性
实验方法：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、土贝母苷乙和土贝母丙，分别用生理盐水溶解，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)。
结果与分析：土贝母苷乙和土贝母苷丙对0.5％兔红细胞的HD50值分别为7.91±0.22μg/ml和4.48±0.11μg/ml，相同条件下Quil A的HD50值为4.60±0.02μg/ml。说明土贝母苷乙的溶血性显著低于Quil A (P<0.01)，而土贝母苷丙与Quil A的溶血性无显著差异。
实施例5土贝母苷乙和土贝母苷丙的毒性
清洁级ICR小鼠分为5组，每组5只。土贝母苷乙和土贝母苷丙以单剂量1.0mg和Quil A以单剂量150和200μg分别肌内注射小鼠。生理盐水处理的动物作为对照。给药后每天观察体征，称重，监测一周。
结果与分析：土贝母苷乙试验组小鼠死亡2只，土贝母苷丙试验组小鼠全部健活，而注射Quil A 150和200μg试验组小鼠分别死亡2和3只小鼠。说明土贝母苷乙和土贝母苷丙的毒性显著低于Quil A。
实施例6：土贝母总皂苷对OVA的免疫佐剂作用
实验方法：清洁级ICR小鼠随机分成6组，每组5只。生理盐水对照组：每只皮下注射生理盐水0.2ml；卵清蛋白(OVA)对照组：每只皮下注射OVA溶液(0.125mg/ml)0.2ml；Quil A对照组：每只皮下注射0.2ml含10μg Quil A的OVA溶液(0.125mg/ml)；土贝母总皂苷(BPS)试验组：每只注射0.2ml含土贝母总皂苷(50、100或200μg)的OVA溶液(0.125mg/ml)。首免后二周加强免疫。二免后14天，取血，分离制备血清；处死小鼠，剖取脾脏，制备脾细胞悬液。采用ELISA和MTT法检测血清中抗原特异性抗体效价、脾细胞增殖反应和NK细胞活性。
结果与分析：土贝母总皂苷(BPS)能显著提高免疫小鼠血清中OVA特异性IgG、IgG1、IgG2a和IgG2b抗体效价(图8)；增强OVA受免小鼠Con A、LPS和OVA刺激脾细胞增殖反应(图9)；促进免疫小鼠NK细胞对K562细胞的杀伤活性(图10)。说明土贝母总皂苷不仅能促进免疫小鼠对OVA的体液免疫和细胞免疫应答，而且可诱导平衡的Th1/Th2免疫应答，均有显著的佐剂活性。
实施例7：土贝母苷乙和土贝母苷丙对OVA的免疫佐剂作用
实验方法：清洁级ICR小鼠随机分9组，每组5只。生理盐水对照组：每只皮下注射生理盐水0.2ml；卵清蛋白(OVA)对照组：每只皮下注射OVA溶液(0.125mg/ml)0.2ml；Quil A对照组：每只皮下注射0.2ml含10μg Quil A的OVA溶液(0.125mg/ml)；土贝母苷乙试验组：每只注射0.2ml含土贝母苷乙(25、50或100μg)的OVA溶液(0.125mg/ml)；土贝母苷丙试验组：每只注射0.2ml含土贝母苷丙(25、50或100μg)的OVA溶液(0.125mg/ml)。第一次免疫和第二次免疫间隔二周。二免后14天，取血，分离制备血清；处死小鼠，取脾脏，制备脾细胞悬液。采用ELISA、MTT法和RT-PCR检测OVA免疫小鼠血清中特异性抗体效价、脾细胞增殖反应、NK细胞活性、OVA刺激脾细胞培养上清中细胞因子含量、以及OVA刺激脾细胞Th1和Th2细胞因子mRNA表达水平。
结果与分析：土贝母苷乙和土贝母苷丙均能显著提高免疫小鼠血清中OVA特异性IgG、IgG1、IgG2a和IgG2b抗体效价(图11)；增强OVA受免小鼠Con A、LPS和OVA刺激脾细胞增殖反应(图12)；促进免疫小鼠NK细胞对K562细胞的杀伤活性(图13)；增强免疫小鼠抗原刺激脾细胞分泌IL-2、IL-10和IFN-γ的能力(图14)；上调免疫小鼠OVA刺激脾细胞IL-2、IL-4、IL-10和IFN-γ等细胞因子mRNA表达水平(图15)。说明土贝母苷乙和土贝母苷丙均有显著的佐剂活性，不仅能促进免疫小鼠对OVA的体液免疫和细胞免疫应答，而且可诱导平衡的Th1/Th2免疫应答。
实施例8：土贝母苷乙和土贝母苷丙对OVA致敏小鼠迟发型超敏反应的影响
实验方法：清洁级ICR小鼠随机分9组，每组5只。生理盐水对照组：每只皮下注射生理盐水0.2ml；卵清蛋白(OVA)对照组：每只皮下注射OVA溶液(0.05mg/ml)0.2ml；Quil A对照组：每只皮下注射0.2ml含10μg Quil A的OVA溶液(0.05mg/ml)；土贝母苷乙试验组：每只注射0.2ml含土贝母苷乙(25、50或100μg)的OVA溶液(0.05mg/ml)；土贝母苷丙试验组：每只注射0.2ml含土贝母苷丙(25、50或100μg)的OVA溶液(0.0mg/ml)。致敏后5天，于右后足垫皮下注射OVA溶液(2mg/ml)50μl，左后足垫皮下注射等体积的PBS。于激发后24h h，分别游标卡尺测量脚垫厚度，计算肿胀度(Δd)，即左后脚垫厚度减去右后脚垫厚度。
结果与分析：如图16所示，与OVA单独致敏组相比，激发24h后，Quil A、土贝母苷乙和土贝母苷丙(25、50、100μg)均可极显著增强致敏小鼠对OVA的DTH反应(P<0.001)。说明土贝母苷乙和土贝母苷丙能增强免疫小鼠细胞介导免疫反应。
实施例9土贝母苷乙和土贝母苷丙对口蹄疫疫苗的佐剂作用
实验方法：清洁级ICR小鼠随机分为9组，每组5只。生理盐水对照组：每只注射生理盐水0.2ml；口蹄疫疫苗对照组：每只注射含7.5μg/ml口蹄疫病毒颗粒146S(FMDV146S)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；土贝母苷乙试验组：每只注射含土贝母苷乙(50、100或200μg)和FMDV146S(1.5μg)的生理盐水0.2ml；土贝母苷丙试验组：每只注射含土贝母苷丙(50、100或200μg)和FMDV146S(1.5μg)的生理盐水0.2ml。首次免疫后二周进行加强免。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：土贝母苷乙和土贝母苷丙均能显著或极显著提高口蹄疫疫苗免疫小鼠血清中FMDV 146S特异性IgG、IgG1、IgG2a和IgG2b抗体滴度(P<0.001)。然而，氢氧化铝对口蹄疫疫苗免疫小鼠血清中FMDV 146S特异性IgG2a和IgG2b抗体滴度无显著影响(P>0.05)。说明土贝母苷乙和土贝母苷丙对口蹄疫疫苗具有显著的佐剂作用，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10土贝母苷乙和土贝母苷丙对重组乙肝疫苗的佐剂作用
实验方法：清洁级ICR小鼠随机分9组，每组5只。生理盐水对照组：每只注射生理盐水0.2ml；重组乙肝疫苗对照组：每只注射浓度为20μg/ml的重组乙肝表面抗原(rHBsAg)生理盐水溶液0.2ml；氢氧化铝对照组：每只注射0.2ml含200μg氢氧化铝的rHBsAg溶液(20μg/ml)；土贝母苷乙试验组：每只注射0.2ml含土贝母苷乙(50、100或200μg)的rHBsAg溶液(20μg/ml)；土贝母苷丙试验组：每只注射0.2ml含土贝母苷丙(50、100或200μg)的rHBsAg溶液(20μg/ml)。首免后二周加强免疫。二免后14天，采血，分离血清，进行rHBsAg特异性抗体效价检测。
结果与分析：土贝母苷乙和土贝母苷丙均能显著或极显著提高重组乙肝疫苗免疫小鼠血清中rHBsAg特异性IgG、IgG1、IgG2a和IgG2b抗体滴度，且免疫小鼠血清中rHBsAg特异性IgG1、IgG2a和IgG2b抗体效价显著高于氧氧化铝免疫组。然而，氢氧化铝对口蹄疫疫苗免疫小鼠血清中FMDV 146S特异性IgG2a和IgG2b抗体滴度无显著影响(P>0.05)。说明土贝母苷乙和土贝母苷丙对重组乙肝疫苗均有显著的佐剂作用，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。
综上所述，本发明涉及的土贝母苷乙、土贝母苷丙、以及包含有这2种皂苷化合物的土贝母总皂苷)具有显著的佐剂活性，可诱导机体对抗原同时产生Th1型和Th2型免疫应答，显示出比现有技术中已知的铝胶佐剂产生更强的细胞免疫和体液免疫反应。可以作为疫苗的免疫佐剂，发挥理想的免疫效果。同时，本发明涉及的土贝母苷乙、土贝母苷丙、以及土贝母总皂苷的制备方法，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乙、土贝母苷丙、以及土贝母总皂苷的制备方法是一种简便、经济、适合大规模生产的制备工艺。
Claims (3)
1.土贝母皂苷在制备疫苗免疫佐剂中的应用，其特征是，所述土贝母皂苷是从中药土贝母中制备的土贝母苷乙或土贝母苷丙，所述疫苗为口蹄疫疫苗或重组乙肝疫苗，所述土贝母皂苷促进机体对抗原的细胞免疫和体液免疫应答，诱导机体同时产生Th1型和Th2型免疫应答。
2.如权利要求1所述的应用，其特征在于, 所述土贝母皂苷的制备方法包括以下步骤：
a. 土贝母药材粉碎，以70%乙醇回流提取；
b. 提取液减压回收乙醇，浓缩，得到醇提取物；
c. 醇提取物上D101大孔吸附树脂柱，以水和10%-95%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，回收乙醇，浓缩，干燥，得土贝母总皂苷；
e. 土贝母总皂苷经HPLC，流动相无水乙醇与水的比例为40∶60，流速为10 mL/min，收集土贝母苷乙或土贝母苷丙对应色谱峰流出液，减压回收溶剂，冷冻干燥，得土贝母苷乙或土贝母苷丙。
3. 根据权利要求2所述的应用，其特征在于，所述步骤b中醇提取物的密度为1.02-1.35 g/mL。
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Citations (2)
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| CN102180934A (zh) * | 2011-01-24 | 2011-09-14 | 南京泽朗医药科技有限公司 | 一种土贝母苷甲的制备方法 |
| CN102342973A (zh) * | 2011-10-11 | 2012-02-08 | 北京中医药大学 | 土贝母总皂苷的提取富集方法 |
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|---|---|---|---|---|
| CN102180934A (zh) * | 2011-01-24 | 2011-09-14 | 南京泽朗医药科技有限公司 | 一种土贝母苷甲的制备方法 |
| CN102342973A (zh) * | 2011-10-11 | 2012-02-08 | 北京中医药大学 | 土贝母总皂苷的提取富集方法 |
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| Structure-activity relationship of tubeimosides in anti-inflammatory, antitumor, and antitumor-promoting effects;T X Yu等;Acta Pharmacol Sin;第22卷(第5期);第463-468页 * |
| 高效液相制备色谱制备土贝母中土贝母苷甲、乙、丙对照品的研究;金晓东等;现代药物与临床;第29卷(第12期);正文第1段，第1至3节,图1 * |
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