CN1748792A - 木鳖子含三萜皂甙提取物的用途 - Google Patents
木鳖子含三萜皂甙提取物的用途 Download PDFInfo
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- CN1748792A CN1748792A CN 200510060433 CN200510060433A CN1748792A CN 1748792 A CN1748792 A CN 1748792A CN 200510060433 CN200510060433 CN 200510060433 CN 200510060433 A CN200510060433 A CN 200510060433A CN 1748792 A CN1748792 A CN 1748792A
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Abstract
本发明公开了一种木鳖子含三萜皂甙提取物的用途。将中药木鳖子粉碎后,用乙醇回流提取、乙醚萃取除去脂溶性成分、正丁醇萃取除去不溶于正丁醇的成分、吸附性大孔树脂纯化,获得一种淡黄色的木鳖子含三萜皂甙提取物,该木鳖子含三萜皂甙提取物毒性低,肌肉注射小鼠、猪、牛后无局部刺激性,口服和注射使用安全,可作为佐剂应用于疫苗制作,经验证,用木鳖子含三萜皂甙提取物作为佐剂制作的疫苗可有效提高免疫效果,在疫苗制作领域具有广泛的应用前景。
Description
技术领域
本发明属于中药有效成分应用技术,涉及一种中药木鳖子含三萜皂甙提取物在疫苗制作方面的用途。
背景技术
木鳖子(Semen momordicae)为葫芦科植物木鳖Momordica Cochinchinesis(Lour.)Spreng.的干燥成熟种子。木鳖为多年生藤本,主要分布在东南亚国家、蒙古、以及我国南方的大部分地区,如广西、四川、湖北、湖南、贵州、云南、广东、安徽等省。一般冬季采收成熟果实,剖开,晒至半干,除去果肉,取出种子,晒干备用。木鳖子为常用中药,味苦、微甘,性凉,具有散结消肿,攻毒疗疮的功能。北宋《开宝本草》(公元973年)记载木鳖子治“妇人乳痈,肛门肿痛”。现在该药已收载于《中国药典》和《中国兽药典》。人医和兽医临床上常用于治疗乳痈、腹泻、肿毒、瘰疬等感染性疾病。
木鳖子作为一个抗感染中药已经在临床上应用了1000多年。木鳖子可以单味应用,也可以和其他药物配伍组成方剂使用。
化学分析表明木鳖子含脂肪酸、皂甙、蛋白质以及α-菠菜甾醇、齐墩果酸、木鳖子酸等成分。最近的研究发现木鳖子的一种水提取物具有抗肿瘤(Tien et al.,2005;Tsoi et al.,2004;Wonget al.,2004)以及抗氧化活性(Tsoi et al.,2005)。Iwamnoto等(1985)从木鳖子中分离出两种皂甙,momordica saponin I和II,并确认I为丝石竹皂甙,II为一种含皂皮酸的皂甙。丝石竹皂甙为含双糖链的三萜皂甙。
目前免疫界常用的佐剂有如下几种:
氢氧化铝铝胶佐剂:该类佐剂是迄今美国食品和药物管理局(FDA)准许用于人类疫苗的唯一佐剂。铝佐剂在疫苗中吸附抗原形成复合物,注射后在局部形成抗原贮存库,缓慢释放出抗原发挥佐剂作用。铝佐剂主要促进体液免疫应答,适用于以抗体为保护性免疫的疾病疫苗,如白喉、破伤风、乙肝、麻疹等。铝佐剂虽然在人用或兽用疫苗上广泛应用,仍存在许多缺点,如轻度局部反应,形成肉芽肿,甚至发生局部无菌性脓肿;人的一种肌肉的局部组织损伤可能和铝佐剂有关;此外,铝胶冷冻后胶体状态被破坏,不能发挥佐剂作用,因此不能冷冻保存;不同制备批次的制备铝胶佐剂,胶体状态不同,难以获得相同佐剂效果。
油乳佐剂:含油和乳化剂。在油乳佐剂疫苗中抗原包被在油相形成的微结构内,在注射部位缓慢释放发挥佐剂作用。使用的油有佐制-65、白油Span佐剂、MF-59等。这类佐剂局部刺激性大,可引起肉芽肿和无菌性脓肿;稳定性差,难以长期保存。故仅限用于兽用疫苗。
细胞因子佐剂:是由免疫效应细胞和相关细胞产生的,具有重要生物学活性的细胞调节蛋白。近年来,随着研究的深入,发现许多细胞因子具有明显的免疫佐剂效应。如白细胞介素2和γ干扰素等。细胞因子用于佐剂的缺点是大多数细胞因子在的体内半衰期较短,其活性易受内环境如pH值、各种水解酶及血浆蛋白的影响;同时,大剂量的使用细胞因子还可导致发热、炎症等副作用;目前的技术水平,纯化、制备细胞因子的成本比常规佐剂要高。
Quil A和QS21佐剂:Quil A是从南美的皂树Quillaia Saponaria Molina的树皮中提取出来的一种皂素。Quil A的主要缺点是有较大的局部刺激反应。进一步分析Quil A,从中分离出QS21。QS21佐剂主要促进IgG2a抗体亚型(Thl型)产生;QS21作为佐剂免疫动物后,用相同细菌或病毒攻击,抗原特异的抗体分泌细胞数增加,细胞毒T淋巴细胞(CTL)的杀伤作用也明显增强。但制备Quil A和QS21佐剂的原料仅产于南美,我国缺乏这种原料;该佐剂从树皮中提取,树皮剥离后,影响树的存活,过度开发,会造成资源紧缺。
虽然木鳖子的药用价值在医学文献中的记载已经有1000多年的历史,但有关该药免疫活性方面的研究尚未见报道。木鳖在我国分布广泛,资源丰富,种子采收后,植物仍然存活,可以继续利用。因此,开发和利用中药木鳖子不会对该物种的生存造成威胁。
本发明的目的在于充分开发木鳖子的药用价值,开拓木鳖子含三萜皂甙提取物的应用,以及提供一种新的免疫佐剂。
发明内容
本发明公开了一种中药木鳖子含三萜皂甙提取物的新用途。木鳖子含三萜皂甙提取物作为佐剂与疫苗混合用于疫苗的制作,可以提高疫苗的免疫效果。本发明中药木鳖子含三萜皂甙提取物为从木鳖子中提取的去除毒性成分的含三萜皂甙的水溶液,或将该水溶液浓缩、干燥后得到的含三萜皂甙的黄色粉末状提取物。
木鳖子含三萜皂甙提取物作为佐剂与疫苗混合的使用方法为:在疫苗的制造阶段或者在疫苗注射之前,将木鳖子提取物和抗原混合。
附图说明
图1:ECMS(木鳖子提取物)和氢氧化铝铝胶对抗原卵清白蛋白(OVA)的佐剂作用。OVA(10微克)和生理盐水(组1),ECMS(10微克,组2;50微克,组3;100微克,组4)或铝胶(50微克,组5)混合,皮下注射免疫小鼠。3周后进行二免。二免2周后眼眶静脉采血,分离血清。用ELISA法检测血清抗OVA抗体滴度。具有不同英文字母的矩形图表明两者之间具有显著性差异(P<0.05)。
图2:ECMS和氢氧化铝铝胶对IgG亚类产生的影响。OVA(10微克)和生理盐水(组1),ECMS(10微克,组2;50微克,组3;100微克,组4)或铝胶(50微克,组5)混合,皮下注射免疫小鼠。3周后进行二免。二免2周后眼眶静脉采血,分离血清。用ELISA法检测血清抗OVA的IgG1和IgG2a。具有不同英文字母的矩形图表明两者之间具有显著性差异(P<0.05)。
图3:ECMS和氢氧化铝铝胶对淋巴细胞增殖的影响。OVA(10微克)和生理盐水,ECMS(50微克)或铝胶(50微克)混合,皮下注射免疫小鼠。3周后进行二免。二免2周后分离脾细胞。用MTT法检测脾淋巴细胞对ConA和LPS刺激的增殖能力。具有不同英文字母的矩形图表明两者之间具有显著性差异(P<0.05)。
具体实施方式
将木鳖子含三萜皂甙的提取物作为佐剂制备商品灭活疫苗,如猪或牛口蹄疫疫苗。经验证,该疫苗免疫动物,可以使动物的抗体效价提高,增强抗病力。
一、鳖子含三萜皂甙提取物溶液的配制
(1)将原料木鳖子粉碎后在40~50%乙醇中浸泡,回流提取2~5次,提取液过滤后减压浓缩;
(2)在浓缩液中加入澄清剂去除杂质,静置后取上清液,用乙醚萃取去除脂溶性成分,保留水层的水溶液;
(3)水溶液用饱和正丁醇萃取,减压浓缩正丁醇萃取液至干,得黄色粉末状木鳖子粗提物;
(4)用50~80℃热水溶解粗提物,调节pH至6~7,充分溶解后,将水溶解的木鳖子粗提物加入吸附性大孔树脂柱,静置24小时后,水洗去除非有效成分的杂质;
(5)用40~50%乙醇洗脱,减压浓缩洗脱液,将浓缩的洗脱液真空干燥,得黄色粉末状木鳖子精提物。
经检测,黄色粉末状木鳖子精提物中主要含有三萜皂甙成分,经动物试验,测定其已去除毒性,局部注射无刺激反应。
将含三萜皂甙成分的黄色粉末状木鳖子提取物配制成1mg/ml的水溶液,0.2μm滤膜过滤。按说明书用鲎试剂(湛江安度斯生物有限公司,批号0502060)检测木鳖子含三萜皂甙提取物水溶液中的内毒素。检测结果表明木鳖子含三萜皂甙提取物溶液的内毒素含量小于0.5EU/ml,可以排除内毒素对实验结果的影响。该溶液用于以下免疫实验。
二、小鼠免疫学实验
1.实验动物和免疫程序
将SPF条件下饲养的4~6周龄BALB/c雌性小鼠30只(购自上海国家啮齿类实验动物中心)随机分成5组,每组6只。动物皮下注射模式抗原卵清白蛋白(OVA)(上海伯奥生物科技有限公司,批号020902)或卵清白蛋白和木鳖子含三萜皂甙提取物或氢氧化铝铝胶(中牧实业股份有限公司江西生物药厂,批号20030726)的混合物。各组抗原和佐剂剂量见表1。共免疫二次,间隔21天。分别于首免前,二免前和二免后二周对每只小鼠称重。二免后2周眼眶静脉采血,取脾脏,分离脾淋巴细胞。
表1每个动物一次注射的OVA和佐剂类型及剂量
组别 | 小鼠只数 | 模式抗原OVA的剂量(μg) | 佐剂类型和剂量 |
12345 | 66666 | 1010101010 | 无佐剂10μg木鳖子含三萜皂甙提取物50μg木鳖子含三萜皂甙提取物100μg木鳖子含三萜皂甙提取物50μg氢氧化铝 |
2.血清抗OVA抗体效价的测定
在96孔酶标板(浙江拱东医用塑料厂)每孔加入100ul包被液(含5μg OVA/ml的0.05mol/L碳酸盐缓冲液),置4℃孵育18小时后,用洗涤液洗涤3次,每次3min。每孔加入300ul含1%小牛血清的PBS封闭液(0.01mol/L,pH7.4),于37℃孵育1h后,用洗涤液洗涤3次,每次3min。在96孔酶标板每孔加入100ul稀释液,然后在第1孔内加入96ul稀释液,再加待检血清4ul,使血清稀释度为1∶50,然后依次各孔做对倍稀释。于37℃孵育2h后,用洗涤液洗涤3次,每次3min。加入经1∶500稀释的辣根过氧化物酶标记的羊抗小鼠IgG抗体(CHEMICON International,Inc),100μl/孔,37℃孵育2h,用洗涤液洗涤3次,每次3min。加入TMB(四甲基联苯二胺,SIGMA-ALDRICH公司)底物溶液显色,100ul/孔,37℃孵育15min,加2N H2SO450ul/孔终止反应。用酶标仪(DIALAB,GMBH,Austria)在450nm波长测定OD(吸光度)值。根据相邻稀释度的OD值之差最大值确定为拐点,根据拐点OD值的平均值作为临界值,大于或者等于临界值的最大稀释度判为样品的抗体效价。
3.血清抗OVA IgG1和IgG2a水平的测定
在96孔酶标板(浙江拱东医用塑料厂)每孔加入100ul包被液(含5μg OVA/ml的0.05mol/L碳酸盐缓冲液),置4℃孵育18小时后,用洗涤液洗涤3次,每次3min。每孔加入300ul含1%小牛血清的PBS封闭液(0.01mol/L,pH7.4),于37℃孵育1h后,用洗涤液洗涤3次,每次3min。加入1∶800倍稀释的待检血清,100u1/孔,37℃孵育1h,用洗涤液洗涤3次,每次3min。加入经1∶600稀释的生物素标记的山羊抗小鼠IgG1或IgG2a(SantaCruz Biotechnology,Inc),100ul/孔,37℃孵育1h,用洗涤液洗涤3次,每次3min。加入经1∶4000稀释的辣根过氧化物酶标记的抗生物素(BD Biosciences Pharmingen)100ul/孔,37℃孵育1h,用洗涤液洗涤3次,每次3min。加入TMB(四甲基联苯二胺,SIGMA-ALDRICH公司)底物溶液显色,100ul/孔,37℃孵育15min,加2N H2SO450ul/孔终止反应。用酶标仪(DIALAB,GMBH,Austria)在450nm波长测定OD(吸光度)值。
4.淋巴细胞增殖能力的测定
无菌条件下取出小鼠脾脏,用无菌注射器取PBS冲洗分离脾细胞。1500rpm离心10min,弃上清,如此重复洗3次。将脾细胞悬浮于1ml RPMI-1640培养液中,用台朌兰拒染法进行脾细胞计数,用培养液将脾细胞浓度配成3×106个/ml;每孔加入100ul脾细胞悬液(3×106细胞/ml),并分别加刀豆蛋白A(concanavalin A,Con A,SIGMA公司产品)溶液,终浓度为15μg/ml、脂多糖(lipopolysaccharide,LPS,SIGMA公司产品)溶液,终浓度为5μg/ml或RPMI-1640培养液至终体积为200ul,另设空白对照,即只加100ul的RPMI-1640培养液。在5%CO237℃培养箱孵育72h。加入2mg/ml MTT(噻唑蓝,3,4,5-dimethylthliazol-2,5-dipenyl tertrazolium bromid)溶液,50ul/孔,继续培养4h。取出培养板,离心2000rpm,离心5min。轻轻倾去孔内液体,吹干。加入异丙醇-盐酸溶液,暗室培养30min,然后充分振荡,用酶标仪(DIALAB,GMBH,Austria)在570nm波长测定OD(吸光度)值。每个样品进行3个重复,变异系数应小于10%。根据下列公式计算刺激指数(stimulation index,SI):
SI=含丝裂原培养物的OD570值/不含丝裂原培养物的OD570值
5、实验结果
(1)木鳖子含三萜皂甙提取物对小鼠增重没有影响,注射部位无刺激反应。
免疫前后各组小鼠平均体重见表2。在同一时间,各组小鼠的平均体重无统计学差异。木鳖子含三萜皂甙提取物在10~100μg剂量注射小鼠不影响增重,注射部位未见刺激反应。
表2免疫前后各组小鼠平均体重(X±SD)
组别 | 首免前* | 二免前* | 二免后二周* |
12345 | 19.7±1.07g20.6±1.55g20.4±0.27g20.5±0.83g20.5±0.94g | 21.6±0.96g21.2±1.53g22.2±0.64g22.3±0.31g21.7±1.37g | 22.3±1.15g22.0±1.92g23.0±0.56g22.8±0.26g22.5±1.37g |
*统计分析表明各组小鼠体重之间无显著差异(P>0.05)。
(2)血清抗OVA抗体效价测定结果表明:木鳖子含三萜皂甙提取物和血清抗体效价之间呈量效关系
二免后血清抗OVA IgG抗体效价检测结果见图1。木鳖子含三萜皂甙提取物和血清抗体效价之间呈量效关系。随着木鳖子含三萜皂甙提取物剂量的增加,血清抗体效价也随之升高。木鳖子含三萜皂甙提取物50微克剂量组的血清抗体效价显著高于对照组(P<0.01),但和铝胶佐剂组比较无显著差异(P>0.05);木鳖子含三萜皂甙提取物100微克剂量组的血清抗体效价显著高于对照组(P<0.01),并高于铝胶佐剂组但无统计学差异(P>0.05)。
(3)血清抗OVA的IgG1和IgG2a抗体水平测定结果表明:以50微克木鳖子含三萜皂甙提取物组为最高。
图1表示不同剂量木鳖子含三萜皂甙提取物和铝胶佐剂对小鼠血清抗OVA的IgG1和IgG2a抗体水平的影响。只有木鳖子含三萜皂甙提取物100μg剂量组血清的IgG1抗体水平显著高于对照组(P<0.05);其余各组个对照组比较无显著性差异(P>0.05)。木鳖子含三萜皂甙提取物50μg和100μg剂量组以及铝胶佐剂组血清IgG2a抗体水平显著高于对照组(P<0.05),以50微克鳖子含三萜皂甙提取物组为最高;木鳖子含三萜皂甙提取物10μg剂量组的血清IgG2a抗体水平和对照组比较无显著性差异(P>0.05)。
(4)淋巴细胞增殖反应测定结果显示:和不加佐剂的空白对照组铝胶相比,木鳖子提取物有显著增强了淋巴细胞对Con A和LPS刺激引起的增殖反应。
图3表示木鳖子含三萜皂甙提取物和铝胶可显著增强小鼠淋巴细胞对Con A刺激引起的增殖反应(P<0.05);但对LPS引起的淋巴细胞增殖反应,只有木鳖子含三萜皂甙提取物有显著增强作用(P<0.05)。
三、木鳖子含三萜皂甙提取物提高奶牛对W疫苗免疫反应的持续时间的试验
1、试验方法
将杭州市郊区CQ奶牛场的12头黑白花奶牛作为随机分为二组,每组6头。免疫时,每头奶牛肌肉注射2mlW病疫苗。第1组奶牛每次注射时疫苗中混入木鳖子含三萜皂甙提取物1mg,对照组奶牛不加木鳖子提取物。分别于免疫前,免疫后一个月采血,以后每两个月采一次血,连续采二次。共采血4次,分离血清,-20℃保存待用。检测W病抗体采用正向间接血凝试验,检测试剂使用中国农业科学院兰州兽医研究所提供的口蹄疫O型正向血凝抗原。被检血清在检测前经56℃水浴30分钟。检测方法根据兰州兽医研究所提供方法。
2、试验结果
表3结果表明,木鳖子含三萜皂甙提取物和W疫苗混合同时注射奶牛可以延长抗体的持续时间,因此可以减少免疫次数,减少对奶牛的应激。注射后未见注射部位局部刺激反应,因此,木鳖子含三萜皂甙提取物在试验剂量下对奶牛是安全的。
表3木鳖子提取物的延长奶牛对W疫苗免疫反应的持续时间
组别 | 动物数量 | 免疫前 | 免疫后时间 | ||
1个月 | 3个月 | 5个月 | |||
疫苗+木鳖子提取物 | 6 | 50% | 100% | 100% | 100% |
疫苗 | 6 | 0% | 100% | 33.3% | 50% |
四、木鳖子含三萜皂甙提取物提高奶牛对W疫苗免疫反应并减少疫苗用量
1、试验方法
将AJ牛场24头黑白花奶牛随机分为三组,每组8头。第1组每头牛注射3mlW病疫苗,第2组每头牛注射3ml疫苗并加木鳖子提取物1mg,第3组每头牛注射一半剂量(1.5ml)疫苗并加木鳖子提取物1mg。分别于免疫前,首免后2个月采血,分离血清,-20℃保存待用。检测W病抗体采用正向间接血凝试验,检测试剂使用中国农业科学院兰州兽医研究所提供的口蹄疫O型正向血凝抗原。被检血清在检测前经56℃水浴30分钟。检测方法根据兰州兽医研究所提供的说明方法。
2、试验结果
见表4,常规或一半疫苗剂量加木鳖子提取物组免疫合格率高于不加木鳖子提取物的常规剂量组。注射后未见注射部位局部刺激反应。
表4木鳖子提取物的提高奶牛对W疫苗免疫反应并减少疫苗用量的作用
组别 | 动物数 | 合格率 | |
免疫前 | 免疫后2个月 | ||
常规剂量 | 8 | 0% | 62.5% |
常规剂量组+木鳖子提取物 | 8 | 0% | 100% |
半剂量+木鳖子提取物 | 8 | 12.5% | 75% |
五、木鳖子含三萜皂甙提取物增强猪W疫苗的免疫佐剂作用
1、试验方法
将ZJ实验牧场23头60日龄仔猪进行W病疫苗免疫试验,猪随机分为2组。第1组11头,每头猪免疫时注射2ml疫苗;第2组12头,注射前疫苗中混合木鳖子含三萜皂甙提取物1mg。分别于免疫前、免疫3周后采血,分离血清,-20℃保存待用。检测抗体采用正向间接血凝试验,检测试剂使用中国农业科学院兰州兽医研究所提供的口蹄疫O型正向血凝抗原。被检血清在检测前经56℃水浴30分钟。检测方法根据兰州兽医研究所提供的说明方法。
2、试验结果
疫苗中混合木鳖子含三萜皂甙提取物1mg免疫仔猪可以提高免疫合格率。注射后未见注射部位局部刺激反应。
表5试验猪免疫前后免疫合格率
组别 | 免疫前 | 免疫后 |
W疫苗2ml | 36.4% | 36.4% |
W疫苗2ml+木鳖子提取物1mg | 33.3% | 50% |
木鳖子含三萜皂甙提取物可以同时促进体液免疫和细胞免疫反应,并以促进细胞免疫反应为主;而常用的氢氧化铝铝胶佐剂主要促进体液免疫反应。对于抗病毒感染而言,细胞免疫反应更为重要。
本发明用木鳖子含三萜皂甙提取物作为制作疫苗的佐剂,具有取材广泛、不引起资源匮缺、安全有效的优点,与铝胶佐剂相比,不会引起局部反应,可以加热消毒,也可冷冻保存,可明显增强生物体细胞免疫反应。
Claims (3)
1、木鳖子含三萜皂甙提取物作为佐剂与疫苗混合用于疫苗的制作。
2、按权利要求1,其特征在于:所述木鳖子含三萜皂甙提取物为从木鳖子中提取的去除毒性成分的含三萜皂甙的水溶液,或将该水溶液浓缩、干燥后得到的含三萜皂甙的黄色粉末状提取物。
3、按权利要求1和2,其特征在于:木鳖子含三萜皂甙提取物作为佐剂与疫苗混合的使用方法为:在疫苗制造阶段或者在疫苗注射之前,将木鳖子提取物和抗原混合。
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