CN102781457B - 免疫球蛋白a分泌促进剂 - Google Patents
免疫球蛋白a分泌促进剂 Download PDFInfo
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- CN102781457B CN102781457B CN201180012014.XA CN201180012014A CN102781457B CN 102781457 B CN102781457 B CN 102781457B CN 201180012014 A CN201180012014 A CN 201180012014A CN 102781457 B CN102781457 B CN 102781457B
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Abstract
迄今已知的IgA抗体的分泌促进剂中大多数与肠内IgA抗体产生有关,而与口腔、鼻腔和呼吸道等呼吸系统的器官中的IgA分泌促进作用和感染抑制作用有关的仅有极少数。因此,本发明的目的是提供不仅在肠内而且在口腔和呼吸道等呼吸系统器官中也具有IgA分泌促进作用的医药组合物。含有虞美人提取物的IgA分泌促进剂不仅具有肠内的IgA分泌促进效果,在口腔和呼吸道内也具有IgA分泌促进作用,因此,通过摄取所述IgA分泌促进剂,可以抑制病毒和细菌的感染,此外也能预防过敏的发生。
Description
技术领域
本发明涉及可望具有对细菌和病毒等感染预防效果和过敏预防效果的IgA分泌促进剂,特别是可望具有在上呼吸道的IgA分泌促进效果的组合物。
背景技术
在口腔、呼吸道、消化道等生物体内部与外界相连的表面上构筑有为了防止病毒和细菌等从外界侵入生物体内的被称为粘膜免疫的强有力的防御体系。在粘膜表面,从唾液腺和粘液腺等源源不断地分泌粘液,在对病毒和细菌的侵入进行物理防御的同时,通过粘液中存在的抗体、抗菌肽、酶等的作用,防御生物体受到外敌入侵。在所述防御体系中承担中心作用的是免疫球蛋白A(IgA)抗体,具有除去病毒和细菌等以及防止它们在粘膜表面粘附等的作用(非专利文献1)。
关于IgA抗体和感染症的关系,报道了在动物实验中使对于中耳炎的病因菌的特异性IgA抗体增加以及病因菌的感染被抑制的研究结果(非专利文献2)。此外,报道了如果将某种感冒的病因病毒的特异性IgA抗体经由鼻腔给药,病毒的感染被抑制的研究结果(非专利文献3)。
此外,认为IgA抗体除了具有预防生物体受到细菌和病毒等的感染的作用之外,还能阻止诸如引起过敏反应的外来抗原从粘膜侵入生物体内(非专利文献4)。
从以上的背景可知,如果能够促进IgA抗体的分泌,对于细菌和病毒等的感染症预防和过敏的预防是有效的。
迄今为止已公开的具有IgA抗体的分泌促进作用的有各种乳酸菌和担子菌(专利文献1~4)、多糖类和低聚糖类(专利文献5~9、非专利文献5),多数与肠内的IgA抗体产生有关,而与口腔、鼻腔和呼吸道等呼吸系统的器官的IgA分泌促进作用和感染抑制作用有关的仅是少数(专利文献2、专利文献10)。
现有技术文献
专利文献
专利文献1:日本专利第2968374号公报
专利文献2:日本专利第3818319号公报
专利文献3:日本专利特开平11-92389号公报
专利文献4:日本专利特开2005-97133号公报
专利文献5:日本专利第4162147号公报
专利文献6:日本专利特开2001-64181号公报
专利文献7:日本专利特开2003-201239号公报
专利文献8:日本专利特开2006-70217号公报
专利文献9:日本专利特开2006-213671号公报
专利文献10:日本专利特开2009-161447号公报
专利文献11:日本专利特开2002-370993号公报
非专利文献
【非专利文献1】Brandtzaeg P.、International Journal of MedicalMicrobiology(《医学微生物学国际期刊》)、2003年、293卷、1号、3~15页、Role of secretory antibodies in the defence against infections(分泌抗体在防御感染中的作用).
【非专利文献2】Hotomi M.等、Vaccine(《疫苗》)、1998年、16卷、20号、1950~1956页、Specific mucosal immunity and enhancednasopharyngeal clearance of nontypeable Haemophilus influenzae afterintranasal immunization with outer membrane protein P6and cholera toxin(非典型流感嗜血杆菌在鼻内用外膜蛋白P6和霍乱毒素免疫后的特殊粘膜免疫和增强的鼻咽清洁).
【非专利文献3】Heikkinen T.等、Pediatric Infectious DiseaseJournal(《儿科感染病医学期刊》)、1998年、17卷、5号、367~372页、Intranasally administered immunoglobulin for the prevention of rhinitisin children(用于预防儿童鼻炎的鼻腔内给药免疫球蛋白).
【非专利文献4】池泽善郎、低アレルギー食品の開発(《低能量食品的开发》)、シーエムシー(CMC)出版、41-49页
【非专利文献5】Scholtens P.A.等、Journal of Nutrition(《营养学杂志》)、2008年、138卷、6号、1141~1147页、Fecal secretoryimmunoglobulin A is increased in healthy infants who receive a formulawith short-chain galacto-oligosaccharides and long-chainfructo-oligosaccharides(粪便分泌免疫球蛋白A在服用短链半乳糖-低聚糖和长链果糖低聚糖处方的健康婴儿中被促进).
发明内容
鉴于上述课题,本发明的目的在于提供不仅在肠内而且在口腔和呼吸道内也具有IgA分泌促进作用的医药组合物。
发明人深入研究,结果发现,虞美人(ヒナゲシ)提取物具有IgA分泌促进作用,并由此完成了本发明。
本发明的含有虞美人提取物的IgA分泌促进剂不仅具有肠内的IgA分泌促进效果,在口腔和呼吸道内也具有IgA分泌促进作用,因此,通过摄取所述IgA分泌促进剂,可以抑制病毒和细菌的感染,此外也能预防过敏的发生。
具体实施方式
本发明中具有IgA分泌促进作用的虞美人(学名:Papaver rhoeas L.)是罂粟科植物,别名为虞美人草。据称具有镇静、催眠、止痛作用等,用于止咳药和镇静剂。
与含有草药的免疫赋活剂有关的专利(专利文献11)中,草药的例子仅记载了罂粟,但是缺乏发明的具体性,也没有关于IgA的记载。
此外,本发明中使用的虞美人提取物可以使用水或水与亲水性有机溶剂的混合物提取虞美人的花、叶、茎,优选虞美人的花。
提取方法和溶剂的种类没有特别限定。作为溶剂,除了水之外,可以使用乙醇、甲醇、丙醇、甘油、丙二醇等醇类,醚、丙酮、乙酸乙酯、氯仿、己烷等有机溶剂,或它们的混合溶剂。但出于安全性方面的考虑,理想的是使用乙醇或其混合溶液提取。
作为获得提取物的提取条件,可以在高温、室温、低温的任一种温度下提取,优选在40~90℃提取0.5~5小时左右。
提取物可以以溶液的状态直接使用,也可以进一步过滤、蒸馏除去提取溶剂后,通过减压干燥或冷冻干燥制成粉末状,或浓缩后使用。此外,也可以使用通过有机溶剂分配、柱色谱法等将这些提取物分离纯化而得的制品。进而,也可以加入除臭、脱色等纯化处理。
虞美人提取物的含量没有特别限定,优选相对于饮食物或制剂,换算成干燥重量含有0.01重量%以上,更优选0.1~90重量%。
虞美人提取物的摄取方法也没有特别限定,可以是口服摄取、通过舌下给药摄取、通过鼻腔给药摄取、皮下或血管内等局部直接给药等所有公知的方法。
含有本发明的虞美人提取物的医药组合物可以作为药品、医药部外品(准药物,介于药品和化妆品之间的一类)或饮食品等使用。
为药品时,以口服制剂的形式给药。作为口服制剂的剂型,可例举片剂、胶囊剂、散剂、糖浆剂、饮剂等。为医药部外品时,以片剂、胶囊剂、饮剂等剂型使用。为饮食品时,除了饮料等饮品和糖果、脆饼、饼干等食品以外,可例举烟草和口香糖等嗜好品、饲料和饲料类、漱口剂和牙膏等化妆品类等。这些只不过是例举,本发明可以用于任何物品。
为药品或医药部外品时,可以含有药理学上可接受的一种或两种以上的载体,还可以根据需要含有其它用于治疗的有效成分。除此之外,还可以含有赋形剂、粘合剂、崩解剂、润滑剂、分散剂、表面活性剂、增塑剂、混悬剂、乳化剂、稀释剂、缓冲剂、抗氧化剂、细菌抑制剂等。
此外,饮食品也可以含有食品制造上可接受的基材、载体、赋形剂、添加剂、增量剂、着色剂、香料等。
虞美人提取物以外的成分也不受上文的限定,本发明可以任意含有公知的任何成分。
含有本发明的虞美人提取物的药品、医药部外品或饮食品等可以通过各技术领域内公知的任意方法制造。其制造过程中可以通过公知的任何方法添加虞美人提取物。
本发明的含有虞美人提取物的医药组合物不仅可以对人使用,也可以对人以外的动物(以下简称为非人动物)使用。作为非人动物,可例举哺乳类、爬行类、两栖类、鱼类等人以外的动物。
实施例
以下利用实施例对本发明进行说明,但这些实施例不对本发明的范围造成任何限定。
[实施例1]
热水提取物的制备
粉碎100g虞美人花后,加入相对于试样重量的10倍量的水,在70℃下提取2小时。过滤不溶物后,冷冻干燥提取液,得到热水提取物37.5g。
[实施例2]
使用来自派尔集合淋巴结(パイエル板;Peyer's patch)的淋巴球细胞的IgA抗体产生促进作用的体外评价
摘取小鼠(BALB/c系、雌、6周龄)的小肠,采集派尔集合淋巴结,浸渍于含有2%牛胎儿血清(FCS)的RPMI1620培养基。用剪刀剪断派尔集合淋巴结,用セルストレーナー(BD生物科技公司(BD Biosciences)制、孔径70μm)回收淋巴球细胞。离心分离回收的细胞悬浊液,吸去上清液后,加入完全培养基,将淋巴球细胞悬浊液的细胞浓度调整为4×106细胞/毫升。完全培养基使用含有10%FCS、50μM 2-巯基乙醇、1mM丙酮酸钠、100U/ml青霉素、100μg/ml链霉素的RPMI1620培养基。在96孔盘的各孔内各加入溶解了淋巴球细胞悬浊液和试样的培养基(试样调整为最终浓度100ppm)0.05ml,在37℃、5%C02的条件下培养1周。回收培养液离心分离后,通过夹心ELISA法测定上清液中的总IgA抗体量。以不添加试样培养的对照的总IgA抗体量为1,以相对值算出添加试样培养时的总IgA抗体量。
通过夹心ELISA法测定总IgA抗体量
采用上述夹心ELISA法进行的测定按照如下顺序进行。
将1μ/ml的抗鼠源免疫球蛋白抗体溶液加入96孔ELISA板,各100μl,4℃下使其反应1晚,使抗体吸附在板上。从板上除去溶液,加入含有1%牛血清白蛋白(BSA)的磷酸盐缓冲液(PBS)各180μl,在室温下培养1小时,阻断目的外蛋白质的非特异性吸附。用含有0.05%吐温20的PBS(PBST)清洗各孔,然后加入试样溶液,在室温下培养2小时。用PBST清洗各孔,在各孔内加入过氧化酶结合抗鼠源IgA抗体各25ng,在室温下培养1小时,然后加入过氧化酶基质使其反应2分钟。加入0.5N盐酸使反应停止,测定450nm处的吸光度。使用鼠源IgA抗体作为标准物质制作校准曲线,测定IgA抗体量。
结果如表1所示,以100ppm的浓度添加虞美人(花)热水提取物时培养基中IgA抗体量是什么都不添加时的2.6倍。
[表1]
[实施例3]
向小鼠反复给与混合饲料引起的IgA分泌促进作用的评价-1
以5%(w/w)的比例在普通饲料(MF,东方酵母公司(オリエンタル酵母))中混合虞美人(花)热水提取物,配制试验饲料。将6只7周龄的小鼠(BALB/c系)分为对照组和试验组(每组3只),使对照组自由摄取普通饲料,使试验组自由摄取试验饲料,饲养6周。饲养开始后每周回收粪便,使其分别悬浊在磷酸缓冲液中以使浓度为100mg/ml,离心分离后,与实施例2同样,用夹心ELISA法测定上清液中的总IgA抗体量。
0周的总IgA抗体量为1,将各个体相对于它的相对值结果示于表2。以平均值作比较,给药组的粪便中总IgA抗体量的相对值高于对照组。
[表2]
IgA水平测定结果
[实施例4]
向小鼠反复给予混合饲料引起的IgA分泌促进作用的评价-2
以5%(w/w)的比例在普通饲料(MF,东方酵母公司(オリエンタル酵母))中混合虞美人(花)热水提取物,配制试验饲料。将12只7周龄的小鼠(BALB/c系)分为对照组和试验组(每组6只),使对照组自由摄取普通饲料,使试验组自由摄取试验饲料,饲养。在第4周经鼻腔给予50μg的磷酸胆碱-钥孔血兰蛋白(PC-KLH、对细菌的共同抗原)免疫。然后依旧每周经鼻腔免疫,从第3周的鼻腔免疫开始一周后回收鼻腔粘液(100~200μl/只)和鼻腔粘膜固有层淋巴球细胞(1×105~5×105细胞/只),测定总IgA抗体量和总IgA抗体产生细胞数、PC特异性IgA抗体效价和PC特异性IgA抗体产生细胞数。此外,回收粪便,使其悬浊在磷酸缓冲液中以成为100mg/ml的浓度,离心分离后,测定上清液中的总IgA抗体量。总IgA抗体量和PC特异性IgA抗体效价的测定采用夹心ELISA法进行,PC特异性IgA抗体产生细胞数的测定采用酶联免疫斑点检测(ELISPOT)法进行。从各动物采集的鼻腔粘膜固有层淋巴球细胞的数目非常少,因此,将每3只的细胞汇集成一组,连续2次测定IgA抗体产生细胞数。
通过夹心ELISA法测定总IgA抗体量
上述总IgA抗体量的测定与实施例2同样采用夹心ELISA法进行。此外,上述PC特异性IgA抗体量的测定,除了最初使用PC-BSA代替吸附在ELISA板上的抗鼠源免疫球蛋白抗体之外,与实施例2同样地通过夹心ELISA法进行。
通过ELISPOT法测定IgA抗体产生细胞数
上述通过ELISPOT法测定PC特异性IgA抗体产生细胞数按照以下顺序进行。
在市售的96孔ELISPOT用硝基纤维素膜板上添加抗鼠源免疫球蛋白抗体(5μg/ml PBS)或PC-BSA(5μg/ml PBS)100μl,在4℃下使其反应1晚并吸附。除去抗体溶液,用完全培养基封闭后,加入适当稀释的来自鼻腔粘膜固有层的淋巴球细胞悬浊液,在37℃、5%CO2的条件下培养4小时。用PBS清洗后,加入100ng的过氧化酶结合抗鼠源IgA抗体,使其在4℃下反应1晚。用PBS清洗后,加入发色基质(3-氨基-9-乙基咔唑)溶液,使其在室温、避光条件下发色。用水清洗并使其干燥后,在实体显微镜下计数斑点作为抗体产生细胞。
粪便中的总IgA量测定
回收粪便,使其悬浊在PBS中以使浓度为100mg/ml,离心分离后,与实施例2同样,用夹心ELISA法测定上清液中的总IgA抗体量。
结果如表3和表4所示。鼻腔粘液、粪便测定结果都显示,总IgA量是给药组高,鼻腔粘膜固有层的总IgA产生细胞数也是给药组高。认为,通过给予虞美人提取物,在肠管和呼吸系统的IgA分泌被促进。
[表3]
IgA水平测定结果
[表4]
IgA产生细胞数测定结果
[实施例5]
向小鼠反复给予混合饲料引起的IgA分泌促进作用的评价-3
以5%(w/w)的比例在普通饲料(MF,东方酵母公司(オリエンタル酵母))中混合虞美人(花)热水提取物,配制试验饲料。将20只7周龄的小鼠(BALB/c系)分为对照组和试验组(每组10只),用与实施例4同样的方法测定鼻腔粘液的总IgA抗体量和PC特异性IgA抗体效价、粪便的总IgA抗体量。
结果如表5所示。给药组的鼻腔粘液总IgA量为高值,认为通过给予虞美人提取物的混合饲料,呼吸系统的IgA分泌被促进。
[表5]
IgA水平测定结果
[实施例6]
向小鼠反复舌下给药引起的IgA分泌促进作用的评价-1
将7周龄的小鼠(BALB/c系)以每组4只分为对照组和给药组,用普通饲料(MF,东方酵母公司)饲养。给药组从饲养开始时以4mg/天的用量每天舌下给予虞美人(花)热水提取物的水溶液,对照组仅舌下给予同量的水。在第4周经鼻腔给予50μg的PC-KLH免疫,此后依旧每周经鼻腔免疫,从第3次的经鼻腔免疫开始1周后回收鼻腔粘液和鼻腔粘液固有层淋巴球细胞,测定总IgA抗体量和总IgA抗体产生细胞数。总IgA抗体量的测定与实施例2同样用夹心ELISA法,IgA抗体产生细胞数的测定与实施例4同样采用酶联免疫斑点(ELISPOT)检测法进行。能够从各动物采集的鼻腔粘膜固有层淋巴球细胞的数目非常少,因此,将每4只的细胞汇集在一起,连续2次测定IgA抗体产生细胞数。
结果如表6和表7所示,给药组的鼻腔粘膜的总IgA量、总IgA产生细胞数为高值,认为通过舌下给予虞美人提取物,在呼吸系统的IgA分泌被促进,IgA产生细胞增加。
[表6]
[表7]
[实施例7]
向小鼠反复舌下给药引起的IgA分泌促进作用的评价-2
将7周龄的小鼠(BALB/c系)以每组15只分为对照组和给药组,用普通饲料(MF,东方酵母公司)饲养。给药组从饲养开始时以4mg/天的用量每天舌下给予虞美人(花)热水提取物的水溶液,对照组仅舌下给予同量的水。在第4周经鼻腔给予50μg的PC-KLH免疫,此后依旧每周经鼻腔免疫,从第3次的经鼻腔免疫开始1周后回收鼻腔粘液和鼻腔粘液固有层淋巴球细胞,测定总IgA抗体量和总IgA抗体产生细胞数、PC特异性IgA抗体效价和PC特异性IgA抗体产生细胞数。此外,回收粪便,使其悬浊在磷酸缓冲液中,离心分离后,测定上清液中的总IgA抗体量。总IgA抗体量和PC特异性IgA抗体效价的测定与实施例2同样用夹心ELISA法,IgA抗体产生细胞数的测定与实施例4同样采用酶联免疫斑点(ELISPOT)检测法进行。能够从各动物采集的鼻腔粘膜固有层淋巴球细胞的数目非常少,因此,将每3只的细胞汇集在一起,连续2次测定IgA抗体产生细胞数。
结果如表8和表9所示,给药组的各项值都比对照组高,认为通过舌下给予虞美人花提取物,IgA产生细胞数和IgA分泌量在肠管和呼吸系统中均增加。
[表8]
IgA水平测定结果
[表9]
IgA产生细胞数测定结果
[实施例8]
向小鼠反复经鼻腔给药引起的IgA分泌促进作用的评价-1
将7周龄的小鼠(BALB/c系)以每组6只分为对照组和给药组,用普通饲料(MF,东方酵母公司)饲养。给药组从饲养开始时以2mg/天的用量每天经鼻腔给予虞美人(花)热水提取物的水溶液,对照组仅经鼻腔给予同量的水。在第4周经鼻腔给予50μg的PC-KLH免疫,此后依旧每周经鼻腔免疫,从第3次的经鼻腔免疫开始1周后回收鼻腔粘液和鼻腔粘液固有层淋巴球细胞,测定总IgA抗体量和总IgA抗体产生细胞数。总IgA抗体量的测定与实施例2同样用夹心ELISA法,IgA抗体产生细胞数的测定与实施例4同样采用酶联免疫斑点(ELISPOT)检测法进行。能够从各动物采集的鼻腔粘膜固有层淋巴球细胞的数目非常少,因此,将每3只的细胞汇集在一起,连续2次测定IgA抗体产生细胞数。
结果如表10和表11所示,给药组的鼻腔粘膜的总IgA量、总IgA产生细胞数为高值,认为通过经鼻腔给予虞美人提取物,在呼吸系统的IgA分泌被促进,IgA产生细胞增加。
[表10]
[表11]
[实施例9]
向小鼠反复经鼻腔给药引起的IgA分泌促进作用的评价-2
将5~6月龄的小鼠(BALB/c系)以每组5只分为对照组和给药组,用普通饲料(MF,东方酵母公司)饲养。给药组从饲养开始时以2mg/天的用量每天经鼻腔给予虞美人(花)热水提取物的水溶液,对照组仅经鼻腔给予同量的水。在第4周经鼻腔给予50μg的PC-KLH免疫,此后依旧每周经鼻腔免疫,从第3次的经鼻腔免疫开始1周后回收鼻腔粘液和鼻腔粘液固有层淋巴球细胞,测定总IgA抗体量和总IgA抗体产生细胞数。总IgA抗体量的测定与实施例2同样用夹心ELISA法,IgA抗体产生细胞数的测定与实施例4同样采用酶联免疫斑点(ELISPOT)检测法进行。能够从各动物采集的鼻腔粘膜固有层淋巴球细胞的数目非常少,因此,将每2~3只的细胞汇集在一起,连续2次测定IgA抗体产生细胞数。
结果如表12和表13所示,给药组的鼻腔粘膜的总IgA量、总IgA产生细胞数为高值,认为通过经鼻腔给予虞美人提取物,在呼吸系统的IgA分泌被促进,IgA产生细胞增加。
[表12]
[表13]
此外,使用实施例1中制备的虞美人提取物依常法配制成药丸、散剂、吸入剂、滴鼻剂、口香糖、糖果、巧克力、饼干、软糖、压片糖、冰淇淋、雪糕、饮料。以下显示为它们的配方。本发明的范围不受这些限制。
[实施例10]
按照以下配方配制药丸。
[实施例11]
按照以下配方配制药丸。
[实施例12]
按照以下配方配制胶囊剂。
[实施例13]
按照以下配方配制散剂。
[实施例14]
按照以下配方配制吸入剂。
[实施例15]
按照以下配方配制滴鼻剂。
[实施例16]
按照以下配方配制口香糖。
[实施例17]
按照以下配方配制口香糖。
[实施例18]
按照以下配方配制口香糖。
[实施例19]
按照以下配方配制糖果。
[实施例20]
按照以下配方配制糖果。
[实施例21]
按照以下配方配制巧克力。
[实施例22]
按照以下配方配制饼干。
[实施例23]
按照以下配方配制软糖。
[实施例24]
按照以下配方配制压片糖。
[实施例25]
按照以下配方配制压片糖。
[实施例26]
按照以下配方配制片剂。
[实施例27]
按照以下配方配制冰淇淋。
[实施例28]
按照以下配方配制雪糕。
[实施例29]
按照以下配方配制饮料。
[实施例30]
按照以下配方配制饮料。
Claims (7)
1.虞美人提取物在制备IgA分泌促进剂中的用途,所述虞美人提取物是通过水、乙醇或其混合溶液在40~90℃对虞美人进行提取而得的提取物。
2.如权利要求1所述的用途,其特征在于,制备成含有换算成干燥重量为0.01%~90%的所述IgA分泌促进剂的医药组合物。
3.如权利要求1所述的用途,其特征在于,口服摄取所述虞美人提取物。
4.如权利要求1所述的用途,其特征在于,通过舌下给药摄取所述虞美人提取物。
5.如权利要求1所述的用途,其特征在于,通过鼻腔给药摄取所述虞美人提取物。
6.如权利要求1所述的用途,其特征在于,制备成含有所述IgA分泌促进剂的食品。
7.如权利要求1所述的用途,其特征在于,制备成含有所述IgA分泌促进剂的医药组合物。
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JP2968374B2 (ja) | 1991-05-16 | 1999-10-25 | 株式会社ヤクルト本社 | IgA産生促進剤 |
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JP2002370993A (ja) | 2001-06-13 | 2002-12-24 | Bussan Biotech Kk | ハーブを含む免疫賦活剤 |
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JP2005097133A (ja) | 2003-09-22 | 2005-04-14 | Unitika Ltd | ハナビラタケ由来IgA産生促進剤 |
JP2006070217A (ja) | 2004-09-03 | 2006-03-16 | Kitasato Inst:The | オウギ属植物地上部由来の多糖および生体防御機能賦活化剤 |
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