CN110300524B - 免疫活化剂的制造方法、免疫活化用饮食品的制造方法以及包含免疫活化剂的药品 - Google Patents
免疫活化剂的制造方法、免疫活化用饮食品的制造方法以及包含免疫活化剂的药品 Download PDFInfo
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Abstract
本发明包含将平卧菊三七在0℃以上且40℃以下干燥的干燥工序和将通过上述干燥工序干燥后的平卧菊三七在保持于0℃以上且40℃以下的同时切断或粉碎的切断/粉碎工序。
Description
技术领域
本发明涉及免疫活化剂的制造方法和免疫活化用饮食品的制造方法。
背景技术
免疫是用于保护身体不受细菌、病毒等抗原入侵到体内所带来的不良影响的防御系统。生物体内的免疫大致分为自然免疫和获得性免疫这两种。
自然免疫会在抗原入侵体内后立即起作用,并且将抗原的信息传递给获得性免疫。在此,自然免疫涉及巨噬细胞、自然杀伤细胞、树突细胞等,进行抗原的通过消化或分解的排除、细胞因子的产生等。
另外,获得性免疫通过由巨噬细胞等自然免疫相关的免疫细胞进行了信号转导的T细胞来进行。在此,对于参与获得性免疫的T细胞而言,未成熟的辅助性T细胞受到细胞因子等的分化诱导刺激,从而变成Th1细胞或Th2细胞。Th1细胞参与细胞免疫,使保护性免疫/抗肿瘤免疫等生物体防御能力增强。另外,Th2细胞参与体液免疫。
另外,对于以增强保护性免疫/抗肿瘤免疫等生物体防御能力、治疗或预防感染症和癌症为目的的免疫活化剂,正在进行各种研究(例如,参照专利文献1和专利文献2)。
专利文献1中对五层龙属植物或其提取物进行了研究,专利文献2中对来自茶叶的儿茶素类的代谢物进行了研究。另外,如专利文献2中也记载了“儿茶素代谢物对NK细胞(自然杀伤细胞)活性的作用尚属未知”那样,认为关于来自天然物质的成分,存在许多未知的作用。
现有技术文献
专利文献
专利文献1:日本特开2010-235544号公报
专利文献2:日本特开2016-160238号公报
发明内容
发明所要解决的问题
本发明的目的在于,提供来自天然物质的新的免疫活化剂的制造方法和免疫活化用饮食品的制造方法。
用于解决问题的方法
本发明人进行深入研究的结果发现,通过使用菊三七(菊科菊三七属),可以达到上述目的,从而完成了本发明。
即,根据本发明,提供:
(1)一种免疫活化剂的制造方法,其包含:将平卧菊三七(Gynura procumbens)在0℃以上且40℃以下干燥的干燥工序;和将通过上述干燥工序干燥后的平卧菊三七在保持于0℃以上且40℃以下的同时切断或粉碎的切断/粉碎工序。
(2)根据(1)所述的免疫活化剂的制造方法,其中,作为上述平卧菊三七,使用平卧菊三七的叶或茎中的至少一者。
(3)根据(1)或(2)所述的免疫活化剂的制造方法,其中,还包含:添加维生素类、橄榄或其提取物以及蕺菜或其提取物中的至少一种的工序。
(4)一种免疫活化用饮食品的制造方法,其包含:在食品或饮料中配合通过(1)~(3)中任一项所述的免疫活化剂的制造方法得到的免疫活化剂的工序。
发明效果
根据本发明,可提供来自天然物质的新的免疫活化剂的制造方法和免疫活化用饮食品的制造方法。
附图说明
图1是示出免疫诱导反应的研究结果的图。
图2是示出免疫诱导反应的研究结果的图。
图3是示出免疫诱导反应的研究结果的图。
图4是示出自然免疫反应的研究结果的图。
图5是示出获得性免疫反应的研究结果的图。
图6是示出对癌细胞的直接性细胞杀伤效果的研究结果的图。
图7是示出杀伤细胞、自然杀伤细胞的活化介导的癌细胞抑制活性的研究结果的图。
具体实施方式
以下对本发明的免疫活化剂的制造方法和免疫活化用饮食品的制造方法进行说明。本发明的免疫活化剂的制造方法包含将菊三七在0℃以上且40℃以下干燥的干燥工序、和将通过上述干燥工序干燥后的菊三七在保持于0℃以上且40℃以下的同时切断或粉碎的切断/粉碎工序。
菊三七属于菊科菊三七属。作为菊三七属,可列举红凤菜(Gynura bicolor)、木耳菜(Gynura cusimbua)、白子菜(Gynura divaricata)、兰屿木耳菜(Gynura eliptica)、白凤菜(Gynura formosana)、菊三七(Gynura japonica(Gynura segetum(土三七))、尼泊尔菊三七(Gynura nepalensis)、平卧菊三七(Gynura procumbens)、狗头七(Gynurapseudochina)等,可以优选使用平卧菊三七。
作为平卧菊三七,可以使用叶、茎、根等中的任一者,优选使用叶或茎,更优选使用叶。
菊三七优选使用采集后干燥并切断或粉碎的菊三七。干燥优选在0℃以上且40℃以下、优选0℃以上且36℃以下、更优选0℃以上且34℃以下的温度条件下进行。另外,优选进行低温除湿干燥。
在比上述温度高的温度下进行干燥时,认为菊三七所含的成分中的发挥本发明效果的成分发生变性或分解,从而得不到本发明的效果。另外,在切断或粉碎之前或之后冷冻保存的情况下,也得不到本发明的效果。
另外,关于进行低温除湿干燥时的湿度,优选以相对湿度计为10%以下。另外,低温除湿干燥优选进行几小时~50小时,更优选进行10小时~30小时。
另外,将菊三七切断或粉碎时的方法可以通过公知的切断或粉碎方法来进行,优选在使菊三七的叶保持于上述干燥条件中的温度范围的同时进行切断或粉碎。为了进行这种方法,优选使用冷却式粉碎机,例如,可以使用边冷却边进行粉碎的珠磨机、均化器、水冷式石磨等。
需要说明的是,从使菊三七的保存状态保持良好的观点出发,不优选将菊三七用榨汁机等处理而以包含水分的状态保存。优选如本发明那样以使菊三七干燥后的状态保存。
以上述方式处理后的菊三七可以作为免疫活化剂使用。
以上述方式处理后的菊三七中,可以添加橄榄或其提取物、蕺菜或其提取物。另外,可以向本发明的包含菊三七或其提取物的免疫活性剂中添加维生素、氨基酸、蛋白质、矿物质成分、脂肪酸、肽等追加成分。作为追加成分,优选添加维生素,作为维生素,可列举维生素A、维生素B、维生素C、维生素E或这些维生素类的衍生物等。
以上述方式处理后的菊三七与橄榄或其提取物、蕺菜或其提取物以及追加成分的合计量的重量比率优选为50:50~100:0、更优选为70:30~100:0、进一步优选为80:20~100:0。
通过本发明的免疫活化剂的制造方法得到的免疫活化剂可以配合于食品、饮料等中。作为食品,可列举面包类、面条类、点心类、肉食加工品、鱼贝加工品、冷冻食品、果冻类、冰激凌类、乳制品、各种调味料等。另外,除了一般食品以外,还可以配合于特定保健用食品、医药部外品、健康食品、补充剂中。作为饮料,可列举清凉饮料水、乳饮料、酒类、茶、红茶饮料、咖啡、果汁饮料、碳酸饮料、矿物质水类、果蔬饮料等。
另外,可以将配合有通过本发明的免疫活化剂的制造方法得到的免疫活化剂的食品、饮料制成与片剂、胶囊剂、糖浆等经口给药制剂同样的形态。
另外,在制造配合有通过本发明的免疫活化剂的制造方法得到的免疫活化剂的食品、饮料时,可以在不妨碍本发明的效果的范围内根据需要添加甜味料、着色料、保存料、增稠剂、稳定剂、凝胶化剂、抗氧化剂、显色剂、漂白剂、乳化剂、膨胀剂、酸味料、光泽剂、香料等添加剂;溶剂;油。这些添加剂可以单独使用一种,也可以组合使用两种以上。
上述食品、饮料中所配合的免疫活化剂的比例可以根据使用目的适当调整,上述食品、饮料中所配合的免疫活化剂的比例优选为0.0001~80重量%、更优选为0.003~50重量%、进一步优选为0.005~30重量%。
另外,通过本发明的制造方法得到的免疫活化剂在药品领域中以治疗或预防感染症、癌症为目的的用途中有用。本发明的免疫活化剂可以单独使用,或者可以与通常制剂上可接受的添加剂一起混合并制剂化。另外,作为给药形态,可列举使用片剂、颗粒剂、胶囊剂、丸剂、散剂、液剂、悬浊剂、乳剂、糖浆剂、酏剂、浸膏剂等经口剂的给药形态或者使用注射剂、液剂、栓剂、软膏剂、贴剂、巴布剂、洗剂等非经口剂的给药形态等,但没有特别限制,可以根据治疗目的等适当选择。
另外,在片剂、颗粒剂、丸剂、胶囊剂、散剂的情况下,可以含有赋形剂、结合剂、崩解剂、润滑剂等添加剂。作为赋形剂,可列举淀粉、羧甲基纤维素、白糖、糊精、玉米淀粉等。
作为结合剂,可列举结晶纤维素、结晶纤维素-羧甲基纤维素钠、甲基纤维素、羟丙基纤维素、低取代度羟丙基纤维素、羟丙基甲基纤维素、羟丙基甲基纤维素邻苯二甲酸酯、醋酸羟丙基甲基纤维素琥珀酸酯、羧甲基纤维素钠、乙基纤维素、羧甲基乙基纤维素、羟乙基纤维素、小麦淀粉、大米淀粉、玉米淀粉、马铃薯淀粉、糊精、α化淀粉、部分α化淀粉、羟丙基淀粉、普鲁兰多糖、聚乙烯基吡咯烷酮、甲基丙烯酸氨基烷基酯共聚物E、甲基丙烯酸氨基烷基酯共聚物RS、甲基丙烯酸共聚物L、甲基丙烯酸共聚物、聚乙烯醇缩乙醛二乙基氨基乙酸酯、聚乙烯醇、阿拉伯胶、阿拉伯胶末、琼脂、明胶、白色虫胶、黄蓍胶、精制白糖、聚乙二醇(Macrogol)。
作为崩解剂,可列举结晶纤维素、甲基纤维素、低取代度羟丙基纤维素、羧甲基纤维素、羧甲基纤维素钙、羧甲基纤维素钠、交联羧甲基纤维素钠、小麦淀粉、大米淀粉、玉米淀粉、马铃薯淀粉、部分α化淀粉、羟丙基淀粉、羧甲基淀粉钠、黄蓍胶。
作为润滑剂,可列举小麦淀粉、大米淀粉、玉米淀粉、硬脂酸、硬脂酸钙、硬脂酸镁、含水二氧化硅、轻质无水硅酸、合成硅酸铝、干燥氢氧化铝凝胶、滑石、硅酸铝镁、磷酸氢钙、无水磷酸氢钙、蔗糖脂肪酸酯、蜡类、氢化植物油、聚乙二醇。
另外,在液剂、糖浆剂、悬浊剂、乳剂、酏剂的情况下,除了水、植物油等通常使用的惰性稀释剂以外,还可以含有着色剂、矫味剂、着香剂等作为添加剂。
另外,在注射剂的情况下,可以含有悬浊液、乳浊液、临用时溶解剂等添加剂。另外,在软膏剂、栓剂的情况下,可以含有脂肪、脂肪油、羊毛脂、凡士林、石蜡、蜡、树脂、塑料、基剂、二醇类、高级醇、水、乳化剂、助悬剂等作为添加剂。另外,在巴布剂的情况下,可以含有甘油、水、水溶性高分子、吸水性高分子等作为添加物。另外,在洗剂的情况下,可以含有溶剂、乳化剂、助悬剂等作为添加剂。
实施例
以下列举实施例来说明本发明,但本发明不受其限定。需要说明的是,本实施例中的份和%在无特别声明时为重量基准。
在本实施例中,对于菊三七,进行(1)免疫诱导反应的研究、(2)自然免疫反应和获得性免疫反应的研究、(3)对癌细胞的细胞抑制活性的研究。
(1)免疫诱导反应的研究
(样本的制备)
作为免疫诱导反应的研究中使用的菊三七的叶的样本,制备下述4种。
样本A.将采集的菊三七(平卧菊三七)的叶在20~30℃、相对湿度10%以下的条件下进行低温除湿干燥。将低温除湿干燥后的菊三七的叶在20~30℃下粉碎,加入到超纯水中,从而得到样本。
样本B.将采集的菊三七(平卧菊三七)的叶在室温(约25℃)下用低速榨汁机处理,使用所得到的液体和纤维质这两者,作为样本。
样本C.将采集的菊三七(平卧菊三七)的叶在室温(约25℃)下用低速榨汁机处理,仅使用所得到的液体和纤维质中的液体,作为样本。
样本D.将样本B冷冻,然后解冻,作为样本。
另外,对于枇杷的叶、山白竹的叶、明日叶的叶、蕺菜的叶,直接将叶作为样本使用。
(免疫诱导反应的研究)
将样本A~D、枇杷的叶、山白竹的叶、明日叶的叶和蕺菜的叶的样本分别稀释至1/10000,添加到小鼠脾脏细胞中,通过定量PCR法研究此时的细胞因子-mRNA量的变动。
更具体而言,对于(i)无免疫诱导反应剂、(ii)使用作为免疫诱导反应剂的伴刀豆球蛋白A(ConA)2μg/mL、(iii)使用脂多糖(LPS)1μg/mL的情况,对6种细胞因子-mRNA的变动进行了研究。另外,对于不加入上述样本地进行上述(i)~(iii)的情况(以下称为“仅药物刺激”。)也进行了研究。
另外,作为细胞因子,对于IL1a、IFN、TNF、IL4、IL12、IL2这6种进行了研究。将结果示于图1~3。需要说明的是,以在细胞内稳定表达的mRNA(GAPDH)为基准,将与其相对表达量之差表示为Delta Ct值(PCR循环数之差)。将结果示于图1~3。
根据图1~3,由IL1a、IFN、IL4的上升程度认为,在菊三七的叶的样本(样本A~D)中,低温除湿干燥后进行粉碎的样本A可以高效地提取或者保持引发免疫反应的有效成分。另外,由IL1a、IFN、IL4的上升程度暗示了,山白竹的叶和蕺菜的叶的样本的作用强。
(2)自然免疫反应和获得性免疫反应的研究
(样本的制备)
关于自然免疫反应和获得性免疫反应的研究中使用的菊三七的叶的样本,使用上述免疫诱导反应的研究中使用的样本A(以下,在该项目中称为“菊三七的叶的样本”)。
另外,对于蕺菜的叶、橄榄的叶,也作为样本使用。另外,对于岩藻多糖,也按照与菊三七的叶的样本的浓度为同等浓度的方式制备样本。
(自然免疫反应的研究)
从小鼠中分离巨噬细胞,将各样本分别稀释至1/100、1/1000、1/10000和1/100000后进行添加,然后添加一定量的荧光珠,培养8小时。然后,用荧光显微镜对培养后的巨噬细胞进行观察,将摄入的荧光珠量以荧光量进行数值化。
作为样本,使用菊三七的叶、蕺菜的叶、橄榄的叶和岩藻多糖的样本、以及上述4种的混合样本。另外,对于不使用这些样本的对照(Control),也进行了研究。
另外,作为数值化,使用荧光显微镜获取表示荧光珠的摄入状态的图像,基于图像中一定的荧光亮度以上的总面积进行数值化处理。将结果示于图4。图4中,以直方图示出对各样本按照各个添加量(稀释倍率)进行数值化而得到的摄入荧光量。
(获得性免疫反应的研究)
分离小鼠脾脏淋巴细胞(CD4+T细胞),将各样本分别稀释至1/100、1/1000和1/10000后进行添加,然后培养1周。如果产生IFN-γ则表示T细胞分化为Th1细胞,如果产生IL-4则表示T细胞分化为Th2细胞。
因此,将培养后产生IFN-γ的淋巴细胞作为Th1,将培养后产生IL-4的淋巴细胞作为Th2,用荧光标记抗体测定各自的存在。根据测定结果,对于各样本按照各个稀释倍率求出Th1/Th2的比率。将结果示于图5。需要说明的是,关于图5的混合样本的稀释倍率为10000倍的数据,Th1/Th2为94.5,但从图5的易懂性的观点考虑省略了表示。
由图4和图5可知,对于菊三七的叶的样本而言,与自然免疫反应相比,更强地引发获得性免疫反应。由此认为,如果活化了杀伤T细胞、自然杀伤细胞等、或者观察到对肿瘤细胞的细胞杀伤效果等,则可观察到抗肿瘤效果。
(3)对癌细胞的细胞抑制活性的研究
(样本的制备)
关于对癌细胞的细胞抑制活性的研究中使用的菊三七的叶的样本,使用上述免疫诱导反应的研究中的样本A。(以下,在该项目中称为“菊三七的叶的样本”)
另外,对于蕺菜的叶、橄榄的叶,也作为样本使用。另外,对于岩藻多糖,也按照与菊三七的叶的样本的浓度为同等浓度的方式制备样本。
(对癌细胞的直接性细胞杀伤效果的研究)
培养癌细胞,对直接添加各样本时的使细胞死亡的细胞杀伤效果进行研究。作为癌细胞,对3种进行研究,使用实体癌、血液癌、表皮癌的代表性小鼠癌细胞株(CT-26:大肠癌、EL-4:淋巴瘤、B16mel:黑素瘤)。需要说明的是,作为样本,使用菊三七的叶、蕺菜的叶、橄榄的叶和岩藻多糖的样本、以及上述4种的混合样本。将结果示于图6。
各样本对EL-4、B16mel均未显示出效果,但对CT-26观察到一些效果。特别是在橄榄的叶和蕺菜的叶的样本中,其效果强。
(杀伤细胞、自然杀伤细胞的活化介导的癌细胞抑制活性的研究)
向从小鼠脾脏分离出的淋巴细胞中添加各种样本,培养1周。然后,将另行培养的3种癌细胞与分别培养的淋巴细胞合并后继续培养。在此,如果杀伤活性被诱导,则淋巴细胞会抑制癌细胞。为了测定这一点,使用细胞抑制活性测定试剂盒(Takara制造的LDH细胞毒性检测试剂盒)进行测定。
需要说明的是,作为样本,使用菊三七的叶、蕺菜的叶、橄榄的叶和岩藻多糖的样本、以及上述4种的混合样本。另外,对于对照,也作为样本使用。作为癌细胞,使用CT-26、EL-4和B16mel。
另外,作为淋巴细胞,分别从与3种癌细胞适应的种和与3种癌细胞不适应的种中分离出。癌细胞被适应性淋巴细胞抑制、且癌细胞不被非适应性淋巴细胞抑制这一点可以认为是癌症特异性杀伤活性的诱导。将结果以相对于对照的比例示于图7。
如图7所示,观察到适应性淋巴细胞的杀伤活性、未观察到非适应性淋巴细胞的杀伤活性的是菊三七的叶、蕺菜的叶和橄榄的叶(果实)的样本,其作用共通地对CT-26较强,另外,对于B16mel而言,仅菊三七的叶的样本观察到效果。另外,整体上作用最强的是菊三七的叶的样本。
需要说明的是,对于4种的混合样本而言,得到了岩藻多糖会抑制其它样本的效果这样的结果。
Claims (5)
1.一种免疫活化剂的制造方法,其包含:
将平卧菊三七(Gynura procumbens)在0℃以上且40℃以下、并且相对湿度为10%以下的条件下进行低温除湿干燥的干燥工序;和
将通过所述干燥工序干燥后的平卧菊三七在保持于0℃以上且40℃以下的同时切断或粉碎的切断/粉碎工序。
2.如权利要求1所述的免疫活化剂的制造方法,其中,作为所述平卧菊三七,使用平卧菊三七的叶或茎中的至少一者。
3.如权利要求1或2所述的免疫活化剂的制造方法,其中,还包含:
添加维生素类、橄榄或其提取物以及蕺菜或其提取物中的至少一种的工序。
4.一种免疫活化用饮食品的制造方法,其包含:
在食品或饮料中配合通过权利要求1~3中任一项所述的免疫活化剂的制造方法得到的免疫活化剂的工序。
5.一种药品,其包含通过权利要求1~3中任一项所述的制造方法得到的免疫活化剂。
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