CN112494569A - 一种提高免疫力的中药组合物及其制备方法和用途 - Google Patents
一种提高免疫力的中药组合物及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种提高免疫力的中药组合物及其制备方法和用途。本发明的中药组合物由下述重量份的中药制成:灵芝3~5份、枸杞子3~5份、杜仲3~5份、人参1~2份和马鹿茸0.5~1份。本发明的中药组合物提高机体免疫力具有高效、安全、适用人群广、药性温和等优点,适用于免疫力低下者长期服用。
Description
技术领域
本发明涉及中药领域,具体涉及一种提高免疫力的中药组合物及其制备方法和用途。
背景技术
中华中医药学会2006年发布的《亚健康中医临床指南》指出,“亚健康是指人体处于健康和疾病之间的一种状态。2016年世界卫生组织报告,全球亚健康人数超过60亿人,占全球总人口的85%。中国亚健康人数占中国总人口70%,约9.5亿人左右。处于亚健康状态者,不能达到健康的标准,表现为一定时间内的活力降低、功能和适应能力减退的症状,但不符合现代医学有关疾病的临床或亚临床诊断标准。”生活不规律、经常熬夜、吸烟、饮酒、摄入高热量高脂肪食物、缺乏运动等都是亚健康的常见原因。亚健康主要表现为人体各***的生理功能紊乱、衰退,或综合体能下降、精神不振等。神经***具体表现为:头晕、头痛、失眠、多梦、记忆力减退、精神不振;循环***具体表现为:心悸、心部隐痛、临界的高血压、血脂异常;消化***具体表现为:食欲不振、胃部隐痛、腹胀、消化不良、便秘等等;呼吸***具体表现为:憋气、气短、咽喉干涩或者堵塞感;感官***具体表现为:耳鸣、听力减退、眼睛干涩、酸胀;代谢***具体表现为:体重超标、肥胖;运动***具体表现为:动作迟缓,关节运动欠灵活、肌肉酸痛;内分泌***具体表现为:临界的甲状腺功能亢进、血糖增高或低血糖;综合体能具体表现为:工作效率低、容易疲劳、体力透支、手足冰冷、体质虚弱、性功能减退、自然衰老加速。
现代医学认为,免疫力是人体自身的天然防御机制,是机体抵抗外来侵袭、维护体内环境稳定性的能力,免疫功能下降是亚健康状态的普遍现象,提高免疫***功能是增强机体抵御多种不适症状、改善机体亚健康状态的有效手段。
具有免疫增强作用的中药药味众多,如人参、党参、桂圆、燕窝、鱼腥草、金银花、猪苓、大青叶、大蒜、板蓝根、南五加皮、女贞子、天麻、灵芝、枸杞子、黄芪、淫羊藿、绞股蓝、紫河车、桑椹、白术、菟丝子、干姜、龙胆草、三七、乌梅、白芍、地耳草、防风、香薷、黄精、蒲公英、金荞麦、茯苓、夏枯草、柴胡、山药、山楂、广枣、云芝、甘草、龙眼肉、当归、麦冬、杜仲、何首乌、青木香、杏仁、荠菜、鹿茸、酸枣、桑枝、黄荆叶、仙茅等,但单方药通常只能控制或缓解某些症状,存在疗效欠佳等缺陷。中药复方制剂通过君、臣、佐、使的中药配伍理论,使各药味之间产生协同作用,进而达到提高疗效、降低毒副作用等效果,是未来中药复方制剂的主要发展方向。
现代医学的免疫功能与中医领域的体虚理论密切相关。正所谓,“正气存内,邪不可干;邪之所凑,其气必虚”。益气、养血、滋阴、助阳等是现有中医药领域增强机体免疫力的基本组方策略。但是,免疫力低下往往是由多种综合因素导致,各个环节紧密相连,仅提高某些机体器官功能或一味以滋补为主,仅改善某一个环节并不适宜,不能满足免疫力低下人群的需求。而且,在不同的配伍理论指导下,众多的免疫力增强中药复方产品普遍存在疗效弱、副作用多、适用人群受限等缺陷,未见高效、安全、适用人群广、药性温和且适合长期服用的中药复方。例如:“四君子汤”为补气基础方,可提高巨噬细胞吞噬能力,用于治疗脾胃气虚,阴虚内热,不适于脘腹胀满者;“四物汤”为补血理血经典方,该处方偏温性,适于血虚型机体的免疫调节,不适于阴虚内热者;“六味地黄丸”为补阴方,可增强单核巨噬细胞吞噬能力和T、B淋巴细胞增殖,主治肾阴虚或肝肾阴虚,但处方中多为滋阴药,阳虚患者不宜使用。
因此,开发出一种高效、安全、适用人群广、药性温和、适合长期服用的提高机体免疫力的中药复方是迫切需要本领域技术人员解决的技术问题。
发明内容
发明人认为,免疫力低下属中医“虚劳”范畴。“虚劳”主要是由于机体气血亏损、阴阳失衡、五脏失养所致。“五脏之伤,穷必及肾”,肾为先天之本,主骨生精,藏真阴而寓元阳,为机体动力所在。
发明人以传统中医理论为基础,深入探究免疫力低下者的各种“虚劳”表现及内在原因,开创性地使用补气安神、滋补肝肾与生津养血的配伍理论,提出了以灵芝为君药,以枸杞子、杜仲共为臣药,人参与马鹿茸为佐使药的创新性组方,并运用现代医药免疫调节理论及研究成果对上述组方的安全性、有效性进行了科学验证。实验证明,本发明提供的提高免疫力的中药组合物具有高效、安全、适用人群广、药性温和、适合长期服用等诸多优点。
本发明的提高机体免疫力的中药组合物主要基于以下构思进行组方:
灵芝:味甘,性平,归肾、肝、心、肺经,具有补气安神的功效,《神农本草经》认为“益心气”、“安精魂”、“补肝益气”、“坚筋骨”等并列为上品;《本草纲目》也记载“滋补强壮”、“延年益寿”,久服使人“好颜色”等,为本方之君药。
枸杞子与杜仲:枸杞子味甘,性平,归肝肾经,有补肾益精,养肝明目之功效。杜仲味甘,性温,归肝、肾经,有补益肝肾,强壮筋骨的功效,“久服轻身耐老”(《神农本草经》),“能入肝补肾,补中益精气,坚筋骨,强志,治肾虚腰痛,久服,轻身耐老”(《本草纲目》)。枸杞子与杜仲,一补肝肾之精血,一补肝肾之气,共为本方之臣药。
人参与马鹿茸:人参味甘、微苦、微温,归脾、肺、心、肾经,具有大补元气、复脉固脱,补脾益肺,生津止渴,安神益智的作用。马鹿茸味甘、咸,性温,归肝、肾经,具有补肾阳,益精血,强筋骨,调冲任,托疮毒的作用。人参与马鹿茸,一补气,一补阳,为本方之佐使药。
本发明以灵芝为君药,补气安神,滋补强壮。枸杞子补肾益精,养肝明目;杜仲补益肝肾,强壮筋骨;枸杞子与杜仲,一补肝肾之精血,一补肝肾之气,共为本发明之臣药。人参善补元气,有补气生津作用;马鹿茸补肾阳,益精血;人参与马鹿茸,一补气,一补阳,为本发明之佐使药。灵芝、枸杞子、杜仲、人参与马鹿茸五药合用,相使互补,通过滋补肝肾、益气健脾、生津养血,阴阳气血双补,五脏六腑皆益,使机体强健,机体免疫力得以高效提升,且药性温和、安全,适合所有免疫力低下者长期服用。
本发明以中医君臣佐使理论和临床用药实践为基础,结合药理研究成果证实,与同样选用灵芝、枸杞子、杜仲、人参、马鹿茸的其他配伍构思相比,本发明在补气安神、滋补肝肾与生津养血的配伍理论指导下形成的以灵芝为君药、枸杞子与杜仲为臣药、人参与马鹿茸为佐使药的中药组合物在提高机体免疫力、使用安全性及适用人群范围等方面具有明显的优势,基于此,完成了本发明。
具体地,
本发明提供了一种中药组合物,其能够高效、安全地提高机体免疫力,并具有药性温和、适用人群广、适合长期服用等优点,其由下述重量份的中药制成:
其中,所述的灵芝为君药,枸杞子与杜仲为臣药,人参与马鹿茸为佐使药。
其中,所述的马鹿茸优选马鹿茸粉。
其中,所述的人参优选人参粉。
其中,作为本发明中药组合物的臣药,枸杞子与杜仲的重量比优选为1:1。
其中,作为本发明中药组合物的佐使药,人参与马鹿茸的重量比优选为1~2:1。
优选地,本发明提供了一种提高机体免疫力的中药组合物,其由下述重量份的中药制成:
其中,灵芝优选多孔菌科真菌赤芝的干燥子实体,全年采收,除去杂质,剪除附有朽木、泥沙或培养基质的下端菌柄,阴干,进行前处理(除去杂质、洗净、干燥);枸杞子优选茄科植物宁夏枸杞的干燥成熟果实,于夏、秋二季果实呈红色时采收,晾至皮皱后,晒干,除去果梗;杜仲优选杜仲科植物杜仲的干燥树皮,于4-6月剥取,刮去粗皮,堆置“发汗”至内皮呈紫褐色,晒干,进行前处理(刮去残留粗皮、洗净、切块、干燥);人参优选五加科植物人参的干燥根和根茎,洗净,干燥,进行前处理(润透、切薄片、干燥);马鹿茸为鹿科动物马鹿的雄鹿未骨化密生茸毛的幼角,优选经干燥、粉碎后所得粉末。
进一步地,本发明还涉及一种所述中药组合物的制备方法,包括如下步骤:
1)将灵芝、枸杞子、杜仲加水煎煮,煎液滤过,滤液浓缩、干燥、粉碎成混合粉;
2)将人参干燥、粉碎成人参粉,或直接称取人参粉;
3)将马鹿茸干燥、粉碎成马鹿茸粉,或直接称取马鹿茸粉;
4)将前述混合粉、人参粉与马鹿茸粉混匀,即得所述中药组合物。
优选地,其中所述混合粉、人参粉、马鹿茸粉应全部通过五号筛,并含能通过六号筛不少于95%的粉末。
优选地,其中所述步骤1)中有机溶剂为乙醇。
优选地,其中所述步骤1)中灵芝、枸杞子、杜仲加水煎煮至少2次,每次加5-15倍量水(更优选10倍量水),各煎煮1-3小时(更优选2小时),合并煎液,滤过;其中,滤过步骤采用150-300目筛,更优选200目筛。
优选地,其中所述步骤1)中滤液减压浓缩得浸膏,真空干燥得干膏,再粉碎成混合粉,过筛;其中,滤液减压浓缩的条件为-0.065~-0.075Mpa(75℃),浸膏相对密度为1.10~1.35(60℃),优选1.10~1.25(60℃),更优选1.10~1.20(60℃);真空干燥的条件为-0.085Mpa、65℃;过筛优选80目筛;滤液浓缩优选采用中药浓缩罐,以免有效成分长时间受热破坏。
优选地,其中所述步骤2)中人参的干燥温度为60℃,人参粉的粒度优选过100目筛,更优选将所述人参粉进行辐照灭菌,辐照条件优选5kGy剂量的60Co。
优选地,其中所述步骤3)中马鹿茸的干燥温度为60℃,马鹿茸粉的粒度优选过100目筛,马鹿茸粉在经过辐照灭菌后使用,辐照条件优选5kGy剂量的60Co。
进一步地,本发明还涉及所述的中药组合物在制备具有提高免疫力作用的产品中的应用,所述产品优选药物或保健食品。
进一步地,本发明还涉一种提高免疫力的中药制剂,其可以按照本领域的常规制剂制备技术,将所述中药组合物制备成胶囊剂、颗粒剂、片剂、丸剂、散剂或口服液等其他剂型。例如:对于胶囊剂,可以将本发明的中药组合物用85%乙醇制软材,16目筛制粒,55-60℃干燥,14目筛整粒,按0.35g/粒装入0号胶囊,经抛光、检验、包装,即可制得本发明的胶囊剂。
本发明中药制剂胶囊的推荐剂量为:口服2次/日,每次3粒(1.05g),或遵医嘱。
本发明的中药组合物取得了如下有益效果:
(1)本发明以补气安神、益心气、补肝益气的灵芝为君药,主攻机体“虚劳”所致气血亏损。
(2)本发明开创性地为君药灵芝辅以臣药枸杞子与杜仲的双倍滋补肝肾效用,有效激发并保持机体的肝肾精血与肝肾之气,机体气血得以双补。特别是,臣药枸杞子、杜仲与君药灵芝近乎等量用药,且共同进行水煎煮提取等,君臣二药协同作用,达到了机体气血亏损恢复迅速,效用持久的技术效果。
(3)本发明以生津养血的佐使药人参、马鹿茸进一步补益五脏,与君药、臣药相比,药量近乎减半,既激发灵芝、枸杞子、杜仲迅速发挥气血双补之效,又制约灵芝、枸杞子与杜仲之气血双补之烈,并解机体阴阳失衡、五脏失养之症,成本亦有降低。
(4)科学实验表明,本发明的中药组合物经口连续给予小鼠30天,对小鼠体重、脏/体比值、碳廓清能力均无明显影响;对迟发型***反应、抗体生成细胞数、HC50、巨噬细胞吞噬鸡红细胞能力、NK细胞活性及ConA诱导的小鼠淋巴细胞转化均有增强或促进作用,即本发明利用现代医学手段证实了本发明的中药组合物对机体的细胞免疫、体液免疫、单核-巨噬细胞功能及NK细胞活性均有明显的促进作用,具有安全、高效增强机体免疫力的功能。
(5)本发明利用中医药君臣佐使理论,结合机体免疫力低下之“虚劳”内因,巧妙地以灵芝为君药,以枸杞子、杜仲为臣药,以人参、马鹿茸为佐使药,使得全方高效、安全、药性温和、适用者众、适合免疫力低下者长期服用。
(6)与其他构思的组方,如以生津养血的人参、马鹿茸为君药,以补气安神的灵芝为臣药,以滋补肝肾的枸杞子、杜仲为佐使药的组方(对照例1);以滋补肝肾的枸杞子为君药,以生津养血的人参为臣药,以补气安神的灵芝、滋补肝肾的杜仲为佐药,以生津养血的马鹿茸为使药的组方(对照例2);以滋补肝肾的枸杞子、杜仲为君药,以补气安神的灵芝、生津养血的马鹿茸为臣药,以生津养血的人参为佐药的组方(对照例3)相比,本发明的中药组合物在用药安全性、有效性及适用人群范围等方面具有明显优势。即,与其他组方策略或构思相比,本发明以补气安神、滋补肝肾为主,并辅以生津养血的组方(实施例1~3)在提高机体免疫力方面更有效、适用人群范围更广。
(7)人参、马鹿茸名贵,成粉性好。本发明的中药组合物以粉末入药,既可兼做赋形剂,避免胶囊内容物吸潮、结块,又可最大程度保留其有效成分;既提高了产品稳定性,又减少了辅料使用,降低了生产成本。
具体实施方式
下面结合具体实施例、对照例对本发明的技术方案作详细说明。本发明所用中药原料均购自西安藻露堂药业集团西京医药有限公司。
实施例1本发明中药组合物的制备
(1)灵芝、枸杞子、杜仲水煎煮
取灵芝5.0kg、枸杞子3.5kg、杜仲3.5kg,加水煎煮2次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.30~1.35(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得混合粉。
(2)人参干燥、粉碎
取人参1.5kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得人参粉。
(3)马鹿茸干燥、粉碎
取马鹿茸1.0kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得马鹿茸粉。
(4)中药组合物
将混合粉、人参粉、马鹿茸粉混匀,得本发明的中药组合物4.50kg。
实施例2本发明中药组合物的制备
(1)灵芝、枸杞子、杜仲水煎煮
取灵芝5.0kg、枸杞子3.5kg、杜仲4.5kg,加水煎煮3次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.10~1.20(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得混合粉。
(2)人参干燥、粉碎
取人参1.0kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得人参粉。
(3)马鹿茸干燥、粉碎
取马鹿茸0.5kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得马鹿茸粉。
(4)中药组合物
将混合粉、人参粉、马鹿茸粉混匀,得本发明的中药组合物3.37kg。
实施例3本发明中药组合物的制备
(1)灵芝、枸杞子、杜仲水煎煮
取灵芝4.5kg、枸杞子4.5kg、杜仲3.5kg,加水煎煮2次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.20~1.25(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得混合粉。
(2)人参粉、马鹿茸粉
取人参粉1.0kg和马鹿茸粉1.0kg,过100目筛,辐照灭菌(5kGy,60Co)。
(3)中药组合物
将混合粉、人参粉、马鹿茸粉混匀,得本发明的中药组合物3.95kg。
实施例4本发明中药组合物的制备
(1)灵芝、枸杞子、杜仲水煎煮
取灵芝3.5kg、枸杞子4.0kg、杜仲4.0kg,加水煎煮3次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.10~1.20(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得混合粉。
(2)人参粉、马鹿茸粉
取人参粉2.0kg和马鹿茸粉1.0kg,过100目筛,辐照灭菌(5kGy,60Co)。
(3)中药组合物
将混合粉、人参粉、马鹿茸粉混匀,得本发明的中药组合物4.71kg。
实施例5本发明中药组合物胶囊的制备
取实施例1制备的中药组合物4.5kg,用85%乙醇制软材,16目筛制粒,55-60℃干燥,14目筛整粒,按0.35g/粒装入0号胶囊,抛光,检验,包装,制得本发明的中药组合物胶囊剂11712粒。
对照例1对照中药组合物的制备
(1)人参、马鹿茸水煎煮
取人参4.5kg、马鹿茸4.5kg,加水煎煮2次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.30~1.35(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得人参与马鹿茸混合粉。
(2)灵芝干燥、粉碎
取灵芝3.0kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得灵芝粉。
(3)枸杞子、杜仲干燥、粉碎
取枸杞子1.0kg、杜仲1.5kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得枸杞子与杜仲混合粉。
(4)中药组合物
将人参与马鹿茸混合粉、灵芝粉、枸杞子与杜仲混合粉混匀,制得对照中药组合物6.57kg。
对照例2对照中药组合物的制备
(1)枸杞子、人参水煎煮
取枸杞子5.0kg、人参4.0kg,加水煎煮3次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.20~1.25(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得枸杞子与人参混合粉。
(2)灵芝、杜仲干燥、粉碎
取灵芝2.0kg、杜仲2.0kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得灵芝与杜仲粉。
(3)马鹿茸干燥、粉碎
取马鹿茸1.5kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得马鹿茸粉。
(4)中药组合物
将枸杞子与人参混合粉、灵芝与杜仲粉、马鹿茸粉混匀,制得对照中药组合物6.62kg。
对照例3对照中药组合物的制备
(1)枸杞子、杜仲水煎煮
取枸杞子4.0kg、杜仲5.0kg,加水煎煮2次,每次加入10倍量水,各煎煮2小时,煎液滤过(200目不锈钢筛),滤液合并,减压浓缩(-0.065~-0.075Mpa,75℃)至相对密度为1.10~1.20(60℃)的浸膏,真空干燥(-0.085Mpa,65℃)得干膏后粉碎,过80目筛得枸杞子与杜仲混合粉。
(2)灵芝、马鹿茸干燥、粉碎
取灵芝2.0kg、马鹿茸2.5kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得灵芝与马鹿茸混合粉。
(3)人参干燥、粉碎
取人参1.0kg,60℃干燥后粉碎,过100目筛,辐照灭菌(5kGy,60Co)后得人参粉。
(4)中药组合物
将枸杞子与杜仲混合粉、灵芝与马鹿茸混合粉、人参粉混匀,制得对照中药组合物6.84kg。
本发明中药组合物安全性、有效性实验方法
取Balb/C小鼠384只(中国人民解放军军事医学科学院实验动物中心提供,合格证号0008159,SPF级,雌雄各半,体重18g-22g),随机分为4批(每批96只),每批随机分为8组(每组12只),即每批设4个实验组、3个对照组和1个空白对照组。连续灌胃30日后,第1批进行脏器/体重比值、小鼠碳廓清试验,第2批进行迟发型***反应、抗体生成细胞检测、HC50测定,第3批进行小鼠腹腔巨噬细胞吞噬鸡红细胞试验,第4批进行ConA诱导的小鼠淋巴细胞转化实验、NK细胞活性测定。
各组的受试液配置方法及给药方式、剂量等如下表所示:
实验例1本发明中药组合物对小鼠体重、脏器/体重比值的影响
实验组、对照组、空白对照组小鼠的初始体重、末期体重是均衡的,本发明的中药组合物对小鼠体重无明显影响,详见下表1~2。
表1各组小鼠的初始体重
组别 | 小鼠数量 | 第1批 | 第2批 | 第3批 | 第4批 |
实验组1 | 12只 | 19.3±0.9g | 19.0±0.7g | 19.3±1.1g | 19.3±1.0g |
实验组2 | 12只 | 19.4±1.3g | 19.3±0.9g | 19.2±1.0g | 19.0±0.8g |
实验组3 | 12只 | 19.3±1.1g | 19.2±1.0g | 20.0±1.1g | 19.4±0.9g |
实验组4 | 12只 | 19.3±0.7g | 19.1±1.2g | 20.1±1.0g | 19.6±1.0g |
对照组1 | 12只 | 19.4±0.8g | 19.1±0.8g | 19.6±0.8g | 19.8±1.1g |
对照组2 | 12只 | 19.5±1.0g | 19.3±0.8g | 19.2±1.0g | 19.3±0.9g |
对照组3 | 12只 | 19.3±1.2g | 19.1±1.1g | 19.5±0.9g | 19.7±0.6g |
空白对照组 | 12只 | 19.3±0.9g | 19.1±0.5g | 19.6±1.2g | 19.5±0.6g |
表2各组小鼠的末期体重
将小鼠称重后脱臼处死,取脾脏与胸腺,滤纸吸干表面血污称重,计算脾脏/体重比值和胸腺/体重比值。结果显示,实验组、对照组、空白对照组的小鼠脏/体比值无明显差异,本发明的中药组合物对小鼠脏/体比值无明显影响,详见下表3。
表3本发明中药组合物对小鼠脏器/体重比值的影响
组别 | 小鼠数量 | 脾脏/体重(mg/g) | 胸腺/体重(mg/g) |
实验组1 | 12只 | 3.46±0.35 | 2.10±0.31 |
实验组2 | 12只 | 3.43±0.27 | 2.04±0.18 |
实验组3 | 12只 | 3.27±0.19 | 2.37±0.26 |
实验组4 | 12只 | 3.35±0.19 | 2.35±0.25 |
对照组1 | 12只 | 3.36±0.25 | 2.15±0.21 |
对照组2 | 12只 | 3.40±0.20 | 2.10±0.15 |
对照组3 | 12只 | 3.37±0.15 | 2.27±0.20 |
空白对照组 | 12只 | 3.48±0.29 | 2.21±0.51 |
实验例2本发明中药组合物对小鼠碳廓清能力的影响
小鼠尾静脉注射1:4稀释的印度墨汁,按每10g体重0.1ml计算,待墨汁注入,立即计时。墨汁注入后2、10min,分别从内眦静脉丛取血20μl,并立即将其加到2ml 0.1%碳酸钠溶液中。用半自动生化分析仪在600nm波长处测定光密度(OD),以碳酸钠溶液作空白对照。将小鼠处死,取肝脏和脾脏,用滤纸吸干脏器表面血污,称重,按下式计算吞噬指数a:
K=(lgOD1-lgOD2)/(t2-t1),a=体重÷(肝重+脾重)×K1/3
结果表明,对照组小鼠的碳廓清能力(吞噬指数a)虽优于空白对照组,但明显弱于实验组,而实验组小鼠的碳廓清能力则明显优于对照组和空白对照组。可以预期,本发明的中药组合物可以显著增强机体的非特异免疫功能,详见下表4。
表4本发明中药组合物对小鼠碳廓清能力(吞噬指数a)的作用
实验例3本发明中药组合物对小鼠迟发型***反应能力的影响
绵阳红细胞诱发小鼠DTH(足跖增厚法):取羊血,用生理盐水洗涤3次(2000r/min,10分钟),每只鼠经腹腔注射2%(v/v,用生理盐水配制)压积SRBC 0.2ml,致敏后4天,测量左后足跖部厚度,同一部位测量三次,取平均值。然后在测量部位皮下注射20%(v/v,用生理盐水配制)压积SRBC 20μl,于注射后24小时测量左后足跖部厚度,以攻击前后足跖厚度的差值来表示DTH的程度。
结果表明,对照组小鼠的24h足跖肿胀度高于空白对照组,而实验组小鼠的24h足跖肿胀度明显高于对照组和空白对照组,本发明的中药组合物对小鼠的迟发型***反应能力具有增强作用,详见下表5。
表5本发明中药组合物对绵羊红细胞(SRBC)诱导小鼠DTH的作用
组别 | 小鼠数量 | 24h足跖肿胀度(mm) |
实验组1 | 12只 | 0.31±0.09 |
实验组2 | 12只 | 0.30±0.07 |
实验组3 | 12只 | 0.32±0.12 |
实验组4 | 12只 | 0.34±0.10 |
对照组1 | 12只 | 0.25±0.05 |
对照组2 | 12只 | 0.28±0.14 |
对照组3 | 12只 | 0.23±0.10 |
空白对照组 | 12只 | 0.21±0.09 |
实验例4本发明中药组合物对小鼠抗体生成细胞的影响
抗体生成细胞检测(Jerne改良载片法):将压积SRBC用生理盐水配成2%的细胞悬液,每只鼠腹腔注射0.2ml进行免疫,五天后,将小鼠颈椎脱臼处死,取出脾脏,放在盛有Hank’s液的研磨器内,轻轻磨碎脾脏,制成细胞悬液,经200目筛网过滤,离心(1000r/min)10min,用Hank’s液洗2遍,最后将细胞悬浮在8ml RPMI 1640培养液中。将表层培养基(1g琼脂糖加双蒸水至100ml)加热溶解后,放45℃水浴保温,与等量pH7.2-7.4、2倍浓度的Hank’s液混合,分装小试管,每管0.5ml,再向管内加50μ1 10%SRBC(v/v,用SA液配制),25μl脾细胞悬液,迅速混匀,倾倒于已刷琼脂糖薄层的玻片上,做平行片,待琼脂凝固后,将玻片水平扣放在片架上,放入二氧化碳培养箱中温育1.5h,然后用SA缓冲液稀释的补体(1:6)加入到玻片架凹槽内,继续温育1.5h后,计数溶血空斑数。
结果表明,对照组小鼠的溶血空斑数明显高于空白对照组,而实验组小鼠的溶血空斑数明显高于对照组和空白对照组,本发明的中药组合物对小鼠抗体生成细胞的生成具有显著的促进作用。可以预期,本发明的中药组合物可以显著提高机体的体液免疫功能,详见下表6。
表6本发明中药组合物对抗体生成细胞数的影响
组别 | 小鼠数量 | 溶血空斑数(个) |
实验组1 | 12只 | 166.5±30.9 |
实验组2 | 12只 | 153.6±25.8 |
实验组3 | 12只 | 154.9±30.2 |
实验组4 | 12只 | 158.3±28.6 |
对照组1 | 12只 | 122.6±22.6 |
对照组2 | 12只 | 133.6±24.4 |
对照组3 | 12只 | 124.9±31.8 |
空白对照组 | 12只 | 103.0±15.3 |
实验例5本发明中药组合物对小鼠半数溶血值的影响
半数溶血值(HC50)的测定:将压积SRBC用生理盐水配成2%的细胞悬液,每只小鼠腹腔注射0.2ml进行免疫,五天后,摘眼球取血,分离血清。血清用SA缓冲液稀释300倍,取稀释后的血清1ml,依次加入10%SRBC 0.5ml,补体1ml(用SA缓冲液按1:6稀释),另设不加血清对照管(用SA缓冲液代替)。置37℃恒温水浴25min,冰浴终止反应。2000r/min离心10min,取上清液1ml,都氏剂3ml于试管内,同时取10%SRBC(v/v)0.25ml,加都氏剂至4ml于另一试管内,充分混匀,放置10min后,于540nm处测定各管光密度值。
样品HC50=(样品光密度值/SRBC半数溶血时的光密度值)×稀释倍数
结果表明,实验组小鼠的HC50明显高于对照组和空白对照组,本发明的中药组合物对小鼠半数溶血值有增高作用,详见下表7。
表7本发明中药组合物对小鼠血清溶血素(半数溶血值HC50)的影响
组别 | 小鼠数量 | HC<sub>50</sub> |
实验组1 | 12只 | 187.7±19.3 |
实验组2 | 12只 | 190.2±19.6 |
实验组3 | 12只 | 196.7±26.9 |
实验组4 | 12只 | 192.3±22.5 |
对照组1 | 12只 | 169.9±16.7 |
对照组2 | 12只 | 160.2±17.6 |
对照组3 | 12只 | 176.7±25.4 |
空白对照组 | 12只 | 140.6±24.7 |
实验例6本发明中药组合物对小鼠腹腔巨噬细胞吞噬鸡红细胞能力的影响
小鼠腹腔巨噬细胞吞噬鸡红细胞试验(体外法):实验前4天给每只小鼠腹腔注射2%压积绵羊红细胞0.2ml。用颈椎脱臼法处死小鼠,经腹腔注射给予小鼠含有5%小牛血清的Hank’s液5ml/只,轻揉腹部20次后抽取腹腔清洗液2ml,取0.5ml腹腔清洗液加入盛有Hank’s液配制成1%鸡RBC悬液0.5ml的试管内,混匀,取混悬液0.5ml涂片,放入垫有湿纱布的瓷盘内,置37℃温箱内25min,取出后用生理盐水从玻片背面冲洗,固定、染色、阅片。计算吞噬率和吞噬指数。
结果表明,对照组与空白对照组小鼠的巨噬细胞吞噬鸡红细胞吞噬百分率及吞噬指数无明显差异,而实验组小鼠的巨噬细胞吞噬鸡红细胞吞噬百分率及吞噬指数均明显高于对照组和空白对照组,本发明的中药组合物对小鼠巨噬细胞吞噬鸡红细胞能力具有显著的升高作用,详见下表8。
表8本发明中药组合物对小鼠腹腔巨噬细胞吞噬鸡红细胞能力的影响
组别 | 小鼠数量 | 吞噬率(%) | 吞噬指数 |
实验组1 | 12只 | 31.7±2.3 | 0.44±0.04 |
实验组2 | 12只 | 36.5±3.7 | 0.50±0.05 |
实验组3 | 12只 | 38.4±4.0 | 0.54±0.05 |
实验组4 | 12只 | 35.8±3.5 | 0.51±0.04 |
对照组1 | 12只 | 21.7±2.5 | 0.32±0.05 |
对照组2 | 12只 | 26.3±3.2 | 0.37±0.04 |
对照组3 | 12只 | 28.5±3.8 | 0.40±0.04 |
空白对照组 | 12只 | 24.3±2.5 | 0.35±0.04 |
实验例7本发明中药组合物对小鼠淋巴细胞转化的影响
ConA诱导的小鼠脾淋巴细胞转化实验(MTT法):无菌取脾,置于盛有适量无菌Hank’s液的研磨器内,轻轻磨碎脾脏,制成细胞悬液,经200目筛网过滤。用Hank’s液洗2次,每次离心10min(1000r/min)。然后将细胞悬浮于2mL的完全培养液中,计数活细胞数,调整细胞浓度为3×105个/ml。再将细胞悬浮液分两孔加入24孔培养板中,每孔1ml,在其中一孔加75μl ConA液(相当于7.5μg/ml),另一孔作为对照,置5%CO2,37℃CO2培养箱中培养72h。培养结束前4h,每孔轻轻吸去上清液0.7ml,加入0.7ml不含小牛血清的RPMI 1640培养液,同时加入MTT(5mg/ml)50μl/孔,继续培养4h。培养结束后,每孔加入1ml酸性异丙醇,吹打混匀,使紫色结晶完全溶解。用半自动生化分析仪以570nm处测光密度值。淋巴细胞的增值能力用加ConA孔的光密度值减去不加ConA孔的光密度值表示。
结果表明,对照组ConA诱导的小鼠淋巴细胞的增殖能力稍优于空白对照组,而实验组ConA诱导的小鼠淋巴细胞的增殖能力明显优于对照组和空白对照组,本发明的中药组合物对ConA诱导的小鼠淋巴细胞转化具有显著的增强作用,详见下表9。
表9本发明中药组合物对ConA诱导的小鼠脾淋巴细胞转化的影响
实验例8本发明中药组合物对小鼠NK细胞活性的影响
NK细胞活性测定:无菌取脾,置于盛有适量无菌Hank’s液的研磨器内,轻轻磨碎脾脏,制成细胞悬液,经200目筛网过滤,用Hank’s液洗2次,每次离心10min(1000r/min),最后用RPMI 1640完全培养液调整细胞浓度为2×107个/ml,取靶细胞和效应细胞各100μl(效靶比50:1),加入U型96孔培养板;靶细胞自然释放孔加靶细胞和培养液各100μl,靶细胞最大释放孔加靶细胞和1%NP40各100μl;上述各项均设三个复孔,于37℃、5%CO2培养箱中培养4h,然后将96孔培养板以1500r/min离心5min,每孔吸取上清100μl置平底96孔培养板,同时加入LDH基质液100μl,反应3min,每孔加入1mol/l的盐酸30μl,在酶标仪490nm处测定光密度值(OD)。
NK细胞活性%=[(反应孔OD-自然释放孔OD)/(最大释放孔OD-自然释放孔OD)]×100%
结果表明,实验组小鼠的NK细胞活性明显高于对照组和空白对照组,本发明的中药组合物对小鼠NK细胞活性具有显著的促进作用,详见下表10。
表10本发明中药组合物对小鼠NK细胞活性的影响
组别 | 小鼠数量 | NK细胞活性% |
实验组1 | 12只 | 51.2±5.8 |
实验组2 | 12只 | 49.5±4.3 |
实验组3 | 12只 | 52.5±4.6 |
实验组4 | 12只 | 52.7±4.0 |
对照组1 | 12只 | 32.2±4.2 |
对照组2 | 12只 | 35.5±5.3 |
对照组3 | 12只 | 37.5±5.7 |
空白对照组 | 12只 | 21.4±2.6 |
Claims (10)
1.一种中药组合物,由下述重量份的中药制成:
灵芝 3~5份,
枸杞子 3~5份,
杜仲 3~5份,
人参 1~2份,
马鹿茸 0.5~1份。
2.根据权利要求1所述的中药组合物,其中所述的灵芝为君药,枸杞子与杜仲为臣药,人参与马鹿茸为佐使药。
3.根据权利要求1所述的中药组合物,其中所述的马鹿茸为马鹿茸粉。
4.一种权利要求1-3任一项所述中药组合物的制备方法,包括如下步骤:
1)将灵芝、枸杞子、杜仲加水煎煮,煎液滤过,滤液浓缩、干燥、粉碎成混合粉;
2)将人参干燥、粉碎成人参粉,或直接称取人参粉;
3)将马鹿茸干燥、粉碎成马鹿茸粉,或直接称取马鹿茸粉;
4)将前述混合粉、人参粉与马鹿茸粉混匀,即得所述中药组合物。
5.根据权利要求4所述中药组合物的制备方法,其中所述混合粉、人参粉、马鹿茸粉为全部通过五号筛,并含通过六号筛不少于95%的粉末。
6.根据权利要求4所述中药组合物的制备方法,其中所述步骤1)中灵芝、枸杞子、杜仲加水煎煮至少2次。
7.一种中药制剂,其由权利要求1-3任一项所述的中药组合物制成。
8.根据权利要求7所述的中药制剂,其为胶囊剂、颗粒剂、片剂、丸剂、散剂或口服液。
9.根据权利要求8所述的中药制剂,其中所述胶囊剂的内容物含量为0.35g/粒。
10.权利要求1-3任一项所述中药组合物或权利要求7-9任一项所述的中药制剂在制备具有提高免疫力作用的产品中的应用。
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