CN110897160A - 一种蛹虫草龟鹿蛋白肽口服液及其制备方法 - Google Patents
一种蛹虫草龟鹿蛋白肽口服液及其制备方法 Download PDFInfo
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Abstract
本发明公开了本发明一种蛹虫草龟鹿蛋白肽口服液及其制备方法,其技术方案要点是包括如下重量比组分:蛹虫草提取物10%~12%,鹿肉20%~30%,龟肉20%~30%,蓝莓提取物16%~20%,鹿角3.0%~4.0%,龟甲提取物3.0%~4.0%,人参0.6%~0.8%,枸杞子1.4%~1.8%,柠檬酸0.16%~0.2%,柠檬酸钠0.16%~0.2%,三氯蔗糖0.016%~0.02%,其余为纯化水。本发明的一种蛹虫草龟鹿蛋白肽口服液,口感好、服用方便、可充分吸收利用、效果更为显著的蛹虫草龟鹿蛋白肽口服液,满足现代人的营养健康需求。
Description
【技术领域】
本发明涉及功能性保健食品技术领域,具体是一种蛹虫草龟鹿蛋白肽口服液及其制备方法。
【背景技术】
医学认为肾藏精为先天之本。肾之精是人生命活动之源,是构成人体的基本物质,精能化气,气能生精、肾生精、精生血、精少则病,精尽则亡,精衰气则虚弱,精足气则强健,气衰也则弱,气盛血则旺,气为血之帅,血为气之母,可见精、气、血是相互依存的。阴精是阳气之根本。阳化气、阴成形,阴精与血同源,如无阴精之形,便不足以载阳气,所以物之生,生于阳;物之成,成于阴,只有阴精充足,阳气旺盛,且二者协调平衡,人体才能康健。
而根据阴阳互根的原理,凡阴虚者,于补阴之时需伍以补阳之品,使“阴得阳升而泉源不竭;凡阳虚者,于补阳之时又需伍以补阴之品,使“阳得阴助而生化无穷”。由于精、气、血相依互用(精生气血,气生精血,血养精气),三者之中,一方虚损常累及其余,即精损可及气血、气损可及精血、血损可及精气,治疗应以补其一方为主而兼顾其余,其意义在于:①虚损一方及受累者可经受其补;②即使一方虚损未累及其余,其余受补之后,通过气化可以加快虚损一方的恢复。因此,本方除用于治疗阴阳两虚及精气血俱虚外,也可以用于治疗阴、阳及精、气、血等任何一方虚损。
鹿角、龟甲为方中主药,均归肝、肾二经。“鹿得天地之阳气最全,善通督脉”(《古今名医方论-龟鹿二仙膏》),其角为胶,味咸,性微温,能补肾阳、生精血。“龟得天地之阴气最厚,善通任脉”(《古今名医方论·龟鹿二仙膏》),其腹甲为胶,味咸、甘、性平,能滋阴潜阳、补血。“二物气血之属,又得造化之玄微,异类有情”(《古今名医方论-龟鹿二仙膏》),与人则同气相求,作为“竹破竹补”之用,非它草木可比。人参,味甘、微苦,性平,归脾、肺、心经,大补元气而生津,‘善于固气”(《医方考-虚损劳瘵门》)。枸杞子,味甘,性平,归肝、肾经,益精生血,“善于滋阴”(《医方考-虚损劳瘵门》)。四药合用,性味平和,入五脏而以肝、肾为主,又善通奇经之任、督,生精、益气、养血,阴阳并朴,且补阴而无凝滞之弊,补阳而无燥热之害。李士材所言“是方也.一阴一阳,无偏胜之忧;人气人血,有和平之美”(《古今名医方论·龟鹿二仙膏》),正是对本方特点的概括。
蛹虫草[Cordyceps mditaris(L)Link],也称北虫草,是虫草属药用真菌中唯一获得人工培养子实体的菌种,因与冬虫夏草同为麦角菌科虫草属真菌,其化学成分大体相同,且虫草素含量超过了天然虫草的含量。蛹虫草分布广泛,蛹虫草子实体可以大面积人工栽培,质量可控,商品价格低,更适合大众消费,被世界学者和消费者所公认,近年来研究表明,蛹虫草中虫草素含量高于天然冬虫夏草,其蛋白质、氨基酸、维生素和微量元素的含量均达到甚至也超过了天然冬虫夏草的水平。蛹虫草富含多种生物活性物质,如虫草多糖、虫草素、虫草酸、SOD酶、腺苷、类胡萝卜素、多肽/氨基酸和甘露醇等。其中,虫草素:抗病毒、抗菌、明显抑制肿瘤生长;虫草酸:预防治疗脑血栓、脑溢血、肾功能衰竭,利尿;虫草多糖:提高免疫力,延缓衰老,扶正固本,保护心脏、肝脏,抗痉;超氧化物歧化酶(SOD):抑制或消除催人衰老的超氧自由基形成及抗癌、防衰、减毒;腺苷:抗病毒、抗菌,消除面斑,抗衰防皱;麦角甾醇:抗癌、防衰、减毒。
龟甲须经过长时间的炮制及熬炼,将其有效成分提炼出来,提取方法极为耗时,且溶出的胶体主要为胶原蛋白,它们属于高分子生物活性物质,不易被人体吸收利用。胶原蛋白肽由胶原蛋白水解而成,分子量较小,易于被人体吸收,不仅有蛋白质的营养特性,而且具有独特的生理活性,有保护胃黏膜、抗氧化、抗衰老、降血压、降胆固醇、增强骨强度、促进皮肤胶原代谢、免疫调节等独特的生理功能。
蓝莓(Blueberry),蓝莓一直在欧美地区流行,殊不知,早在我国明代,李时珍就已经掌握了蓝莓的功效。蓝莓果实中除了常规的糖、酸和Vc外,富含VE、VA、VB、熊果苷、蛋白质、花青苷、食用纤维以及丰富的K、Fe、Zn、Ca等矿物质元素。蓝莓中蕴藏着大量紫色成分,就是花青素。花青素是纯天然的抗衰老营养补充剂,研究证明是当今人类发现有效的抗氧化剂。花青素的抗氧化性能比维生素E高五十倍,比维生素C高二十倍。
随着人们生活压力的增加及饮食起居的不规律,使越来越多的人们身体处于亚健康状态,免疫力低下,进而出现各种因气血亏虚,肝肾不足等原因引起的头晕耳鸣,腰膝酸软,潮热盗汗,情志异常等的或轻或重的一些症状。
常规的龟鹿二仙胶组成为鹿角,龟甲,人参,枸杞子,能够滋阴填精,益气壮阳,用于体质虚弱,腰膝酸软等证。但由于处方中的主要成份是由龟甲及鹿角中提取的胶原蛋白,它们属于高分子生物活性物质,这种胶原蛋白普遍存在着消化、吸收率低的缺陷,人体无法对其充分利用,功效平缓,效果不明显。另外龟鹿二仙胶口感差,在食用龟鹿二仙胶时,有些有浓浓的腥味,让人很难下咽,患者依从性较低。另外其剂型也不适用于现代人们的生活需要,且活性组分保持时间有限,不利于长期储存或运输。
所以非常有必要将龟鹿二仙胶开发成一款口感好、服用方便、可充分吸收利用、效果更为显著的口服液,此为我们要达到的目标。
【发明内容】
本发明的一种蛹虫草龟鹿蛋白肽口服液,口感好、服用方便、可充分吸收利用、效果更为显著的蛹虫草龟鹿蛋白肽口服液,满足现代人的营养健康需求。
本发明还提供一种蛹虫草龟鹿蛋白肽口服液的制备方法。
本发明一种蛹虫草龟鹿蛋白肽口服液,包括如下重量比组分:
蛹虫草:味甘,性温,入肺肾二经,既能补肺阴,又能补肾阳,主治肾虚,阳痿遗精,腰膝酸痛,病后虚弱,久咳虚弱,劳咳痰血,自汗盗汗等;
鹿角:性咸温,温补肾阳,生精益血,强筋健骨;鹿常回头看其尾,故能通督脉,取其角以补命、补精、补气以养阳,且鹿得天地之阳最全,足以养精,能蓄精力而长寿;
龟甲:味甘而平,补心益肾,滋阴资智,龟因常将手脚缩入腹甲中,故能通任脉,可取其甲以益精,补肾养血;且龟得天地之阴气最具,足以养气,能伏息而长寿;
鹿肉:鹿是我国最重要的传统药用经济动物之一,具有极高的药用价值和食用价值。在中华文化中,鹿常常是与神仙相联系的,传说中的许多神仙就是以鹿作为坐骑,因而鹿在古代被视为神物,认为能给人们带来吉祥、幸福和长寿。鹿肉味甘,性温,能益气血,补虚羸,补肾益精。鹿肉补益肾气之功为所有肉类之首,故对于新婚夫妇和肾气日衰的老人,吃鹿肉是很好的补益食品。鹿肉以高蛋白、低脂肪、易消化、营养丰富、味道鲜美而著称,鹿肉还具有提高人体代谢强度和抵抗力的功能,一直受到人们的青睐。我国传统中医学认为,鹿肉具有补脾中、益气力、强五脏的作用,还可补虚劳羸瘦、壮阳益精、养血益容,是中药中的滋补佳品;
龟肉:味甘,咸平,性温,有强肾补心壮阳之功,主治痨瘵骨蒸、久咳咯血、血痢、筋骨疼痛、病后阴虚血弱,尤其对小儿虚弱和产后体虚、脱肛、子宫下垂及性功能低下等有较好的疗效。龟肉营养丰富,味道鲜美,所谓“龟身五花肉”,即是指龟肉含有牛、羊、猪、鸡、鱼等5种动物肉的营养和味道。现代研究表明,每100克龟肉含蛋白质16.5克,脂肪1.0克、糖类1.6克,并富含维生素A、维生素B1、维生素B2、脂肪酸、肌醇、钾、纳等人体所需的各种营养成分;自古以来就将龟作为高级滋补品和防止疾病的食疗佳品。尤以龟肉为主料烹饪的食品,以成为高档筵席上的时尚佳味珍肴;
人参甘温,大补元气,开心益智,通行血脉;
枸杞甘平,滋肾益气,生精助阳,润肺清肝去风明目;
蓝莓提取物:蓝莓提取物中含有丰富的花青素,花青素是纯天然的抗衰老的营养补充剂,它的抗氧化性能比维生素E高出五十倍,比维生素C高出二十倍;它对人体的生物有效性是100%,服用后二十分钟就能在血液中检测到;蓝莓提取物能抗氧化、预防血管硬化、促进血液循环,在改善性功能障碍方面具有辅助作用。
优选的,所述蓝莓提取物中蛋白酶的含量高于35mg/g。
优选的,所述龟甲提取物的主要成分为酶解龟甲胶原蛋白肽。龟甲经过长时间的炮制及熬炼,将其有效成分提炼出来,通过蓝莓提取物进行生物酶解,使得难以溶出的高分子胶原蛋白,分解为小分子的胶原蛋白肽,易于被人体吸收,不仅有蛋白质的营养特性,而且具有独特的生理活性,有保护胃黏膜、抗氧化、抗衰老、降血压、降胆固醇、增强骨强度、促进皮肤胶原代谢、免疫调节等独特的生理功能。
优选的,所述鹿角需切片,并粉碎至5目以下。
一种蛹虫草龟鹿蛋白肽口服液的制备方法,包括以下步骤:
1)、制备蛹虫草提取物:
①将蛹虫草加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为3.5:1,之后在30~50℃下进行超声浸提后,经固液分离,得到滤渣和第一粗提液;
②将所述滤渣加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为8.5:1,在60~80℃进行真空浸提后,固液分离,得到第二粗提液;
③将所述第一粗提液和第二粗提液合并,进行减压蒸馏,浓缩至相对密度1.15~1.20,得到蛹虫草提取物;
2)、制备活性肽浓缩液:
①鹿肉与龟肉分别切片,洗净,滤干后剁碎成肉糜;
②加入2~4倍纯化水,搅拌均匀,于60~80℃水浴锅中预处理20~40min,取出,放冷至常温;
③加入肉糜重量0.002~0.006倍的蓝莓提取物,搅拌均匀,于30~50℃水浴锅中酶解3~6h,取出;
④于70~90℃灭活20~40min,取出;
⑤冷藏静置24~48h,过滤,滤液过200~300目筛;
⑥收集过滤液,减压蒸馏,浓缩至相对密度1.15~1.20,得活性肽浓缩液备用;
3)、制备龟甲提取物:
①将龟甲洗净,破碎至1mm粒径以下;
②投入纯化水中浸泡2~4H;
③按5~10万U/g龟甲的量加入蓝莓提取物,搅拌混匀,酶解1~4小时;
④加热至70~90℃,保持30min使蛋白酶灭活;
⑤趁热过滤,滤液过200~300目筛,然后减压蒸馏,浓缩至相对密度1.15~1.20,得到龟甲提取物;
4)、鹿角切片,粉碎至5目以下;
5)、药剂浓缩液的制备:
①人参、枸杞、鹿角加入8倍量食用酒精,室温下密闭浸泡10min~30min,然后加热回流浸提40min~60min,分离得到浸提液和原料渣;
②将浸提液浓缩回收乙醇后,得到浓缩液体A;
③将原料渣回收乙醇后,按照回收乙醇后的原料渣与水质量比1:8~1:10加入到多功能提取罐内,经减压回流提取或加热回流浸提2次~3次,每次的提取时间为40min~60min,得到提取液B;
④合并两次提取液,滤液过200~300目筛,浓缩至相对密度1.05~1.10,得药剂浓缩液;
6)、配制口服液:将上述所得材料投入配液罐,按配方称取柠蓝莓提取物、檬酸、柠檬酸钠、三氯蔗糖,投入配液罐,补足纯化水,配液罐打开蒸汽阀,搅拌中加热至75~80℃,保温20min,搅拌溶解均匀,通过胶体磨进行胶磨1~2次,然后进行灌装,灌装后用湿热灭菌法进行灭菌,工艺参数为灭菌温度121℃±2℃,时间15min,即得口服液。
优选的,所述蓝莓提取物的制备方法如下:
1)、原料处理:将蓝莓,清洗、晾干、破碎、打浆;
2)、原料调配:向蓝莓浆液中加入蔗糖和菊芋粉,搅拌均匀,同时调节其pH为4.5~5.0;蔗糖添加量分别为蓝莓浆液质量的1%~2%,菊芋粉添加量分别为蓝莓浆液质量的6%~10%;
3)、发酵:将调配好的原料装满发酵容器,进行超声波处理并立即水封,控制温度在35℃~42℃进行第一阶段无氧发酵;当pH降为4.0~4.5时,降低温度至30℃~35℃,并保持此温度继续进行第二阶段无氧发酵;当pH降为3.5~4.0时,倒罐、搅拌,降低温度至25℃~30℃,并在此温度进行有氧发酵;当还原糖含量小于4g/L时,发酵结束;其中,第一阶段无氧发酵的时间为2~3天,第二阶段无氧发酵的时间为5~7天,有氧发酵的时间为4~6天;
4)、分离:发酵结束后压榨分离得蓝莓提取物。
与现有技术相比本发明具有如下的有益效果:
本发明的蛹虫草龟鹿蛋白肽口服液,是在龟鹿二仙胶的基础上,添加阴阳双补的蛹虫草,并添加用鹿肉、龟肉、鹿角、龟甲提取物通过现代生物酶解技术制备的小分子鹿活性肽、龟活性肽,吸收率高,效果更显著,同时为了改善传统龟鹿二仙胶口感差(具有浓烈腥味)、服用不方面、患者依从性较低等缺点,本发明添加了蓝莓提取物、柠檬酸、柠檬酸钠、三氯蔗糖等,将其制备成了一款口感好、服用方便的口服液,可充分吸收利用,从而补养气血,滋阴补肾,温通经脉,舒缓人体情志异常,从而达到提高人体自身免疫力及防病抗病能力,改善人体的亚健康状态的目的;
本发明的蛹虫草龟鹿蛋白肽口服液的制备方法,采用特殊方法制备的蓝莓提取物,蓝莓在天然发酵过程中抗氧化能力有明显提高,特别是蛋白酶的含量大幅提高,作为天然酶能够有效促进口服液组分中的多种蛋白肽酶解,降解为小分子肽,通过现代生物酶解技术制备的小分子鹿活性肽、龟活性肽、龟甲胶原蛋白肽,吸收率高,原料取自天然,反应温和,有效发挥各种药效组分的效果,且活性保持时间长久,利于长时间储存或运输,适于批量生产,另外蓝莓提取物也带有蓝莓的风味,可调节口服液的口感,使得本产品风味宜人。
【具体实施方式】
下面的实施例有助于本领域更全面的理解本发明,但不以任何方式限制本发明。
实施例1:
原料配比(按重量计):蛹虫草提取物12%,鹿肉20%,龟肉20%,鹿角4.0%,龟甲提取物4.0%,人参0.8%,枸杞子1.6%,蓝莓提取物18%,柠檬酸0.16%,柠檬酸钠0.16%,三氯蔗糖0.016%,剩余为纯化水。
实施例2:
原料配比(按重量计):蛹虫草提取物10%,鹿肉30%,龟肉30%,鹿角3.0%,龟甲提取物3.0%,人参0.6%,枸杞子1.4%,蓝莓提取物20%,柠檬酸0.17%,柠檬酸钠0.17%,三氯蔗糖0.02%,剩余为纯化水。
实施例3:
原料配比(按重量计):蛹虫草提取物12%,鹿肉25%,龟肉25%,鹿角3.5%,龟甲提取物3.5%,人参0.7%,枸杞子1.8%,蓝莓提取物16%,柠檬酸0.2%,柠檬酸钠0.2%,三氯蔗糖0.018%,剩余为纯化水。
实施例1-3的蛹虫草龟鹿蛋白肽口服液的制备方法,包括以下步骤:
1)、制备蛹虫草提取物:
①将蛹虫草加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为3.5:1,之后在30~50℃下进行超声浸提后,经固液分离,得到滤渣和第一粗提液;
②将所述滤渣加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为8.5:1,在60~80℃进行真空浸提后,固液分离,得到第二粗提液;
③将所述第一粗提液和第二粗提液合并,进行减压蒸馏,浓缩至相对密度1.15~1.20,得到蛹虫草提取物;
2)、制备活性肽浓缩液:
①鹿肉与龟肉分别切片,洗净,滤干后剁碎成肉糜;
②加入2~4倍纯化水,搅拌均匀,于60~80℃水浴锅中预处理20~40min,取出,放冷至常温;
③加入肉糜重量0.002~0.006倍的蓝莓提取物,搅拌均匀,于30~50℃水浴锅中酶解3~6h,取出;
④于70~90℃灭活20~40min,取出;
⑤冷藏静置24~48h,过滤;
⑥收集过滤液,减压蒸馏,浓缩至相对密度1.15~1.20,得活性肽浓缩液备用;
3)、制备龟甲提取物:
①将龟甲洗净,破碎至1mm粒径以下;
②投入纯化水中浸泡2~4H;
③按5~10万U/g龟甲的量加入蓝莓提取物,搅拌混匀,酶解1~4小时;
④加热至70~90℃,保持30min使蛋白酶灭活;
⑤趁热过滤,滤液过200~300目筛,然后减压蒸馏,浓缩至相对密度1.15~1.20,得到龟甲提取物;
4)、鹿角切片,粉碎至5目以下;
5)、药剂浓缩液的制备:
①人参、枸杞、鹿角加入8倍量食用酒精,室温下密闭浸泡10min~30min,然后加热回流浸提40min~60min,分离得到浸提液和原料渣;
②将浸提液浓缩回收乙醇后,得到浓缩液体A;
③将原料渣回收乙醇后,按照回收乙醇后的原料渣与水质量比1:8~1:10加入到多功能提取罐内,经减压回流提取或加热回流浸提2次~3次,每次的提取时间为40min~60min,得到提取液B;
④合并两次提取液,滤液过200~300目筛,浓缩至相对密度1.05~1.10,得药剂浓缩液;
6)、配制口服液:将上述所得材料投入配液罐,按配方称取柠蓝莓提取物、檬酸、柠檬酸钠、三氯蔗糖,投入配液罐,补足纯化水,配液罐打开蒸汽阀,搅拌中加热至75~80℃,保温20min,搅拌溶解均匀,通过胶体磨进行胶磨1~2次,然后进行灌装,灌装后用湿热灭菌法进行灭菌,工艺参数为灭菌温度121℃±2℃,时间15min,即得口服液。
优选的,所述蓝莓提取物的制备方法如下:
1)、原料处理:将蓝莓,清洗、晾干、破碎、打浆;
2)、原料调配:向蓝莓浆液中加入蔗糖和菊芋粉,搅拌均匀,同时调节其pH为4.5~5.0;蔗糖添加量分别为蓝莓浆液质量的1%~2%,菊芋粉添加量分别为蓝莓浆液质量的6%~10%;
3)、发酵:将调配好的原料装满发酵容器,进行超声波处理并立即水封,控制温度在35℃~42℃进行第一阶段无氧发酵;当pH降为4.0~4.5时,降低温度至30℃~35℃,并保持此温度继续进行第二阶段无氧发酵;当pH降为3.5~4.0时,倒罐、搅拌,降低温度至25℃~30℃,并在此温度进行有氧发酵;当还原糖含量小于4g/L时,发酵结束;其中,第一阶段无氧发酵的时间为2~3天,第二阶段无氧发酵的时间为5~7天,有氧发酵的时间为4~6天;
4)、分离:发酵结束后压榨分离得蓝莓提取物。
对上述实施例1-3所得蛹虫草龟鹿蛋白肽口服液,不同时间段的标志性组分含量检测如下:
表1:实施例1-3中不同时间段的标志性组分含量检测如下:
由表1可知,本方案方法制备的蛹虫草龟鹿蛋白肽口服液具有较高的肽含量,而且其中适于人体吸收的小分子肽含量比例很高,而且在制备完成后,能够长时间保持其活性组分稳定,便于长时间保存。
进一步通过试验对实施例所得口服液效果做验证:
一、毒理试验:
试验目的:验证本产品是否有毒性。
试验材料:
样品:优选实施例3所得的蛹虫草龟鹿蛋白肽口服液,人体口服推荐量为每日20mL,成人体重按60kg计算,折合剂量0.33mL/kg.bw,取5倍浓缩液进行实验;
实验动物:清洁级昆明种小鼠、SD大鼠及饲料;
实验条件:为屏障环境,实验期间环境温度22-25℃,湿度52%-58%;
试验方法:
通过小鼠急性毒性试验(取蛹虫草龟鹿蛋白肽口服液5倍浓缩液以0.2mL/10g.bw的灌胃体积给小鼠一次性经口灌胃)、Ames试验(试验设五个剂量,分别为5000ug/皿、1000ug/皿、200ug/皿、40ug/皿、8ug/皿,同时设自发回变、溶剂对照和阳性突变剂对照)、小鼠骨髓嗜多染红细胞微核试验(以40mg/kg.bw剂量的环磷酰胺为阳性对照,蒸馏水为阴性对照,试验组3个剂量分别为10.0mL/kg.bw、5.0mL/kg.bw、2.5mL/kg.bw,取蛹虫草龟鹿蛋白肽口服液5倍浓缩液50.0mL、25.0mL、12.5mL加蒸馏水至100mL,配成相应剂量的受试液给小鼠灌胃)、小鼠精子畸形试验(以40mg/kg.bw剂量的环磷酰胺为阳性对照,蒸馏水为阴性对照,试验组3个剂量分别为10.0mL/kg.bw、5.0mL/kg.bw、2.5mL/kg.bw,取蛹虫草龟鹿蛋白肽口服液5倍浓缩液50.0mL、25.0mL、12.5mL加蒸馏水至100mL,配成相应剂量的受试液给小鼠灌胃)、30天喂养试验(取蛹虫草龟鹿蛋白肽口服液16.5mL、33mL、66mL加蒸馏水定容至100mL,对照组给予等体积蒸馏水,每日灌胃一次,连续30天)来进行毒理学试验评定;
试验结果:
1.急性经口毒性试验结果:
雌、雄昆明种小鼠的最大耐受量(MTD)均大于20.0mL/kg.bw,属无毒级;
2.三项遗传毒性试验结果:
三项遗传毒性试验(Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠精子畸形试验)结果均为阴性;
3、30天喂养试验结果:
以8.25mL/kg.bw、16.5mL/kg.bw、33.0mL/kg.bw剂量(相当于人体推荐剂量的25、50、100倍)的蛹虫草龟鹿蛋白肽口服液给大鼠灌胃30天,实验期间,动物生长发育良好,各剂量组体重、增重、食物利用率、血常规指标、血生化指标、脏器重量及脏器/体重比值与对照组比较,无显著性差异(P>0.05);大体解剖和组织病理检查未见与样品有关的异常改变。
二、功能试验:
试验目的:检验产品是否具有增强免疫力的保健功能;
试验材料:
样品:优选实施例2所得的蛹虫草龟鹿蛋白肽口服液,20mL/支,密封,置阴凉、通风、干燥处保存;
试验动物:清洁级昆明种雌性小鼠200只,体重为18-22g;
实验环境条件:屏障系统。温度22-25℃,湿度52-58%;
试验方法与结果:
实施例2所得的蛹虫草龟鹿蛋白肽口服液,人体口服推荐量为每日20mL,成人体重按60kg计算,折合剂量0.33mL/kg.bw;设蛹虫草龟鹿蛋白肽口服液低、中、高剂量分别为1.67mL/kg.bw、3.33mL/kg.bw、10.00mL/kg.bw(分别相当于人体推荐剂量的5、10、30倍);低、中、高剂量受试液配制时,分别取16.7mL、33.3mL、100mL蛹虫草龟鹿蛋白肽口服液加蒸馏水至200mL,对照组予以等体积的蒸馏水,分别给予受试动物灌胃,每天灌胃一次,灌胃体积为0.2mL/10g.bw,连续30天。
1、二硝基氟苯(DNFB)诱导迟发型变态反应(DTH)试验:
依实验设计剂量给各组试验小鼠连续灌胃30d后,小鼠腹部去毛,用1%DNFB丙酮麻油溶液50uL涂抹致敏,致敏后5d,小鼠右耳均匀涂抹1%DNFB丙酮麻油溶液10uL进行攻击,左耳涂抹丙酮麻油溶液作对照,攻击后24h处死小鼠,剪下左右耳壳,于同一部位取直径8mm的耳片称重,左右耳片重量之差作为肿胀度。
表2:蛹虫草龟鹿蛋白肽口服液对小鼠迟发型变态反应的影响(n=10)
组别 | 剂量(mL/kg.bw) | 耳肿胀度(mg) |
对照组 | 0 | 2.91±1.15 |
低剂量组 | 1.67 | 3.62±1.03 |
中剂量组 | 3.33 | 4.89±3.18 |
高剂量组 | 10.00 | 5.15±2.04 |
由表2可知,与对照组相比,蛹虫草龟鹿蛋白肽口服液中剂量和高剂量对小鼠耳肿胀度有明显促进升高作用,差异有显著性(P<0.05);这说明蛹虫草龟鹿蛋白肽口服液对小鼠迟发型变态反应的耳肿胀度具有促进作用,即具有增强小鼠细胞免疫功能的作用。
2、小鼠碳廓清试验:
依实验设计剂量给各组试验小鼠连续灌胃30d后,从小鼠的尾静脉按0.1mL/10g量注射浓度为20%的墨汁,间隔2min、15min后,分别从小鼠内眦静脉从取血20uL,加入到2mL的1%的Na2C03溶液中。用光度计在600nm波长处分别测定值0D值,以Na2C03溶液作空白对照。然后颈椎脱臼处死小鼠,取出肝脏和脾脏,用滤纸吸干脏器表面的血污,称重。将测出的OD值、肝脏和脾脏重量代入公式,算出吞噬指数(以吞噬指数表示小鼠碳廓清的能力)。
表3蛹虫草龟鹿蛋白肽口服液对小鼠巨噬细胞碳廓清能力的影响(n=10)
组别 | 剂量(mL/kg.bw) | 吞噬指数(a) |
对照组 | 0 | 4.47±0.06 |
低剂量组 | 1.67 | 5.43±0.53 |
中剂量组 | 3.33 | 5.76±0.81 |
高剂量组 | 10.00 | 5.98±0.78 |
由表3可见,与对照组比较,实施例2所得的蛹虫草龟鹿蛋白肽口服液低、中、高剂量组对小鼠巨噬细胞碳廓清能力的吞噬指数均具有明显的升高作用,差异有显著性(P<0.05);这说明蛹虫草龟鹿蛋白肽口服液具有促进小鼠噬细胞的吞噬作用,可增强其非特异性免疫功能。
3、抗体生成细胞检测:
依实验设计剂量给各组试验小鼠连续灌胃30d后,每鼠腹腔注射2%(v/v)压积SRBC 0.2mL致敏。免疫5d后,将小鼠颈椎脱臼处死。取出脾脏,制成单细胞悬液,进行溶血空斑试验。计数溶血空斑数,用空斑数/106脾细胞来表示抗体生成细胞数。
表4保健食品对小鼠脾抗体生成细胞量的影响(n=10)
由表4可知,与对照组比较,与对照组比较,蛹虫草龟鹿蛋白肽口服液低、中、高剂量组对小鼠脾抗体生成细胞量均具有明显的升高作用,其中高、中剂量组空斑数与对照组值相比有统计学差异(P<0.05)。这表明蛹虫草龟鹿蛋白肽口服液具有增强小鼠脾抗体生成细胞的能力,即具有增强小鼠体液免疫功能的作用。
根据《保健食品检验与评价技术规范实施手册》(2003版)中的有关增强免疫力试验结果判定标准,本实验结果:(1)本发明的保健食品具有促进细胞免疫的功能的作用(迟发型变态反应实验结果阳性);(2)本发明的保健食品具有促进体液免疫的功能的作用(抗体生成细胞检测试验结果阳性);(3)本发明的保健食品具有促进单核一巨噬细胞功能的作用(小鼠碳廓清实验与小鼠腹腔巨噬细胞吞噬鸡红细胞实验结果均阳性);因此,这表明本发明的保健食品具有增强动物免疫力的作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
2.根据权利要求1所述的蛹虫草龟鹿蛋白肽口服液,其特征在于:所述蛹虫草提取物的制备方法包括如下步骤:
①将蛹虫草加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为3.5:1,之后在30~50℃下进行超声浸提后,经固液分离,得到滤渣和第一粗提液;
②将所述滤渣加入90%质量份数的乙醇溶液中,乙醇溶液与蛹虫草重量比为8.5:1,在60~80℃进行真空浸提后,固液分离,得到第二粗提液;
③将所述第一粗提液和第二粗提液合并,进行减压蒸馏,浓缩至相对密度1.15~1.20,得到蛹虫草提取物。
3.根据权利要求1所述的蛹虫草龟鹿蛋白肽口服液,其特征在于:所述蓝莓提取物中蛋白酶的含量高于35mg/g。
4.根据权利要求1所述的蛹虫草龟鹿蛋白肽口服液,其特征在于:所述龟甲提取物的主要成分为酶解龟甲胶原蛋白肽。
5.根据权利要求1所述的蛹虫草龟鹿蛋白肽口服液,其特征在于:所述鹿角需切片,并粉碎至5目以下。
6.一种如权利要求1至5任一项所述的蛹虫草龟鹿蛋白肽口服液的制备方法,其特征在于包括以下步骤:
1)、制备活性肽浓缩液:
①鹿肉与龟肉分别切片,洗净,滤干后剁碎成肉糜;
②加入2~4倍纯化水,搅拌均匀,于60~80℃水浴锅中预处理20~40min,取出,放冷至常温;
③加入肉糜重量0.002~0.006倍的蓝莓提取物,搅拌均匀,于30~50℃水浴锅中酶解3~6h,取出;
④于70~90℃灭活20~40min,取出;
⑤冷藏静置24~48h,过滤,滤液过200~300目筛;
⑥收集过滤液,减压蒸馏,浓缩至相对密度1.15~1.20,得活性肽浓缩液备用;;
2)、制备龟甲胶原蛋白肽:
①将龟甲洗净,破碎至1mm粒径以下;
②投入纯化水中浸泡2~4H;
③按5~10万U/g龟甲的量加入蓝莓提取物,搅拌混匀,酶解1~4小时;
④加热至70~90℃,保持30min使蛋白酶灭活;
⑤趁热过滤,滤液过200~300目筛,然后减压蒸馏,浓缩至相对密度1.15~1.20,得到龟甲提取物;
3)、鹿角切片,粉碎至5目以下;
4)、制备药剂浓缩液:
①人参、枸杞、鹿角加入8倍量食用酒精,室温下密闭浸泡10min~30min,然后加热回流浸提40min~60min,分离得到浸提液和原料渣;
②将浸提液浓缩回收乙醇后,得到浓缩液体A;
③将原料渣回收乙醇后,按照回收乙醇后的原料渣与水质量比1:8~1:10加入到多功能提取罐内,经减压回流提取或加热回流浸提2次~3次,每次的提取时间为40min~60min,得到提取液B;
④合并两次提取液,滤液过200~300目筛,浓缩至相对密度1.05~1.10,得药剂浓缩液;
5)、配制口服液:将上述所得材料投入配液罐,按配方称取柠蓝莓提取物、檬酸、柠檬酸钠、三氯蔗糖,投入配液罐,补足纯化水,配液罐打开蒸汽阀,搅拌中加热至75~80℃,保温20min,搅拌溶解均匀,通过胶体磨进行胶磨1~2次,然后进行灌装,灌装后用湿热灭菌法进行灭菌,工艺参数为灭菌温度121℃±2℃,时间15min,即得口服液。
7.根据权利要求3所述的蛹虫草龟鹿蛋白肽口服液,其特征在于:所述蓝莓提取物的制备方法如下:
1)、原料处理:将蓝莓,清洗、晾干、破碎、打浆;
2)、原料调配:向蓝莓浆液中加入蔗糖和菊芋粉,搅拌均匀,同时调节其pH为4.5~5.0;蔗糖添加量分别为蓝莓浆液质量的1%~2%,菊芋粉添加量分别为蓝莓浆液质量的6%~10%;
3)、反应:将调配好的原料装满发酵容器,进行超声波处理并立即水封,控制温度在35℃~42℃进行第一阶段无氧发酵;当pH降为4.0~4.5时,降低温度至30℃~35℃,并保持此温度继续进行第二阶段无氧发酵;当pH降为3.5~4.0时,倒罐、搅拌,降低温度至25℃~30℃,并在此温度进行有氧发酵;当还原糖含量小于4g/L时,发酵结束;其中,第一阶段无氧发酵的时间为2~3天,第二阶段无氧发酵的时间为5~7天,有氧发酵的时间为4~6天;
4)、分离:发酵结束后压榨分离得蓝莓提取物。
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