CN111358831A - 一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用 - Google Patents
一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用 Download PDFInfo
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- CN111358831A CN111358831A CN202010169466.XA CN202010169466A CN111358831A CN 111358831 A CN111358831 A CN 111358831A CN 202010169466 A CN202010169466 A CN 202010169466A CN 111358831 A CN111358831 A CN 111358831A
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- chinese medicine
- traditional chinese
- lactobacillus salivarius
- fermentation liquor
- enzymolysis
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Abstract
本发明公开了一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用。该中药发酵液的制备方法包括下述步骤:1)将中药组合物先粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化的中药;所述中药组合物由下述质量份的药材组成:菊花3份、决明子3份、珍珠粉2.5份、薄荷叶1.0份、冰片0.5份;2)将微米化的中药分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶进行酶解;酶解结束后,向所述酶解体系中接种唾液乳杆菌进行发酵,收集上清液,即得到所述中药发酵液。药效试验表明,本发明提供的中药发酵液具有优异的缓解视疲劳作用的功效。
Description
技术领域
本发明属于医药领域,具体涉及一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用。
背景技术
微米中药是指微米级中药有效成分、有效部位、原药及其复方制剂。将中药微米化后既能有效保留中药的有效成分不被破坏,同时由于制得的颗粒比表面积增大,能有效增进药物有效成分的溶出,提高药物的生物利用度;其次它可以提高药物的靶向性,有助于控制药物在体内的分布等。
微生物发酵法的生产工艺技术主要是通过对微生物进行大规模的生长培养,使底物发生生物转化和化学变化,从而产生和积累大量人们发酵所需要的代谢产物的过程。中药经过发酵后,可以提高中药药效、消除中药毒副作用、改变中药药性。
因此,将两种技术结合对促进中医药发展具有里程碑的意义。
发明内容
本发明的目的是提供一种具有缓解视疲劳作用的中药发酵液。
本发明所提供的具有缓解视疲劳作用的中药发酵液是按照包括下述步骤的方法制备得到的:
1)将中药组合物先粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化的中药;所述中药组合物由下述质量份的药材组成:菊花3份、决明子3份、珍珠粉2.5份、薄荷叶1.0份、冰片0.5份;
2)将微米化的中药分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶进行酶解;酶解结束后,向所述酶解体系中接种唾液乳杆菌进行发酵,收集上清液,即得到所述微米中药发酵液。
上述方法步骤1)中,所述的超微粉碎的条件为:粉碎温度20-40℃,粉碎压力10-15Mpa,气流速度300-400m/s,粉碎的时间1-2h;具体的粉碎条件为:粉碎温度25℃,粉碎压力12Mpa,气流速度350m/s,粉碎的时间1.5h。
上述方法步骤2)中,所述微米化的中药与水的质量比为1:3-4,具体质量比可为1:4。
所述果胶酶与微米化的中药的用量比为800-1200U:1kg,具体可为1000U:1kg。
所述一次酶解的条件为:pH值4.5-5.0,温度40-50℃,时间1.5-2小时。
上述方法步骤2)中,所述唾液乳杆菌是唾液乳杆菌(Lactobacillus salivarius)LJ,该菌株已于2016年08月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC No.12857。
本发明中所述的唾液乳杆菌(Lactobacillus salivarius)LJ CGMCC No.12857简称唾液乳杆菌LJ。
所述唾液乳杆菌以唾液乳杆菌菌液的形式接种;所述唾液乳杆菌菌液中唾液乳杆菌的浓度为1.6×109cfu/mL。
所述发酵培养的条件:42℃培养24h。
所述唾液乳杆菌菌液的接种量为所述分散液体积分数的5-10%。
所述唾液乳杆菌菌液的制备方法如下:将唾液乳杆菌接种至乳酸菌培养基中进行培养,获得菌液;
其中,所述乳酸菌培养基可为MRS液体培养基。所述MRS液体培养基的制备方法具体可如下:将蛋白胨10.00g、牛肉膏10.00g、酵母膏5.00g、柠檬酸氢二铵2.00g、乙酸钠5.00g、葡萄糖20.00g、吐温-80 1mL、磷酸氢二钾2.00g、硫酸镁0.58g和硫酸锰0.28g溶于1L蒸馏水,调节pH值至6.4;然后121℃灭菌15min。所述培养可置于二氧化碳培养箱进行,培养条件为可为:42℃静置培养24h。
本发明还提供了上述中药发酵液的应用。
所述应用为所述中药发酵液在制备预防和/或缓解和/或治疗视疲劳产品中的应用。
所述产品可为药品和/或保健品和/或化妆品。
本发明还保护一种具有预防和/或缓解和/或治疗视疲劳产品的产品。
所述产品的活性成分包括本发明所提供的中药发酵液。
所述产品可为药品和/或保健品和/或化妆品。
需要的时候,在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
所述药物可以制成悬浮剂、粉剂、片剂、颗粒剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明所述化妆品还包含化妆品中常用的成分,其包括但不限于活性成分、稀释剂、载体、表面活性剂和辅料等本领域已知的任何成分,这些是本领域技术人员已知的,且可根据需要具体选择其类型和用量。
所述活性成分还可包括除了本发明所述中药发酵液外的,其它本领域已知和常用的任何活性物质,其中包括例如润肤剂、保湿剂、皮肤调理剂等。
所述化妆品不限定形态,例如,可以制成霜剂、乳剂、精华水、凝胶剂以及面膜、眼膜等的形式。上述各种形式的化妆品可通过本领域已知的任何合适的方法进行制备。
药效试验表明,本发明提供的中药发酵液具有优异缓解视疲劳的功效。
本发明具有如下有益效果:
本发明将中药经过超微粉碎、酶解以及唾液乳杆菌发酵后,中药有效成分进行体外转化,化学成分在组成和含量上发生了变化。以决明子为例,应用液相色谱法,对发酵前后蒽醌成份进行检测,发现其药效活性成分大黄酚和大黄素甲醚增加至2.15倍和1.34倍(表示1)。用电泳法测定,发酵液中的蛋白质80%转化为肽和氨基酸。
具体实施方式
下面通过具体实施例对本发明进行说明,但本发明并不局限于此,凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中所用的菊花、决明子、珍珠粉、薄荷叶和冰片均符合《中国药典》(2015年版)一部正文各药材项下的有关规定。投料前,通过鉴定,各味药材实物与名称相符,质量符合标准。
下述实施例中所使用的药材均为净制后的药材。
MRS液体培养基:将蛋白胨10.00g、牛肉膏10.00g、酵母膏5.00g、柠檬酸氢二铵2.00g、乙酸钠5.00g、葡萄糖20.00g、吐温-80 1mL、磷酸氢二钾2.00g、硫酸镁0.58g和硫酸锰0.28g溶于1L蒸馏水,调节pH值至6.4;然后121℃灭菌15min。
MRS固体培养基:将蛋白胨10.00g、牛肉膏10.00g、酵母膏5.00g、柠檬酸氢二铵2.00g、乙酸钠5.00g、葡萄糖20.00g、吐温-80 1mL、磷酸氢二钾2.00g、硫酸镁0.58g、硫酸锰0.28g和琼脂15.00g溶于1L蒸馏水,调节pH值至6.4;然后121℃灭菌15min。
下述实施例中所使用的唾液乳杆菌是唾液乳杆菌(Lactobacillus salivarius)LJ,该菌株已于2016年08月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号),保藏编号为CGMCC No.12857。
对比例1中使用的唾液乳杆菌是唾液乳杆菌(Lactobacillus salivarius)JCM 1231,其保藏编号为CGMCC1.1881。
果胶酶:宁夏夏盛实业集团有限公司,20000U/g;酶活力定义:每小时每毫升酶液在50℃、pH=4.8条件下产生1.0mg还原糖(半乳糖醛酸为准)所需的酶量。
诺维信Alcalase 2.4L蛋白酶:诺维信(中国)生物技术有限公司,2.4AU/g;酶活力定义:在最适条件下,每分钟内催化1微摩尔底物转化为产物所需的酶量。
实施例1、制备具有缓解视疲劳作用的中药发酵液
1)取净制后的菊花、决明子、珍珠粉、薄荷叶、冰片按3:3:2.5:1:0.5比例,混合均匀。先用球磨机粉碎成粗粉,再通过气流式超微粉碎机粉碎至10μm以下,得到微米化的中药;其中,超微粉碎的条件为:粉碎温度25℃,粉碎压力12Mpa,气流速度350m/s,粉碎的时间1.5h。
2)将步骤1)得到的微米化的中药按20%质量比例分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶(每kg中药加入1000U果胶酶),调节pH至4.8,在45℃搅拌酶解2小时;然后95℃保温10min灭酶。
将唾液乳杆菌LJ的单菌落接种于装有10mL MRS液体培养基的试管中,然后置于二氧化碳培养箱。42℃静置培养24h后,得到唾液乳杆菌LJ的菌液(唾液乳杆菌的浓度为1.6×109cfu/mL)。
然后向上述酶解液中接入体积分数10%的唾液乳杆菌LJ的菌液,42℃静置培养24h后,获得发酵液即为微米中药发酵液。
进一步可将上述中药发酵液采用常规方法制备成眼贴膜,每个眼贴膜含中药发酵液0.5ml。
以决明子为例,应用液相色谱法,对发酵前后蒽醌成份进行检测,结果见表1。
表1决明子发酵前后蒽醌成份含量测定结果(mg/g)
大黄酚 | 大黄素甲醚 | |
发酵前样品 | 0.193 | 0.150 |
发酵后样品 | 0.416 | 0.202 |
对比例1、采用其它唾液乳杆菌作为发酵菌制备纳米中药乳酸菌素
基本方法同实施例1,区别在于将其中的唾液乳杆菌LJ替换成唾液乳杆菌(Lactobacillus salivarius)JCM 1231,其保藏编号为CGMCC1.1881。
进一步可将上述中药发酵液采用常规方法制备成眼贴膜,每个眼贴膜含中药发酵液0.5ml。
对比例2、采用蛋白酶制备中药发酵液
基本方法同实施例1,区别在于将其中的果胶酶替换成蛋白酶(诺维信A1calase 2.4L蛋白酶),所述诺维信Alcalase 2.4L蛋白酶与微米化的中药的用量比为25AU:1kg。酶解的条件为:pH值8.5,在55℃搅拌酶解4h,然后95℃保温10min灭酶。
进一步可将上述中药发酵液采用常规方法制备成眼贴膜,每个眼贴膜含中药发酵液0.5ml。
实施例2、缓解视疲劳效果评价
临床病例
资料与方法
1、一般资料选取90例视疲劳患者,年龄15-45岁之间,男性47人,女43人。
2、药物采用试验药物1(按实施例1方法制备);
3诊断标准
3.1西医诊断标准拟定:有眼胀、眼痛、眼干、视物模糊、眼烧灼感、流泪、眼异物感、眼眶痛症状。
3.2中医辨证标准拟定:阴虚火旺、脉络瘀滞证:主症:视物模糊、眼干涩灼热;次症:眼胀痛、眉棱骨痛(眼眶痛)、头痛、头晕。具备主症2项,次症2项或以上,即可诊断。
4入选病例标准符合上述诊断标准。
5治疗方法每人每天早晚使用两次中药发酵液眼贴膜(根据分组,分别为:含实施例1中药发酵液眼贴膜、含对比例1中药发酵液眼贴膜、含对比例2中药发酵液眼贴膜),每次眼部贴敷15min,连续不间断使用2周。
6疗效评价
6.1评价方法
治疗前后对视物模糊、眼痛、眼胀、眼干、眼烧灼感、流泪、异物感、眼眶痛进行评分:0分,无症状;1分,视物有时出现症状(每周≤3次),休息后缓解,与用眼多少有关;2分,视物2h以上出现症状,与用眼多少有关;3分,症状经常出现,与用眼多少无明显关系;4分,症状持续存在,严重影响工作及生活,与用眼多少无关。治疗过程中观察药物不良反应。
6.2评价标准
痊愈:中医症状、体征消失或基本消失,症状积分减少≥95%;
显效:中医症状、体征明显改善,70%≤症状积分减少<95%;
有效:中医症状、体征均有改善,30%≤症状积分减少<70%;
无效:中医症状、体征无改善或加重,积分减少<30%。
表1患者者测试结果
无效 | 有效 | 显效 | 痊愈 | |
实施例1 | 1/30 | 5/30 | 14/30 | 10/30 |
对比例1 | 3/30 | 10/30 | 11/30 | 6/30 |
对比例2 | 3/30 | 12/30 | 10/30 | 5/30 |
Claims (9)
1.一种制备中药发酵液的方法,包括下述步骤
1)将中药组合物先粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化的中药;所述中药组合物由下述质量份的药材组成:菊花3份、决明子3份、珍珠粉2.5份、薄荷叶1.0份、冰片0.5份;
2)将微米化的中药分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶进行酶解;酶解结束后,向所述酶解体系中接种唾液乳杆菌进行发酵,收集上清液,即得到所述中药发酵液。
2.根据权利要求1所述的方法,其特征在于:所述步骤1)中,所述的超微粉碎的条件为:粉碎温度20-40℃,粉碎压力10-15Mpa,气流速度300-400m/s,粉碎的时间1-2h。
3.根据权利要求1或2所述的方法,其特征在于:所述步骤2)中,所述微米化的中药与水的质量比为1:3-4。
4.根据权利要求1-3中任一项所述的方法,其特征在于:所述步骤2)中,所述果胶酶与微米化的中药的用量比为800-1200U:1kg;
所述酶解的条件为:pH值4.5-5.0,温度40-50℃,时间1.5-2小时。
5.根据权利要求1-4中任一项所述的方法,其特征在于:所述步骤2)中,所述唾液乳杆菌是唾液乳杆菌(Lactobacillus salivarius)LJ,,其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.12857。
6.根据权利要求1-5中任一项所述的方法,其特征在于:所述步骤2)中,所述唾液乳杆菌以唾液乳杆菌菌液的形式接种;所述唾液乳杆菌菌液中唾液乳杆菌的浓度为1.6×109cfu/mL;
所述发酵培养的条件:42℃培养24h;
所述唾液乳杆菌菌液的接种量为所述分散液体积分数的5-10%。
7.权利要求1-6中任一项所述方法制备得到中药发酵液。
8.权利要求7所述中药发酵液在制备预防和/或缓解和/或治疗视疲劳产品中的应用;所述产品为药品和/或保健品和/或化妆品。
9.一种具有预防和/或缓解和/或治疗视疲劳产品的产品,其活性成分权利要求7所述的中药发酵液;所述产品为药品和/或保健品和/或化妆品。
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