CN113171398A - 一种具有防脱发育发作用的中药发酵液及其制备方法与应用 - Google Patents
一种具有防脱发育发作用的中药发酵液及其制备方法与应用 Download PDFInfo
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- CN113171398A CN113171398A CN202110483712.3A CN202110483712A CN113171398A CN 113171398 A CN113171398 A CN 113171398A CN 202110483712 A CN202110483712 A CN 202110483712A CN 113171398 A CN113171398 A CN 113171398A
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- traditional chinese
- lactobacillus plantarum
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了一种具有防脱发育发作用的中药发酵液。该发酵液的制备方法如下1)将由侧柏叶4份、苦参4份、积雪草3份、粉防已2份、咖啡1份组成的中药组合物粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化中药;2)将羊胎盘加水匀浆;3)将微米化中药分散于水中,形成胶体溶液;向胶体溶液中加入果胶酶进行酶解;酶解结束后灭酶,然后向体系中加入改良MRS培养基、羊胎盘匀浆,再接种植物乳杆菌和啤酒酵母菌发酵,离心,收集上清液和沉淀;4)将沉淀加乙醇水溶液浸提,离心收集上清,浓缩为醇提取物;5)将微米中药发酵液与醇提取物按体积比5:1混合,即得。药效试验表明,本发明提供的中药发酵液具有优异防脱发和育发功效。
Description
技术领域
本发明属于医药领域,具体涉及一种具有防脱发育发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防脱发育发作用的中药发酵液。
本发明所提供的具有防脱发育发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)将植物中药组合物先粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化中药;所述植物中药组合物由下述质量份的药材组成:侧柏叶4份、苦参4份、积雪草3份、粉防已2份、咖啡1份;
2)将离体的羊胎盘加入纯水后进行匀浆,得到羊胎盘匀浆;
3)将微米化中药分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶进行酶解;酶解结束后灭酶,然后向酶解体系中加入改良MRS培养基、羊胎盘匀浆,再向其中接种植物乳杆菌和啤酒酵母菌进行发酵,离心,分别收集上清液和沉淀,所述上清液即为微米中药发酵液;
4)将收集的所述沉淀加入体积分数为60-80%的乙醇水溶液浸提,浸提结束后离心收集上清,再将上清蒸发去除乙醇,浓缩成原体积的1/3为醇提取物;
5)将步骤3)得到的微米中药发酵液与步骤4)得到的醇提取物按体积比5:1混合,即为最后的中药发酵液。
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,所述离体的羊胎盘与水的质量比为1:1-2,具体质量比可为1:1。
上述方法步骤3)中,所述微米化中药与水的质量比为1:4-8,具体质量比可为 1:5。
所述果胶酶与微米化中药的用量比为800-1200U:1kg,具体可为1000U: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。
所述果胶酶具体可购自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。
上述方法步骤3)中,所述灭酶的条件为:95℃保温10min。
上述方法步骤3)中,所述酶解体系(即酶解后的中药液)与羊胎盘匀浆的质量比为5:1;所述改良MRS培养基的质量与所述酶解体系和羊胎盘匀浆的质量之和的比为1:1。
上述方法步骤3)中,所述植物乳杆菌是植物乳杆菌(Lactobacillus planta-rum)Rs0228,该菌株已于2016年08月18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CGMCCNo.12881。
本发明中所述的植物乳杆菌(Lactobacillus plantarum)Rs0228 CGMCCNo.12881简称植物乳杆菌。
所述植物乳杆菌以植物乳杆菌菌液的形式接种;接种量为5-10%。
所述啤酒酵母菌为通过市售途径购买的啤酒酵母菌,如购自北纳创联生物科技有限公司。
所述啤酒酵母菌以啤酒酵母菌菌液的形式接种;接种量为5-10%。
所述发酵培养的条件:42℃培养24h。
所述植物乳杆菌种子菌液的制备方法如下:将植物乳杆菌接种至MRS培养基中进行培养,获得菌液;进一步的,所述菌液中植物乳杆菌的浓度可为 1.6×109cfu/mL。
其中,所述MRS液体培养基的制备方法具体可如下:将酵母粉5g、胰蛋白胨10.00g、氯化钠10g、葡萄糖20g,蒸馏水1000ml,调节pH值至6.4;然后121℃灭菌15min。所述培养可置于恒温培养箱进行,培养条件为可为:42℃静置培养24h。
所述啤酒酵母菌种子菌液的制备方法如下:将啤酒酵母菌接种至MRS培养基中进行培养,获得菌液;进一步的,所述菌液中啤酒酵母菌的浓度为1.6×109cfu/mL。
其中,所述MRS液体培养基的制备方法具体可如下:将酵母粉5g、胰蛋白胨10.00g、氯化钠10g、葡萄糖20g,蒸馏水1000ml,调节pH值至6.4;然后121℃灭菌15min。所述培养可置于恒温培养箱进行,培养条件为可为:42℃静置培养24h。
上述方法步骤3)中,所述发酵培养基为改良的MRS培养基,即在标准MRS培养基中分别加入质量含量0.01%维生素H、维生素B6、叶酸、维生素原B5、维生素 B6。
上述方法步骤4)中,所述体积分数为60-80%的乙醇水溶液,具体可为体积分数为70%的乙醇水溶液。
上述方法步骤4)中,所述沉淀与所述乙醇水溶液的质量比为1:10。所述浸提的条件为:30℃浸提24小时。
上述方法步骤4)中,所述蒸发的条件为在90℃蒸发。
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防脱发和/或育发产品中的应用。
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防脱发和/或育发功效的产品。
所述产品的活性成分包括本发明所提供的中药发酵液。
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防脱发和育发功效。
本发明具有如下有益效果:
本发明将中药经过超微粉碎、酶解以及植物乳杆菌和啤酒酵母菌混合发酵后,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。混合菌发酵后比单纯一种菌发酵,用标准曲线法测定发酵液中蛋白质转化为肽的比例,双菌发酵肽转化率提高了20%。以苦参为例,应用液相色谱法,对发酵前后苦参碱进行检测,苦参碱由0.4225g/L增加至0.5127g/L。
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苦参、侧柏叶、积雪草、粉防乙、咖啡均符合《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。
改良的MRS培养基:将酵母粉5g、胰蛋白胨10.00g、氯化钠10g、葡萄糖 20g,0.1g、维生素H0.1g、维生素B6 0.1g、叶酸0.1g、维生素原B5 0.1g。加蒸馏水至1000ml,,调节pH值至6.4;然后121℃灭菌15min。
下述实施例中所使用的植物乳杆菌是植物乳杆菌(Lactobacillus plantarum)Rs0228,该菌株已于2016年08月18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.12881。
下述实施例中所使用的啤酒酵母菌(Saccharomyces cerevisiae)购自北纳创联生物科技有限公司,产品编号BNCC340642。
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。
下述实施例中所使用的羊胎盘为屠宰场获取的离体羊胎盘。
实施例1、制备具有防脱发和育发作用的中药发酵液
1)取净制后的苦参、侧柏叶、积雪草、粉防乙、咖啡按质量比4:4:3:2:1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。
3)将羊胎盘用蒸馏水清洗干净后,加入1倍的蒸馏水进行匀浆。匀浆的条件是 10分钟。
4)将植物乳杆菌Rs0228的单菌落接种于装有100mL MRS液体培养基的试管中,42℃静置培养24h后,得到植物乳杆菌Rs0228的菌液(植物乳杆菌的浓度为 1.6×109cfu/mL)。
5)将啤酒酵母菌的单菌落接种于装有100mL MRS液体培养基的试管中,42℃静置培养24h后,得到啤酒酵母菌菌液(啤酒酵母菌的浓度为1.6×109cfu/mL)。
6)改善MRS培养基的制备:将酵母粉5g、胰蛋白胨10.00g、氯化钠10g、葡萄糖20g,0.1g、维生素H0.1g、维生素B6 0.1g、叶酸0.1g、维生素原B50.1g。加蒸馏水至1000ml,调节pH值至6.4;然后121℃灭菌15min。
7)将酶解后的中药液加入改良MRS培养基,再加入羊胎盘匀浆(其中,所述酶解后的中药液与羊胎盘匀浆的质量比为5:1;所述改良MRS培养基的加入量与酶解后的中药液与羊胎盘匀浆质量之和的比为1:1),分别接种10%植物乳杆菌RsO的菌液和啤酒酵母菌液,42℃静置培养24h后,离心,分别收集上清液和沉淀,所述上清液即为微米中药发酵液;
8)将发酵后的沉淀按1:10加入体积分数70%的乙醇30℃浸提24小时,离心收集上清。将上清90℃蒸发去除乙醇,浓缩成原体积的1/3为醇提取物。
9)将微米中药发酵液与醇提取物按体积比5:1混合,即为最后的中药发酵提取液。
可以用上述得到的中药发酵提取液为活性成分(含量在10-15%)做成洗护发用品。
以苦参为例,应用液相色谱法,对发酵前后苦参碱进行检测,苦参碱由0.4225 g/L增加至0.5127g/L(表1)。
表1苦参发酵前后苦参碱含量测定结果(g/L)
苦参碱/(g/L) | |
发酵前样品 | 0.4225 |
发酵后样品 | 0.5127 |
表1中所述发酵前样品是步骤2)灭酶后得到的“酶解后的中药液”;所述发酵后样品是步骤7)所述的微米中药发酵液。
对比例1、制备具有防脱发和育发作用的中药发酵液
该对比例基本同实施例1,区别在于使用“植物乳杆菌(Lactobacillusplantarum) Rs 0228”替换实施例1中的“植物乳杆菌和啤酒酵母菌”。
用标准曲线法(y=0.028x+0.011,相关系数为R2=0.998),分别测定实施例1和对比例1制备的发酵液中蛋白质转化为肽的比例;实施例1的转化比例为35%;对比例1的转化比例为25%,结果表明混合菌发酵后比单纯一种菌发酵,发酵液中蛋白质转化为肽的比例提高了10%。
实施例2、防脱发和育发效果评价
1、动物生发实验
1)小鼠脱毛模型建立
健康成年昆明雌性SD小鼠40只,用基础饲料喂养3~5d,将松香与石蜡按 1:1混合加热溶化,冷却至合适的温度后(以免烫伤小鼠皮肤),在小鼠麻醉条件下均匀涂于其背部,涂抹面积约为2cm×3cm;待凝固变硬后剥去,拔掉所有毛发,
2)试验动物给药
选取脱毛区光滑且无皮肤破损的小鼠,随机分为2组,每组20只,一组用于促进生发作用试验;另一组用作对照实验。
3)试验小鼠为正常饲养,从脱毛后第2天起,对药物观察组其背部涂抹10%的中药发酵液(用水稀释),对照组涂抹生理盐水,连续涂抹给药12d。
4)实验结果
对照组第6天出现小绒毛,第8天明显出现毛发,第10天毛发生长良好;药物组第5天出现小绒毛,第7天出现毛发,第9天毛发生长良好。提示中药发酵液有促进毛发生长作用。
注:“-”代表无毛发生长;“+”代表少量细小绒毛出现;“++”代表代表较明显毛发出现;“++ +”代表毛发生长良好;“++++”代表毛发生长同正常小鼠无异;“*”代表所述状况显著。
2、头发/头皮微观成像测试
选取21名符合条件的志愿者(7名女性,14名男性)参加测试。测试期间志愿者每天或间隔1天使用含有中药发酵液的洗发露(中药发酵液的含量10%)和护发素(含量10%)洗头发,连续使用4周。在头顶1.5cm×1cm区域剃发并对该区域进行头发/头皮微观成像测试(每一时间点间隔一天分别测试一次)。试验数据均采用SPSS 13.0统计学软件分析。
21名志愿者使用含中药发酵液的洗发露和护发素4周后的头发总密度和生长期头发比例的数据如表1所示。产品使用4周后所测得的生长期头发比例较0周增加 4.76%,有显著性差异(p≤0.05);说明产品有一定的防脱发育发效果。
表2产品使用前(T0w)和4周后(T4w)测试结果
Claims (10)
1.一种中药发酵液的制备方法,包括下述步骤:
1)将植物中药组合物先粉碎成粗粉,再进行超微粉碎至10μm以下,得到微米化中药;所述植物中药组合物由下述质量份的药材组成:侧柏叶4份、苦参4份、积雪草3份、粉防已2份、咖啡1份;
2)将离体的羊胎盘加入纯水后进行匀浆,得到羊胎盘匀浆;
3)将微米化中药分散于水中,形成胶体溶液;向所述胶体溶液中加入果胶酶进行酶解;酶解结束后灭酶,然后向所述酶解体系中加入改良MRS培养基、羊胎盘匀浆,再向其中接种植物乳杆菌和啤酒酵母菌进行发酵,离心,分别收集上清液和沉淀,所述上清液即为微米中药发酵液;
4)将收集的所述沉淀加入体积分数为60-80%的乙醇水溶液浸提,浸提结束后离心收集上清,再将上清蒸发去除乙醇,浓缩成原体积的1/3作为醇提取物;
5)将步骤3)得到的微米中药发酵液与步骤4)得到的醇提取物按体积比5:1混合,即为所述中药发酵液。
2.根据权利要求1所述的制备方法,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所述的制备方法,其特征在于:所述步骤2)中,所述离体的羊胎盘与水的质量比为1:1-2。
4.根据权利要求1-3中任一项所述的制备方法,其特征在于:所述步骤3)中,所述微米化中药与水的质量比为1:4-8。
5.根据权利要求1-4中任一项所述的制备方法,其特征在于:所述步骤3)中,所述果胶酶与微米化中药的用量比为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;
所述灭酶的条件为:95℃保温10min。
6.根据权利要求1-5中任一项所述的制备方法,其特征在于:所述步骤3)中,所述酶解体系与羊胎盘匀浆的质量比为5:1;所述改良MRS培养基的质量与所述酶解体系和羊胎盘匀浆的质量之和的比为1:1;
所述植物乳杆菌是植物乳杆菌(Lactobacillus plantarun)Rs0228,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.12881;
所述植物乳杆菌以植物乳杆菌菌液的形式接种;接种量为5-10%;
所述啤酒酵母菌以啤酒酵母菌菌液的形式接种;接种量为5-10%;
所述发酵培养的条件:42℃培养24h;
所述步骤3)中,所述发酵培养基为改良的MRS培养基,即在标准MRS培养基中分别加入质量含量0.01%维生素H、维生素B6、叶酸、维生素原B5、维生素B6。
7.根据权利要求1-6中任一项所述的制备方法,其特征在于:所述步骤4)中,所述体积分数为60-80%的乙醇水溶液为体积分数为70%的乙醇水溶液;
所述沉淀与所述乙醇水溶液的质量比为1:10;所述浸提的条件为:30℃浸提24小时;
所述蒸发的条件为在90℃蒸发。
8.权利要求1-7中任一项所述方法制备得到的中药发酵液。
9.权利要求8所述的中药发酵液在制备防脱发和/或育发产品中的应用;所述产品为药物和/或保健品和/或化妆品。
10.一种具有防脱发和/或育发功效的产品,其活性成分包括权利要求8所述的中药发酵液。
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