CN105878554A - 一种益生菌发酵八珍汤组合物及其制备方法和应用 - Google Patents
一种益生菌发酵八珍汤组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种益生菌发酵八珍汤组合物，将中药人参、白术、茯苓、当归、白芍、川芎、地黄、甘草经烘干，粉碎，水浸泡，调整pH值，淀粉酶酶解，灭菌，接种多株益生菌，发酵，提取，浓缩，干燥或不干燥，灭菌制成。本发明进行了体外益生菌生长实验和对低白小鼠造血功能的影响实验，结果显示：本发明的益生菌发酵八珍汤组合物在体外对人肠道中的主要益生菌具有明显的生长促进作用；能够提高低白小鼠白细胞的水平，特别是提高中性粒细胞和淋巴细胞的数量和比例，在解毒的同时，提高了小鼠个体的免疫能力。
Description
技术领域
本发明涉及一种益生菌发酵中药组合物及其制备方法和应用，尤其涉及一种益生菌发酵八珍汤组合物及其制备方法和应用，属于医药领域。
背景技术
八珍汤源自《正体类要》，作为中医古方气血双补的经典方，由人参、地黄、白术、茯苓、当归、白芍、川芎、甘草组成,具有益气补血之功效。主治气血两虚证。八珍汤是四君子汤和四物汤的复方，方中人参与地黄相配，益气养血，共为君药；白术、茯苓健脾渗湿，协人参益气健脾，当归、白芍养血和营，助熟地补益阴血，均为臣药；佐以川芎活血行气，使之补气而不滞；甘草益气和中，调和诸药，为使药。
祖国医学认为，气为血帅，血为气母，精生血，精血同源，互相资生，互为依存。四君子汤健脾益气、四物汤补血养血，八珍汤汇两方之功，奏两方之效。故八珍汤常用于病后失调、久病失治、失血过多等慢性消耗性疾病。
癌症是现在最常见的慢性消耗性疾病，并且手术、放疗、化疗是临床常用的治疗癌症的方法，由于多程治疗后患者耗气伤血，属中医虚证。
放疗、化疗药物在应用的过程中有较强的毒副作用，它不但对癌细胞和肿瘤细胞起到了杀伤和抑制作用，对机体的正常细胞，特别是造血系统的祖细胞产生了抑制，存在着免疫及造血的双重抑制，从而在个体水平上表现为骨髓抑制现象，外周血白细胞、血小板和中性粒细胞数减少。并且常伴有营养不良和免疫功能低下，免疫功能低下可增加感染和肿瘤复发的危险性。
实验表明:八珍汤能改善白细胞减少症，改善严重的骨髓抑制，并且可降低中性粒细胞减少的发生率，提高机体免疫，改善中医证候及生活质量。但是，中药中的许多成分需要在肠道菌群的作用下才能转化为有效成分，因此肠道环境的差异，导致个体性差异明显。尤其是癌症病人，肠道环境状况不佳。
同时，癌症术后早期肠内营养优于肠外营养，可调节机体的代谢和免疫功能，纠正手术创伤所致的免疫抑制，改善预后。但常规营养支持对于提高癌术后机体免疫力，纠正营养不良等方面的作用存在局限性，近年提出了“肠内免疫营养”，其目的是改善营养状况，调节免疫功能和炎症反应，减少术后并发症的发生。这方面的研究目前集中于GH、谷氨酰胺、精氨酸、ω一3脂肪酸、核苷和核苷酸、中链甘油三酯等。但由于价格昂贵、副作用较多等原因，在临床上应用不多。
发明内容
为了克服上述不足，本发明提供了一种益生菌发酵八珍汤组合物。本发明所述的益生菌发酵八珍汤组合物，具体涉及中药人参、白术、茯苓、当归、白芍、川芎、地黄、甘草，经烘干，粉碎，水浸泡，调整pH值，酶解，灭菌，接种多株益生菌，发酵，提取，浓缩，干燥或不干燥，灭菌，制得发酵组合物。
所述中药在益生菌发酵八珍汤组合物中的重量份为：人参1～10份；白术4～20份；茯苓4～20份；当归4～30份；白芍2～20份；川芎2～20份；地黄4～30份、甘草1～10份。
优选的，各中药成分在益生菌发酵八珍汤组合物中的重量份为：人参5份；白术10份；茯苓10份；当归15份；白芍10份；川芎8份；地黄15份、甘草5份。
所述的酶为高温淀粉酶，添加量为中药材重量的0.1～1.0%
所述的益生菌为双歧杆菌和乳杆菌。
双歧杆菌选自两歧双歧杆菌、短双歧杆菌、青春双歧杆菌和婴儿双歧杆菌；所述的乳杆菌选自嗜酸乳杆菌、德氏乳杆菌、植物乳杆菌、干酪乳杆菌和鼠李糖乳杆菌。
菌种来源于中国典型培养物保藏中心（CCTCC）（中国，武汉）或中国普通微生物菌种保藏管理中心（CGMCC）（中国，北京）或者从正常人的粪便或肠粘膜组织中分离得到这些细菌菌株。
本发明的益生菌发酵八珍汤组合物的制备方法，包括以下步骤：
a. 取中药复方中的人参、白术、茯苓、当归、白芍、川芎、地黄、甘草，50-80℃烘干，粉碎，加水5～10倍量，水浸泡0.5～1.0小时，调整pH6.0±0.2，加热至80～90℃，加入高温淀粉酶，酶解至使碘试液不变蓝色；
b．将步骤a得到的中药酶解物，调整pH7.0±0.5，并于110～121℃下灭菌30～60分钟，温度降至37℃时，同时接种预培养的体积百分比0.5～2.0%的一种或两种双歧杆菌的菌液和预培养的体积百分比各0.2～1.0%的两种或者三种乳杆菌的菌液；常规发酵16～48小时，当pH降至4.0以下时结束发酵；
C.将步骤b得到的发酵物分离药渣和发酵液，药渣再加乙醇或水5～10倍量，加热至60～80℃，保持0.5～2.0小时，滤过，提取液回收乙醇或浓缩，合并浓缩发酵液，定容或喷雾干燥，灭菌，即得到益生菌发酵八珍汤组合物。
本发明中使用的双歧杆菌和乳杆菌属于乳酸菌，为常见益生菌菌株，主要是基于它们在人体肠道内分布的总体广泛性，以及它们生理学活性的互补性。乳酸菌发酵可直接产生有机酸、氨基酸等营养物质，并且乳酸菌参与多种维生素代谢，能产生维生素B、叶酸、烟酸、生物素等供机体所需。并且，中药和肠道菌群的相互作用对中药药效的发挥非常重要。
八珍汤配方中许多中药成分都是借助肠道细菌的作用转化为有效成分而达到治疗作用，尤其是具有水溶性糖部分的葡萄糖苷成分。这类化合物通常在肠道内难以吸收，生物利用度低。其原形物药理活性较小，被认为是“天然前体药物”，经肠菌代谢后被水解生成苷元而发挥其药理作用。例如：天然人参中含有人参皂苷Rb1，Rb2，Re，Rg1和20(S)-原人参二醇等成分。人参皂苷Rb1在肠菌作用下其代谢途径为人参皂苷Rb1→人参皂苷Rd→人参皂苷F2→ Compound K→20(s)-原人参二醇。人参皂苷Rg1在人体内代谢途径为：人参皂苷Rg1→人参皂苷Rh1→原人参三醇。人参皂苷Re的人体肠内菌代谢次生产物有6个，分别为人参皂苷Rg1、人参皂苷 Rg2、人参皂苷Rh1、20，25-环-人参三醇、20(R)，25-羟基人参皂苷Rg2、原人参三醇。Rh1可作用于T细胞和腹腔巨噬细胞而产生免疫调节作用。原人参二醇类皂苷成分的肠菌主要代谢产物Compound K对人组织瘤细胞(THP-1)分泌与炎症细胞因子(IL-Iα，TNF-α，IL-8)产生影响，显示抗癌活性。甘草酸(glyeyrrhizin)在肠道内难以吸收，与肠道菌群作用产生18a-甘草次酸的中间产物，最终代谢为甘草次酸 (glycyrrhetie acid)而被机体吸收产生药理活性。甘草苷、 异甘草苷也转化为甘草素和异甘草素产生药理活性。芍药苷(paeoniflorin)为双环单萜苷类，在与肠道菌群作用下产生4个代谢产物芍药苷代谢素Ⅰ，Ⅱ， Ⅲ，Ⅳ。芍药苷口服吸收率极低，经口服摄入的芍药苷需在肠道细菌分泌的β-葡萄糖苷酶和β-葡萄糖酯酶的催化下转化成其相应的芍药盐酸硫胺苷元才能发挥药理作用，在Caco-2细胞上的转运研究中发现芍药苷的生物利用度比其苷元低40倍。
中医认为“脾胃为后天之本”，要恢复身体机能，必须在进补的同时，适当强化人体的消化吸收功能，八珍汤与益生菌共同发酵后能增加药物成分的转化和吸收。并且发酵过程中产生大量小分子短链脂肪酸，不但可以刺激肠道蠕动，并且利于有益细菌的生长繁殖，从而可以改善胃肠道内环境，增强药物疗效。发酵组合物进入肠道可刺激消化道壁引发免疫激活产生生物效应；刺激肠内微生态引发生物活性物质分泌产生生物效应；促进肠内微生态新的平衡系统建立。
本发明所述的益生菌发酵八珍汤组合物可用于制备预防或治疗癌症患者术后或放化疗过程中白细胞降低的保健食品、药品。
可按照制药工业中已知的方法，将本发明的发酵组合物与一种或多种医药上可接受的载体或赋形剂或稀释剂按适当的比例混合，并按已知方法除菌后制成含本发明发酵组合物的制剂。
根据给药途径的不同，可按照已知的常规方法，将本发明的发酵八珍汤组合物配制成适于口服给药的合剂、乳剂、散剂、颗粒剂、片剂、丸剂、胶囊剂。
为了制备适于口服给药的合剂、乳剂或片剂、散剂、胶囊剂、丸剂、颗粒剂，可以使用蔗糖、乳糖、半乳糖等二糖，或甘露醇、山梨醇等六碳多羟基醇，以及糊精、β-环糊精、淀粉、明胶、脂质、微晶纤维素等作为载体或赋形剂。必要时，也可在这些口服制剂中加入缓冲盐、着色剂、甜味剂、香料、分散剂及表面活性剂。
根据使用目的的不同，本发明的发酵八珍汤组合物制剂除含有作为基本活性成分的发酵组合物外，还可含有一种或多种具有相同、相似或不同生物学活性，并对基本活性成分表现有辅助或协同作用，但彼此又互不拮抗的天然或合成的其他药物成份或其混合物。
可将本发明的发酵组合物直接制成保健食品或药物，适于癌症患者术后使用或放化疗期间预防或治疗白细胞减少。
一般说来，本发明的发酵组合物的口服给药剂量可根据待治疗的病症或病理状态的性质、严重程度、病人的年龄、体重、一般健康状态，以及病人对所用药物的敏感性、耐受性和给药方式等因素，按照个体化的原则由临床医生确定。
有益效果：为检测本发明的益生菌发酵八珍汤组合物具有调节有益菌群的作用，同时能改善白细胞减少症，本发明分别进行了体外益生菌生长实验和对低白小鼠造血功能的影响实验，结果显示：本发明的益生菌发酵八珍汤组合物在体外对人肠道中的主要益生菌具有明显的生长促进作用；并且具有明显提高低白小鼠白细胞的功能，特别是提高中性粒细胞和淋巴细胞数量和比例，作用优于未发酵八珍汤。
具体实施方式
实施例1：益生菌发酵八珍汤组合物a的制备
取中药人参50g；白术100g；茯苓100g；当归150g；白芍100g；川芎80g；地黄150g、甘草50g。70℃烘干，粉碎过20目，加水5倍量，水浸泡1.0小时，调整pH6.0，加热至80℃，加入1.2g高温淀粉酶，酶解0.5小时，至使碘试液不变蓝色；
将中药酶解物，调整pH7.0，并于115℃下灭菌50分钟，温度降至37℃时，同时接种预培养的体积百分比1.0%的短双歧杆菌（CGMCC 1.2213）的菌液和体积百分比各0.5%的嗜酸乳杆菌（CGMCC 1.1854）、植物乳杆菌（CGMCC 1.19）及德氏乳杆菌（CGMCC 1.1480）的菌液，37℃发酵24小时检测pH值，当pH值降至4.0以下时结束发酵；分离药渣和发酵液，药渣再加水8倍量，加热至80℃，保持1.0小时，滤过，滤液合并发酵液，减压浓缩,定容至1000ml；灭菌，即得到益生菌发酵八珍汤组合物。
实施例2：益生菌发酵八珍汤组合物b的制备
取中药人参1000g；白术800g；茯苓800g；当归2000g；白芍2000g；川芎400g；地黄1000g、甘草600g。80℃烘干，粉碎过40目，加水10倍量，水浸泡1.0小时，调整pH6.0，加热至80℃，加入15g高温淀粉酶，酶解1.0小时，至使碘试液不变蓝色；
将中药酶解物，调整pH7.2，并于121℃下灭菌30分钟，温度降至37℃时，同时接种预培养的体积百分比1.0%的两歧双歧杆菌（CGMCC 1.5090）的菌液和体积百分比各0.5%的干酪乳杆菌（CGMCC 1.3206）、植物乳杆菌（CGMCC 1.19）及鼠李糖乳杆菌（CGMCC 1.26）的菌液，37℃发酵40小时检测pH值，当pH值降至4.0以下时结束发酵，分离药渣和发酵液，药渣再加80%乙醇8倍量，加热至80℃，保持2.0小时，滤过，滤液回收乙醇，添加β-环糊精包合，合并发酵液，浓缩至5000ml，喷雾干燥，辐射灭菌，即得到益生菌发酵八珍汤组合物b。
实验例1：本发明的益生菌发酵八珍汤组合物对肠道益生菌的体外生长促进作用
本实施例旨在举例说明按本发明方法制备的发酵八珍汤组合物对选自长双歧杆菌（B.longum）、两歧双歧杆菌（B.bifidum）和嗜酸乳杆菌（L.acidophilus）等人肠道益生菌的体外生长促进作用。
使用长双歧杆菌、两歧双歧杆菌和嗜酸乳杆菌作为实验菌，实验菌经活化后按每管0.1ml（接种后培养基细菌浓度为106个/ml）分别接种于（1）常规PTYG培养基（每升培养基含有胰蛋白胨5g、大豆蛋白胨5g、酵母提取物10g、葡萄糖10g、盐溶液40ml、半胱氨酸盐酸盐0.5g、吐温80 0.1ml、0.1%刃天青1ml）；（2）本发明的实施例1制备的发酵八珍汤组合物a稀释2倍；本发明的实施例2制备的发酵八珍汤组合物b水溶解稀释10倍（分别使用氢氧化钠调pH值至7并灭菌）。接种后将试管置于37℃下恒温培养24小时。培养后收集培养液并稀释，然后进行菌落计数。结果见表1：
表1 发酵八珍汤组合物对双歧杆菌及嗜酸乳杆菌生长的影响
从表1中所示的数据可以看出，将双歧杆菌和嗜酸乳杆菌等已知的益生菌，接种于稀释的本发明发酵八珍汤组合物中并培养24小时后，被接种的细菌数均增殖到108cfu/ml以上。
由以上结果可以看出，按本发明方法制备的发酵组合物对人体肠道中的主要益生菌具有明显的生长促进作用。
实验例2：比较八珍汤发酵前后对低白小鼠白细胞的影响
本实施例旨在利用注射环磷酰胺使小鼠造血功能受到抑制，造成血虚模型，观察八珍汤发酵前后对血虚型小鼠的造血功能影响。
取实施例1制备的发酵组合物a和按相同配方制备的未经益生菌发酵的八珍汤A，分别浓缩制成相同浓度（药材：药液=1：1）；取实施例2制备的发酵组合物b和按相同配方制备的未经益生菌发酵的八珍汤B，分别加水溶解稀释制成相同浓度（药材：药液=1：1），作为供试样品。
昆明种小鼠，雌雄各半，随机分为六组：正常组、模型组、八珍汤A组、八珍汤B组、发酵组合物a组、发酵组合物b组。正常组和模型组每只小鼠每日用生理盐水O．2ml/lOg体重灌胃，八珍汤A组、八珍汤B组分别用八珍汤A、八珍汤B O. 2ml／lOg体重灌胃，发酵组合物a组、发酵组合物b组分别用发酵八珍汤组合物a、发酵八珍汤组合物b O. 2ml／lOg体重灌胃，连续10天。第8 天正常组腹腔注射生理盐水0.lml／10g体重，模型组、八珍汤A组、八珍汤B组、发酵组合物a组、发酵组合物b 组分别腹腔注射环磷酰胺CY 0.1ml／10g，连续3天。
小鼠于注射环磷酰胺（CY）后第4天，分组取尾静脉血，用全自动血象分析仪对外周血象进行分析检测，测定白细胞计数、血小板计数。结果用生物统计学方法处理。实验结果见表2：
表2 对小鼠外周血象的影响(x±s)
*与模型组相比，p<0.05；**与模型组比较，p<0.01；※与八珍汤组相比，p<0.05。
外周血象结果说明环磷酰胺显著降低了小鼠外周血中的白细胞和血小板水平，模型组较之正常组，其自细胞计数下降了 28.99％，血小板计数下降了20．08％，说明造模成功；八珍汤A组、八珍汤B组使其得到一定恢复。发酵组合物a组、发酵组合物b组使其基本恢复到正常水平。说明八珍汤能对抗化疗药物CY的毒副作用，一定程度抑制CY所致小鼠外周血象的降低，发酵八珍汤组合物能极显著抑制CY所致小鼠外周血象的降低，效果明显优于未发酵八珍汤。
另外，对白细胞中淋巴细胞及粒细胞进行更详细的数据检测和分析，实验结果见表3 ：
表3对小鼠白细胞的影响(x±s)
*与模型组比较，P<0.05； **与模型组比较，P<0.01；
※与八珍汤组比较，P<0.05； ※※与八珍汤组比较，P<0.01。
实验结果充分说明了发酵八珍汤组合物不但能够提高白细胞的水平，也能提高中性粒细胞和淋巴细胞的数量，在解毒的同时，提高了小鼠个体的免疫能力。并且益生菌发酵八珍汤组合物提高中性粒细胞和淋巴细胞效果优于未发酵八珍汤。
Claims (7)
1.一种益生菌发酵八珍汤组合物，其特征在于，将中药人参、白术、茯苓、当归、白芍、川芎、地黄、甘草经烘干，粉碎，水浸泡，调整pH值，淀粉酶酶解，灭菌，接种多株益生菌，发酵，提取，浓缩，干燥或不干燥，灭菌，即得；
所述中药在益生菌发酵八珍汤组合物中的重量份为：人参1～10份；白术4～20份；茯苓4～20份；当归4～30份；白芍2～20份；川芎2～20份；地黄4～30份、甘草1～10份；
所述的益生菌为双歧杆菌和乳杆菌。
2.根据权利要求1所述的益生菌发酵八珍汤组合物，其特征在于，所述中药在益生菌发酵八珍汤组合物中的重量份为：人参5份；白术10份；茯苓10份；当归15份；白芍10份；川芎8份；地黄15份、甘草5份。
3.根据权利要求1所述的益生菌发酵八珍汤组合物，其特征在于，所述的酶为高温淀粉酶，添加量为中药材重量的0.1-1.0%。
4.根据权利要求1所述的益生菌发酵八珍汤组合物，其特征在于，所述的双歧杆菌选自两歧双歧杆菌、短双歧杆菌、青春双歧杆菌和婴儿双歧杆菌；所述的乳杆菌选自嗜酸乳杆菌、德氏乳杆菌、植物乳杆菌、干酪乳杆菌和鼠李糖乳杆菌。
5.权利要求1-4任一项所述的益生菌发酵八珍汤组合物的制备方法，其特征在于，步骤如下：
a.取中药复方中的人参、白术、茯苓、当归、白芍、川芎、地黄、甘草，50-80℃烘干，粉碎，加水5～10倍量，水浸泡0.5～1.0小时，调整pH6.0±0.2，加热至80～90℃，加入高温淀粉酶，酶解至使碘试液不变蓝色；
b.将步骤a得到的中药酶解物调整pH7.0±0.5，并于110～121℃下灭菌30～60分钟，温度降至37℃时，同时接种预培养的体积百分比0.5～2.0%的一种或两种双歧杆菌的菌液和预培养的体积百分比各0.2～1.0%的两种或者三种乳杆菌的菌液；常规发酵16～48小时，当pH降至4.0以下时结束发酵；
c.将步骤b得到的发酵物分离药渣和发酵液，药渣再加乙醇或水5～10倍量，加热至60～80℃，保持0.5～2.0小时，滤过，提取液回收乙醇或浓缩，合并浓缩发酵液，定容或喷雾干燥，灭菌，即得。
6.权利要求1-4任一项所述的益生菌发酵八珍汤组合物在制备预防或治疗癌症患者术后或放化疗过程中白细胞降低的保健食品、药品中的应用。
7.根据权利要求6所述的应用，其特征在于，将所述的益生菌发酵八珍汤组合物与一种或多种医药上可接受的载体或赋形剂或稀释剂，按照已知的常规方法，制成适于口服给药的合剂、乳剂、散剂、颗粒剂、片剂、丸剂、胶囊剂。
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