CN112521447A - 一种化合物、中药益生菌发酵产物及其在制备具有延缓衰老作用的药物或保健品中的应用 - Google Patents
一种化合物、中药益生菌发酵产物及其在制备具有延缓衰老作用的药物或保健品中的应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域,具体公开了一种化合物、中药益生菌发酵产物及其在制备具有延缓衰老作用的药物或保健品中的应用。所述的中药益生菌发酵产物,包含式(Ⅰ)化合物;其制备方法包含如下步骤:(1)取中药原料用益生菌进行发酵,得发酵液;(2)将发酵液进行干燥后即得所述的中药益生菌发酵产物;步骤(1)中所述的中药原料为包含紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的中药原料。本发明所述的式(Ⅰ)化合物以及中药益生菌发酵产物具有优异的抗氧化以及延缓衰老的作用;可以用于制备药物和保健品。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种化合物、中药益生菌发酵产物及其在制备具有延缓衰老作用的药物或保健品中的应用。
背景技术
人的个体随着年龄的增长,会出现头发发白、牙齿脱落、肌肉萎缩、血管硬化、感觉反应迟钝、记忆力衰退、代谢功能下降等衰老情况。通常人们将体内各种器官、组织和细胞随着年龄的增加而伴随出现的不可逆转的功能衰退、逐渐趋向死亡的现象称之为机体的衰老。集体的衰老都有其细胞生物学基础。在衰老过程中,组织器官的细胞也经历了形态结构和生理功能逐渐衰退的现象。随着年龄的增长,细胞自我更新和增殖分化的能力下降,导致组织器官损伤难以修复,正常生理功能难以维系,机体衰老必然发生。如何延缓衰老这一课题亟待探索和研究。
中医基础理论对衰老机制的认识以脏腑为核心,其中肾在中医衰老机制中占有重要的地位。抗氧化、抗衰老中药(包括单味中药、复方制剂以及中药提取物),对其选用既要符合综合性、早期性和长期性原则,又要从个体的实际出发,因人制宜。2000多年前,中国最早的中医典籍《内经》就已经有了对人类衰老过程的记载。《灵枢·天年篇》记载:“五十岁,肝气始衰,肝叶始薄,胆汁始减,目始不明。六十岁,心气始衰,苦忧悲,血气懈坠,故好卧。七十岁,脾气虚,皮肤枯。八十岁,肺气衰,魄离,魄离故言善误。九十岁,肾气焦,四脏经脉空虚”。
中医基础理论对衰老机制的认识以脏腑为核心,包括肾虚衰老、肝郁衰老、脾胃虚弱衰老、气滞血瘀痰浊衰老等学说。中医认为,衰老首要是因肾虚,其次是脾虚,再次是气血两虚。故补肾、健脾、益气是延缓衰老的基本途径,活血化瘀是延缓衰老的主要方法。具有补气、扶正、固本、补益等作用的一些中药,通过抗氧化等原理能达到抗衰老的目的。
百合,药性甘、平。归脾、肺、心经。其效:大补元气,复脉固脱,补脾益肺,生津,安神。主治:用于体虚欲脱,肢冷脉微,脾虚食少便溏,气短乏力,肺虚喘咳,津伤口渴,内热消渴,久病虚羸,惊悸失眠,阳痿宫冷;心力衰竭,心原性休克。对中枢神经***有特殊作用,可使大脑的兴奋和抑制保持平衡,增强体力,有显著的抗疲劳作用,可改善情绪和睡眠状况,消除全身无力及头痛症状。
薄荷,药性微甘、温。归肝、肾经。其效:祛风湿,补肝肾,强筋骨,活血脉。主治:风寒湿痹,腰膝疼痛,筋骨痿软,小数点儿行迟,体虚赢弱,跌打伤,骨折,水肿,脚气,阴下湿痒。用于肝肾不足,加强身体对有害刺激因素的抵抗能力,有良好的抗疲劳作用,能明显提高耐缺氧能力和增强机体的非特异性免疫功能。
芡实,药性甘、温。归脾、肺经。其效:益卫固表,补气升阳,托毒生肌,利水消肿。主治:用于气虚乏力、食少便溏,中气下陷,久泻脱肛,自汁盗汗,血虚萎黄,阴疽漫肿,气虚水肿,内热消渴。能补气升阳、固表止汗、托毒排脓、利水消肿,有增强免疫、促进代谢、降血压、利尿、抗菌等作用。
银杏,药性甘、平。有小毒。归心、肺经。其效:活血化瘀,通络止痛,敛肺平喘,化浊降脂。主治:用于瘀血阻络,胸痹心痛,中风偏瘫,肺虚咳喘,高脂血症。作为传统的中药材,一直以其对大脑的返老还童作用而闻名于世。银杏还是一种高效的抗氧化物。有实验显示,银杏在清除自由基方面的效率比维生素E高,可以有效地防止高脂肪的细胞膜被氧化。银杏能在细胞被自由基破坏之后,重新恢复细胞膜的完整。此外,银杏可以恢复大脑细胞接受来自神经传导***指导大脑进行工作的信号的能力。
其它如生熟地黄、天冬、麦冬、当归、丹皮、三七、党参、白术、灵芝、山楂、何首乌、茯苓、黄精等中药均可用作抗衰老药。目前有少量将百合、薄荷、银杏、芡实制成饮料或制剂的报道。这些方法虽然有一定的创新,但都仍沿用传统提取制备方法,人体吸收利用率低,存在一定的局限性。抗衰老药物不可乱用,其应用既要符合综合性、早期性和长期性原则,又要从个体的实际出发,因人制宜选用抗衰老药物。延缓衰老是目前生命科学研究中的重点和难点之一。因此开发出具有自主知识产权的抗氧化、抗衰老药物也成为中药研发的一个热点。
发明内容
鉴于此,本发明首先提供一种新化合物,经进一步研究表明,该新化合物具有抗氧化作用和延缓衰老作用。
此外,本发明还提供一种中药益生菌发酵产物,该中药益生菌发酵产物同样具有抗氧化作用和延缓衰老作用。
另外,本发明还提供了一种上述新化合物和中药益生菌发酵产物的制备方法以及应用。
本发明的详细技术方案如下:
一种式(Ⅰ)化合物或其可药用盐;
式(Ⅰ)化合物命名为:
Benzyl O-benzyl-N-(2,4-dimethoxybenzoyl)serylglycinate。
优选地,所述可药用盐为可药用酸加成盐,其中所述酸选自:柠檬酸、酒石酸、乳酸、乙酸、琥珀酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、丙酮酸、富马酸、马来酸、盐酸、氢溴酸、硝酸、磷酸和硫酸。
本发明提供一种式(Ⅰ)化合物的制备方法,其包含如下步骤:
(1)取中药原料用益生菌进行发酵,得发酵液;
(2)将发酵液进行干燥后得粗产物;
(3)将粗产物用色谱技术进行分离,即得式(Ⅰ)化合物;
步骤(1)中所述的中药原料为包含紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的中药原料;
步骤(1)中所述的益生菌选自双歧杆菌、乳杆菌菌液、青春双歧杆菌或婴儿双歧杆菌中的一种或二种以上的混合。
优选地,步骤(1)中所述的中药原料中紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的重量用量比为1~5:1~5:1~5:1~5:1~5:1~5:1~5:1~5:1~5。
最优选地,步骤(1)中所述的中药原料中紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的重量用量比为1:1:1:1:1:1:1:1:1。即各中药原料的重量用量相等。
优选地,步骤(1)的具体方法为:取中药原料加水提取,浓缩提取液得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为35~40℃、pH为6.5~7.5的条件下发酵24~48h,得发酵液。
进一步优选地,步骤(1)中水的用量为中药原料的8~15倍量。
进一步优选地,步骤(1)中浓缩至提取液体积的1/3~1/4得浓缩液。
进一步优选地,步骤(1)中发酵营养剂包括葡萄糖0.5%(重量/体积)、酵母粉0.1%(重量/体积)、酵母膏0.2%(重量/体积)和蛋白胨0.2%(重量/体积);所述的百分比是以浓缩液体积计算。
更进一步优选地,步骤(1)中发酵营养剂还包括磷酸二氢钾0.2%(重量/体积)、硫酸镁0.05%(重量/体积)、硫酸亚铁0.01%(重量/体积);所述的百分比是以浓缩液体积计算。
所述的益生菌为选自双歧杆菌和乳杆菌中的一种与青春双歧杆菌和婴儿双歧杆菌中的一种的组合。所述的益生菌可以以干粉形式加入,也可以通过常规方法进行活化,以菌液形式加入。
优选地,步骤(3)中所述的色谱技术为制备型HPLC技术;所述的制备型HPLC技术的具体条件为:采用反相色谱柱;选用紫外检测器,检测波长为220~230nm;以0.1~0.3体积%的三氟乙酸水溶液为流动相A,以0.1~0.3体积%的三氟乙酸乙腈溶液为流动相B,流动相A:流动相B=78:22;收集13.0~13.7min色谱峰对应的部位,浓缩干燥后即得式(Ⅰ)化合物。
本发明还提供一种中药益生菌发酵产物,其包含式(Ⅰ)化合物。
本发明还提供一种上述中药益生菌发酵产物的制备方法,其包含如下步骤:
(1)取中药原料用益生菌进行发酵,得发酵液;
(2)将发酵液进行干燥后即得所述的中药益生菌发酵产物;
步骤(1)中所述的中药原料为包含紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的中药原料;
步骤(1)中所述的益生菌选自双歧杆菌、乳杆菌菌液、青春双歧杆菌或婴儿双歧杆菌中的一种或二种以上的混合。
优选地,步骤(1)中所述的中药原料中紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的重量用量比为1~5:1~5:1~5:1~5:1~5:1~5:1~5:1~5:1~5。
最优选地,步骤(1)中所述的中药原料中紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的重量用量比为1:1:1:1:1:1:1:1:1。即各中药原料的重量用量相等。
优选地,步骤(1)的具体方法为:取中药原料加水提取,浓缩提取液得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为35~40℃、pH为6.5~7.5的条件下发酵24~48h,得发酵液。
进一步优选地,步骤(1)中水的用量为中药原料的8~15倍量。
进一步优选地,步骤(1)中浓缩至提取液体积的1/3~1/4得浓缩液。
进一步优选地,步骤(1)中发酵营养剂包括葡萄糖0.5%(重量/体积)、酵母粉0.1%(重量/体积)、酵母膏0.2%(重量/体积)和蛋白胨0.2%(重量/体积);所述的百分比是以浓缩液体积计算。
更进一步优选地,步骤(1)中发酵营养剂还包括磷酸二氢钾0.2%(重量/体积)、硫酸镁0.05%(重量/体积)、硫酸亚铁0.01%(重量/体积);所述的百分比是以浓缩液体积计算。
所述的益生菌为选自双歧杆菌和乳杆菌中的一种与青春双歧杆菌和婴儿双歧杆菌中的一种的组合。所述的益生菌可以以干粉形式加入,也可以通过常规方法进行活化,以菌液形式加入。
本发明还提供一种上述式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物在制备药物或保健品中的应用。
优选地,所述的药物为具有抗氧化作用和/或延缓衰老作用的药物;所述的保健品为具有抗氧化作用和/或延缓衰老作用的保健品。
优选地,所述的药物或保健品含有式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物和载体;所述式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物的质量分数为5%~95%。
进一步优选地,所述载体包括溶剂、聚合物和脂质体中的至少一种。更进一步优选地,所述溶剂包括但不限于水、生理盐水,以及其他非水性溶剂。更进一步优选地,所述聚合物包括但不限于为聚赖氨酸、聚乙烯亚胺及其改性物、壳聚糖、聚乳酸、明胶。更进一步优选地,所述脂质体可以但不限于为胆固醇、豆卵磷脂、蛋黄卵磷脂。
更进一步优选地,所述载体还包括稀释剂和赋形剂中的一种或多种。具体地,所述稀释剂包括淀粉类、糖类、纤维素类和无机盐中的一种或多种。具体地,所述赋形剂包括片剂中的黏合剂、填充剂、润滑剂,半固体制剂软膏剂、霜剂中的基质部分,液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、着色剂中的一种或多种。
进一步优选地,所述式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物的质量分数为10%~90%、15%~85%或30%~80%。
优选地,所述保健品或药物的形式包括片剂、胶囊、粉剂、颗粒剂、丸剂、糖浆剂、溶液剂、混悬剂或气雾剂。
优选地,所述保健品的形式还包括、凝胶状或水剂状。
进一步优选地,所述保健品还包括辅料基质,所述辅料基质包括单糖、寡糖、多糖、氨基酸、防腐剂和pH调节剂中的一种或多种。
有益效果:(1)本发明提供了一种全新的式(Ⅰ)化合物、中药益生菌发酵产物及其制备方法;所述的式(Ⅰ)化合物、中药益生菌发酵产物具有优异的抗氧化以及延缓衰老作用;可以用于制备药物和保健品;(2)在本发明实验显示,中药益生菌代谢产物可明显改善因D-半乳糖诱导造成的氧化损伤,明显提高小鼠血清和肝脏中SOD、GSH-Px以及T-AOC的活性,降低小鼠血清和肝脏中脂质过氧化物的MDA含量,提升小鼠的抗氧化能力。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例的附图,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明式(Ⅰ)化合物的质谱图。
图2是本发明式(Ⅰ)化合物的氢谱图。
图3是本发明式(Ⅰ)化合物的碳谱图。
图4为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠血清GSH-Px活性的测定图。
图5为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠血清T-AOC活性的测定图。
图6为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠血清SOD活性的测定图。
图7为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠血清MDA含量的测定图。
图8为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠肝脏GSH-Px活性的测定图。
图9为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠肝脏T-AOC活性的测定图。
图10为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠肝脏SOD活性的测定图。
图11为中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对小鼠肝脏MDA含量的测定图。
图12中药益生菌发酵产物及式(Ⅰ)结构的单体化合物为N2秀丽隐杆线虫寿命的影响。
图13中药益生菌代谢产物对氧化应激条件下N2秀丽隐杆线虫生存率的影响。
图14中药益生菌代谢产物对氧化应激条件下N2秀丽隐杆线虫ROS含量的影响。
图15中药益生菌代谢产物对氧化应激条件下N2秀丽隐杆线虫MDA活性的影响。
图16中药益生菌代谢产物对氧化应激条件下N2秀丽隐杆线虫SOD活性的影响。
具体实施方式
下面将结合实施例对本发明技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1式(Ⅰ)化合物的制备
(1)取中药原料加10倍水提取,浸泡60分钟,超声提取60分钟;浓缩提取液至1/3体积得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为37℃、pH为7.0的条件下发酵36h,得发酵液;
(2)将发酵液于8000r/min离心30min,除去沉淀并保留上清液,对上清液进行冷冻干燥,得粗产物;
(3)将粗产物用色谱技术进行分离,即得式(Ⅰ)化合物;
步骤(1)中所述的中药原料由紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷和芡实按重量比1:1:1:1:1:1:1:1:1组成(即各中药原料用量相等);
步骤(1)中发酵营养剂的加入量按浓缩液的体积计算:其包括葡萄糖0.5%(重量/体积)、酵母粉0.1%(重量/体积)、酵母膏0.2%(重量/体积)和蛋白胨0.2%(重量/体积);磷酸二氢钾0.2%(重量/体积)、硫酸镁0.05%(重量/体积)、硫酸亚铁0.01%(重量/体积);
步骤(1)中所述的益生菌为乳杆菌干粉和婴儿双歧杆菌干粉;按浓缩液的体积计算,所述的乳杆菌干粉的加入量为5%(重量/体积),婴儿双歧杆菌干粉的加入量为2%(重量/体积);
步骤(3)中所述的色谱技术为制备型HPLC技术;所述的制备型HPLC技术的具体条件为:采用ODS反相色谱柱;选用紫外检测器,检测波长为220nm;以0.2体积%的三氟乙酸水溶液为流动相A,以0.2体积%的三氟乙酸乙腈溶液为流动相B,流动相A:流动相B=78:22;收集13.0~13.7min色谱峰对应的部位,浓缩干燥后即得式(Ⅰ)化合物。
式(Ⅰ)化合物的质谱、氢谱及碳谱数据如下(具体谱图见图1~3):
质谱数据显示[M+Na]+为529.2,分子式为C28H30N2O7,1H NMR(500MHz,DMSO)δ:8.64(t,1H),8.55(d,1H),7.92(d,1H),7.31(m,10H),6.68(m,2H),5.12(s,2H),4.75(m,1H),4.51(s,2H),3.95(d,2H),3.56(m,2H),3.37(s,6H),3.07(m,1H).13C NMR(126MHz,DMSO)δ:170.26,169.52,163.68,163.25,158.98,138.16,135.87,132.83,128.40,128.21,128.05,127.91,127.44,127.40,113.69,105.97,98.68,72.13,70.09,65.88,56.25,55.57,52.82,40.94.
经质谱、氢谱及碳谱解析,可以确定上述方法制备得到了式(Ⅰ)化合物Benzyl O-benzyl-N-(2,4-dimethoxybenzoyl)serylglycinate。
实施例2中药益生菌发酵产物的制备
(1)取中药原料加10倍水提取,浸泡60分钟,超声提取60分钟;浓缩提取液至1/3体积得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为37℃、pH为7.0的条件下发酵36h,得发酵液;
(2)将发酵液于8000r/min离心30min,除去沉淀并保留上清液,对上清液进行冷冻干燥,得中药益生菌发酵产物;
步骤(1)中所述的中药原料由紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷和芡实按重量比1:1:1:1:1:1:1:1:1组成(即各中药原料用量相等);
步骤(1)中发酵营养剂的加入量按浓缩液的体积计算:其包括葡萄糖0.5%(重量/体积)、酵母粉0.1%(重量/体积)、酵母膏0.2%(重量/体积)和蛋白胨0.2%(重量/体积);磷酸二氢钾0.2%(重量/体积)、硫酸镁0.05%(重量/体积)、硫酸亚铁0.01%(重量/体积);
步骤(1)中所述的益生菌为乳杆菌干粉和婴儿双歧杆菌干粉;按浓缩液的体积计算,所述的乳杆菌干粉的加入量为5%(重量/体积),婴儿双歧杆菌干粉的加入量为2%(重量/体积)。
对比例1中药提取物
取中药原料加10倍水提取,浸泡60分钟,超声提取60分钟;将提取液浓缩干燥得中药提取物。所述的中药原料由紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷和芡实按重量比1:1:1:1:1:1:1:1:1组成(即各中药原料用量相等)。
实验例1中药益生菌发酵产物及式(Ⅰ)结构的单体化合物对由D-半乳糖诱导造成的小鼠氧化损伤实验
(1)动物分组及喂养
取健康小鼠70只,饲养温度为18-22℃,自然光照,自由进食饮水,在此条件下用普通饲料喂养5d。将小白鼠随机分成5组,(正常对照组(Control)、及模型对照组(DG)、对比例1制备得到的中药提取物组(TCM)、实施例2制备得到的中药益生菌发酵产物组(TCMPM)及式(Ⅰ)结构的单体化合物组(TPMSC),除正常对照组外,其余各组进行腹部注射700mg/(kg·d)D-半乳糖建立氧化损伤模型,中药提取物组、中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组按照200mg/(kg·d)的剂量灌胃相应的化合物,正常对照组注射等体积的生理盐水,灌胃等体积的蒸馏水,每2d称质量1次,持续28d。眼球取血处死小鼠,测定血清、肝脏中的超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量和总抗氧化能力(T-AOC),研究中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物的体内抗氧化活性。
(2)样品指标检测
小鼠在最后1d灌胃后,禁食12h,眼眶取血,5000r/min离心20min,分离血清,取上清液,按照试剂盒说明书检测血清的MDA含量、GSH-Px、SOD、T-AOC的活性。解剖小鼠,迅速取出肝脏,生理盐水冲洗干净后用吸水纸吸去水分,称取0.5g肝组织,置于匀浆机中,加入10倍体积的预冷生理盐水,制成10%的肝组织匀浆,4000r/min离心15min,取上清液,参照试剂盒说明书测定肝组织样品的GSH-Px、T-AOC、SOD活性以及MDA含量。
(3)实验结果
由图4,图5,图6,图7可知,在注射D-半乳糖之后,小鼠血清中的GSH-Px、SOD、T-AOC活性与正常对照组相比显著下降,MDA含量和正常对照组相比显著升高。而经中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物灌胃之后,小鼠血清的GSH-Px与T-AOC活性与模型损伤组相比极显著升高;与模型损伤组相比经中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物灌胃之后,SOD的活性显著提高;经中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物灌胃之后,与模型损伤组相比,MDA含量显著下降;其中式(Ⅰ)结构的单体化合物组SOD的活力为模型组的1.26倍,GSP-Px的活力为模型组的1.36倍,T-AOC的活力为模型组的1.61倍,MDA的含量为模型组的69.6%。
由图8,图9,图10,图11可知,在注射D-半乳糖之后,小鼠肝脏中的GSH-Px活性与SOD活性和正常对照组相比显著下降,T-AOC活性也显著下降,MDA含量与模型损伤组相比则显著升高;中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组中小鼠肝脏的T-AOC活性与模型损伤组相比均显著升高,但实验结束时仍低于正常对照组;与模型损伤组相比,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组中小鼠肝脏的GSH-Px、SOD活性显著提高;与模型损伤组相比,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组显著降低了小鼠肝脏的MDA含量;其中式(Ⅰ)结构的单体化合物组中,SOD的活力为模型组的1.73倍,GSP-Px的活力为模型组的1.31倍,T-AOC的活力的上升为模型组的1.74倍,MDA的含量为模型组的69.1%。
上述实验表明,中药益生菌发酵产物及式(Ⅰ)结构的单体化合物的抗氧化作用大于单纯的中药提取物;这说明在中药提取物基础上经本发明所述的方法制备得到的中药益生菌发酵产物及式(Ⅰ)结构的单体化合物具有更优异的抗氧化作用;尤其是分离得到的式(Ⅰ)结构的单体化合物,其具有更强的抗氧化作用。
实验例2中药益生菌发酵产物及式(Ⅰ)结构的单体化合物抗衰老作用评价
(1)将N2野生型秀丽隐杆线虫在20℃进行培养,并随机分成4组(每组数量不小于60条),每组3个平行对照组,分别是正常对照组(Control)、对比例1制备得到的中药提取物组(TCM)、实施例2制备得到的中药益生菌发酵产物组(TCMPM)及式(Ⅰ)结构的单体化合物组(TPMSC)。实验组按照50μg/mL的剂量预培养4天,正常对照组用磷酸缓冲盐溶液代替中药益生菌发酵代谢产物,每天计数1次,直至所有虫体死亡。结果如图12所示,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组可显著的提高N2野生型线虫的寿命,中药提取物组,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组的线虫寿命与正常对照组相比,分别提高17.9%、25.9%、34.0%。
(2)N2野生型线虫在20℃进行培养,随机分成5组(每组数量不小于60条),每组3个平行对照组,分别是正常对照组(简写为Control)、模型损伤组(Par)、对比例1制备得到的中药提取物组(TCM)、实施例2制备得到的中药益生菌发酵产物组(TCMPM)及式(Ⅰ)结构的单体化合物组(TPMSC),同时实验组按照50μg/mL的剂量预培养4天,除了正常对照组外,其余各组线虫用百草枯处理,正常对照组用磷酸缓冲盐溶液代替中药,中药益生菌发酵产物,中药益生菌代谢产物单体化合物,每天计数1次,直至所有虫体死亡。结果如图13所示,百草枯处理后线虫的寿命显著降低,百草枯处理后线虫寿命降低23.9%;经中药益生菌发酵产物及式(Ⅰ)结构的单体化合物培养的线虫其寿命明显提高,中药提取物组,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组线虫的寿命分别提高10.3%、13.5%、23.5%。
(3)将N2野生型线虫在20℃进行培养,并随机分成5组(每组数量不小于60条),每组3个平行对照组,分别是正常对照组(简写为Control)、模型损伤组(Par)、对比例1制备得到的中药提取物组(TCM)、实施例2制备得到的中药益生菌发酵产物组(TCMPM)及式(Ⅰ)结构的单体化合物组(TPMSC),同时实验组按照50μg/mL的剂量预培养4天,除了正常对照组外,其余各组线虫用百草枯处理,正常对照组用磷酸缓冲盐溶液代替中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物,每天计数1次,继续培养4天,测定其体内相关抗氧化酶活性(超氧化物歧化酶SOD和丙二醛MDA)及活性氧簇(ROS)含量。线虫在中药提取物、中药益生菌发酵产物及式(Ⅰ)结构的单体化合物预培养及百草枯处理后,继续培养4天后分别记录存活率。结果如图14~16所示,其中图14为ROS含量测定图,图15为MDA含量测定图,图16为SOD含量测定图。百草枯处理后,线虫体内的相关抗氧化酶活性与正常对照组相比显著下降,ROS含量和正常对照组相比显著升高;经50μg/mL中药益生菌发酵产物及式(Ⅰ)结构的单体化合物预处理后,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组中线虫体内的抗氧化酶活性与模型损伤组相比显著升高;对于SOD的活性,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组与模型损伤组相比均显著升高;对于ROS含量,中药益生菌发酵产物组及式(Ⅰ)结构的单体化合物组与模型损伤组相比显著下降,其中式(Ⅰ)结构的单体化合物组中,SOD的活力为模型损伤组的1.26倍,MDA的含量为模型损伤组的68.8%。
上述实验表明,中药益生菌发酵产物及式(Ⅰ)结构的单体化合物的抗衰老作用大于单纯的中药提取物;这说明在中药提取物基础上经本发明所述的方法发酵得到的中药益生菌发酵产物及式(Ⅰ)结构的单体化合物具有更优异的抗衰老作用;尤其是分离得到的式(Ⅰ)结构的单体化合物,其具有更强的抗衰老作用。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,本领域普通技术人员可以理解实现上述实施例的全部或部分流程,并依本发明权利要求所作的等同变化,仍属于发明所涵盖的范围。
Claims (10)
2.权利要求1所述的式(Ⅰ)化合物的制备方法,其特征在于,包含如下步骤:
(1)取中药原料用益生菌进行发酵,得发酵液;
(2)将发酵液进行干燥后得粗产物;
(3)将粗产物用色谱技术进行分离,即得式(Ⅰ)化合物;
步骤(1)中所述的中药原料为包含紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的中药原料;
步骤(1)中所述的益生菌选自双歧杆菌、乳杆菌菌液、青春双歧杆菌或婴儿双歧杆菌中的一种或二种以上的混合。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)的具体方法为:取中药原料加水提取,浓缩提取液得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为35~40℃、pH为6.5~7.5的条件下发酵24~48h,得发酵液。
4.根据权利要求2所述的制备方法,其特征在于,步骤(3)中所述的色谱技术为制备型HPLC技术;所述的制备型HPLC技术的具体条件为:采用反相色谱柱;选用紫外检测器,检测波长为220~230nm;以0.1~0.3体积%的三氟乙酸水溶液为流动相A,以0.1~0.3体积%的三氟乙酸乙腈溶液为流动相B,流动相A:流动相B=78:22;收集13.0~13.7min色谱峰对应的部位,浓缩干燥后即得式(Ⅰ)化合物。
5.一种中药益生菌发酵产物,其特征在于,包含式(Ⅰ)化合物。
6.权利要求5所述的中药益生菌发酵产物的制备方法,其特征在于,包含如下步骤:
(1)取中药原料用益生菌进行发酵,得发酵液;
(2)将发酵液进行干燥后即得所述的中药益生菌发酵产物;
步骤(1)中所述的中药原料为包含紫苏、黄芪、百合、红景天、女贞子、三七、银杏叶、薄荷、芡实的中药原料;
步骤(1)中所述的益生菌选自双歧杆菌、乳杆菌菌液、青春双歧杆菌或婴儿双歧杆菌中的一种或二种以上的混合。
7.根据权利要求5所述的制备方法,其特征在于,步骤(1)的具体方法为:取中药原料加水提取,浓缩提取液得浓缩液;然后向浓缩液中加入发酵营养剂;再加入益生菌在温度为35~40℃、pH为6.5~7.5的条件下发酵24~48h,得发酵液。
8.权利要求1所述的式(Ⅰ)化合物或其可药用盐或权利要求6所述的中药益生菌发酵产物在制备药物或保健品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述的药物为具有抗氧化作用和/或延缓衰老作用的药物;
所述的保健品为具有抗氧化作用和/或延缓衰老作用的保健品。
10.根据权利要求9所述的应用,其特征在于,所述的药物或保健品含有式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物和载体;所述式(Ⅰ)化合物或其可药用盐或中药益生菌发酵产物的质量分数为5%~95%。
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