CN107670002A - 一种降血糖组合物及其制备方法和应用 - Google Patents
一种降血糖组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于降血糖保健品技术领域,尤其涉及一种降血糖组合物及其制备方法和应用。本发明公开的降血糖组合物包括:天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物,其中,所述天然植物组分提取物为天然植物组分经酶解、水提和过滤制成;所述真菌组分提取物为真菌组分醇提和水提制成。本发明还公开了降血糖组合物的制备方法,包括以下步骤:a1)制备天然植物组分提取物、制备真菌组分提取物和制备天然植物组分提取物经细菌发酵的产物;b1)将步骤a1)的产物按3:1:1的比例混合,制得产品。本发公开的组合物具有良好的降血糖功效和对机体的综合调理作用。
Description
技术领域
本发明属于降血糖制品技术领域,尤其涉及一种降血糖组合物及其制备方法和应用。
背景技术
糖尿病是由遗传和环境因素相互作用而引起的一组代谢异常综合征。长期的糖尿病可引起多个系统器官的慢性并发症,导致功能障碍和衰竭,成为致残、致死的主要原因。目前,我国人群中糖尿病的患病率近几年有显著上升的趋势。由于糖尿病的病因和发病机制尚未完全明了,因此目前对糖尿病还缺乏有效的治疗方法。现在临床上对糖尿病的治疗多采用胰岛素治疗和口服降糖药物治疗,然而糖尿病属于一种终身性疾病,需要长期用药,胰岛素的注射给药方法往往会给患者带来许多不便,而临床常用的口服降糖药物多属于化学药品,长期服用也会引发一系列的不良反应。
当前,国内外对糖尿病的治疗方面研究较多,口服降糖药除原有的磺脲类、双胍类外又研制出新一代降糖药如α糖苷酶抑制剂、快速胰岛素促分泌剂、胰岛素增敏剂等。口服降糖药有许多不尽人意之处,磺脲类易致低血糖,双胍类易引起消化道反应、乳酸酸中毒,以及某些药物价格昂贵,胰岛素临床应用虽已有几十年的历史,但由于治疗条件、用药途径以及对胰岛素认识不足等诸方面因素,尚不能被部分患者所接受。糖尿病是一种综合性的慢性疾病,传统口服降糖药的治疗作用往往只针对机体糖代谢中的某个环节,并没有起到对机体进行整体调理的作用,这种“头痛医头脚痛医脚”的治疗方法在治疗以糖尿病为代表的慢性疾病的疗效往往不尽人意。
目前研究发现,很多天然植物或药材中含有具有降糖作用的物质,但现有的由天然植物、药材制得的糖尿病口服液或制剂降血糖的效果往往不理想,或疗效不持续。因此,研发一种能对机体进行综合调理、具有更好降血糖功效的组合物及其制备方法,是本领域技术人员亟待解决的技术问题。
发明内容
有鉴于此,本发明提供了一种降血糖组合物,本发明具有良好的降血糖功效和对机体的综合调理作用,本发明还提供了所述降血糖组合物的制备方法及在制备降血糖药物及保健品中的应用。
本发明提供了一种降血糖组合物,由以下原料制成:
天然植物组分提取物、真菌组分提取物及天然植物组分提取物经细菌发酵的产物;
所述天然植物组分提取物为天然植物组分经酶解、水提和过滤制成;所述天然植物组分包括:薄荷、赤芍、大豆、丹皮、覆盆子、葛根、枸杞、甘草、胡芦巴种子、黄精、黄连、黄芪、姜、咖啡豆、苦瓜、灵芝、芦荟、麦门冬、木瓜、奶蓟草、人参、肉桂、桑叶、山药、山茱萸、生地黄、首乌、熟地黄、太子参、天门冬、田七、菟丝子、五味子、武靴叶、西洋参、益母草、玉竹和知母;
所述真菌组分提取物为真菌组分醇提和水提制成;所述真菌组分包括:冬虫夏草、茯苓、桦褐孔菌、桑黄菌、蛹虫草和云芝;
所述天然植物组分提取物为天然植物组分经细菌发酵得到的产物;所述细菌包括:瑞士乳杆菌、乳酸链球菌、嗜热乳酸链球菌、动物双歧杆菌、短双歧杆菌和地衣芽孢杆菌。
其中,瑞士乳杆菌(编号为CGMCC 1.1877)、乳酸链球菌(编号为GIM 1.198)、嗜热乳酸链球菌(编号为GIM 1.540)、动物双歧杆菌(编号为GIM 1.169)、短双歧杆菌(编号为GIM 1.206)及地衣芽孢杆菌(编号为GGMCC 1.1858)。
作为优选,按照重量份计,所述天然植物组分包括:
薄荷1~3份、赤芍2~5份、大豆3~6份、丹皮2~5份、覆盆子1~4份、葛根2~5份、枸杞4~7份、甘草1~3份、胡芦巴种子1~3份、黄精2~4份、黄连1~3份、黄芪5~8份、姜1~3份、咖啡豆2~6份、苦瓜2~6份、灵芝12~16份、芦荟2~4份、麦门冬3~6份、木瓜6~10份、奶蓟草3~6份、人参7~9份、肉桂2~5份、桑叶1~3份、山药2~4份、山茱萸1~4份、生地黄1~3份、首乌1~3份、熟地黄1~3份、太子参8~10份、天门冬3~6份、田七2~5份、菟丝子1~3份、五味子1~4份、武靴叶3~6份、西洋参6~8份、益母草1~4份、玉竹1~3份和知母2~4份;
按照重量份计,所述真菌组分包括:冬虫夏草20~30份、茯苓16~20份、桦褐孔菌15~18份、桑黄菌13~16份、蛹虫草12~16份和云芝10~15份。
作为优选,所述降血糖组合物还包括缓释微球,所述缓释微球包裹所述降血糖组合物的多肽提取物;其中,所述缓释微球为海藻酸钠和壳聚糖制备得到。
作为优选,所述降血糖组合物还包括蚕蛹和蜂蛹的提取物,所述蚕蛹和蜂蛹的提取物为蚕蛹粉和蜂蛹粉末的酶解的产物;
所述蚕蛹的重量份数为15~20份,所述蜂蛹的重量份数为18~25份。
作为优选,所述降血糖组合物还包括:矫味剂及防腐剂,所述矫味剂为苹果醋,所述防腐剂为山梨酸钾。
矫味剂用于改善或屏蔽组合物的味道,所述矫味剂还可以为蔗糖、甘露醇、山梨醇、阿斯巴甜中的一种或多种。
所述防腐剂还可以为苯甲醇、苯甲酸钠、对羟基苯甲酸乙酯中的一种或多种。
为促进所述降血糖组合物的口服吸收效果,作为优选,所述降血糖组合物还包括胃肠道酶抑制剂和促吸收剂;
所述胃肠道酶抑制剂包括:大豆胰蛋白酶抑制剂、抑肽酶和杆菌肽中的一种或多种;
所述促吸收剂包括:壳聚糖、卡波姆、十二烷基硫酸钠、聚乙烯吡咯烷酮和乙二胺四乙酸钠中的一种或多种。
本发明公开了所述降血糖组合物的制备方法,包括以下步骤:
a1)制备天然植物组分提取物:按重量份称取天然植物组分,粉碎过筛后进行纤维素酶和蛋白水解酶进行酶解处理,然后经水提、过滤制得的滤液为天然植物组分提取物;
制备真菌组分提取物:按重量份称取的真菌组分,粉碎后过40~60目筛,加入重量/体积比为3倍的醇溶液加热回流提取后得到真菌成分粉末,然后对真菌成分粉末进行水提后过滤制得的滤液为真菌组分提取物;
制备天然植物组分提取物经细菌发酵的产物:对步骤a)制得的天然植物组分提取物加入体积百分比0.5%的瑞士乳杆菌、体积百分比0.3%的乳酸链球菌、体积百分比0.3%的嗜热乳酸链球菌、体积百分比0.3%的动物双歧杆菌、体积百分比0.3%的短双歧杆菌和体积百分比0.2%的地衣芽孢杆菌后,置于37℃,发酵16小时得到天然植物组分提取物经细菌发酵的产物;
b1)将步骤a1)的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合物,制得降血糖组合物。
进一步的,所述降血糖组合物具体为所述天然植物组分提取物、所述真菌组分提取物和所述天然植物组分提取物经细菌发酵的产物按3:1:1的体积比例混合。
作为优选,所述步骤a)还包括蚕蛹和蜂蛹的提取物,所述蚕蛹和蜂蛹的提取物的制备方法如下:
按重量份称取蚕蛹粉和蜂蛹粉末,加重量/体积比为10倍的饮用水配成溶液,往溶液中加入所述蚕蛹和蜂蛹粉末的总干重的2%的碱性蛋白酶和1%的复合蛋白酶,在pH 8.5下酶解4h,过滤后的滤液为蚕蛹和蜂蛹的提取物;
将蚕蛹和蜂蛹的提取物、天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物的1:3:1:1的体积比例混合。
进一步的,所述蚕蛹和蜂蛹的提取物、所述天然植物组分提取物、所述真菌组分提取物和所述天然植物组分提取物经细菌发酵的产物经过浓缩后在混合制成所述降血糖组合物。
作为优选,所述步骤a1)后还包括:缓释微球多肽提取物,所述缓释微球多肽提取物为将缓释微球包裹所述降血糖组合物的多肽提取物;
所述缓释微球多肽提取物的制备方法为:将所述步骤a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合形成混合物,从1/2体积的混合物中分离得到多肽提取物后,利用海藻酸钠和壳聚糖将所述多肽提取物包裹形成缓释微球多肽提取物;
进一步的,所述缓释微球多肽提取物、所述天然植物组分提取物、所述真菌组分提取物和所述天然植物组分提取物经细菌发酵的产物混合制备得到降血糖组合物。
其中,按照重量份计,所述天然植物组分包括:
薄荷1~3份、赤芍2~5份、大豆3~6份、丹皮2~5份、覆盆子1~4份、葛根2~5份、枸杞4~7份、甘草1~3份、胡芦巴种子1~3份、黄精2~4份、黄连1~3份、黄芪5~8份、姜1~3份、咖啡豆2~6份、苦瓜2~6份、灵芝12~16份、芦荟2~4份、麦门冬3~6份、木瓜6~10份、奶蓟草3~6份、人参7~9份、肉桂2~5份、桑叶1~3份、山药2~4份、山茱萸1~4份、生地黄1~3份、首乌1~3份、熟地黄1~3份、太子参8~10份、天门冬3~6份、田七2~5份、菟丝子1~3份、五味子1~4份、武靴叶3~6份、西洋参6~8份、益母草1~4份、玉竹1~3份和知母2~4份;
按照重量份计,所述真菌组分包括:冬虫夏草20~30份、茯苓16~20份、桦褐孔菌15~18份、桑黄菌13~16份、蛹虫草12~16份和云芝10~15份;
所述蚕蛹的重量份数为15~20份,所述蜂蛹的重量份数为18~25份。
进一步的,本发明公开了降血糖组合物的制备方法,包括以下步骤:
a1)制备天然植物组分提取物:按重量份称取天然植物组分,粉碎过筛后进行纤维素酶和蛋白水解酶进行酶解处理,然后经水提、过滤制得的滤液为天然植物组分提取物;
制备真菌组分提取物:按重量份称取的真菌组分,粉碎后过40~60目筛,加入重量/体积比为3倍的醇溶液加热回流提取后得到真菌成分粉末,然后对真菌成分粉末进行水提后过滤制得的滤液为真菌组分提取物;
制备天然植物组分提取物经细菌发酵的产物:对步骤a)制得的天然植物组分提取物加入体积百分比0.5%的瑞士乳杆菌、体积百分比0.3%的乳酸链球菌、体积百分比0.3%的嗜热乳酸链球菌、体积百分比0.3%的动物双歧杆菌、体积百分比0.3%的短双歧杆菌和体积百分比0.2%的地衣芽孢杆菌后,置于37℃,发酵16小时得到天然植物组分提取物经细菌发酵的产物;
a2)将所述步骤a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合形成混合物,从1/2体积的混合物中分离得到多肽提取物后,利用海藻酸钠和壳聚糖将所述多肽提取物包裹形成缓释微球多肽提取物;
b1)将所述a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物的混合物和所述步骤a2)的缓释微球多肽提取物混合制备得到降血糖组合物。
进一步的,所述a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物的混合物和所述步骤a2)的缓释微球多肽提取物按3:1的体积比例混合,制得降血糖组合物。
b1)将步骤a1)的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合物;其中,所述天然植物组分提取物、所述真菌组分提取物和所述天然植物组分提取物经细菌发酵的产物按3:1:1的体积比例混合,制得降血糖组合物。
进一步的,将所述步骤a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合形成混合物,从1/2体积的混合物中分离得到多肽提取物,分离方法为为凝胶过滤层析、离子交换层析、液相色谱柱分离和超滤和膜分离中的一种或多种。
本发明还公开了上述降血糖组合物或所述制备方法制得的降血糖组合物在制备降血糖药物及保健品中的应用。
本发明公开的降血糖组合物使用了多种具有降血糖功效的天然植物成分,并提取其有效成分,发挥综合调理的功效。
以下为本发明公开的组合物中使用的几种天然植物组分。
葛根:味甘辛,性平无毒,食之可生津止渴,清热解毒,同时因它含有大量的黄酮素化合物,不仅含有雌激素、对女性具有养颜保健作用;葛根还可以降压退火、抗病解毒、并且提高肝细胞的再生能力,恢复正常肝脏机能,促进胆汁分泌,防止脂肪在肝脏堆积,促进人体新陈代谢。现代医学研究表明,葛根对高血脂形成的冠状动脉硬化,通过改善心肌缺血状态,防治冠心病、心绞痛、心肌梗塞等心血管疾病有一定的辅助效果。如以葛根配伍天花粉则有生津止渴、清肺胃之燥热,又能养阴生津以止渴,为清热生津之良药;二者配伍使用特具清热生津止渴之效;用于治疗热病口渴及消渴(糖尿病)等症状效果较好。
玉竹:性味甘、平;具有清热润肺、益脾养胃、生津增液、消渴糖的功效,对小便频繁、消谷易饥等症作用明显。玉竹根状茎含玉竹粘多糖,由D-果糖,D-甘露糖,D-葡萄糖及半乳糖醛酸所组成。现代医学研究指出,玉竹有滋养、镇静及强心作用,适用于治疗心悸、心绞痛;玉竹有降低血脂和血糖作用,对肾上腺素引起的高血糖有显著抑制作用,可用于糖尿病;玉竹中有抗氧化作用的成分,可调节人体免疫力,抑制肿瘤的生长。
黄精(别名:鸡头参):《本草纲目》记载黄精入脾、肺、肾三经,填精髓、补血滋阴、益脾气胃、生津耐寒、补肾、助筋骨。用于脾胃虚弱,体倦乏力,腰膝酸软、肾虚精亏、口干食少,肺虚燥咳,精血不足,内热消渴等。现代医学研究表明,黄精具消渴生津、抑制血糖升高的作用,并且抗病原微生物作用明显,对肾上腺素引起的血糖失衡呈显著抑制作用;并在抗疲劳、延缓衰老和抗氧化方面也有明显特性。
苦瓜(又名凉瓜):为一年生攀缘草本植物。明代《救荒草本》、《本草纲目》具有记载,性甘味平、养血滋肝、润脾补肾,滋养作用显著,果实含苦瓜甙,以及5-羟基色胺在内的10多种对人体有益的氨基酸。现代医学研究表明,苦瓜不但降压降血糖的作用显著还具有清热祛暑、明目解毒、利尿凉血、解疲清心、益气壮阳之功效。
本发明制备的降血糖组合物经国际权威的第三方机构SGS检测进行菌落总数测定、大肠菌群计数检验、沙门氏菌检验、志贺氏菌检验、金黄色葡萄球菌检验、食品中总砷及无机砷的测定、食品中铅的测定、食品中总汞及有机汞的测定,其中,按照GB4789.2-2010的食品安全国家标准进行食品菌落总数的检验,按照GB4789.3-2010的食品安全国家标准进行大肠菌群计数检验,按照GB4789.4-2010的食品安全国家标准进行沙门氏菌检验,按照GB4789.5-2012的食品安全国家标准进行志贺氏菌检验,按照GB4789.10-2010的食品安全国家标准进行金黄色葡萄球菌检验,按照GB/T5009.11-2003的食品中总砷及无机砷的测定,按照GB5009.12-2010的食品安全国家标准进行食品中铅的测定,按照GB/T5009.17-2003的食品中总汞及有机汞的测定。
菌落总数、大肠菌群计数检验、沙门氏菌检验、志贺氏菌检验、金黄色葡萄球菌检验、白色念珠菌、食品中总砷及无机砷的测定、食品中铅的测定、食品中总汞及有机汞的测定,均达到食品级要求。3种重金属检测汞、砷、铅、均未检出。产品农药残留、重金属、生物毒素的含量远低于国家标准,没有发现任何毒副作用。
本发明公开的降血糖组合物可制成多种剂型,可制成口服液、汤剂、粉剂。
经试验,本发明公开的降血糖组合物及由本发明公开的制备方法制得的降血糖组合物,包括天然植物组分提取物、真菌组分提取物及天然植物组分提取物经细菌发酵的产物,其中,天然植物组分提取物是用38种中药通过酶解后提取得到,将这38种中药的有效成分大大浓缩在提取液中,该天然植物组分提取物含有丰富的活性分子,各种多肽成分和寡糖等活性糖成分;真菌组分提取物是由6种真菌通过提取得到活性物质,真菌的细胞壁和真菌内含物含有多种降糖的活性成分;天然植物组分提取物经细菌发酵的产物是通过38种天然植物组分经复合的细菌发酵得到,通过多种复合的细菌发酵天然植物后,能从天然植物中得到更多的降糖物质。因此,本发明的降血糖组合物有以下有益效果:
1.双向调节血糖,平衡血糖效果明显稳定
2.组合物成分来源于多种组分,对机体具有综合调理的效果
3.组合中含有对天然植物提取物进行发酵的成分,有助于改善药性,增强对机体综合调理的效果
4.组分中含有活性肽类物质,能增强对机体的调理效果
5.组分中含有活性肽类物质的缓释成分,增加口服吸收的效果
6.具有修复肝脏功能、提升肝脏排毒能力的效果
7.具有加强细胞代谢功能的效果
8.改善消化道机能,促进消化道吸收能力提升
9.健脾养胃,协助脾脏运化,缓解运动疲劳
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。
图1示含有不同浓度的本发明实施例2制备的组合物的细胞培养基对HepG2细胞葡萄糖消耗量的影响;
图2示含有不同浓度的本发明实施例2制备的组合物的细胞培养基对HepG2细胞增殖的抑制作用(横坐标为本发明的降糖组合物在细胞培养基的浓度)。
具体实施方式
本发明提供了一种降血糖组合物及其制备方法和应用,所述降血糖组合物具有良好的降血糖功效及对机体的综合调理作用。
下面将对本发明实施例中的技术方案进行清楚、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做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。
其中,薄荷、赤芍、大豆、丹皮、覆盆子、葛根、枸杞、甘草、胡芦巴种子、黄精、黄连、黄芪、姜、咖啡豆、苦瓜、灵芝、芦荟、麦门冬、木瓜、奶蓟草、人参、肉桂、桑叶、山药、山茱萸、生地黄、首乌、熟地黄、太子参、天门冬、田七、菟丝子、五味子、武靴叶、西洋参、益母草、玉竹、知母、冬虫夏草、茯苓、桦褐孔菌、桑黄菌、蛹虫草和云芝均为市售;瑞士乳杆菌(编号为CGMCC 1.1877)、乳酸链球菌(编号为GIM 1.198)、嗜热乳酸链球菌(编号为GIM 1.540)、动物双歧杆菌(编号为GIM 1.169)、短双歧杆菌(编号为GIM 1.206)及地衣芽孢杆菌(编号为GGMCC 1.1858)均为从广东省微生物菌种保藏中心或中国微生物菌种保藏中心获得。
实施例1制备降血糖组合物
1、天然植物组分提取物,具体操作步骤如下:
称取以下植物的药用部分:薄荷200g、赤芍300g、大豆500g、丹皮300g、覆盆子300g、葛根400g、枸杞600g、甘草200g、胡芦巴种子200g、黄精300g、黄连200g、黄芪650g、姜200g、咖啡豆400g、苦瓜500g、灵芝1300g、芦荟300g、麦门冬500g、木瓜800g、奶蓟草500g、人参800g、肉桂400g、桑叶200g、山药300g、山茱萸300g、生地黄200g、首乌200g、熟地黄250g、太子参900份、天门冬450g、田七400g、菟丝子200g、五味子300g、武靴叶500g、西洋参700g、益母草300g、玉竹200g和知母300g;
将称取的植物组分粉碎后混合,过100目筛,用100mg/kg的纤维素酶和100mg/kg的蛋白水解酶在45℃下处理1h。处理完后加8倍(重量/体积比)的饮用水,浸泡2小时,并于100℃提取1小时,过滤,再向滤渣中加6倍(重量/体积比)的水,100℃提取1小时后,过滤后得植物成分提取物。其中,1/2的滤液为天然植物组分提取物。
2、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:称取冬虫夏草2500g、茯苓1800g、桦褐孔菌1700g、桑黄菌1450g、蛹虫草1400g和云芝1300g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液。
3、植物提取液的细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入蛋白胨200g,于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵5天制得植物提取液细菌发酵产物。
4、将步骤1、步骤2和步骤3的产物按3:1:1的比例混合,制得降血糖组合物。
实施例2制备降血糖组合物
1、天然植物组分提取物,具体操作步骤如下:
称取以下植物的药用部分:薄荷100g、赤芍200g、大豆300g、丹皮200g、覆盆子100g、葛根200g、枸杞400g、甘草100g、胡芦巴种子100g、黄精200g、黄连100g、黄芪500g、姜100g、咖啡豆200g、苦瓜200g、灵芝1200g、芦荟200g、麦门冬300g、木瓜600g、奶蓟草300g、人参700g、肉桂200g、桑叶100g、山药200g、山茱萸100g、生地黄100g、首乌100g、熟地黄100g、太子参800g、天门冬300g、田七200g、菟丝子100g、五味子100g、武靴叶300g、西洋参600g、益母草100g、玉竹100g和知母200g;
将称取的植物组分粉碎,过100目筛,用120mg/kg的纤维素酶和150mg/kg的蛋白水解酶在40℃下处理1h。加重量/体积比6倍的饮用水,浸泡3小时后,并于100℃提取2小时,过滤,再向滤渣中加重量/体积比5倍的水,100℃提取2小时后,过滤后得植物成分提取物。取滤液的1/2进行细菌发酵。
2、蚕蛹及蜂蛹组分提取,具体操作步骤如下:
称取蚕蛹粉末1800g,蜂蛹粉末2000g,加重量/体积比10倍的饮用水配成溶液,往溶液体中加入蚕蛹和蜂蛹粉末总干重2%的碱性蛋白酶和1%的复合蛋白酶,在pH 8.5下酶解4h,过滤后的蚕蛹和蜂蛹成分提取物。
3、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:冬虫夏草2000g、茯苓1600g、桦褐孔菌1500g、桑黄菌1300g、蛹虫草1200g和云芝1000g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液。
4、植物提取液细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入葡萄糖(重量/体积比0.6%)、蛋白胨(重量/体积比0.3%)、无机盐(MgSO4·7H2O 50mg/100ml、KH2PO450mg/100ml),调整pH为7.0左右,并于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM 1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵4天制得植物提取液细菌发酵产物。
5、制备缓释微球多肽提取物,具体操作步骤如下:
将步骤1~4得到的产品混合,得混合液。将1/2混合液用sephadex G-25凝胶柱进行分离,然后对结合在柱上的蛋白进行洗脱,得洗脱液。在37℃下配制1.5%的海藻酸钠溶液1000ml,加入洗脱液200ml混合均匀;加入橄榄油120ml,加入15ml司盘-80和4ml吐温-80,混合均匀。1000r/min搅拌5min,搅拌中滴入0.15mol/100ml氯化钙溶液10ml,形成W/O乳剂;然后用磁力搅拌器慢速搅拌30min,使其交联固化;将乳剂转移至试管中,静置8h,取下层2000r/min冷冻离心3min,分离得到缓释微球多肽提取物。
6、将混合液进行减压浓缩,浓缩至比重为1.1,与缓释微球多肽提取物按3:1的比例混合,制得降血糖组合物。
实施例2得到的降血糖组合物含有了缓释微球多肽提取物,通过利用缓释微球将天然植物提取物的多肽成分包裹起来,使得胃肠道酶抑制剂和促吸收剂起作用前,避免了胃肠道的蛋白酶或多肽酶将降血糖组合物的多肽成分降解,当胃肠道酶抑制剂和促吸收剂在胃肠道起作用后,缓释微球多肽提取物中的多肽提取物释放出来,有利于胃肠道的细胞迅速将缓释微球内的多肽提取物吸收利用,大大提高本发明的降血糖组合物的有效性。
实施例3制备降血糖组合物
1、植物成分提取,具体操作步骤如下:
称取以下植物的药用部分:薄荷100g、赤芍200g、大豆300g、丹皮200g、覆盆子100g、葛根200g、枸杞400g、甘草100g、胡芦巴种子100g、黄精200g、黄连100g、黄芪500g、姜100g、咖啡豆200g、苦瓜200g、灵芝1200g、芦荟200g、麦门冬300g、木瓜600g、奶蓟草300g、人参700g、肉桂200g、桑叶100g、山药200g、山茱萸100g、生地黄100g、首乌100g、熟地黄100g、太子参800g、天门冬300g、田七200g、菟丝子100g、五味子100g、武靴叶300g、西洋参600g、益母草100g、玉竹100g和知母200g。
将称取的植物组分粉碎,过100目筛,用80mg/kg的纤维素酶、80mg/kg半纤维素酶和150mg/kg的蛋白水解酶在55℃下处理30min。加重量/体积比7倍的饮用水,浸泡2小时后,并于100℃提取1小时,过滤,再向滤渣中加重量/体积比6倍的水,100℃提取1小时后,过滤后得植物成分提取物。取滤液的1/2进行细菌发酵。
2、蚕蛹及蜂蛹组分提取,具体操作步骤如下:
称取蚕蛹粉末1800g,蜂蛹粉末2000g,加重量/体积比10倍的饮用水配成溶液,往溶液体中加入蚕蛹和蜂蛹粉末总干重2%的碱性蛋白酶和1%的复合蛋白酶,在pH 8.5下酶解4h,过滤后的蚕蛹和蜂蛹成分提取物。
3、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:冬虫夏草2000g、茯苓1600g、桦褐孔菌1500g、桑黄菌1300g、蛹虫草1200g和云芝1000g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液。
4、植物提取液细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入葡萄糖(重量/体积比0.6%)、蛋白胨(重量/体积比0.3%)、酵母粉(重量/体积比0.2%)、三氯蔗糖(重量/体积比0.02%)、无机盐(FeSO4·7H2O 1.0mg/100ml、ZnSO 4·7H 2O 4.4mg/100ml、Na 2SeO 3·5H 2O 0.024mg/100ml、MgSO4·7H 2O 70mg/100ml、K 2HPO 4·3H 2O 100mg/100ml、KH 2PO 4100mg/100ml)及三氯化铬(CrCl 3·6H 2O)0.77mg/100ml,调整pH为7.0左右,并于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC 1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM 1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵16小时检测pH值,当pH值降至4.0以下时结束发酵,70℃稳定化处理4小时,即得到植物提取液细菌发酵产品。
5、制备缓释微球多肽提取物,具体操作步骤如下:
将步骤1~4得到的产品混合,得混合液。将1/2混合液用sephadex G-50凝胶柱进行分离,然后对结合在柱上的蛋白进行洗脱,得洗脱液。在37℃下配制0.8mg/mL的壳聚糖溶液1000ml,加入洗脱液300ml混合均匀,80℃、1000r/min搅拌3h制得缓释微球多肽提取物。
6、将混合液用旋转蒸发仪进行浓缩,与步骤5制得的缓释微球多肽提取物按3:1的比例混合,加入0.2%的抑酶肽、0.1%的山梨酸钠和2%的聚乙烯吡咯烷酮,并用苹果醋调至微甜口味,制得降血糖组合物。
实施例4制备降血糖组合物
1、植物成分提取,具体操作步骤如下:
称取以下植物的药用部分:薄荷200g、赤芍300g、大豆500g、丹皮300g、覆盆子300g、葛根400g、枸杞600g、甘草200g、胡芦巴种子200g、黄精300g、黄连200g、黄芪650g、姜200g、咖啡豆400g、苦瓜500g、灵芝1300g、芦荟300g、麦门冬500g、木瓜800g、奶蓟草500g、人参800g、肉桂400g、桑叶200g、山药300g、山茱萸300g、生地黄200g、首乌200g、熟地黄250g、太子参900份、天门冬450g、田七400g、菟丝子200g、五味子300g、武靴叶500g、西洋参700g、益母草300g、玉竹200g和知母300g。
将称取的植物组分粉碎,过100目筛,加重量/体积比8倍的饮用水,浸泡2小时后,并于100℃提取1小时,过滤,再向滤渣中加重量/体积比6倍的水,100℃提取1小时后,过滤后得植物成分提取物。取滤液的1/2进行细菌发酵
2、蚕蛹及蜂蛹组分提取,具体操作步骤如下:
称取蚕蛹粉末1800g,蜂蛹粉末2000g,加重量/体积比10倍的饮用水配成溶液,往溶液体中加入蚕蛹和蜂蛹粉末总干重2%的碱性蛋白酶和1%的复合蛋白酶,在pH 8.5下酶解4h,过滤后的蚕蛹和蜂蛹成分提取物。
3、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:称取冬虫夏草2500g、茯苓1800g、桦褐孔菌1700g、桑黄菌1450g、蛹虫草1400g和云芝1300g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液。
4、植物提取液的细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入蛋白胨200g,于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC 1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM 1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵5天制得植物提取液细菌发酵产物。
5、将步骤1、步骤2和步骤3的产物浓缩至1.15,按3:1:1:1的体积比例混合,制得降血糖组合物。
实施例5制备降血糖组合物
1、植物成分提取,具体操作步骤如下:
称取以下植物的药用部分:薄荷100g、赤芍200g、大豆300g、丹皮200g、覆盆子100g、葛根200g、枸杞400g、甘草100g、胡芦巴种子100g、黄精200g、黄连100g、黄芪500g、姜100g、咖啡豆200g、苦瓜200g、灵芝1200g、芦荟200g、麦门冬300g、木瓜600g、奶蓟草300g、人参700g、肉桂200g、桑叶100g、山药200g、山茱萸100g、生地黄100g、首乌100g、熟地黄100g、太子参800g、天门冬300g、田七200g、菟丝子100g、五味子100g、武靴叶300g、西洋参600g、益母草100g、玉竹100g和知母200g。
将称取的植物组分粉碎,过100目筛,加重量/体积比6倍的饮用水,浸泡3小时后,并于100℃提取2小时,过滤,再向滤渣中加重量/体积比5倍的水,100℃提取2小时后,过滤后得植物成分提取物。取滤液的1/2进行细菌发酵。
2、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:冬虫夏草2000g、茯苓1600g、桦褐孔菌1500g、桑黄菌1300g、蛹虫草1200g和云芝1000g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液。
3、植物提取液细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入葡萄糖(重量/体积比0.6%)、蛋白胨(重量/体积比0.3%)、无机盐(MgSO4·7H2O 50mg/100ml、KH2PO450mg/100ml),调整pH为7.0左右,并于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM 1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵4天制得植物提取液细菌发酵产物。
4、制备缓释微球多肽提取物,具体操作步骤如下:
将步骤1~3得到的产物混合,得混合液。将1/2混合液用截留分子量为8000的超滤膜在0.6MPa、45℃的条件下进行超滤,得超滤液。在37℃下配制2%的海藻酸钠溶液1000ml,加入超滤液200ml混合均匀;加入橄榄油120ml,加入15ml司盘-80和4ml吐温-80,混合均匀。1000r/min搅拌5min,搅拌中滴入0.15mol/100ml氯化钙溶液10ml,形成W/O乳剂;然后用磁力搅拌器慢速搅拌30min,使其交联固化;将乳剂转移至试管中,静置8h,取下层2000r/min冷冻离心3min,分离得到缓释微球多肽提取物。
将步骤1、2、3的提取产物进行真空浓缩与缓释微球多肽提取物按3:1的比例混合,加入0.2%乙二胺四乙酸钠,制得降血糖组合物。
实施例6制备降血糖组合物
1、植物成分提取,具体操作步骤如下:
称取以下植物的药用部分:薄荷100g、赤芍200g、大豆300g、丹皮200g、覆盆子100g、葛根200g、枸杞400g、甘草100g、胡芦巴种子100g、黄精200g、黄连100g、黄芪500g、姜100g、咖啡豆200g、苦瓜200g、灵芝1200g、芦荟200g、麦门冬300g、木瓜600g、奶蓟草300g、人参700g、肉桂200g、桑叶100g、山药200g、山茱萸100g、生地黄100g、首乌100g、熟地黄100g、太子参800g、天门冬300g、田七200g、菟丝子100g、五味子100g、武靴叶300g、西洋参600g、益母草100g、玉竹100g和知母200g。
将称取的植物组分粉碎,过100目筛,用90mg/kg的纤维素酶和120mg/kg的蛋白水解酶在46℃下处理40min。加重量/体积比8倍的饮用水,浸泡2小时后,并于100℃提取1小时,过滤,再向滤渣中加重量/体积比6倍的水,100℃提取1小时后,过滤后得植物成分提取物。取滤液的1/2进行细菌发酵2、真菌成分提取,具体操作步骤如下:
称取以下真菌的药用部分:冬虫夏草2000g、茯苓1600g、桦褐孔菌1500g、桑黄菌1300g、蛹虫草1200g和云芝1000g;
将称取的真菌组分粉碎后过40目筛,加入重量/体积比为3倍的70%的乙醇溶液,在70℃条件下加热回流3h,提取2次,然后3000r/min离心,20min,弃去上清液,沉淀在60℃真空干燥箱中干燥12,得到预处理的真菌成分粉末。往真菌成分粉末加入重量/体积比为3倍量的水,浸泡5小时后,于85℃提取3小时,过滤后得到真菌成分提取液
3、植物提取液细菌发酵,具体操作步骤如下:
将步骤1制得的1/2的滤液加入葡萄糖(重量/体积比0.6%)、蛋白胨(重量/体积比0.3%)、酵母粉(重量/体积比0.2%)、三氯蔗糖(重量/体积比0.02%)、无机盐(FeSO4·7H2O 1.0mg/100ml、ZnSO 4·7H 2O 4.4mg/100ml、Na 2SeO 3·5H 2O 0.024mg/100ml、MgSO4·7H 2O 70mg/100ml、K 2HPO 4·3H 2O 100mg/100ml、KH 2PO 4100mg/100ml)及三氯化铬(CrCl 3·6H 2O)0.77mg/100ml,调整pH为7.0左右,并于110℃下灭菌30分钟。当温度降至38℃接种预培养的体积百分比0.5%的瑞士乳杆菌(CGMCC 1.1877)、体积百分比0.3%的乳酸链球菌(GIM 1.198)、体积百分比0.3%的嗜热乳酸链球菌(GIM 1.540)、体积百分比0.3%的动物双歧杆菌(GIM 1.169)、体积百分比0.3%的短双歧杆菌(GIM 1.206)和体积百分比0.2%的地衣芽孢杆菌(GGMCC 1.1858),37℃发酵16小时检测pH值,当pH值降至4.0以下时结束发酵,70℃稳定化处理4小时,即得到植物提取液细菌发酵产品。
4、制备缓释微球多肽提取物,具体操作步骤如下:
将步骤1~3得到的产物混合,得混合液。将1/2混合液用截留分子量为5000的超滤膜在0.5MPa、45℃的条件下进行超滤,得超滤液。在37℃下配制1.0mg/mL的壳聚糖溶液1000ml,加入超滤液300ml混合均匀,80℃、1000r/min搅拌3h制得。
5、将步骤1~3得到的产物混合的混合液进行减压浓缩至原体积的1/2,与步骤4制得的缓释微球多肽提取物按3:1的比例混合,加入0.2%的抑酶肽、0.1%的山梨酸钠,并用苹果醋调至微甜口味,制得降血糖组合物。
实施例7多糖含量测定
多糖含量的测定,具体测定步骤如下:
采用硫酸-苯酚法,首先是标准曲线的制作:精确称取干燥的葡萄糖10mg,用蒸馏水定容至100mL,分别吸取0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8mL,各以蒸馏水补至2mL,然后加入6%苯酚水溶液1.0mL,再迅速加入浓硫酸5.0mL,水溶15分钟后显色彻底,在冷水中冷却,至室温后在波长490nm处测定吸光值,以水代替糖作为空白对照,以吸光度为纵坐标,葡萄糖含量为横坐标制作标准曲线。测得的标准曲线为:Y=7.92X+0.012,R2=0.99
对实施例1~6制备的样品用硫酸-苯酚法测定吸光度,根据标准曲线进行多糖含量计算,测定结果如表1所示。表1显示本发明提供的实施例1~6的降血糖组合物均含有丰富的多糖。
表1
样品 | 实施例1 | 实施例2 | 实施例3 | 实施例4 | 实施例5 | 实施例6 |
多糖含量 | 4.3% | 4.5% | 4.1% | 4.2% | 3.9% | 4.1% |
实施例8多肽含量测定
测定多肽含量,具体测定步骤如下:
取50mg考马斯亮蓝G-250溶于25mL 95%乙醇中,待完全溶解后加入50mL 85%磷酸,最后用蒸馏水定容至500mL,制得考马斯亮蓝G-250染液。取BSA标准溶液(100mg/L)0mL、0.1mL、0.2mL、0.4mL、0.6mL、0.8mL、1mL,分别加水补至1mL,依次加入5mL考马斯亮蓝G-250染液,测定595nm吸光值,测得的标准曲线为y=6.5x+0.0062,R2=0.997
对实施例1~6制备的样品用考马斯亮蓝G-250染液测定吸光度,根据标准曲线进行肽类含量计算,测定结果如表2所示。表2显示本发明提供的实施例1~6的降血糖组合物均含有丰富的多肽。
表2
样品 | 实施例1 | 实施例2 | 实施例3 | 实施例4 | 实施例5 | 实施例6 |
肽类含量 | 2.2% | 2.4% | 2.1% | 2.2% | 2.5% | 2.3% |
实施例9对葡萄糖代谢相关酶的抑制试验
葡萄糖代谢相关酶的抑制试验,具体测定步骤如下:
酯酶(Lipase),α-糖苷酶(α-Glucosidase)及α-淀粉酶(α-Amylase)是影响人体血糖(特别是餐后血糖)的主要消化酶。降血糖产品中的酶类可通过竞争性抑制小肠上段上皮细胞的酯酶,α-糖苷酶及α-淀粉酶活性,使双糖向单糖(主要为葡萄糖)的转化减少,从而延缓糖类的吸收,降低餐后高血糖;还能延缓肠道碳水化合物的吸收,降低餐后高血糖,减轻餐后高血糖对胰岛B细胞的刺激作用,增加胰岛素敏感性。目前主要通过消化酶抑制达到控制血糖目的的药物包括阿卡波糖(如拜唐苹)和倍欣。
本实施例9的实验测试在体外环境中降血糖组合物对三种消化酶的抑制作用,从而评估糖尿病患在饮用被测饮液后血糖控制的效果。本实验使用阿卡波糖作为阳性对照;效果对比中使用的阿卡波糖浓度,为正常的口服剂量(每日50~300毫克,分2~3次口服);效果对比中使用的被测饮液剂量,为每日口服400mL
一、α-淀粉酶活性抑制试验,具体检测步骤如下:
1、在500μL含有浓度为0.5mg/mLα-淀粉酶的缓冲液中加入500μL的样品,以25℃温孵10分钟。
2、温孵后,加入0.5%的淀粉溶液500μL,以40℃水浴温孵30分钟。
3、加入1.0mL的DNS试剂终止反应,随后将试管以90℃水浴10分钟,冷却至室温。
4、随后将反应液转移至10mL的蒸馏水中。
5、用紫外光可见光分光光度计/酶标仪于波长540nm处测吸光度(A)
6、取不同倍数浓缩的样品重复以上操作,记录数据。
7、以缓冲液代替样品作为空白对照组,重复以上步骤。
8、将样品数据与空白对照数据进行对比,得出抑制活性,结果如表3所示。
表3 实施例1与实施例2制备得到的降血糖组合物的α-淀粉酶活性抑制
表3的试验结果表明,实施例1和实施例2制备的组合物对α-淀粉酶的活性有抑制作用。用实施例3~6制备的组合物重复上述试验,得到相似的结果。二、脂酶活性抑制试验,具体检测步骤如下:
1、准备好96微孔板,将25μL含有浓度为16.7U/mL脂酶的Tris-HCl缓冲液分别注入到微孔中。
2、往微孔中加入25μL的不同浓度的样品。
3、加入对硝基苯基月桂酸酯底物溶液50μL。
4、将微孔板放入酶标仪中,以波长405nm观察75分钟内的反应,每分钟记录一次吸光度。
5、将得到的数据整理,计算出每组反应的曲线下面积,结果如表4所示。
表4 实施例1与实施例2制备得到的降血糖组合物的脂酶活性抑制数据
表4的试验结果表明,实施例1和实施例2制备的组合物对脂酶的活性有抑制作用。用实施例3~6制备的组合物重复上述试验,得到相似的结果。
三、α-葡萄糖苷酶活性抑制实验,具体检测步骤如下:
1、在50μL含有浓度为1U/mLα-葡萄糖苷酶的磷酸缓冲液中加入50μL的样品,以37℃一边水浴一边震荡,温孵3分钟。
2、温孵后,加入PNPG溶液200μL,保持37℃温孵。
3、在一系列反应时间点(5min,15min,25min,35min,45min,55min,65min,75min)吸取30μL反应液,分别加入已有100μL终止液Na2CO3的离心管中。
4、溶液混匀后,吸取100μL,加到96微孔板中,用紫外光可见光分光光度计/酶标仪于波长405nm处测吸光度(A)。
5、取不同倍数浓缩的样品重复以上操作,记录数据。
6、设立以缓冲液代替样品作为空白对照,重复以上操作。设立以阿卡波糖溶液代替样品为阳性对照组,重复以上操作。
7、抑制率(%)=[(A空白-A样品)/A空白]×100%A空白:不加样品反应后的吸收值,结果无表5所示。
表5实施例1与实施例2制备得到的降糖组合物的α-葡萄糖苷酶活性抑制实验数据
上述的表3-表5可知,在实验条件下,被测样品对酯酶、α-糖苷酶及α-淀粉酶均有明显抑制作用;被测样品对酯酶的最大抑制率为对照组活性的31.20%;被测样品对α-糖苷酶的最大抑制率为对照组活性的65.89%;同等实验条件下,2mg/mL的阿卡波糖溶液对α-糖苷酶的最大抑制率为86.96%;被测样品对α-淀粉酶的最大抑制率为对照组活性的26.56%。说明被测样品对上述三种影响人体餐后血糖的主要消化酶有抑制作用。
实施例10细胞试验
HepG2细胞源于人的肝胚胎瘤细胞,系一种表型与肝细胞极为相似的肝胚胎瘤细胞株,在高水平的胰岛素条件下,HepG2细胞表面胰岛素受体的数目下降,下降程度与胰岛素水平及刺激持续的时间呈正相关,因此HepG2是体外研究胰岛素抵抗发病机制和降糖药物作用机制的理想细胞模型。如果被测样品能够使HepG2细胞在高糖环境下葡萄糖消耗程度明显增加,表明该被测样品能够有效减少血液中的葡萄糖浓度,从而达到控制血糖的目的。
在本试验中,使用实施例2制备的降血糖组合物作为被测样品,采用高糖培养基培养的HepG2细胞葡萄糖消耗速率将被测量;同时,HepG2细胞本身的增殖可以通过MTT方法进行检测。
细胞试验,具体操作步骤如下:
1、HepG2细胞的培养,具体检测步骤如下:细胞复苏后用含10%灭活胎牛血清的DMEM高糖培养液转入细胞培养瓶中,37℃、5%CO2条件下培养。当细胞贴壁长满后,弃去培养液,用PBS溶液轻轻洗涤2次,用0.25%胰蛋白酶消化,每3d按1∶3比例传代1次,取对数生长期的细胞用于实验。
2、HepG2细胞葡萄糖消耗及MTT实验,具体检测步骤如下:状态良好的细胞经0.25%胰蛋白酶消化,终止消化后洗涤2次,再用含10%小牛血清DMEM培养液配制成单个细胞悬液,以每孔1×104个细胞接种于96孔培养板内,细胞贴壁后更换无血清的高糖DMEM培养液,饥饿12h,使细胞同步化。吸弃细胞上清液,加入本发明实施例2制备的降血糖组合物的高糖DMEM培养液,使高糖DMEM培养液含所述组合物的浓度梯度为0%、10%、20%、30%、40%、50%,同一水平6孔并列为一组,分别于培养24h后采用葡萄糖氧化酶法测定培养液中葡萄糖的变化,结果如图1所述。
3、HepG2细胞MTT实验,具体检测步骤如下:将步骤2的细胞在葡萄糖消耗试验24h孵育结束,每孔加入2.5g/mL的MTT溶液20μL,继续培养4h终止培养后吸弃孔内培养上清液,并每孔加入150μL二甲亚砜(DMSO)终止反应,微型震荡器震荡10min,使结晶物充分溶解,立即用酶标仪在波长490m处进行光密度(OD)检测,以光密度值反映细胞活性和数量的多少,结果如图2所述。
从图1和图2可知,根据图1的HepG2细胞对含有不同浓度的本发明的降糖组合物的细胞培养基时葡萄糖的消耗变化可以得知,本发明实施例2制备的降血糖组合物在细胞培养基的浓度为10%-50%时具有明显的降糖作用;被测饮液能够大幅度提高HepG2细胞消耗利用葡萄糖的速率。当实施例2降血糖组合物在细胞培养基的浓度为50%时,细胞葡萄糖消耗率是不添加所述组合物的20倍左右;且所述组合物对HepG2细胞无增殖作用。用实施例1、实施例3~6制备的组合物重复上述试验,得到相似的结论。
实施例11毒理试验
一、小鼠急性毒性试验
选用健康、成熟、体重18~22g昆明小鼠108只,雌雄各半,购自南方医科大学实验动物中心。分为3个组别:1、正常浓度组;2、1.5倍浓度组:将产品的浓度浓缩至正常浓度的1.5被;3、2倍浓度组:将产品的浓度浓缩至正常浓度的2倍。每个试验组别均对实施例1~6制备的降血糖组合物进行试验,每个试验组中每个实施例的降血糖组合物使用的小鼠数量为6只,雌雄各半,使用剂量为0.3mL/10g。在灌胃前,小鼠隔夜禁食16小时,分两次灌胃,间隔6小时,连续观察5天,并记录小鼠的症状。
结果与分析:试验期间,小鼠存活状态良好,未出现明显中毒症状,也无一例死亡
二、小鼠慢性及遗传性毒性试验
选用健康、成熟、体重18~22g昆明小鼠108只,雌雄各半,购自南方医科大学实验动物中心。同样分成3个组别:1.正常浓度组;2.1.5倍浓度组;3.2倍浓度组。每个试验组别同样对实施例1~6制备的降血糖组合物进行试验,每个试验组中每个实施例降血糖组合物使用的小鼠数量为6只,雌雄各半,使用剂量为0.2mL/10g。每天早上9:00-10:00给药,连续给药12周,结束给药后对受试小鼠的外观体征、行为活动、体重、血液生化指标和脏器病理学进行检测。
结果与分析:
各组别小鼠给药前后被毛平顺、反应机敏、步态和饮食正常,口腔黏膜和粪便没有出现异常,说明本发明实施例制备的降血糖组合物对小鼠一般行为和体重无显著影响。
各组别小鼠的血常规和生化指标均未出现异常,脏器外观及脏器系数均未出现异常,说明本发明实施例制备的组合物对小鼠的血液生化指标和脏器无显著影响
实施例12动物降血糖试验
动物降血糖试验,具体操作步骤如下:
选用用四氧嘧啶造模的高血糖小鼠42只,雌雄各半,购自南方医科大学实验动物中心。将高血糖小鼠分成7组,每组6只,雌雄各半,2~7组为试验组,喂服实施例1~6制备的降血糖组合物,使用剂量为0.2mL/10g,每天一次。第1组为对照组,喂服等量的生理盐水;第7组为阿卡波糖组,用作治疗效果对照。连续喂服30天,观察试验前和试验后各组受试小鼠的体重和空腹血糖变化。检测的结果如表6所示,所测的体重和空腹血糖值均为平均值。
表6
结果与分析:如表6所示,试验前后,各组别的体重无显著变化(P>0.05),对照组试验前后的空腹血糖值无显著变化(P>0.05),各实施例的试验组喂服30天后的空腹血糖值较试验前均显著下降(P<0.05),说明本发明实施例制备的降血糖组合物具有良好的降血糖功效。
实施例13肝功能
测定肝功能,具体操作步骤如下:
选择华侨医院2015~2016年符合肝功能受损诊断标准的中医门诊病人60例,其中男性39例,女性21例;平均病程8个月,年龄40~65岁,平均年龄52.3,所有病人均签署知情同意书。
病例选择标准:1.谷丙转氨酶(ALT)和谷酰转肽酶(GGT)轻度(小于或等于正常值的2倍)或中度(大于正常值的2倍)升高;2.肝区隐痛;3.病程超过3个月。所选择的受试病人同时符合以上标准
观测指标如下:
1、临床症状、体征;2、谷丙转氨酶(ALT)和谷酰转肽酶(GGT)。
疗效评定标准如下:
显效:主要症状消失或基本消失,肝功能谷丙转氨酶(ALT)和谷酰转肽酶(GGT)降至正常范围;有效:主要症状部分消失或明显减轻,肝功能谷丙转氨酶(ALT)和谷酰转肽酶(GGT)原值下降50%以上;无效:主要症状改善不明显,肝功能谷丙转氨酶(ALT)和谷酰转肽酶(GGT)下降未达到有效标准。
将所选病例随机分为治疗组和对照组。治疗组31例,男性20例,女性11例;对照组29例,男性19例,女性10例。
给药方法:
治疗组给予实施例1制备的降血糖组合物,每天早晚各服用一次,每次200mL,连续给药30天。对照组给予生理盐水,生理盐水的给予量和时间和治疗组一致。
结果如表7所示:
表7
分组 | 显效 | 有效 | 无效 | 总有效率 |
治疗组 | 20 | 9 | 2 | 93.5% |
对照组 | 0 | 0 | 29 | 0 |
如表7所示,两组均以显效加有效的总人数来计算总有效率,治疗组总有效率为93.5%,对照组的总有效率为0,说明本发明制备的组合物具有良好的肝功能治疗功效(P<0.05)。
实施例14改善消化
测定消化水平,具体操作步骤如下:
选择珠海人民医院2015~2016年符合消化不良诊断标准的中医门诊病人50例,其中男性24例,女性26例;平均病程3个月,年龄20~65岁,平均年龄35.7,所有病人均签署知情同意书。
病例选择标准:1、有上腹部疼痛或不适、饱胀感、反酸;2、食欲减退;3、排便异常,便秘或拉稀;无确切的器质性疾病。符合以上标准选择为受试者。
观测指标:1、体征及症状是否改善;2、食欲是否恢复正常;3、排便是否正常。
疗效评定标准:
显效:体征及症状显著改善,食欲及排便恢复正常,心情舒畅,精神状态好;有效:体征及症状有改变,食欲有提高,排便较正常,心情舒畅,精神状态好;无效:体征及症状无改善,食欲和排便无改善。
将所选病例随机分为治疗组和对照组。治疗组25例,男性12例,女性13例;对照组25例,男性12例,女性13例。
给药方法:
治疗组给予实施例1制备的降血糖组合物,每天早晚各服用一次,每次200mL,连续给药30天。对照组给予生理盐水,生理盐水的给予量和时间和治疗组一致。
结果如表8所示。
表8
分组 | 显效 | 有效 | 无效 | 总有效率 |
治疗组 | 14 | 8 | 3 | 88% |
对照组 | 0 | 1 | 24 | 0 |
如表8所示,两组均以显效加有效的总人数来计算总有效率,治疗组总有效率为88%,对照组的总有效率为4%,治疗组的总有效率相比于对照组具有显著性差异(P<0.05),说明本发明制备的降血糖组合物具有良好的改善消化功效。
实施例15健脾养胃
参考文献及专利:《健脾养胃粉》、《一种健脾养胃颗粒剂及配制方法》。
选择广东省中医院2015~2016年符合脾胃虚弱诊断标准的中医门诊病人50例,其中男性24例,女性26例;平均病程3个月,年龄20~65岁,平均年龄35.7,所有病人均签署知情同意书。
病例选择标准:1、脾胃虚弱、气血不足;2、食欲不振、面色苍白、肌瘦、精神不佳、形神怯弱、四肢乏力;3、出现腹泄或腹痛时作时止等腹部症状。
疗效评定标准:
显效:体征及症状显著改善,食欲及排便恢复正常,心情舒畅,精神状态好;有效:体征及症状有改变,食欲有提高,排便较正常,心情舒畅,精神状态好;无效:体征及症状无改善,食欲和排便无改善。
将所选病例随机分为治疗组和对照组,治疗组25例,对照组25例。
给药方法:
治疗组给予实施例2制备的降血糖组合物,每天早晚各服用一次,每次200mL,连续给药20天。对照组给予生理盐水,给药量和给药时间同治疗组。
结果如表9所示。
表9
分组 | 显效 | 有效 | 无效 | 总有效率 |
治疗组 | 15 | 6 | 4 | 84% |
对照组 | 0 | 2 | 23 | 0 |
如表9所示,两组均以显效加有效的总人数来计算总有效率,治疗组总有效率为84%,对照组的总有效率为8%,治疗组的总有效率相比于对照组具有显著性差异(P<0.05),说明本发明制备的降血糖组合物具有良好的健脾养胃功效。
实施例16低血糖治疗试验
低血糖治疗试验具体操作步骤如下:
选择茂名市第一人民医院2015~2016年符合低血糖诊断标准的内分泌科门诊病人30例,其中男性16例,女性14例;平均病程2年,年龄50~70岁,平均年龄66,所有病人均签署知情同意书。
病例选择标准:1、空腹血糖浓度低于2.8mmol/L,若是糖尿病患者,血糖值≤3.9mmol/L;2、时常出现出汗、饥饿、心慌、颤抖、面色苍白症状。所选病例均符合以上标准。
疗效评定标准:
显效:血糖恢复正常,体征及症状显著改善,心情舒畅,精神状态好;有效:血糖接近正常,体征及症状有改变,心情舒畅,精神状态好;无效:血糖、体征及症状无改善。
将所选病例随机分为治疗组和对照组。治疗组15例,对照组15例。
给药方法:
治疗组给予实施例3制备的降血糖组合物,每天早晚各服用一次,每次200mL,连续给药30天。对照组给予生理盐水,给药量和给药时间同治疗组。
结果如表9所示。
表9
分组 | 显效 | 有效 | 无效 | 总有效率 |
治疗组 | 10 | 4 | 1 | 84% |
对照组 | 0 | 0 | 15 | 0 |
如表9所示,两组均以显效加有效的总人数来计算总有效率,治疗组总有效率为93%,对照组的总有效率为0,说明本发明制备的降血糖组合物具有良好的对低血糖的治疗作用。
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 (10)
1.一种降血糖组合物,其特征在于,由以下原料制成:
天然植物组分提取物、真菌组分提取物及天然植物组分提取物经细菌发酵的产物;
所述天然植物组分提取物为天然植物组分经酶解、水提和过滤制成;所述天然植物组分包括:薄荷、赤芍、大豆、丹皮、覆盆子、葛根、枸杞、甘草、胡芦巴种子、黄精、黄连、黄芪、姜、咖啡豆、苦瓜、灵芝、芦荟、麦门冬、木瓜、奶蓟草、人参、肉桂、桑叶、山药、山茱萸、生地黄、首乌、熟地黄、太子参、天门冬、田七、菟丝子、五味子、武靴叶、西洋参、益母草、玉竹和知母;
所述真菌组分提取物为真菌组分醇提和水提制成;所述真菌组分包括:冬虫夏草、茯苓、桦褐孔菌、桑黄菌、蛹虫草和云芝;
所述天然植物组分提取物为天然植物组分经细菌发酵得到的产物;所述细菌包括:瑞士乳杆菌、乳酸链球菌、嗜热乳酸链球菌、动物双歧杆菌、短双歧杆菌和地衣芽孢杆菌。
2.根据权利要求1所述的降血糖组合物,其特征在于,按照重量份计,所述天然植物组分包括:
薄荷1~3份、赤芍2~5份、大豆3~6份、丹皮2~5份、覆盆子1~4份、葛根2~5份、枸杞4~7份、甘草1~3份、胡芦巴种子1~3份、黄精2~4份、黄连1~3份、黄芪5~8份、姜1~3份、咖啡豆2~6份、苦瓜2~6份、灵芝12~16份、芦荟2~4份、麦门冬3~6份、木瓜6~10份、奶蓟草3~6份、人参7~9份、肉桂2~5份、桑叶1~3份、山药2~4份、山茱萸1~4份、生地黄1~3份、首乌1~3份、熟地黄1~3份、太子参8~10份、天门冬3~6份、田七2~5份、菟丝子1~3份、五味子1~4份、武靴叶3~6份、西洋参6~8份、益母草1~4份、玉竹1~3份和知母2~4份;
按照重量份计,所述真菌组分包括:冬虫夏草20~30份、茯苓16~20份、桦褐孔菌15~18份、桑黄菌13~16份、蛹虫草12~16份和云芝10~15份。
3.根据权利要1所述的降血糖组合物,其特征在于,还包括缓释微球,所述缓释微球包裹所述降血糖组合物的多肽提取物;其中,所述缓释微球为海藻酸钠和壳聚糖制备得到。
4.根据权利要求1所述的降血糖组合物,其特征在于,还包括蚕蛹和蜂蛹的提取物,所述蚕蛹和蜂蛹的提取物为蚕蛹和蜂蛹的酶解的产物;
所述蚕蛹的重量份数为15~20份,所述蜂蛹的重量份数为18~25份。
5.根据权利要求1所述的降血糖组合物,其特征在于,还包括:矫味剂和防腐剂;
所述矫味剂为苹果醋;
所述防腐剂为山梨酸钾。
6.根据权利要求1所述的降血糖组合物,其特征在于,还包括胃肠道酶抑制剂和促吸收剂;
所述胃肠道酶抑制剂包括:大豆胰蛋白酶抑制剂、抑肽酶和杆菌肽中的一种或多种;
所述促吸收剂包括:壳聚糖、卡波姆、十二烷基硫酸钠、聚乙烯吡咯烷酮和乙二胺四乙酸钠中的一种或多种。
7.一种降血糖组合物的制备方法,其特征在于,包括以下步骤:
a1)制备天然植物组分提取物:按重量份称取天然植物组分,粉碎过筛后进行纤维素酶和蛋白水解酶的酶解处理,然后经水提、过滤制得的滤液为天然植物组分提取物;
制备真菌组分提取物:按重量份称取的真菌组分,粉碎后过40~60目筛,加入重量/体积比为3倍的醇溶液加热回流提取后得到真菌成分粉末,然后对真菌成分粉末进行水提后过滤制得的滤液为真菌组分提取物;
制备天然植物组分提取物经细菌发酵的产物:对步骤a)制得的天然植物组分提取物加入体积百分比0.5%的瑞士乳杆菌、体积百分比0.3%的乳酸链球菌、体积百分比0.3%的嗜热乳酸链球菌、体积百分比0.3%的动物双歧杆菌、体积百分比0.3%的短双歧杆菌和体积百分比0.2%的地衣芽孢杆菌后,置于37℃,发酵16小时得到天然植物组分提取物经细菌发酵的产物;
b1)将步骤a1)的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合制备得到降血糖组合物,制得降血糖组合物。
8.根据权利要求7所述的制备方法,其特征在于,步骤a)还包括蚕蛹和蜂蛹的提取物,所述蚕蛹和蜂蛹的提取物的制备方法如下:
按重量份称取蚕蛹和蜂蛹,加重量/体积比10倍的饮用水配成溶液,往溶液中加入所述蚕蛹和蜂蛹的总干重的2%的碱性蛋白酶和1%的复合蛋白酶,在pH 8.5下酶解4h,过滤后的滤液为蚕蛹和蜂蛹的提取物;
将所述蚕蛹和蜂蛹的提取物、所述天然植物组分提取物、所述真菌组分提取物和所述天然植物组分提取物经细菌发酵的产物按照1:3:1:1的体积比例混合。
9.根据权利要求7所述的制备方法,其特征在于,所述步骤a1)后还包括:缓释微球多肽提取物,所述缓释微球多肽提取物为将缓释微球包裹所述降血糖组合物的多肽提取物;
所述缓释微球多肽提取物的制备方法为:将所述步骤a1)得到的天然植物组分提取物、真菌组分提取物和天然植物组分提取物经细菌发酵的产物混合形成混合物,从1/2体积的混合物中分离得到多肽提取物后,利用海藻酸钠和壳聚糖将所述多肽提取物包裹形成缓释微球多肽提取物。
10.根据权利要求1~6所述的降血糖组合物或权利要求7~9所述的制备方法制备制得的降血糖组合物在制备降血糖药物及保健品中的应用。
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