CN114773495B - 一种制备具有降糖、降脂功能的茯砖茶茶多糖的方法 - Google Patents
一种制备具有降糖、降脂功能的茯砖茶茶多糖的方法 Download PDFInfo
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- CN114773495B CN114773495B CN202210466052.2A CN202210466052A CN114773495B CN 114773495 B CN114773495 B CN 114773495B CN 202210466052 A CN202210466052 A CN 202210466052A CN 114773495 B CN114773495 B CN 114773495B
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- fuzhuan tea
- polysaccharide
- tea polysaccharide
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/82—Theaceae (Tea family), e.g. camellia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Diabetes (AREA)
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- Materials Engineering (AREA)
- Biochemistry (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种制备具有降糖、降脂功能的茯砖茶茶多糖的方法。将茯砖茶粉碎后以料液比为1:30g/mL加入去离子水,随后加入2%酶浓度的复合酶及醋酸‑醋酸盐缓冲溶液,在55℃水浴锅内加热酶解60min,酶解提取液过滤后浓缩、去蛋白、醇沉,沉淀用去离子水复溶后冷冻干燥,将冻干的样品溶解后在DEAE‑52纤维素柱中分离,洗脱液经透析、浓缩、冷冻干燥,得茯砖茶茶多糖。本发明制备的茯砖茶茶多糖,具有得率高、纯度高、提取时间短、减少能源消耗、工艺条件温和、可有效保存多糖的生物活性等优点,具有良好降糖、降脂功效。
Description
技术领域
本发明涉及活性茶多糖的提取技术,具体涉及一种茯砖茶茶多糖的制备方法。
背景技术
茯砖茶是以黑毛茶为原料,经过汽蒸、渥堆、压制成型、发花等工艺而制成的一种黑茶。茯砖茶属于后发酵茶,以茶叶为培养基生长繁殖产生金黄色闭囊壳,形成茶砖内部肉眼可见的金黄色颗粒,俗称“金花菌”,同时通过“金花菌”的代谢活动转化茶叶的各种成分,形成独特的色香味品质特征,茶汤呈橙红或黄色,味道醇厚甘甜,散发浓郁的“菌花香”。长期饮用茯砖茶,能够帮助消化,有效促进人体新陈代谢,对人体起着一定的保健和病理预防作用。
茯砖茶的活性成分提取和功效受到了越来越广泛的关注。其中,茶多糖的常用提取方法是热水浸提法,但这种提取方法存在提取率低、耗时、提取的有效成分易失活等问题,例如中国专利CN113812481A中提取得到了以茶褐素为主要成分的组合物(还含有茶多糖、钾、钙、氨基酸等),并且经过实验并未发现茶多糖本身具有显著降糖、降脂效果。除了热水浸提法,还有报道采用复合酶水解、醇沉、离子交换层析的组合工艺提取多糖成分,例如中国专利CN108424470A。但由于酶解的物料在结构和组成上的复杂性,导致制备具有降糖、降脂功能的茯砖茶茶多糖仍属于技术难题。
发明内容
本发明的目的在于提供一种制备具有降糖、降脂功能的茯砖茶茶多糖的方法。
为达到上述目的,本发明采用了以下技术方案:
一种茯砖茶茶多糖的制备方法,包括以下步骤:
(1)将茯砖茶茶粉与去离子水、纤维素酶、木瓜蛋白酶以及pH为4-8的醋酸-醋酸盐缓冲溶液混合后进行酶解,得提取液,其中纤维素酶和木瓜蛋白酶的质量比为1:1-5:1;将提取液过滤,将所得滤液浓缩后采用sevage法去除蛋白质,然后进行醇沉并收集沉淀;
(2)取步骤1中的沉淀在去离子水中复溶后冷冻干燥,得茯砖茶粗多糖,其得率为5.5%-7.5%,多糖含量为45%-55%;
(3)取步骤2中的茯砖茶粗多糖采用离子交换柱分离多糖组分,将多糖组分通过洗脱收集后依次进行透析(作用为除盐、保留有效多糖组分)、浓缩、冷冻干燥,得到茯砖茶茶多糖,其得率为10%-26%,多糖含量为60%-72%。
优选的,所述茯砖茶茶粉是通过将茯砖茶粉碎后过60-100目筛而获得的。
优选的,所述茯砖茶茶粉以料液比为1:10-1:50g/mL加入去离子水,即每1g茯砖茶茶粉与10-50mL去离子水混合。
优选的,所述纤维素酶与木瓜蛋白酶的混合浓度为1%-5%,即纤维素酶与木瓜蛋白酶构成的复合酶在酶解体系中的初始质量分数为1%-5%。
优选的,所述酶解的条件包括:在40-60℃水浴锅中加热40-80min,酶解完成后沸水浴高温灭酶10-15min。
优选的,所述醇沉具体包括以下步骤:将去除蛋白质后的浓缩滤液与该浓缩滤液体积2-6倍的无水乙醇或95%的乙醇溶液混合,然后在0-10℃条件下静置12-20h。
优选的,所述步骤3中,茯砖茶粗多糖采用去离子水溶解后上样于离子交换柱,离子交换柱的填料为阴离子交换树脂(例如DEAE等纤维素类树脂),洗脱液为去离子水以及0.1-0.3mol/LNaCl溶液,洗脱流速为1-8mL/min。
优选的,所述洗脱的过程为:依次用去离子水、0.1mol/L的NaCl溶液、0.2mol/L的NaCl溶液、0.3mol/L的NaCl溶液进行洗脱。
优选的,所述透析采用的截留分子量为8000-14000。
上述茯砖茶茶多糖的制备方法制备的茯砖茶茶多糖在制备具有降糖和/或降脂功效的药物、保健品中的应用。
优选的,以上步骤3所得茯砖茶茶多糖对α-葡萄糖苷酶、α-淀粉酶、胆固醇具有抑制作用及对胆酸盐具有结合作用。
本发明的有益效果体现在:
本发明以茯砖茶为原料,通过优化纤维素酶与木瓜蛋白酶的比例构成以及优化酶解缓冲液的成分和pH,结合对酶解提取液进行去蛋白、醇沉、离子交换层析,使提取所得茯砖茶茶多糖能够有效抑制α-葡萄糖苷酶和α-淀粉酶的活性,以及有效的结合胆酸盐并抑制胆固醇,从而可以作为具有显著的降糖、降脂功能的药物、保健品的有效成分。
进一步的,本发明采用一定浓度的纤维素酶和木瓜蛋白酶的复合酶,对一定料液比茯砖茶进行酶解,有利于加快多糖的溶出速率,使多糖全面析出,提高纯度(多糖含量)。
进一步的,本发明所用的酶解提取工艺条件温和、提取时间短,在最大程度的保留茯砖茶茶多糖的生物活性的同时,还有效避免了因长时间处在高温环境及酸碱性条件中,而使茯砖茶中其它具有降糖、降脂协同作用的活性成分受到破坏的问题。
进一步的,本发明采用纤维柱柱层析实现茯砖茶茶多糖的分离纯化,依次用不同浓度NaCl溶液进行洗脱,洗脱分离效果好,从而获得了具有更高降糖、降脂活性的茯砖茶茶多糖组分。
附图说明
图1为本发明实施例中茯砖茶酶解提取法的茯砖茶粗多糖得率随复合酶中纤维素酶与木瓜蛋白酶比例(a)、复合酶浓度(b)、酶解pH(c)、酶解温度(d)、酶解时间(e)和料液比(f)的变化。
图2为本发明实施例提取的茯砖茶粗多糖及不同浓度NaCl溶液洗脱(盐洗)的茯砖茶茶多糖对α-葡萄糖苷酶(a)、α-淀粉酶(b)的抑制效果。
图3为本发明实施例提取的茯砖茶粗多糖及不同浓度NaCl溶液洗脱(盐洗)的茯砖茶茶多糖对胆酸盐(a)的结合效果、胆固醇(b)的抑制效果。
具体实施方式
下面结合附图和实施例对本发明作进一步详细描述。所述实施例仅用于解释本发明,而非对本发明保护范围的限制。
(一)茯砖茶茶多糖的制备工艺
针对现有提取技术难以对茯砖茶中的多糖进行充分提取、浪费原料,同时茯砖茶茶多糖提取纯度不高的问题,本发明提出了一种基于茯砖茶酶解提取、纤维柱柱层析分离的茯砖茶茶多糖制备方法。
1.1茯砖茶的来源、产地
茯砖茶来源于植物黑茶,产自陕西。
1.2醋酸-醋酸盐缓冲溶液的配制
醋酸-醋酸钠缓冲液(pH 5.0-6.0):取醋酸钠54.6g,加1mol/L醋酸溶液20mL溶解后,加水稀释至500mL,即得。
通过添加0.1mol/L的醋酸、0.1mol/L的氢氧化钠调节实验所需的pH。
1.3复合酶
由纤维素酶和木瓜蛋白酶构成。
1.4茯砖茶酶解提取的工艺参数优化
图1a的实验中,固定复合酶浓度为2%、酶解pH 4.0、酶解温度60℃、酶解时间50min和料液比1:20g/mL。
图1b的实验中,固定纤维素酶与木瓜蛋白酶比例3:1、酶解pH 4.0、酶解温度60℃、酶解时间50min和料液比1:20g/mL。
图1c的实验中,固定纤维素酶与木瓜蛋白酶比例3:1、复合酶浓度2%、酶解温度60℃、酶解时间50min和料液比为1:20g/mL。
图1d的实验中,固定纤维素酶与木瓜蛋白酶比例3:1、复合酶浓度2%、酶解pH4.0、酶解时间50min和料液比为1:20g/mL。
图1e的实验中,固定纤维素酶与木瓜蛋白酶比例3:1、复合酶浓度2%、酶解pH4.0、酶解温度60℃和料液比为1:20g/mL。
图1f的实验中,固定纤维素酶与木瓜蛋白酶比例3:1、复合酶浓度2%、酶解pH4.0、酶解温度60℃和酶解时间50min。
1.5茯砖茶茶多糖的制备实例
依照图1,选择茶粗糖得率较高的工艺参数完成茯砖茶酶解提取,在纤维柱柱层析分离中采用不同浓度NaCl溶液进行了多糖的盐洗实验。
实例1
(1)将茯砖茶粉碎后,过80目筛获得茯砖茶茶粉;
(2)将茯砖茶粉碎后按料液比向1g茯砖茶茶粉中加入30mL去离子水,再加入4mL含有复合酶(纤维素酶:木瓜蛋白酶的质量比为3:1)的醋酸-醋酸盐缓冲溶液,将混合而成的酶解体系(复合酶浓度为2%,质量分数)置于水浴锅中,在酶解pH 5和酶解温度55℃的条件下酶解60min,酶解完成后沸水浴高温灭酶15min,得提取液,将提取液用真空抽滤装置进行减压过滤,以除去提取液中的不溶物,随后采用旋转蒸发仪进行减压浓缩(60℃水温条件),以减少提取液中的水分,向浓缩液中加入相对于浓缩液体积1/3的sevage试剂(氯仿:正丁醇=4:1,v/v),经振荡、离心后去除蛋白质,随后向离心所得上清中加入四倍体积无水乙醇进行醇沉(在4℃条件下静置过夜),采用旋转蒸发仪减压浓缩醇沉液(60℃水温条件)以去除溶液中的乙醇,通过低温(4℃)离心机离心,收集沉淀;
(3)取步骤2中经醇沉所得的沉淀,在去离子水中复溶后冷冻干燥,得茯砖茶粗多糖,得率(茯砖茶粗多糖质量比上茶粉质量)为6.52%,采用苯酚-硫酸法测定多糖含量为57.11%;
(4)取步骤3中的粗多糖0.50g,用50mL去离子水溶解后上样于DEAE-52纤维素柱进行分离,依次用去离子水、0.1mol/LNaCl溶液、0.2mol/LNaCl溶液、0.3mol/LNaCl溶液进行洗脱,流速为5mL/min,采用苯酚-硫酸法检测洗脱液中多糖的吸光值,用自动收集器收集洗脱液,每2min收集1管洗脱液,收集去离子水洗脱的第1-200管的洗脱液、0.1mol/L NaCl溶液洗脱的第201-300管的洗脱液、0.2mol/L NaCl溶液洗脱的第301-400管的洗脱液和0.3mol/L NaCl溶液洗脱的第401-500管的洗脱液,将各洗脱液置于截流分子量为14000的透析袋中,在去离子水中透析48h,采用旋转蒸发仪减压浓缩(60℃水温条件)透析后的洗脱液,以减少洗脱液中的水分,随后于-50℃条件下冷冻干燥48h,得到茯砖茶茶多糖;
结果表明,采用去离子水、0.1mol/LNaCl溶液、0.2mol/LNaCl溶液、0.3mol/L NaCl溶液依次进行洗脱,茯砖茶茶多糖最终得率(茯砖茶茶多糖质量比上粗多糖质量)分别为10.01%、12.12%、24.07%、20.04%,多糖的含量分别为60.13%、59.84%、72.45%、67.77%。
实例2
(1)将茯砖茶粉碎后,过80目筛获得茯砖茶茶粉;
(2)将茯砖茶粉碎后按料液比向1g茯砖茶茶粉中加入40mL去离子水,再加入5.5mL含有复合酶(纤维素酶:木瓜蛋白酶的质量比为4:1)的醋酸-醋酸盐缓冲溶液,将混合而成的酶解体系(复合酶浓度3%,质量分数)置于水浴锅中,在酶解pH 6和酶解温度50℃条件下酶解50min,酶解完成后沸水浴高温灭酶15min,得提取液,将提取液用真空抽滤装置进行减压过滤,随后采用旋转蒸发仪进行减压浓缩(60℃水温条件),向浓缩液中加入相对于浓缩液体积1/3的sevage试剂(氯仿:正丁醇=4:1,v/v),经振荡、离心后去除蛋白质,随后向离心所得上清中加入四倍体积无水乙醇进行醇沉(在4℃条件下静置过夜),采用旋转蒸发仪减压浓缩(60℃水温条件)醇沉液,通过低温(4℃)离心机离心,收集沉淀;
(3)取步骤2中经醇沉所得的沉淀,在去离子水中复溶后冷冻干燥,得茯砖茶粗多糖,得率(茯砖茶粗多糖质量比上茶粉质量)为6.24%,采用苯酚-硫酸法测定多糖含量为55.27%;
(4)取步骤3中的粗多糖0.50g,用50mL去离子水溶解后上样于DEAE-52纤维素柱进行分离,依次用去离子水、0.1mol/LNaCl溶液、0.2mol/LNaCl溶液、0.3mol/LNaCl溶液进行洗脱,流速为3mL/min,采用苯酚-硫酸法检测洗脱液中多糖的吸光值,用自动收集器收集洗脱液,每3min收集1管洗脱液,收集去离子水洗脱的第1-200管的洗脱液、0.1mol/L NaCl溶液洗脱的第201-300管的洗脱液、0.2mol/L NaCl溶液洗脱的第301-400管的洗脱液和0.3mol/L NaCl溶液洗脱的第401-500管的洗脱液,将各洗脱液置于截流分子量为14000的透析袋中,在去离子水中透析48h,采用旋转蒸发仪减压浓缩(60℃水温条件)洗脱液,随后于-50℃条件下冷冻干燥48h,得到茯砖茶茶多糖;
结果表明,采用去离子水、0.1mol/LNaCl溶液、0.2mol/LNaCl溶液、0.3mol/L NaCl溶液依次进行洗脱,茯砖茶茶多糖最终得率(茯砖茶茶多糖质量比上粗多糖质量)分别为10.27%、11.04%、26.47%、21.28%,多糖的含量分别为62.08%、63.15%、72.33%、65.49%。
(二)茯砖茶茶多糖体外降糖、降脂实验
2.1茯砖茶茶多糖对α-葡萄糖苷酶、α-淀粉酶的抑制效果
实验分组:实例1、2制得的茯砖茶茶粗糖及不同盐洗浓度对应的茯砖茶茶多糖分别为实验药物,以阿卡波糖为阳性药物。
(1)α-葡萄糖苷酶抑制率
分别称取实验药物和阿卡波糖适量,用去离子水配制浓度为0.025、0.05、0.1、0.2、0.4和0.8mg/mL的实验药物溶液和阿卡波糖溶液。在酶标板中依次加入400μL 0.1mol/L pH 6.8磷酸盐缓冲液,取自上述各浓度梯度的药物溶液(样品)各100μL,以及100μL0.8U/mLα-葡萄糖苷酶,混匀,置于37℃水浴10min后,加入100μL 10mmol/L PNPG溶液,于37℃继续反应30min,反应终止后于405nm处测定吸光度,根据公式计算茯砖茶茶多糖对α-葡萄糖苷酶的抑制率。
空白参比(A0):用去离子水代替样品;对照(A1):将α-葡萄糖苷酶换成磷酸盐缓冲液;样品组(A2):加入α-葡萄糖苷酶。
(2)α-淀粉酶抑制率
配制实验药物溶液和阿卡波糖溶液。将1mL样品(实验药物溶液或阿卡波糖溶液)和1mL 15U/mL的α-淀粉酶溶液混合,37℃下反应30min,然后加入2mL 1%的淀粉溶液,37℃下加热10min,加入2mL DNS试剂,在沸水浴中放置5min,冷却,待冷却后于540nm测定吸光度。根据公式计算茯砖茶茶多糖对α-淀粉酶的抑制率:
空白参比(A0):用去离子水代替样品;对照(A1):不含α-淀粉酶;样品组(A2):加入α-淀粉酶。
实验结果:参见图2,采用0.2mol/LNaCl盐洗后所得茯砖茶茶多糖(实例1)对α-葡萄糖苷酶、α-淀粉酶的抑制作用已接近阿卡波糖,即该茯砖茶茶多糖具有显著的降糖作用。实例2的降糖结果与实例1基本相同。
2.2茯砖茶茶多糖对胆酸盐的结合效果、及对胆固醇的抑制效果
实验分组:实例1、2制得的茯砖茶茶粗糖及不同盐洗浓度对应的茯砖茶茶多糖分别为实验药物,以辛伐他汀为阳性药物。
(1)体外结合胆酸盐能力
0.3mM混合母液:用0.1moL/L pH 7.6磷酸盐缓冲液配制0.3mmoL/L甘氨胆酸钠溶液、0.3mmoL/L牛磺胆酸钠溶液和0.3mmoL/L胆酸钠溶液,混匀,备用。
取0.3mM的混合母液0、0.1、0.5、1.0、1.5、2.0、2.5mL,加入磷酸盐缓冲液至2.5mL,然后加入7.5mL 60%的硫酸溶液,70℃水浴20min,置于冰水5min,在387nm处测吸光度,绘出了相应的标准曲线。
称取适量样品(茯砖茶茶多糖、粗多糖或阳性药物)于具塞试管中,加入1mL0.01mol/L的盐酸溶液(模拟人体胃液的酸性环境),在37℃下振动2h,pH值调整到7.6(人体小肠液的pH),加入4mL胆酸盐溶液,在37℃下振动2h。将混合物以4000r/min离心20min,保留上清,取2.5mL上清试样,测量吸光度,并根据标准曲线计算出试样中的胆酸盐浓度,根据公式计算胆酸盐结合率。
c1为样品溶液的胆酸盐浓度(mmol/L),c0为空白溶液的胆酸盐浓度(mmol/L),空白对照为无样品加入。
(2)体外抑制胆固醇能力
胆固醇胶束溶液制备:在5mL甲醇中溶解脂质(油酸、胆固醇和卵磷脂),充分溶解后用氮气吹干脂质溶液。加入氯化钠、pH 7.4的磷酸盐缓冲液和牛磺胆酸钠,混匀后,37℃水浴12h制成胆固醇胶束溶液(胶束溶液中含有132mmol/L氯化钠、pH 7.4浓度为15mmol/L的磷酸缓冲液、5mmol/L胆固醇和油酸、10mmol/L卵磷脂和牛磺胆酸钠)。
在试管中移取1mL胶束溶液,加入10mg样品(茯砖茶茶多糖、粗多糖或阳性药物)混匀,空白对照为无样品加入的胶束溶液,37℃振荡2h,以10000r/min离心10min,取上清,测胆固醇水平,根据公式计算胆固醇抑制率。
c1为样品溶液的胆固醇溶解度(mg/mL),c0为空白溶液的胆固醇溶解度(mg/mL)。
实验结果:参见图3,采用去离子水、0.1mol/L NaCl、0.2mol/L NaCl、0.3mol/LNaCl盐洗后所得茯砖茶茶多糖(实例1)对胆酸盐结合作用接近阳性药物,0.2mol/L NaCl、0.3mol/L NaCl盐洗后所得茯砖茶茶多糖(实例1)对胆固醇的抑制作用接近阳性药物,即该茯砖茶茶多糖具有显著的降脂作用。实例2的降脂结果与实例1基本相同。
总之,本发明在茯砖茶酶解提取中采用优化的酶解条件,将所得提取液通过过滤、浓缩、去蛋白及醇沉、复溶、冷冻干燥,获得茯砖茶粗多糖,对茯砖茶粗多糖采用纤维柱柱层析进行分离纯化,洗脱液经透析、浓缩、冷冻干燥,获得茯砖茶茶多糖。本发明制备的茯砖茶茶多糖具有得率高、纯度高、提取时间短、减少能源消耗、工艺条件温和、可有效保存茯砖茶的多糖成分的生物活性等优点,并且,实验表明该茯砖茶茶多糖具有良好的降糖、降脂功效,为茯砖茶茶多糖的进一步开发提供新的途径。
Claims (9)
1.一种茯砖茶茶多糖的制备方法,其特征在于:包括以下步骤:
1) 将茯砖茶茶粉与水、纤维素酶、木瓜蛋白酶以及pH为4-8的醋酸-醋酸盐缓冲溶液混合后进行酶解,得提取液,其中纤维素酶和木瓜蛋白酶的质量比为1:1-5:1;将提取液过滤,将所得滤液浓缩后采用sevage法去除蛋白质,然后进行醇沉并收集沉淀;
2) 将沉淀在水中复溶后冷冻干燥,得茯砖茶粗多糖;
3) 将茯砖茶粗多糖采用离子交换柱分离多糖组分,将多糖组分通过洗脱收集后依次进行透析、浓缩、冷冻干燥,得到茯砖茶茶多糖;
所述步骤3中,茯砖茶粗多糖采用水溶解后上样于离子交换柱,离子交换柱的填料为阴离子交换树脂,洗脱液为水以及0.1-0.3 mol/L NaCl溶液,洗脱流速为1-8 mL/min;
洗脱的过程为:依次用水、0.1 mol/L NaCl溶液、0.2mol/L NaCl溶液、0.3 mol/L NaCl溶液进行洗脱。
2.根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述茯砖茶茶粉是通过将茯砖茶粉碎后过60-100目筛而获得的。
3.根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述步骤1中,每1g茯砖茶茶粉与10-50mL水混合。
4.根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述步骤1中,由纤维素酶与木瓜蛋白酶构成的复合酶在酶解体系中的初始质量分数为1%-5%。
5. 根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述酶解的条件包括:酶解温度为40-60 ℃,酶解时间为40-80 min。
6. 根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述醇沉具体包括以下步骤:将去除蛋白质后的浓缩滤液与2-6倍体积的无水乙醇或95%的乙醇溶液混合,然后在0-10 ℃条件下静置12-20 h。
7.根据权利要求1所述一种茯砖茶茶多糖的制备方法,其特征在于:所述步骤3中,透析采用的截留分子量为8000-14000。
8. 如权利要求1-7中任意一项所述的茯砖茶茶多糖的制备方法制备的茯砖茶茶多糖,其特征在于:该茶多糖是0.2mol/L NaCl溶液进行洗脱得到的洗脱液依次进行透析、浓缩、冷冻干燥而得到的茯砖茶茶多糖。
9.如权利要求8所述的茯砖茶茶多糖在制备具有降糖和/或降脂功效的药物、保健品中的应用。
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