CN115611994B - 一种睡茄多糖及其制备方法和在降糖中的应用 - Google Patents
一种睡茄多糖及其制备方法和在降糖中的应用 Download PDFInfo
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- CN115611994B CN115611994B CN202211357574.5A CN202211357574A CN115611994B CN 115611994 B CN115611994 B CN 115611994B CN 202211357574 A CN202211357574 A CN 202211357574A CN 115611994 B CN115611994 B CN 115611994B
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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
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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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- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Sustainable Development (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种睡茄多糖及其制备方法和在降糖中的应用,其中睡茄多糖AP‑1是从干燥睡茄根经水提醇沉、超滤、色谱层析得到的多糖组分,分子量为9.21×103Da,总糖含量97.65%,由甘露糖、葡萄糖和半乳糖组成,摩尔比为0.1:1.5:1。本发明获得的睡茄多糖组分AP‑1无明显毒副作用,具有良好的降糖作用,可用于制备降糖食品或药品,在防治糖尿病及其相关慢性疾病方面具有良好的应用前景。
Description
技术领域
本发明涉及一种睡茄多糖及其制备方法和在降糖中的应用,属于生物医药领域。
背景技术
糖尿病是一种代谢紊乱相关疾病,其特征是胰腺无法分泌胰岛素或胰岛素抵抗,被认为是一种危及生命的严重疾病。根据国际糖尿病联盟的最新计算,全球约有4.63亿成年人患有糖尿病,据推测,到2045年这一数字将达到7.83亿,这是我们这个时代最严重的健康问题之一。治疗糖尿病的药物开发需要遵循有效性和安全性原则,目前用于治疗糖尿病的主要药物类别是胰岛素及其类似物、双胍类药物、磺酰脲类药物和噻唑烷二酮类等化学药物,其存在着诸如副作用大、治疗成本高等局限性。近年来,天然植物成分在降糖药物中的应用研究中受到越来越多的关注,许多从传统药物或其他天然材料中提取的成分被报道具有降血糖作用,其中多糖组分由于具有独特的高效性和安全性,使其很有可能成为糖尿病的新候选药物。
睡茄是茄科睡茄属植物,广泛分布在中国、印度、斯里兰卡、阿富汗等国家的干旱地区。睡茄富含多种功能成分,如多糖、酚类物质、生物碱、甾体内酯、皂苷和木脂素酰胺等,具有广泛的生物活性,但目前研究多集中于睡茄粗提物,对其单一活性成分研究较少,目前对于从睡茄根部中分离得到具有降糖作用的多糖组分未见相关报道。
发明内容
本发明提供了一种睡茄多糖及其制备方法和在降糖中的应用。本发明睡茄多糖AP-1具有良好的降糖作用,无毒副作用,可广泛应用于制备降糖食品、药品等领域。
本发明睡茄多糖,简记为AP-1,是由睡茄根部提取分离获得,其总糖含量为97.65%,分子量为9.21×103Da,由甘露糖、葡萄糖和半乳糖组成,摩尔比为0.1:1.5:1。
本发明睡茄多糖AP-1的制备方法,包括如下步骤:
步骤1:取干燥睡茄根超微粉碎后过80目网筛,获得睡茄根粉末。
步骤2:将步骤1获得的睡茄根粉末与无水乙醇按1:10(g/mL)料液比混合,于80℃水浴锅中热回流2h,离心收集沉淀,烘干后得到预处理的睡茄根粉末。
步骤3:将步骤2获得的睡茄根粉末按照1:10(g/mL)的料液比加入去离子水,100℃水浴2h,分离上清液,重复三次;收集三次上清液,浓缩至原体积的1/5-1/10后,与无水乙醇按照1:4的比例混合,4℃静置10-15h,5000-8000×g离心10-20min,收集沉淀物。
步骤4:将步骤3获得的沉淀物用去离子水溶解,采用3%-6%的TCA法除蛋白,除尽蛋白后,于3500Da透析袋中透析,流水透析48h,去离子水透析48h,冷冻干燥后得睡茄多糖组分A1。
步骤5:将步骤4获得的睡茄粗多糖组分A1用去离子水溶解,经1×104Da的超滤膜分离,收集分子量小于1×104Da的多糖溶液,浓缩冻干得到睡茄多糖组分A2。
步骤6:将步骤5获得的睡茄粗多糖组分A2溶于去离子水中,通过DEAE-SepharoseFF柱层析,用去离子水洗脱,收集洗脱峰,浓缩冻干得到睡茄多糖组分A3。
步骤7:将步骤5获得的睡茄粗多糖组分A3溶于去离子水中,利用SuperdexTM-75凝胶柱层析对其进行纯化,收集目标峰,透析冻干后获得睡茄均一组分AP-1。
本发明睡茄多糖AP-1的用途,是以所述睡茄多糖AP-1制备具有降糖功效的食品或药物制剂。
本发明睡茄多糖AP-1的降糖作用,在于对α-淀粉酶和α-葡萄糖苷酶具有显著的抑制效果,半最大抑制浓度分别为0.58mg/mL和0.54mg/mL;对胰岛素抵抗的3T3-L1脂肪细胞葡萄糖消耗能力均明显提高,且呈剂量依赖性。
与现有技术相比,本发明的有益效果体现在:
1、本发明首次从睡茄根中提取分离纯化得到均一多糖AP-1,步骤简单,效率高;
2、本发明提供的睡茄多糖AP-1对α-淀粉酶和α-葡萄糖苷酶具有显著的抑制效果,还能够促进胰岛素抵抗3T3-L1脂肪细胞摄取消耗葡萄糖的能力,表现出显著的降糖效果,在制备降糖食品或药品方面的应用具有良好的开发前景。
附图说明
图1是AP-1相对分子量检测色谱图,可以看出睡茄多糖AP-1是均一组分。根据分子量标准曲线计算得出分子量为9.21×103Da。
图2是AP-1的单糖组成,(A)单糖标准品(Ara-阿拉伯糖,Rha-鼠李糖,Man-甘露糖,Gal-半乳糖,Glc-葡萄糖、GlcA-葡萄糖醛酸、GalA-半乳糖醛酸)(B)是AP-1;从图中可以看出AP-1为中性杂多糖,由甘露糖、葡萄糖和半乳糖组成,摩尔比为0.1:1.5:1。
图3是不同浓度的AP-1对3T3-L1细胞的生长抑制,从图中可以看出,AP-1对3T3-L1细胞无毒性作用,且对其增殖起到了促进作用。
图4是不同浓度的AP-1对胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗量的影响,结果表明多糖AP-1对胰岛素抵抗有明显的改善作用,且呈浓度依赖性。
具体实施方式
实施例1:睡茄多糖AP-1的制备
步骤1:取干燥睡茄根进行超微粉碎后过80目网筛,获得睡茄根粉末。
步骤2:将步骤1获得的睡茄根粉末与无水乙醇按1:10(g/mL)料液比混合,于80℃水浴锅中热回流2h,离心收集沉淀,烘干后得到预处理的睡茄根粉末。
步骤3:将步骤2获得的睡茄根粉末按照1:10(g/mL)的料液比加入去离子水,100℃水浴2h,重复提取三次,分离上清液。将提取液合并浓缩至适量体积后,加入适量耐高温α-淀粉酶,100℃反应60min,8000×g离心10min,收集上清。与无水乙醇按1:4的比例混合,4℃静置12h,8000×g离心15min,收集沉淀物。
步骤4:将步骤3获得的沉淀物中加入去离子水复配为糖溶液,与5%的三氯乙酸等体积混合,4℃静置12h,8000×g离心10min,留取上清液,用3500Da透析袋流水透析48h,去离子水透析48h,浓缩冻干后得睡茄多糖组分A1。
步骤5:将步骤4获得的睡茄多糖组分A1用去离子水溶解,通过1×104Da的超滤膜进行分离,收集分子量小于1×104Da的多糖溶液,浓缩冻干得到睡茄多糖组分A2。
步骤6:将步骤5获得的睡茄粗多糖组分A2溶于去离子水中,通过DEAE-SepharoseFF(35×400mm)柱层析,用去离子水以1mL/min的流速进行洗脱,收集洗脱峰,浓缩冻干得到睡茄多糖组分A3。
步骤7:将步骤6得到的睡茄多糖A3溶于去离子水中,利用SuperdexTM-75(26×700mm)凝胶柱对其进行层析纯化,流速为0.8mL/min,收集目标峰,浓缩冻干后获得睡茄均一组分AP-1。
实施例2:睡茄多糖AP-1的结构特征
分子量测定:采用高效液相色谱结合蒸发光检测器(HPLC-ELSD)测定AP-1相对分子质量。准确配制2mg/mL葡聚糖标准品(T-3、T-10、T-50、T-500、T-1000、T-2000)溶液1mL,同时准确配制2mg/mL的AP-1溶液,过0.22μm滤膜。采用TSK Gel G6000 PWXL色谱柱(300×7.0mm,13μm),载气为N2,气体流速2.5L/min,柱温30℃,漂移管温度100℃,流动相超纯水,流速1.0mL/min,进样量为10μL。检测完成后,根据分子量标准曲线计算出AP-1的相对分子质量为9.21×103Da。
单糖组成分析:本实验采用酸水解-柱前PMP衍生化方法处理样品,高效液相色谱法分析测定。以阿拉伯糖、鼠李糖、葡萄糖、半乳糖、甘露糖、葡萄糖醛酸、半乳糖醛酸作为标准样品进行标准曲线的制作。称取10mgAP-1于30mL具塞试管中,加入5mL 2M的TFA,混合均匀,充N2封口,110℃油浴6h。将水解产物旋蒸干燥,多次加入甲醇旋蒸除酸,至pH为中性,加入1mL去离子水溶解,备用。分别称取不同标准单糖样品2mg,用去离子水配成2mg/mL不同的标准单糖溶液。向标准单糖和已水解的多糖AP-1溶液中依次加入500μL0.5mol/L的PMP甲醇溶液和500μL 0.3mol/L的NaOH溶液,充分混匀后,在70℃的水浴锅中反应30min进行PMP柱前衍生化,然后加入500μL 0.3mol/L的HCl中和至中性,用0.22μm微孔滤膜过滤。所得产物使用高效液相色谱检测,选用DAD检测器。HPLC柱温为30℃,色谱柱为Zorbox Eclipse XDB-C18柱(4.6mm×250mm,5μm),波长为245nm下检测。检测流动相A为乙腈,流动相B为0.05mol/L磷酸盐缓冲溶液。时间梯度洗脱0-60min,初始设置为流动相A:流动相B=17%:83%,最终洗脱到比例为流动相A:流动相B=20%:80%,进样量为10μL。通过与标准样品的保留时间比对分析单糖种类,分析发现AP-1主要由甘露糖、葡萄糖和半乳糖组成,摩尔比为0.1:1.5:1。
实施例3:AP-1对α-淀粉酶活性的抑制
分别吸取80μL的AP-1(50、200、400、800、1000μg/mL)、40μL的2U/mL的α-淀粉酶和1%的淀粉溶液混合均匀,37℃静置10min,加入80μL的DNS,于95℃水浴10min,于540nm处检测吸光值。每组设三组平行,根据如下公式计算各组α-淀粉酶抑制能力:
α-淀粉酶抑制能力(%)=[1-(A2-A3)/(A0-A1)]×100
其中,A0(空白组)表示用磷酸盐缓冲液代替多糖样品溶液;A1(空白对照组)代表用磷酸盐缓冲液代替α-淀粉酶和样品;A2(实验组)代表各浓度AP-1溶液和阿卡波糖溶液;A3(实验对照组)代表用磷酸盐缓冲液代替α-淀粉酶;
实施例4:AP-1对葡萄糖苷酶活性的抑制
取30μL的AP-1(50、200、400、800、1000μg/mL)与10μL的1U/mL的α-葡萄糖苷酶混合均匀,37℃静置10min。加入60μL的PNPG溶液,37℃静置30min,加入120μL的0.2M Na2CO3溶液,于405nm处检测吸光值。每组设三组平行,根据如下公式计算各组α-葡萄糖苷酶抑制能力:
α-葡萄糖苷酶抑制能力(%)=[1-(A2-A3)/(A0-A1)]×100
其中,A0(空白组)表示用磷酸盐缓冲液代替多糖样品的吸光值;A1(空白对照组)代表用磷酸盐缓冲液代替α-葡萄糖苷酶和样品的吸光值;A2(实验组)代表各浓度AP-1溶液和阿卡波糖的吸光值;A3(实验对照组)代表用磷酸盐缓冲液代替α-葡萄糖苷酶的吸光值;
实施例5:AP-1对3T3-L1细胞的生长抑制
选取生长状态良好的3T3-L1细胞,其制成8×104个/mL的细胞悬浮液,按每孔100μL加入九十六孔板中,于细胞培养箱内培养24h。实验分为两组:空白组每孔加入100μL的培养基;实验组每孔加入100μL含不同浓度AP-1(50、200、400、800、1000μg/mL)的培养基,每组设置8个复孔,孵育24h。弃培养基,每孔加入100μL含0.5%的MTT培养基,继续孵育4h,弃培养基,每孔加入100μL的DMSO,用锡箔纸包裹孔板,摇床慢摇10min,于570nm处检测其吸光值并计算样品对细胞生长抑制率。根据以下公式计算睡茄多糖AP-1对细胞生长抑制率:
细胞生长抑制率(%)=(A0-A1)/A0×100
其中,A0表示空白组的吸光值,A1表示实验组各组吸光值
实验结果如图3所示,可以看出AP-1对3T3-L1细胞无毒性作用,且对其增殖起到了促进作用。
实施例6:AP-1对胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗量的影响
当3T3-L1前脂肪细胞已经诱导成为成熟的脂肪细胞,实验设置为对照组和模型组。对照组每个孔加入200μL的DMEM(10%胎牛血清),模型组每个孔加入200μL的含地塞米松(1μM)的DMEM,每组设置6个复孔,于细胞培养箱内孵育48h,收集上清液,根据GOD-POD法检测葡萄糖含量判断葡萄糖摄取量。实验结果显示,与对照组相比,模型组葡萄糖消耗率明显下降27.3%,有差异统计意义,表明成功建立胰岛素抵抗模型。
建模成功后,实验分为四组:正常组每孔加入200μL的DMEM(10%胎牛血清)培养基;模型组每孔加入200μL含地塞米松(1μM)的DMEM培养基;阳性对照组每孔加入200μL含二甲双胍(5mM)DMEM培养基;样品组每孔加入200μL含AP-1(50、200、400、800、1000μg/mL)的DMEM培养基;于细胞培养箱内孵育48h,根据GOD-POD法检测葡萄糖含量来判断葡萄糖摄取量。
由图4结果显示,与模型组相比,阳性对照组葡萄糖消耗量明显增加,不同浓度的样品组葡萄糖消耗量呈浓度依赖性增加,当AP-1的浓度为50μg/mL时,葡萄糖消耗量为108.78%,高于对照组,当AP-1的浓度为1000μg/mL时,葡萄糖消耗量为173.97%,表明多糖AP-1对胰岛素抵抗有明显的改善作用,且呈浓度依赖性。
综上所述,以本发明的参数范围内制备的睡茄多糖AP-1具有良好的降糖作用,能够广泛应用于制备降糖食品、药品等领域。
以上仅为本发明的优选实施例,并非因此限制本发明的专利范围,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包括在本发明的专利保护范围内。
Claims (8)
1.一种睡茄多糖,简记为AP-1,其特征在于:
所述睡茄多糖是由睡茄根部提取分离获得,其总糖含量为97.65%,分子量为9.21×103Da,由甘露糖、葡萄糖和半乳糖组成,摩尔比为0.1:1.5:1。
2.一种权利要求1所述的睡茄多糖AP-1的制备方法,其特征在于包括如下步骤:
步骤1:取干燥睡茄根超微粉碎后过80目网筛,获得睡茄根粉末;
步骤2:将步骤1获得的睡茄根粉末与无水乙醇混合,于80 ℃回流2 h,离心收集沉淀,烘干后得到预处理的睡茄根粉末;
步骤3:将步骤2获得的睡茄根粉末加入去离子水,100 ℃水浴2 h,分离上清液,重复三次;收集三次上清液,浓缩至原体积的1/5- 1/10后,与无水乙醇按照1:4的比例混合,4 ℃静置10-15 h,离心收集沉淀物;
步骤4:将步骤3获得的沉淀物用去离子水溶解,采用3%-6%的TCA法除蛋白,除尽蛋白后透析,冷冻干燥后得睡茄多糖组分A1;
步骤5:将步骤4获得的睡茄粗多糖组分A1用去离子水溶解,经超滤膜分离,收集多糖溶液,浓缩冻干得到睡茄多糖组分A2;
步骤6:将步骤5获得的睡茄粗多糖组分A2溶于去离子水中,通过DEAE-Sepharose FF柱层析,用去离子水洗脱,收集洗脱峰,浓缩冻干得到睡茄多糖组分A3;
步骤7: 将步骤5获得的睡茄粗多糖组分A3溶于去离子水中,利用SuperdexTM-75凝胶柱层析对其进行纯化,收集目标峰,透析冻干后获得睡茄均一组分AP-1。
3.根据权利要求2所述的制备方法,其特征在于:
步骤2中,睡茄根粉末与无水乙醇按1 g:10 mL料液比混合。
4.根据权利要求2所述的制备方法,其特征在于:
步骤3中,睡茄根粉末按照1 g:10 mL的料液比加入去离子水。
5.根据权利要求2所述的制备方法,其特征在于:
步骤4中,所述透析是于3500 Da透析袋中透析,依次流水透析48 h,去离子水透析48h。
6.根据权利要求2所述的制备方法,其特征在于:
步骤5中,将步骤4获得的睡茄粗多糖组分A1用去离子水溶解,经1×104 Da的超滤膜分离,收集分子量小于1×104 Da的多糖溶液,浓缩冻干得到睡茄多糖组分A2。
7.一种权利要求1所述的睡茄多糖AP-1的用途,其特征在于:
以所述睡茄多糖AP-1制备具有降糖功效的食品或药物制剂;
所述食品或药物制剂对α-淀粉酶和α-葡萄糖苷酶具有显著的抑制效果,半最大抑制浓度分别为0.58 mg/mL和0.54 mg/mL。
8.根据权利要求7所述的用途,其特征在于:
所述食品或药物制剂能够促进胰岛素抵抗3T3-L1脂肪细胞摄取消耗葡萄糖的能力,且呈剂量依赖性。
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