CN111875714A - 低分子量硫酸半乳聚糖及其制备方法和应用 - Google Patents
低分子量硫酸半乳聚糖及其制备方法和应用 Download PDFInfo
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- CN111875714A CN111875714A CN202010778404.9A CN202010778404A CN111875714A CN 111875714 A CN111875714 A CN 111875714A CN 202010778404 A CN202010778404 A CN 202010778404A CN 111875714 A CN111875714 A CN 111875714A
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Abstract
本发明公开了一种低分子量硫酸半乳聚糖及其制备方法和应用。本发明以不同种类的红藻为原料,经过一步提取法、降解、分离纯化,制备得到了不同分子量段的低分子量硫酸半乳聚糖。本发明所述的低分子量硫酸半乳聚糖能够选择性抑制PTP1B,提高胰岛素抵抗条件下细胞的葡萄糖消耗,提高胰岛素敏感性,调节能量消耗,维持葡萄糖内稳态。有望开发成预防和/或治疗II型糖尿病和肥胖症的药物,或开发成具有降糖、降脂、减肥功能的食品,具有良好的市场应用前景。
Description
技术领域
本发明涉及硫酸半乳寡糖技术领域,具体涉及一种低分子量硫酸半乳聚糖及其制备方法和应用。
背景技术
糖尿病(Diabetes Mellitus,DM)是一类慢性代谢性疾病,在三种主要的糖尿病类型中,Ⅱ型糖尿病病例占大约90%。Ⅱ型糖尿病是一种慢性进行性代谢疾病,其发病与年龄、生活方式、遗传因素、肥胖和病毒感染等多种因素相关,并受多种关键基因的调控,如PPARG、IRS-1、IGF-1和CDKN2B等。Ⅱ型糖尿病的发病机制包括胰岛素抵抗、糖毒性、脂毒性、氧化应激、遗传缺陷和炎症反应等,主要与胰岛素抵抗有关。胰岛素抵抗的发生是由许多原发的、基因因素和继发的、环境因素综合参与的结果,包括胰岛素分泌缺陷和机体对胰岛素敏感性缺陷,即身体不再产生足够的胰岛素或者不能有效使用胰岛素,最终导致高血糖和碳水化合物、脂肪和蛋白质代谢异常,从而对机体产生一系列不良影响和多种病理生理改变,成为众多疾病的共同发病基础。
目前治疗糖尿病的方法主要有胰岛素及类似物治疗、口服药物治疗以及尚在研究阶段的干细胞治疗。最常见的口服药物包括α-葡萄糖苷酶抑制剂、磺脲类、氯茴酸(列奈)类、双胍类和胰岛素增敏剂。其中,α-葡萄糖苷酶抑制剂能通过抑制肠系膜刷状缘α-葡萄糖苷酶抑制多糖或双糖转变为单糖,从而延缓糖类物质吸收,降低餐后血糖。磺脲类药通过与胰岛β细胞膜磺脲受体结合,关闭β细胞ATP依赖钾通道、导致β细胞去极化,促进钙离子内流增加,触发胰岛素分泌,有利于改善胰岛素抵抗,增加组织对胰岛素敏感性。氯茴酸(列奈)类药物是一种新型非磺脲类口服降糖药,主要用于饮食和运动不能控制高血糖的Ⅱ型糖尿病,能够有效控制餐后血糖,可以使患者的进食时间更随意。双胍类药物是超重或肥胖Ⅱ型糖尿病的首选药物,也是治疗Ⅱ型糖尿病的一线药物,能增加机体对外周葡萄糖的利用,磺脲类药物继发失效者加用二甲双胍可明显改善血糖控制及其它代谢失常,可与α-葡萄糖苷酶抑制剂、噻唑烷酮类和胰岛素联合使用。胰岛素增敏剂能改善胰岛素抵抗状态,提高机体对胰岛素的反应性,主要有噻唑烷酮类(TZDs),也称格列酮类药物。
蛋白酪氨酸磷酸化酶1B(PTP1B)是一个细胞内非受体型的蛋白酪氨酸磷酸化酶(PTP),对胰岛素信号通路和瘦素信号通路具有负向调节作用。通过抑制PTP1B活性,有助于提高外周组织对胰岛素的敏感性,对维持葡萄糖内稳态,能量消耗等生理过程起重要调节作用。因此,它是预防和治疗II型糖尿病和肥胖的潜在、有效的药物新靶点。目前,PTP1B抑制剂的研究备受人们关注,然而尚未有针对该靶点的药物上市,具有很好的临床应用前景。
从临床上看,用于糖尿病治疗的药物在有效性和安全性等方面都有局限性,因此,从自然资源中寻找更有效和更安全的药物是我们需要解决的发明难题。
发明内容
本发明的目的是提供一种低分子量硫酸半乳聚糖及其制备方法和应用,从红藻中分离纯化得到了低分子量硫酸半乳聚糖,发现该低分子量硫酸半乳聚糖对PTP1B靶点具有选择性抑制作用,具有降血糖和防治II型糖尿病的作用。
本发明第一方面提供了低分子量硫酸半乳聚糖,其化学结构由通式(I)表示的结构单元和通式(II)表示的结构单元组成,所述低分子量硫酸半乳聚糖中,通式(I)表示的结构单元的质量百分含量为5%~50%,通式(II)表示的结构单元的质量百分含量为50%~95%;
其中,R1=H或SO3 -,R2=H或SO3 -,R3=H或SO3 -;n表示15~100的整数;
其中,R1=H或SO3 -,R2=H或SO3 -,R3=H或SO3 -;n表示15~100的整数。
在一些优选的实施例中,所述的低分子量硫酸半乳聚糖的分子量为3kDa~20kDa,所述的低分子量硫酸半乳聚糖中硫酸基团的质量百分含量为15%~40%。
在一些优选的实施例中,所述低分子量硫酸半乳聚糖的化学结构是由4-硫酸基-β-1,3-D-半乳糖(D-Gal)残基和6-硫酸基-α-1,4-D-半乳糖(L-Gal)残基,3,6内醚-D-Gal残基,2-硫酸基-3,6-内醚-D-Gal残基,2-硫酸基-β-1,3-D-半乳糖(D-Gal)残基和2,6-二硫酸基-D-半乳糖(D-Gal)残基,2-硫酸基-3,6内醚-D-Gal中的一种或几种组成。
本发明第二方面提供了本发明第一方面提供的硫酸半乳寡糖的制备方法,该硫酸半乳寡糖采用下述制备工艺:
a)提取:清洗藻类、烘干、切碎,分别用水和重量比为5%~10%的碱水溶液提取0.5~2小时;过滤;残渣按上述过程再提取1~2次;合并滤液,浓缩后进行提纯、离心、干燥、粉碎得粗硫酸半乳聚糖粗品;
b)降解:降解所述硫酸半乳聚糖粗品,浓缩,并用有机溶剂沉淀,干燥,得到低分子量硫酸半乳聚糖;
c)分离纯化:将所述低分子量硫酸半乳聚糖粗品用水溶解,超滤分级,然后将得到的的低分子量硫酸半乳聚糖用QFF凝胶色谱柱层析纯化和G10凝胶柱层析除盐,冷冻干燥,即得不同组成含量的低分子量硫酸半乳聚糖。
在一些优选的实施例中,所述藻类为红藻,为勾沙菜、角叉菜、刺麒麟菜、耳突麒麟菜、小杉藻、亚格菜、沟杉藻中的一种或多种。
本发明第三方面提供了本发明第一方面提供的低分子量硫酸半乳聚糖在制备预防和/或治疗II型糖尿病和肥胖的药物或食品中的应用。
本发明第四方面提供了一种用于预防和/或治疗II型糖尿病和肥胖的药物或食品,所述组合物含有;
1)本发明第一方面提供的低分子量硫酸半乳聚糖,和
2)药学上可接受的载体。
本发明还提供了一种受体调节剂,所述受体调节剂中的有效成分为本发明第一方面提供的低分子量硫酸半乳聚糖,所述受体调节剂选择性抑制蛋白酪氨酸磷酸化酶1B。
在一些优选的实施例中,所述的受体调节剂,
1)提高胰岛素抵抗条件下细胞的葡萄糖消耗,提高胰岛素敏感型,或
2)调节能量消耗,或
3)维持葡萄糖内稳态。
在一些优选的实施例中,所述的受体调节剂用于制备:
1)预防和/或治疗II型糖尿病的药物,或
2)预防和/或治疗肥胖的药物,或
3)降糖、降脂、减肥的食品。
本发明的有益效果体现在:
本发明采用一步提取法,从红藻提取物中分离纯化得到了一种硫酸半乳聚糖,并对其进行降解,制备获得系列低分子量硫酸半乳聚糖,对该硫酸半乳聚糖行了药效研究,发现其对PTP1B靶点具有选择性抑制作用,经细胞和动物试验研究发现,其具有良好的降血糖和减肥降脂作用,可以开发成防治II型糖尿病和肥胖症的药物及食品,具有良好的市场前景。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。在所有附图中,类似的元件或部分一般由类似的附图标记标识。附图中,各元件或部分并不一定按照实际的比例绘制。
图1为本发明实施例低分子量硫酸半乳聚糖(GAS)对HepG2胰岛素抵抗细胞摄取2-NBDG影响结果图;其中,纵坐标为2-NBDG的摄取率,A图表示GASK系列低分子量硫酸半乳聚糖对HepG2胰岛素抵抗细胞摄取2-NBDG的影响,B图表示GASI系列低分子量硫酸半乳聚糖对HepG2胰岛素抵抗细胞摄取2-NBDG的影响,C图表示GASL系列低分子量硫酸半乳聚糖对HepG2胰岛素抵抗细胞摄取2-NBDG的影响;
图2为本发明实施例低分子量硫酸半乳聚糖(GAS)作用于胰岛素抵抗HepG2细胞对IRS-1及P-IRS-1蛋白表达水平的Western blot分析结果图;
图3为低分子量硫酸半乳聚糖(GAS)作用于胰岛素抵抗HepG2细胞对Akt及P-Akt蛋白表达水平的Western blot分析结果图。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只作为示例,而不能以此来限制本发明的保护范围。
需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
下面的实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到。以下实施例中的定量试验,均设置三次重复实验,数据为三次重复实验的平均值或平均值±标准差。
实施例1
本实施例提供了一种低分子量硫酸半乳聚糖,其化学结构由通式(I)表示的结构单元和通式(II)表示的结构单元组成,
其中,R1=SO3 -,R2=H,R3=H;15≤n≤100。
上述低分子量硫酸半乳聚糖的制备方法,包括如下步骤:
(1)将勾沙菜用自来水冲洗掉表面盐分后,烘干、切碎,分别用热水和5-10%的碱水溶液(NaOH或KOH)提取1小时;过滤;残渣按上述过程再提取1-2次;合并多次的提取液,浓缩至提取液原始体积的2%,用3倍体积乙醇沉淀,离心、干燥、粉碎得粗硫酸半乳聚糖粗品;
(2)采用0.05mol/L的稀酸溶液将粗多糖溶解,溶液中粗多糖浓度为5%,70℃下加热反应1.5h,2h,3h和4.5h,加入碱液中和,浓缩溶液,醇沉,过滤,干燥沉淀,得到系列低分子量硫酸半乳聚糖;
(3)采用凝胶柱分级纯化,低分子量硫酸半乳聚糖分成4个组分,分别记为GASK-1,GASK-2,GASK-3,GASK-4。
实施例2
本实施例提供了一种低分子量硫酸半乳聚糖,其化学结构由通式(I)表示的结构单元和通式(II)表示的结构单元组成,
其中,R1=H,R2=SO3 -,R3=SO3 -;15≤n≤100。
上述低分子量硫酸半乳聚糖的制备方法,包括如下步骤:
(1)将亚格藻用自来水冲洗掉表面盐分后,烘干、切碎,用热水提取1小时;过滤;残渣按上述过程再提取1-2次;合并多次的提取液,浓缩至提取液原始体积的2%,用3倍体积乙醇沉淀,离心、干燥、粉碎得粗硫酸半乳聚糖粗品;
(2)采用0.05mol/L的稀酸溶液将粗多糖溶解,溶液中粗多糖浓度为5%,60℃下加热反应1.5h,2h,3h和4.5h,加入3mol/L碱液中和,4倍体积乙醇沉淀,4000r/min离心15min,得到的沉淀冷冻干燥,得到系列低分子量硫酸半乳聚糖;
(3)采用超滤分级,将所得低分子量硫酸半乳聚糖纯化,分别记为GASI-1,GASI-2,GASI-3,GASI-4。
实施例3
本实施例提供了一种低分子量硫酸半乳聚糖,其化学结构由通式(I)表示的结构单元和通式(II)表示的结构单元组成,
其中,R1=H,R2=SO3 -,R3=SO3 -;15≤n≤100。
上述低分子量硫酸半乳聚糖的制备方法,包括如下步骤:
(1)将沟杉藻用自来水冲洗掉表面盐分后,烘干、切碎,用热水提取1小时;过滤;残渣按上述过程再提取1-2次;合并多次的提取液,浓缩至提取液原始体积的2%,用3倍体积乙醇沉淀,离心、干燥、粉碎得粗硫酸半乳聚糖粗品;
(2)采用0.05mol/L的稀酸溶液将粗多糖溶解,溶液中粗多糖浓度为5%,90℃下加热反应1.5h,2.5h,4h和5h,加入3mol/L碱液中和,4倍体积乙醇沉淀,4000r/min离心15min,得到的沉淀冷冻干燥,得到系列低分子量硫酸半乳聚糖;
(3)采用超滤分级,将所得低分子量硫酸半乳聚糖纯化,获得4个组分,分别记为GASL-1,GASL-2,GASL-3,GASL-4。
为确定所得组分的理化性质,用GPC法测定所得化合物的分子量,用离子色谱法检测所得化合物的硫酸根含量,测定结果如表1所示。
表1系列低分子量硫酸半乳聚糖分子量及硫酸根含量
样品 | Mw/kDa | 硫酸根% |
GASK-1 | 10.5 | 24.3 |
GASK-2 | 5.0 | 25.0 |
GASK-3 | 2.9 | 24.6 |
GASK-4 | 2.2 | 25.5 |
GASI-1 | 14.0 | 20.7 |
GASI-2 | 5.0 | 24.5 |
GASI-3 | 3.0 | 25.3 |
GASI-4 | 2.2 | 25.7 |
GASL-1 | 7.7 | 28.7 |
GASL-2 | 5.0 | 31.0 |
GASL-3 | 3.2 | 33.0 |
GASL-4 | 2.4 | 33.7 |
实施例4低分子量硫酸半乳聚糖对PTP1B的抑制作用
1.实验方法
称取样品适量,用纯水配制成10mg/mL的母液。分为空白对照组(加水),阳性对照组,样品组,每组设置三个复孔。每个孔分别加入86μL MES缓冲液,10μL pNPP,4μL样品,100μL PTP1B。样品筛选浓度为200μg。混匀后于37℃恒温培养箱反应1分钟,测405nm吸光值。于37℃恒温培养箱反应后测10分钟时在405nm的吸光值。样品对PTP1B酶抑制率计算公式:
V=(OD5min-OD1min)/4*1000或(OD10min-OD1min)/9*1000
抑制率(%)=(V空白-V样品)/V空白*100
2.实验结果
从表2的初步筛选结果显示,分子量大于3kDa,小于20kDa的不同组成的低分子量硫酸半乳聚糖具有显著的PTP1B抑制活性,抑制率均达50%以上。其中GASK-2的PTP1B抑制率IC50为40.86μg/mL,GASI-2的PTP1B抑制率IC50为66.67μg/mL,GASL-1的PTP1B抑制率IC50为121.30μg/mL。如表3所示,在同一浓度下,低分子量硫酸半乳聚糖对PTP1B同家族的蛋白酪氨酸磷酸化酶CPTP,SHP2和LAR的抑制率均较低,说明其对于PTP1B具有良好的选择性抑制作用,具有潜在的降血糖活性,有望开发成为降血糖和减肥药物。
表2 PTP1B抑制剂筛选结果表
分组 | 抑制率(%) | 分组 | 抑制率(%) |
阴性对照 | 0 | 原钒酸钠 | 89.9 |
GASK-1 | 71.5 | GASL-1 | 66.4 |
GASK-2 | 73.3 | GASL-2 | 54.8 |
GASK-3 | 76.4 | GASL-3 | 31.7 |
GASK-4 | 81.6 | GASL-4 | 27.8 |
GASI-1 | 77.6 | GASI-3 | 80.1 |
GASI-2 | 83.3 |
表3低分子量硫酸半乳聚糖对PTP1B同家族酶抑制率的测定结果表
编号 | PTP1B | TCPTP | SHP2 | LAR |
GASK-2 | 78.6% | 9.3% | 36.2% | -- |
GASI-2 | 81.1% | 19.7% | 22.4% | 11.1% |
GASL-1 | 67.8% | 3.2% | 27.6% | -- |
实施例5低分子量硫酸半乳聚糖对胰岛素抵抗HepG2细胞的葡萄糖摄取影响
(1)实验方法:
取对数生长期的HepG2细胞,经0.25%胰蛋白酶-0.02%EDTA消化后按照2×105个/mL均匀接种于96孔板中,每孔200μL溶液。待细胞汇合度约80%~85%,温PBS洗3遍后,更换不含血清的DMEM低糖培养基饥饿处理12h。将培养基换为加入GAS样品的含药无血清的DMEM低糖培养基,使各样品在细胞上的作用浓度为100μg/mL。以1mmol/L的二甲双胍作为阳性对照,空白组给予等量PBS,每孔6个重复。药物孵育24h后,在孵育结束前30min加入100nmol/L的Insulin作用30min;用KRB缓冲溶液洗3遍,加入100μmol/L的2-NBDG溶液100μL,37℃孵育2h,再用冰KRB缓冲溶液洗3遍,加入200μL的KRB缓冲液,利用485nm激发波长,535nm发射波长测定荧光值,检测细胞对荧光标记葡萄糖2-NBDG的摄取量。
(2)实验结果:
如图1所示,经胰岛素刺激后,与对照组(经棕榈酸处理)相比,各受试组分对葡萄糖摄取显著升高,说明低分子量硫酸半乳聚糖能促进胰岛素抵抗条件下细胞对葡萄糖的消耗,与胰岛素具有协同增强葡萄糖摄取的作用。
实施例6低分子量硫酸半乳聚糖对STZ诱导的糖尿病小鼠血糖水平的影响
取昆明小鼠(18-22g)采用12.5mg/kg链脉佐菌素(Streptozotocin,STZ)腹腔注射建立II型糖尿病(T2DM)小鼠模型。模型复制成功后,实验分为正常组(C组)、T2DM组(DM组)、二甲双胍处理组(Y组)和GASI-2处理T2DM小鼠组(GASI-2-L组,100mg/kg;GASI-2-H组,200mg/kg)。Y组和GASI-2组,给予药物灌胃处理3周,同时C组和DM组给予等体积生理盐水灌胃处理3周。通过试验终点小鼠的随机血糖,评价GASI-2对T2DM小鼠的血糖水平影响。
结果如表4所示,低分子量硫酸半乳聚糖GASI-2口服给药200mg/kg,可显著降低STZ诱导引起的小鼠血糖升高,与模型组相比具有显著性差异,表明GASI-2具有降糖作用,提示该化合物对于II型糖尿病具有一定的防治作用。
表4低分子量硫酸半乳聚糖对STZ诱导的II型糖尿病小鼠随机血糖水平的影响
组别 | 血糖/mmol/L |
C | 7.3±0.5 |
DM | 23.7±6.2<sup>##</sup> |
Y | 16.2±3.5* |
GASI-2-L | 20.9±5.4 |
GASI-2-H | 17.3±4.2* |
注:x±SD,n=10,与空白组相比,##p<0.01;与模型组相比,*p<0.05
实施例7低分子量硫酸半乳聚糖对PTP1B下游信号通路的影响
通过Western Blot实验研究GAS对胰岛素信号通路的影响(图2和图3)。实验结果表明,软脂酸诱导可以成功构建胰岛素抵抗细胞模型。当用几类浓度为100μg/mL的GAS干预后,胰岛素抵抗细胞中IRS-1蛋白和磷酸化IRS-1蛋白的含量明显升高,该结果表明,GAS可以使胰岛素抵抗细胞中的IRS-1蛋白激活,使其表达量增加,以及提高IRS-1酪氨酸磷酸化水平,改善胰岛素信号的传递,减轻胰岛素抵抗。
在胰岛素抵抗HepG2细胞模型组中,Akt丝氨酸磷酸化水平明显低于正常对照组,说明利用软脂酸诱导可以成功构建胰岛素抵抗细胞模型。当用几类浓度为100μg/mL的GAS干预后,胰岛素抵抗细胞中磷酸化Ak t蛋白的含量明显升高,该结果表明,GAS可以使胰岛素抵抗细胞中的胰岛素信号通路下游PI3K/Akt途径激活,使Akt蛋白的磷酸化表达量增加,从而可以引起葡萄糖转运蛋白GLUT-1和GLUT-4增加葡萄糖从胞外向胞内转运,即增加了葡萄糖的摄取和消耗,从而明显改善胰岛素抵抗情况,发挥调节糖代谢作用。
实施例8低分子量硫酸半乳聚糖对高脂诱导肥胖小鼠血脂及体重的影响
昆明小鼠(18-22g)30只,基础饲料喂养3天后,按体重随机分为3组:空白组,模型组,GASI-2组(100mg/kg),每组10只小鼠,其中空白组继续饲喂基础饲料,其他组饲喂高脂饲料,喂养4周后,测量动物体重,采血检测血脂(甘油三脂TC,总胆固醇TG和极低密度脂蛋白VLDL)水平。
实验结果如表5所示,低分子量硫酸半乳聚糖GASI-2可以降低高脂饲料诱导的小鼠体重增加,对小鼠摄食量无显著影响;如表6所示,低分子量硫酸半乳聚糖GASI-2可显著降低高脂诱导小鼠血清中TG含量和TC含量,抑制高脂饲料诱导引起的VLDL降低。上述实验结果表明该类低分子量硫酸半乳聚糖具有一定的调节脂代谢及减肥作用。
表5低分子量硫酸半乳聚糖对高脂诱导肥胖小鼠体重的影响
组别 | 初体重/g | 终体重/g | 摄食量(g) |
空白 | 22.6±2.7 | 43.4±2.8 | 14.1±1.8 |
模型 | 23.4±2.9 | 48.0±4.6<sup>#</sup> | 12.6±3.2 |
GASI-2 | 23.5±2.5 | 44.0±4.6* | 12.4±2.1 |
注:x±SD,n=10,与空白组相比,#p<0.05;与模型组相比,*p<0.05
表6低分子量硫酸半乳聚糖对高脂诱导肥胖小鼠血脂的影响
组别 | TG/mmol/L | TC/mmol/L | VLDL/μmol/L |
空白 | 1.52±0.15 | 2.76±0.33 | 48.7±6.7 |
模型 | 2.21±0.31<sup>##</sup> | 4.01±0.62<sup>##</sup> | 35.1±5.0<sup>#</sup> |
GASI-2 | 1.78±0.32* | 3.34±0.44* | 41.5±6.3* |
注:x±SD,n=10,与空白组相比,#p<0.05,##p<0.01;与模型组相比,*p<0.05
经过研究发现,具有本发明实施例制备得到的这些结构特征的低分子量硫酸半乳聚糖能降低肥胖小鼠的体重及血清中总胆固醇和甘油三脂含量,提升极低密度脂蛋白含量。该低分子量硫酸半乳聚糖可调节PTP1B下游信号通路功能,增加IRS-1蛋白的表达量和磷酸化,增强胰岛素受体敏感性;可以使胰岛素抵抗细胞中的胰岛素信号通路下游PI3K/Akt途径激活,使Akt蛋白的磷酸化表达量增加,从而可以引起葡萄糖转运蛋白GLUT-1和GLUT-4增加葡萄糖从胞外向胞内转运,即增加了葡萄糖的摄取和消耗,从而明显改善胰岛素抵抗情况。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围,其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (10)
2.根据权利要求1所述的低分子量硫酸半乳聚糖,其特征在于,所述的低分子量硫酸半乳聚糖的分子量为3kDa~20kDa,所述的低分子量硫酸半乳聚糖中硫酸基团的质量百分含量为15%~40%。
3.根据权利要求1所述的低分子量硫酸半乳聚糖,其特征在于,所述低分子量硫酸半乳聚糖的化学结构是由4-硫酸基-β-1,3-D-半乳糖(D-Gal)残基和6-硫酸基-α-1,4-D-半乳糖(L-Gal)残基,3,6内醚-D-Gal残基,2-硫酸基-3,6-内醚-D-Gal残基,2-硫酸基-β-1,3-D-半乳糖(D-Gal)残基和2,6-二硫酸基-D-半乳糖(D-Gal)残基,2-硫酸基-3,6内醚-D-Gal中的一种或几种组成。
4.权利要求1-3任一项所述的硫酸半乳寡糖的制备方法,其特征在于,该硫酸半乳寡糖采用下述制备工艺:
a)提取:清洗藻类、烘干、切碎,分别用水和重量比为5%~10%的碱水溶液提取0.5~2小时;过滤;残渣按上述过程再提取1~2次;合并滤液,浓缩后进行提纯、离心、干燥、粉碎得粗硫酸半乳聚糖粗品;
b)降解:降解所述硫酸半乳聚糖粗品,浓缩,并用有机溶剂沉淀,干燥,得到低分子量硫酸半乳聚糖;
c)分离纯化:将所述低分子量硫酸半乳聚糖粗品用水溶解,超滤分级,然后将得到的的低分子量硫酸半乳聚糖用QFF凝胶色谱柱层析纯化和G10凝胶柱层析除盐,冷冻干燥,即得不同组成含量的低分子量硫酸半乳聚糖。
5.根据权利要求4所述的硫酸半乳寡糖的制备方法,其特征在于,所述藻类为红藻,为勾沙菜、角叉菜、刺麒麟菜、耳突麒麟菜、小杉藻、亚格菜、沟杉藻中的一种或多种。
6.权利要求1-3任一项所述的低分子量硫酸半乳聚糖在制备预防和/或治疗II型糖尿病和肥胖的药物或食品中的应用。
7.一种用于预防和/或治疗II型糖尿病和肥胖的药物或食品,其特征在于,所述组合物含有;
1)权利要求1-3任一项中所述的低分子量硫酸半乳聚糖,和
2)药学上可接受的载体。
8.一种受体调节剂,其特征在于,所述受体调节剂中的有效成分为权利要求1或2所述的低分子量硫酸半乳聚糖,所述受体调节剂选择性抑制蛋白酪氨酸磷酸化酶1B。
9.根据权利要求8所述的受体调节剂,其特征在于,所述的受体调节剂,
1)提高胰岛素抵抗条件下细胞的葡萄糖消耗,提高胰岛素敏感型,或
2)调节能量消耗,或
3)维持葡萄糖内稳态。
10.根据权利要求9所述的受体调节剂,其特征在于,所述的受体调节剂用于制备:
1)预防和/或治疗II型糖尿病的药物,或
2)预防和/或治疗肥胖的药物,或
3)降糖、降脂、减肥的食品。
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