CN114957387B - 一种具有降血糖作用的巴旦木多肽及其制备方法与应用 - Google Patents
一种具有降血糖作用的巴旦木多肽及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种具有降血糖作用的巴旦木多肽及其制备方法与应用,属于生物活性肽技术领域。所述的具有降血糖作用的巴旦木多肽,包括RGL、LA、WH、LY、VF、LF、VW和FF短肽。本发明具有降血糖作用的巴旦木多肽具有较高的α‑淀粉酶和α‑葡萄糖苷酶抑制活性,无细胞毒性,能增加HepG2细胞活力,改善HepG2细胞在胰岛素抵抗状态下葡糖糖消耗量及糖原含量,因此,本发明具有降血糖作用的巴旦木多肽可以作为潜在的天然降血糖药品或功能性食品,同时也为提高巴旦木的商业价值提供理论支持。
Description
技术领域
本发明属于生物活性肽技术领域,具体涉及一种具有降血糖作用的巴旦木多肽及其制备方法与应用。
背景技术
糖尿病是一种常见代谢疾病之一,当人体内的胰腺无法再产生胰岛素或身体无法再利用胰岛素调节体内血糖平衡时就会发生这种疾病。根据2021年国际糖尿病联合会(IDF)第十版中所发表的数据,全球患糖尿病已接近5.5亿人,并且这一数字仍在快速增长,据估计在2045年,糖尿病患者将增加至7.83亿人。糖尿病的危害在于机体长期处在持续性高血糖状态下所导致的一系列慢性并发症,如心脑血管疾病、慢性肾病、神经系统及血管病变等,这些并发症会严重降低到患者生活质量甚至危及生命健康,因此,探索能够改善糖尿病患者胰岛素抵抗、高血糖,且无毒副作用的活性物质已成为研究的热点。药物治疗虽有一定的效果,但长期服用会带来呕吐、腹泻、胀气等副作用,而天然食品来源的活性肽是研究热点,采用不同蛋白酶降解蛋白质来制备活性肽,生产成本较低,反应温和,产品无毒副作用,是目前制备活性肽最常见的方法之一。
巴旦木作为世界四大坚果(阿月浑子、榛子、核桃)之一,有着“西域珍果”、“坚果之王”的美誉。我国种植巴旦木的历史已长达1300年,从古波斯引入种子,最早史料记载可追溯至唐代《酉阳杂俎》。我国现有的扁桃野生种及栽培种共有7种,包括野生巴丹杏(野扁桃)、西康扁桃、巴旦木等,大多分布于新疆南部喀什地区。巴旦木仁蛋白质相对含量为20%~28%,可被视为优质的蛋白质来源。巴旦木仁中的蛋白质大部分为必需氨基酸。其限制氨基酸为赖氨酸、蛋氨酸和苏氨酸。食物中的蛋白质在人体内经胃蛋白酶、胰蛋白酶、弹性蛋白酶等蛋白酶消化降解成氨基酸,之后由小肠吸收到人体血液。研究巴旦木蛋白肽的活性作用,对明确巴旦木的生物功能活性,充分利用巴旦木蛋白资源,开发巴旦木功能产品具有重要意义。
目前已从茶籽蛋白、核桃蛋白、马鹿茸蛋白、大豆分离蛋白、绿豆分离蛋白、乳清浓缩蛋白等植物蛋白中分离出具有降血糖活性的多肽,但有关巴旦木多肽降血糖活性的研究鲜有报道。
本发明的目的在于提供一种具有降血糖作用的巴旦木多肽。
本发明的另一个目的在于提供上述具有降血糖作用的巴旦木多肽的制备方法。
本发明的再一个目的在于提供具有降血糖作用的巴旦木多肽的应用。
本发明的上述目的通过以下技术方案实现:
一种具有降血糖作用的巴旦木多肽,包括RGL、LA、WH、LY、VF、LF、VW和FF短肽。
RGL的氨基酸序列为Arg-Gly-Leu;LA的氨基酸序列为Leu-Ala;WH的氨基酸序列为Trp-His;LY的氨基酸序列为Leu-Tyr;VF的氨基酸序列为Val-Phe;LF的氨基酸序列为Leu-Phe;VW的氨基酸序列为Val-Trp;FF的氨基酸序列为Phe-Phe。
所述的具有降血糖作用的巴旦木多肽为分子量小于3kDa的巴旦木多肽。
所述的具有降血糖作用的巴旦木多肽的制备方法,包括如下步骤:
(1)将巴旦木脱脂粉制成溶液,调节溶液pH至10,恒温震荡提取,离心,取上清液,调节上清液pH至4.5,静置,离心,取沉淀,将沉淀配置成溶液后调pH至7.0,干燥,得到巴旦木蛋白粉;
(2)将步骤(1)制备的巴旦木蛋白粉溶解,70~90℃保持5~15min,调节pH至7,加入酶酶解,灭酶,离心,干燥,得到酶解液粉;
(3)将酶解液粉制成溶液进行超滤,干燥,得到分子量小于3kDa的巴旦木多肽;
(4)将分子量小于3kDa的巴旦木多肽制成溶液,过滤,分离,即得具有降血糖作用的巴旦木多肽。
步骤(1)中,所述的巴旦木脱脂粉制成溶液中,溶液的溶剂优选为水;所述的巴旦木脱脂粉与溶剂优选按质量体积比(g:mL)80~120:1000计算;更优选按100:1000计算。
步骤(1)中,所述的恒温的温度优选为40~60℃;更优选为50℃。
步骤(1)中,所述的将沉淀配置成溶液中,溶液的溶剂优选为水。
步骤(1)中,所述的干燥优选为冷冻干燥。
步骤(2)中,所述的溶解的试剂优选为水。
步骤(2)中,所述的溶解的条件优选为80℃保持10min。
步骤(2)中,所述的酶优选为胰蛋白酶和复合蛋白酶。
所述的胰蛋白酶的酶活优选为200~300U/mg;更优选为250U/mg。
所述的复合蛋白酶的酶活优选为100~150U/mg;更优选为120U/mg。
步骤(2)中,所述的酶解条件优选为40~50℃酶解3~5h;更优选为45℃酶解4h。
步骤(2)中,所述的灭酶的方法优选为煮沸灭酶。
步骤(2)中,所述的灭酶的时间优选为5~12min;更优选为10min。
步骤(3)中,所述的超滤优选通过截留分子量为3kDa的超滤离心管超滤。
步骤(3)中,所述的干燥优选为冷冻干燥。
步骤(4)中,所述的过滤优选为滤膜过滤;更优选为0.22μm微孔滤膜过滤。
步骤(4)中,所述的分离优选为用SephadexG-25凝胶层析分离;所述的SephadexG-25凝胶层析分离的上样量为50mg/mL,洗脱条件:洗脱液为蒸馏水,流速0.5mL/min,每6min收集一管,分成4个组分(组分I、组分II、组分III、组分IV),收集的组分IV即为具有降血糖作用的巴旦木多肽。
所述的具有降血糖作用的巴旦木多肽在降血糖中的应用。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明所述制备方法制备得到的具有降血糖作用的巴旦木多肽活性好,制备成本低,效率高,适用工业生产。
(2)本发明具有降血糖作用的巴旦木多肽具有较高的α-淀粉酶和α-葡萄糖苷酶抑制活性,无细胞毒性,能增加HepG2细胞活力,改善HepG2细胞在胰岛素抵抗状态下葡糖糖消耗量及糖原含量,因此,本发明具有降血糖作用的巴旦木多肽可以作为潜在的天然降血糖药品或功能性食品,同时也为提高巴旦木的商业价值提供理论支持。
附图说明
图1为实施例1步骤(1)制备得到的巴旦木蛋白粉的SDS-PAGE凝胶结果图;其中,泳道1-6均代表巴旦木蛋白粉。
图2为A-J组酶解液粉对α-淀粉酶的抑制率结果图;其中,A代表碱性蛋白酶和胰蛋白酶、B代表碱性蛋白酶和中性蛋白酶、C代表碱性蛋白酶和复合蛋白酶、D代表碱性蛋白酶和木瓜蛋白酶、E代表胰蛋白酶和中性蛋白酶、F代表胰蛋白酶和复合蛋白酶、G代表胰蛋白酶和木瓜蛋白酶、H代表中性蛋白酶和复合蛋白酶、I代表中性蛋白酶和木瓜蛋白酶、J代表复合蛋白酶和木瓜蛋白酶。
图3为A-J组酶解液粉对α-葡萄糖苷酶的抑制率结果图;其中,A代表碱性蛋白酶和胰蛋白酶、B代表碱性蛋白酶和中性蛋白酶、C代表碱性蛋白酶和复合蛋白酶、D代表碱性蛋白酶和木瓜蛋白酶、E代表胰蛋白酶和中性蛋白酶、F代表胰蛋白酶和复合蛋白酶、G代表胰蛋白酶和木瓜蛋白酶、H代表中性蛋白酶和复合蛋白酶、I代表中性蛋白酶和木瓜蛋白酶、J代表复合蛋白酶和木瓜蛋白酶。
图4为F组胰蛋白酶和复合蛋白酶酶解得到的酶解液粉制成不同浓度酶解液粉溶液后,不同浓度酶解液粉溶液对α-淀粉酶抑制率结果图。
图5为F组胰蛋白酶和复合蛋白酶酶解得到的酶解液粉制成不同浓度酶解液粉溶液后,不同浓度酶解液粉溶液对α-葡萄糖苷酶抑制率结果图。
图6为不同浓度的分子量小于3kDa的巴旦木多肽溶液和分子量大于3kDa的巴旦木多肽溶液对α-淀粉酶抑制率结果图。
图7为不同浓度的分子量小于3kDa的巴旦木多肽溶液和分子量大于3kDa的巴旦木多肽溶液对α-葡萄糖苷酶抑制率结果图。
图8为实施例1制得的4个组分(组分Ⅰ、组分Ⅱ、组分Ⅲ和组分IV)的色谱图。
图9为实施例1制得的4个组分(组分Ⅰ、组分Ⅱ、组分Ⅲ和组分Ⅳ)对α-淀粉酶抑制率结果图。
图10为实施例1制得的4个组分(组分Ⅰ、组分Ⅱ、组分Ⅲ和组分Ⅳ)对α-葡萄糖苷酶抑制率结果图。
图11为实施例1制得的组分Ⅳ(具有降血糖作用的巴旦木多肽)的质谱图。
图12为不同终浓度的具有降血糖作用的巴旦木多肽组分Ⅳ对HepG2细胞活力的影响结果图;a和b表示各组间存在显著差异(p<0.05)。
图13为不同终浓度的具有降血糖作用的巴旦木多肽组分Ⅳ对HepG2细胞形态的影响结果图。
图14为HepG2细胞胰岛素抵抗模型的建立图。
图15为具有降血糖作用的巴旦木多肽对HepG2细胞葡萄糖的消耗影响结果图;其中,*表示与对照组之间的显著差异(*表示p<0.05;**表示p<0.01;***表示p<0.001)。
图16为具有降血糖作用的巴旦木多肽对HepG2细胞内糖原的影响结果图;其中,*表示与对照组之间的显著差异(*表示p<0.05;**表示p<0.01;***表示p<0.001)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
巴旦木脱脂粉由广东南兴天虹果仁制品有限公司提供;木瓜蛋白酶购于上海源叶生物科技有限公司,酶活800U/mg;胰蛋白酶购于上海源叶生物科技有限公司,酶活250U/mg;复合蛋白酶购于上海源叶生物科技有限公司,酶活120U/mg;中性蛋白酶购于上海源叶生物科技有限公司,酶活100U/mg;碱性蛋白酶购于上海源叶生物科技有限公司,酶活200U/mg。
实施例1:
一种具有降血糖作用的巴旦木多肽的制备方法,包括如下步骤:
(1)取100g巴旦木脱脂粉置于锥形瓶中,加入1000mL蒸馏水,调节pH至10,在50℃条件下恒温振荡提取4h,离心,取上清液,调节上清液pH至4.5,室温下静置1h、离心,取沉淀,加水溶解调pH至7.0,冷冻干燥,得到巴旦木蛋白粉,其SDS-PAGE凝胶电泳结果如图1所示。-20℃保存。
从图1可以看出,巴旦木蛋白粉的分子量为18.4-25kDa之间。
(2)取步骤(1)制备的巴旦木蛋白粉,加水溶解,置于80℃保持10min,调节pH至7,以酶活的比为1:1加入表1所述的两种酶,加酶总量为4000U/g巴旦木蛋白粉,于45℃酶解4h,煮沸10min,冷却后离心,取上清酶解液,冷冻干燥,得到酶解液粉。
表1:
组别 | 酶 |
A | 碱性蛋白酶和胰蛋白酶 |
B | 碱性蛋白酶和中性蛋白酶 |
C | 碱性蛋白酶和复合蛋白酶 |
D | 碱性蛋白酶和木瓜蛋白酶 |
E | 胰蛋白酶和中性蛋白酶 |
F | 胰蛋白酶和复合蛋白酶 |
G | 胰蛋白酶和木瓜蛋白酶 |
H | 中性蛋白酶和复合蛋白酶 |
I | 中性蛋白酶和木瓜蛋白酶 |
J | 复合蛋白酶和木瓜蛋白酶 |
通过测定α-淀粉酶抑制率和α-葡萄糖苷酶抑制率表征A-J组酶解液粉的降血糖活性。
1)α-淀粉酶抑制率测定方法:以PBS缓冲液(0.02mol/L,pH=6.9)为溶剂,配制酶解液粉溶液(1mg/mL)、α-淀粉酶溶液(0.2U/mL)、可溶性淀粉溶液(质量分数为1%)。操作步骤如下:
依次在离心管中加入10μLα-淀粉酶溶液与20μL酶解液粉溶液混匀,于37℃温育20min,随后加入500μL可溶性淀粉溶液(质量分数为1%)混匀,于37℃反应5min后,吸取600μL DNS试剂终止反应,煮沸10min,冷却至室温,吸取200μL反应液,在540nm测反应液的吸光值。同时以PBS缓冲液(0.02mol/L,pH=6.9)为空白组进行上述试验。
酶解液粉溶液对α-淀粉酶的抑制率:
α-淀粉酶抑制率(%)=(A1-A0)/A1;
其中,A1为空白组溶液的吸光值;A0为反应液的吸光值;
结果如图2所示,从图2可以看出,胰蛋白酶与复合蛋白酶组合的酶解产物α-淀粉酶抑制率最高,为27.20%±2.06%。
2)α-葡萄糖苷酶抑制率测定方法:
以PBS缓冲液(0.02mol/L,pH=6.9)为试剂,配制酶解液粉溶液(1mg/mL)、α-葡萄糖苷酶溶液(0.2U/mL)、对硝基苯α-D-葡萄糖吡喃苷(PNPG,10mmol/L)。
操作步骤如下:
依次加入10μL α-葡萄糖苷酶溶液与20μL酶解液粉溶液,混匀置于37℃培养箱中孵育20min,之后再加入10μL PNPG,于37℃培养箱中反应20min,加入150μL Na2CO3溶液(1M)终止反应,得到反应液;于405nm测定反应液的吸光值,空白组用20μL PBS缓冲液(0.02mol/L,pH=6.9)代替20μL酶解液粉溶液。
酶解液粉溶液对α-葡萄糖苷酶的抑制率:
α-葡萄糖苷酶抑制率(%)=(A1-A0)/A1
其中,A1为空白组溶液的吸光值;A0为反应液的吸光值。
结果如图3所示,从图3可以看出,胰蛋白酶与复合蛋白酶组合的酶解产物α-葡萄糖苷酶的抑制率最高,为16.70%±0.51%。
以上结果可知,A-J组中,F组胰蛋白酶和复合蛋白酶组合酶解得到的酶解液粉溶液对α-淀粉酶的抑制率和α-葡萄糖苷酶抑制率更好。
3)将F组胰蛋白酶和复合蛋白酶组合酶解得到的酶解液粉分别制备成1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL酶解液粉溶液,分别按照上述方法测定1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL酶解液粉溶液的α-淀粉酶抑制率和α-葡萄糖苷酶抑制率情况。
结果如图4和5所示。从图4和5可以看出,浓度为5mg/mL时,双酶酶解产物的α-淀粉酶和α-葡萄糖苷酶抑制率可以分别达到55.69%±3.92%和46.30%±0.81%。
(3)将步骤(2)F组胰蛋白酶和复合蛋白酶组合酶解得到的酶解液粉溶于水中,得到酶解液粉溶液(1mg/mL);将酶解液粉溶液(1mg/mL)通过截留分子量为3kDa的超滤离心管超滤,冷冻干燥,分别得到分子量小于3kDa的巴旦木多肽和分子量大于3kDa的巴旦木多肽。
将分子量小于3kDa的巴旦木多肽制成1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL分子量小于3kDa的巴旦木多肽溶液。将分子量大于3kDa的巴旦木多肽制成1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL分子量大于3kDa的巴旦木多肽溶液。按照步骤(2)中1)α-淀粉酶抑制率测定方法和2)α-葡萄糖苷酶抑制率测定方法分别测定上述不同浓度的分子量小于3kDa的巴旦木多肽溶液和分子量大于3kDa的巴旦木多肽溶液对α-淀粉酶抑制率和α-葡萄糖苷酶抑制率;
结果分别如图6和图7所示,在浓度为5mg/mL时,分子量小于3kDa的巴旦木多肽对α-淀粉酶抑制率达到62.08%±4.76%,对α-葡萄糖苷酶抑制率达到52.30%±3.42%。
(4)将分子量小于3kDa的巴旦木多肽配制成40mg/mL的巴旦木多肽溶液,过0.22μm微孔滤膜后,用SephadexG-25凝胶层析分离,上样量为50mg/mL,洗脱条件:洗脱液为蒸馏水,流速0.5mL/min,每6min收集一管,收集4个组分(组分I、组分Ⅱ、组分Ⅲ、组分Ⅳ),如图8,经冷冻干燥收集备用。
分别将4个组分(组分Ⅰ、组分Ⅱ、组分Ⅲ和组分Ⅳ)制成1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL溶液。按照步骤(2)中1)α-淀粉酶抑制率测定方法和2)α-葡萄糖苷酶抑制率测定方法分别测定4个组分(组分Ⅰ、组分Ⅱ、组分Ⅲ和组分Ⅳ)制成的溶液对α-淀粉酶抑制率和α-葡萄糖苷酶抑制率;
结果如图9和图10所示,从图9和10可以看出,组分Ⅳ对α-淀粉酶的抑制率为70.57%±3.34%,组分Ⅳ对α-葡萄糖苷酶的抑制率为64.38%±3.39%。
(5)用超高分辨液质联用仪鉴定组分Ⅳ的氨基酸序列,步骤如下,较精密称量0.02~2mg样品(组分Ⅳ)于2~5mL离心管中,加入1.5mL超纯水,过0.22μm滤膜后上机分析。质谱工作于fullMS/DD-MS2模式(正离子采集)下,一级分辨率设置为35000,质荷比范围100~1500m/z,TopN设置为4,轰击能量为梯级能量(20-40-60),二级分辨率设置为35000,动态质荷比采集范围。色谱条件:色谱柱规格:2.1*100mm,2.6μm,Accucore RP-MS。流动相由0.1%(v/v)的甲酸水溶液(A)和乙腈(B)组成,洗脱方法为:0~4.00min 5.0%B,4.00~6.00min5.0~10.0%B,6.00~30.00min 10.0~40.0%B,30.00~34.00min 40.0~90.0%B,34.00~40.00min 90%B,40.00~42.00min 90.0~95.0%B,42.00-50.00min,5.0%B,流速0.25mL/min,进样量1μL,柱温30~60℃。
结果如图11所示。组分Ⅳ由RGL、LA、WH、LY、VF、LF、VW和FF短肽组成;其中,RGL的氨基酸序列为Arg-Gly-Leu;LA的氨基酸序列为Leu-Ala;WH的氨基酸序列为Trp-His;LY的氨基酸序列为Leu-Tyr;VF的氨基酸序列为Val-Phe;LF的氨基酸序列为Leu-Phe;VW的氨基酸序列为Val-Trp;FF的氨基酸序列为Phe-Phe。
实施例2:
(1)HepG2细胞的培养:
从液氮中取出冻存的HepG2细胞(购自中国科学院典型培养物保藏委员会细胞库;下同),迅速放入37℃恒温水浴锅中融解,防止冰晶破坏细胞结构,在无菌条件下操作,将HepG2细胞加入T25细胞瓶中,并添加5mL DMEM完全培养基(含1%青霉素、1%链霉素和10%胎牛血清的DMEM高糖培养基),置于37℃、5%CO2的细胞培养箱中培养,培养24h后,显微镜观察细胞粘附在细胞瓶壁上后,更换培养液,当细胞密度覆盖T25细胞瓶底部80~90%时,用0.25%胰酶-EDTA进行消化,按1:2或1:3进行传代。在实验过程中,细胞最好处于对数生长期的状态。
(2)HepG2细胞毒性实验(MTT)
取对数生长期的HepG2细胞,制成105个细胞/mL的悬液,以每孔100μL接种于96孔板,于37℃、5%CO2条件下培养细胞24h待细胞贴壁,弃去原来的培养基,分别设置对照组和实验组,对照组每孔加入100μL无血清DMEM培养基,实验组每孔加入100μL含不同浓度的具有降血糖作用的巴旦木多肽组分Ⅳ(终浓度分别为0.001mg/mL、0.01mg/mL、0.1mg/mL、1mg/mL、2mg/mL、5mg/mL、10mg/mL)的无血清DMEM培养基,每组设置6个复孔,混匀后于37℃、5%CO2条件下培养细胞24h、48h、72h后,每孔加入10μL的MTT溶液,继续孵育,3-4h后弃上清液,每孔加入50μL DMSO溶出紫色晶体,振荡15min,用酶标仪测定各组在570nm处的吸光度,并在倒置显微镜下观察各组细胞形态。
HepG2的细胞活性计算公式:
细胞活力(%)=[(A实验组-A空白组)/(A对照组-A空白组)]×100%;
其中,A实验组为实验组的吸光值;A对照组为对照组的吸光值;A空白组为调零组的吸光值。
结果如图12和图13所示。MTT试验和细胞形态观察验证了具有降血糖作用的巴旦木多肽组分Ⅳ对HepG2细胞无明显毒副作用。
(3)HepG2细胞胰岛素抵抗模型的建立
将HepG2细胞制成105个细胞/mL的悬液,以每孔100μL接种于96孔板,于37℃、5%CO2条件下培养细胞24h待细胞贴壁,弃去原来的培养基,分别设置对照组和实验组,对照组每孔加入100μL无血清DMEM培养基,实验组每孔加入100μL含不同浓度胰岛素(终浓度分别为10-1mmol/L、10-2mmol/L、10-3mmol/L、10-4mmol/L、10-5mmol/L、10-6mmol/L、10-7mmol/L、10-8mmol/L、10-9mmol/L、10-10mmol/L)的无血清DMEM培养基,每组设6个复孔,培养24h、48h、72h后,得到培养液,离心,得到上清液;用葡萄糖氧化酶法检测培养液中的葡萄糖含量,依据葡萄糖测定试剂盒(葡萄糖氧化酶法)(购于南京建成生物工程研究所有限公司)的说明书测定细胞上清液中葡萄糖的含量,计算葡萄糖消耗量。
如图14所示,HepG2细胞胰岛素抵抗模型的最优条件为:胰岛素终浓度为10-3mmol/L,培养48h。
1)葡萄糖消耗量的测定:
将HepG2细胞制成105个细胞/mL的悬液,以每孔100μL接种于96孔板,弃去原有培养基,分别设置对照组和实验组(模型组、二甲双胍组、含不同浓度的巴旦木多肽组分Ⅳ组),对照组(正常的HepG2细胞),模型组(胰岛素终浓度为10-3mmol/L,培养48h)、二甲双胍组(二甲双胍终浓度为3mM)、具有降血糖作用的巴旦木多肽组分Ⅳ组(终浓度分别为0.1mg/mL、0.2mg/mL、0.4mg/mL、0.6mg/mL、0.8mg/mL、1.0mg/mL),各组物质用量均为100μL,每组设置6个复孔,给药48h后,计算各组葡萄糖消耗量。
结果如图15所示。以上试验表明,具有降血糖作用的巴旦木多肽组分Ⅳ可以促进胰岛素抵抗HepG2细胞对葡萄糖的消耗,进而说明实施例1制备得到的具有降血糖作用的巴旦木多肽组分Ⅳ具有良好的降血糖作用。
2)糖原含量的测定
将HepG2细胞以105/mL接种2mL于六孔培养板中,弃去原有培养基,分别加入设置对照组(正常的HepG2细胞)、模型组(胰岛素终浓度为10-3mmol/L,培养48h)、二甲双胍组(二甲双胍终浓度为3mM)、具有降血糖作用的巴旦木多肽组分Ⅳ组(终浓度分别为0.1mg/mL、0.2mg/mL、0.4mg/mL、0.6mg/mL、0.8mg/mL、1.0mg/mL),各组物质用量均为2mL,每组设置6个复孔,给药48h后,按照糖原含量检测试剂盒(购于艾美捷科技有限公司)进行糖原含量检测。
结果如图16所示。具有降血糖作用的巴旦木多肽组分Ⅳ可以促进胰岛素抵抗HepG2细胞内糖原的合成,进而说明实施例1制备得到的具有降血糖作用的巴旦木多肽具有良好的降血糖作用。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (4)
1.一种具有降血糖作用的巴旦木多肽组合物,其特征在于,其由短肽RGL、LA、WH、LY、VF、LF、VW和FF组成。
2.根据权利要求1所述的具有降血糖作用的巴旦木多肽组合物,其特征在于,所述的具有降血糖作用的巴旦木多肽组合物的分子量小于3kDa。
3.权利要求1或2所述的具有降血糖作用的巴旦木多肽组合物的制备方法,其特征在于,包括如下步骤:
(1)取100g巴旦木脱脂粉加入1000mL蒸馏水,调节溶液pH至10,50℃震荡提取4h,离心,取上清液,调节上清液pH至4.5,静置,离心,取沉淀,将沉淀加水配置成溶液后调pH至7.0,干燥,得到巴旦木蛋白粉;
(2)将步骤(1)制备的巴旦木蛋白粉加水溶解,80℃保持10min,调节pH至7,以酶活的比为1:1加入胰蛋白酶和复合蛋白酶酶解,加酶总量为4000U/g巴旦木蛋白粉,于45℃酶解4h,灭酶,离心取上清,干燥,得到酶解液粉;
(3)将酶解液粉溶于水中制成1mg/mL的酶解液粉溶液,将所述酶解液粉溶液通过截留分子量为3kDa的超滤离心管进行超滤,干燥,得到分子量小于3kDa的巴旦木多肽;
(4)将分子量小于3kDa的巴旦木多肽配制成40mg/mL的巴旦木多肽溶液,过0.22μm微孔滤膜后,用SephadexG-25凝胶层析分离,上样量为50mg/mL,洗脱条件:洗脱液为蒸馏水,流速0.5mL/min,每6min收集一管,收集4个组分:组分I、组分II、组分III、组分IV,收集的组分IV即为所述的具有降血糖作用的巴旦木多肽组合物,所述的组分Ⅳ对α-淀粉酶的抑制率为70.57%±3.34%,对α-葡萄糖苷酶的抑制率为64.38%±3.39%。
4.权利要求1或2所述的具有降血糖作用的巴旦木多肽组合物在制备降血糖药物中的应用。
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Non-Patent Citations (5)
Title |
---|
Determination of Antihypertensive Peptides from an Izumi Shrimp Hydrolysate;Yoshitaka NII等;《Biosci. Biotechnol. Biochem.》;第72卷(第3期);861–864 * |
Dipeptide self-assembly into water-channels and gel biomaterial;Ottavia Bellotto等;《Org Biomol Chem.》;6211–6218 * |
Discovery of kokumi peptide fromyeast extract by LC-Q-TOF-MS/MS and sensomics approach;Jianbin Liu等;《J Sci Food Agric》;3183–3194 * |
HIV-1-infected Blood Mononuclear Cells Form an Integrin- and Agrin-dependent Viral Synapse to Induce Efficient HIV-1 Transcytosis across Epithelial Cell Monolayer;Annette Alfsen等;《Molecular Biology of the Cell》;第16卷;4267–4279 * |
The di-peptide Trp-His activates AMP-activated protein kinase and enhances glucose uptake independently of insulin in L6 myotubes;Minoru Soga等;《FEBS Open Bio》;898–904 * |
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