CN115919834A - Sclerotiorin作为α-葡萄糖苷酶抑制剂的应用 - Google Patents
Sclerotiorin作为α-葡萄糖苷酶抑制剂的应用 Download PDFInfo
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Abstract
本发明属于微生物次生代谢产物应用技术领域,提供sclerotiorin作为α‑葡萄糖苷酶抑制剂的应用,本发明化合物属于红树林内生真菌的活性次级代谢产物,当sclerotiorin浓度为25.0μM时,对α‑葡萄糖苷酶的抑制率达93.3%;采用倍半梯度稀释法对样品进行稀释后进一步评价化合物sclerotiorin的α‑葡萄糖苷酶抑制活性,实验结果显示,其IC50值为3.3μM。本发明涉及的sclerotiorin化合物能够利用微生物的扩大化发酵进行工业化生产,提取工艺简单,成本廉价,对环境友好;本发明发现的新用途对新型α‑葡萄糖苷酶抑制剂的开发和降血糖药物的新药研发具有十分重要的意义。
Description
技术领域
本发明涉及微生物次生代谢产物应用技术领域,具体涉及sclerotiorin作为α-葡萄糖苷酶抑制剂的应用。
背景技术
糖尿病(Diabetes mellitus,DM)是继肿瘤、心血管疾病之后第三大威胁人类健康的疾病。最新的全球糖尿病地图(第9版)数据显示,2019年全球约4.63亿20-79岁成人患糖尿病;预计到2045年糖尿病患者会突破7亿。根据世界卫生组织的估计,2型糖尿病(T2DM)占所有糖尿病病例的90%-95%,约占总人口数的6%,被认为是全球首要的公共卫生负担。因此,T2DM的防治,是糖尿病预防和治疗的关键和重点。
作为治疗T2DM最主要的手段之一,口服降糖药在治疗糖尿病过程中发挥着举足轻重的作用。α-葡萄糖苷酶抑制剂是二十世纪70年代研发的一类口服降糖药,可以通过可逆性结合占据酶的催化位点,延缓多糖、双糖向单糖转化,降低餐后血糖且不影响空腹血糖,从而达到预防和治疗糖尿病的目的。另有研究发现,α-葡萄糖苷酶抑制剂能够促进部分未被消化的碳水化合物进入小肠,刺激该部位的胰高糖素样肽-1(GLP-1)大量分泌,GLP-1分泌水平的提高有助于进一步控制血糖。此外,α-葡萄糖苷酶抑制剂还能够有效地改善并预防一些T2DM并发症的发生和发展,且无明显肝肾毒副作用。基于上述理由,中国2型糖尿病防治指南(2017年版)将α-葡萄糖苷酶抑制剂列入一线降糖药物的备选。已经上市的α-葡萄糖苷酶抑制剂阿卡波糖、米格列醇和伏格列多糖存在有肝肾损害和低血糖等副作用。因此,研发新型的α-葡萄糖苷酶抑制剂对降血糖药物的开发十分重要。
Sclerotiorin为红树林内生真菌Penicillium sclerotiorum sp.303的活性次级代谢产物,其结构式如下:
其结构已被解析,可通过大规模发酵分离,来源丰富,生产成本低。文献“海洋真菌抗结核分枝杆菌靶向先导物的筛选,陆勇军等,全国第九届海洋生物技术与创新药物学术会议摘要集,2014.08.06-10”公开了sclerotiorin具有较好地抑制PknG酶活性的效果。公开号为CN106420755A的发明专利申请公开了化合物sclerotiorin在制备抗结核病药物中的应用,通过研究发现化合物sclerotiorin能够抑制结核分枝杆菌在细胞内的增殖,且与利福平或者异烟肼联合,能够有效地减少巨噬细胞内结核分枝杆菌的增殖。目前,没有sclerotiorin作为α-葡萄糖苷酶抑制剂的应用。
发明内容
本发明的发明目的在于:针对上述存在的问题,提供sclerotiorin作为α-葡萄糖苷酶抑制剂的应用。
为了实现上述目的,本发明采用的技术方案如下:
本发明提供sclerotiorin作为α-葡萄糖苷酶抑制剂的应用。
更具体低,sclerotiorin作为α-葡萄糖苷酶抑制剂在制备降血糖的食品、医药产品中的应用。
其中,sclerotiorin的结构式为:
综上所述,由于采用了上述技术方案,本发明的有益效果是:
1、本发明通过活性研究发现,微生物来源化合物sclerotiorin能够有效抑制α-葡萄糖苷酶,当sclerotiorin浓度为25.0μM时,能有效抑制α-葡萄糖苷酶,抑制率达93.3%;其IC50值为3.3μM;sclerotiorin可以作为新型α-葡萄糖苷酶抑制剂用于开发降血糖食品和医药产品。
2、α-葡萄糖苷酶抑制剂按照来源可将其分为天然产物中提取、化学合成以及从微生物的代谢产物中得到三大类。然而,天然产物分离原料稀少,纯度低,很少能够进入工业化生产;化学合成的α-葡萄糖苷酶抑制剂产品对人体的毒副作用大。本发明研究的sclerotiorin属于红树林内生真菌的活性次级代谢产物,该化合物的立体结构已经被确证,且来源广泛,能够利用微生物的扩大化发酵进行工业化生产,提取工艺简单,成本廉价,对环境友好;本发明对新型α-葡萄糖苷酶抑制剂的开发和降血糖药物的新药研发具有十分重要的意义。
附图说明
图1为sclerotiorin的浓度-抑制率曲线
具体实施方式
下面将结合本发明实施例,对本发明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付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。
实施例1Sclerotiorin对α-葡萄糖苷酶的抑制作用
l、溶液的配置:
(1)磷酸缓冲液(0.01mol/L):分别精密称取磷酸氢二钾0.8g,磷酸二氢钾0.68g,均加入蒸馏水溶解,定容至500mL。取定容的磷酸氢二钾溶液和磷酸二氢钾溶液适量,混合均匀后用pH仪调至pH为7.00。
(2)pNPG溶液(3mg/mL):精密称取3.0mg PNPG(对硝基苯-α-D-葡萄糖苷),置于5mL离心管中,精密量取1.0mL的磷酸缓冲溶液将其溶解,现配现用。
(3)2U/mLα-葡萄糖苷酶溶液:精密称取1.0mgα-葡萄糖苷酶(50U/mg)放置于25mL棕色容量瓶中,利用0.01mol/L磷酸缓冲液溶解并准确定容,保存于-20℃冰箱中。
(4)sclerotiorin溶液(25.0μM):精密称取3.9mg sclerotiorin固体粉末,转移至2mL的容量瓶中,加入1mL DMSO超声溶解,用DMSO准确定容至2mL。
2、抑制率测试方法:将sclerotiorin溶解于DMSO并配成50μM的浓缩液,往96孔板中每孔加1μL的sclerotiorin样品,再依次加入49μL磷酸缓冲溶液和10μL 2U/mLα-葡萄糖苷酶,将96孔板置于37℃培养箱保温10分钟。然后往每个孔加入20μL pNPG,继续于37℃培养箱孵育。20分钟后,通过酶标仪测定λ405处的吸光度值。并通过下述公式计算sclerotiorin对α-葡萄糖苷酶的抑制率,其中Asample blank为不加入α-葡萄糖苷酶的吸光度值,Asample为加入α-葡萄糖苷酶的吸光度值,Acontrol为不加入sclerotiorin样品的吸光度值,Ablank为不加入样品sclerotiorin和α-葡萄糖苷酶的吸光度值。
(3)IC50值的测得:将sclerotiorin溶解于DMSO分别配成50μM的浓缩液,采用倍半梯度稀释法对样品进行稀释后,往96孔板中每孔加1μL的样品,再依次加入49μL磷酸缓冲溶液和10μL 2U/mLα-葡萄糖苷酶,将96孔板置于37℃培养箱保温10分钟。然后往每个孔加入20μL pNPG,继续于37℃培养箱孵育。20分钟后,通过酶标仪测定λ405处的吸光度值。并通过公式(1)计算sclerotiorin对α-葡萄糖苷酶的抑制率(结果见表1)。将抑制率和浓度数据导入绘图软件Origin 8.0。通过三次多项式回归方程拟合得到浓度-抑制率曲线(结果见图1),计算得到IC50值。
表1不同浓度的sclerotiorin对α-葡萄糖苷酶的抑制率
Sclerotiorin浓度/μM | 抑制率/% |
0.78 | 22.1 |
1.56 | 23 |
3.12 | 53.9 |
6.25 | 73.0 |
12.5 | 82.9 |
25.0 | 93.3 |
50.0 | 95.7 |
从表1可以看出,化合物sclerotiorin具有较强的α-葡萄糖苷酶抑制活性。当sclerotiorin浓度为25.0μM时,对α-葡萄糖苷酶的抑制率达93.3%。
采用倍半梯度稀释法对样品进行稀释后进一步评价化合物sclerotiorin的α-葡萄糖苷酶抑制活性,实验结果显示,化合物sclerotiorin的IC50值为3.3μM。
上述说明是针对本发明较佳可行实施例的详细说明,但实施例并非用以限定本发明的专利申请范围,凡本发明所提示的技术精神下所完成的同等变化或修饰变更,均应属于本发明所涵盖专利范围。
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