CN114591286A - 新型大环内酯化合物acautalides A-C及其制备方法和应用 - Google Patents
新型大环内酯化合物acautalides A-C及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及医药技术领域,具体涉及无茎真菌属Acaulium sp.H-JQSF等菌株产生的三种新型大环内酯(被发明人命名为:acautalides AC)及其制备方法和应用。
背景技术
帕金森病也称震颤麻痹,其发生发展步骤包括:中脑黑质致密区多巴胺能神经元丢失、导致纹状体中多巴胺含量降低,造成乙酰胆碱系统功能相对亢进,从而引起神经系统的变性或变病。此病目前有些治疗方法和手段,包括药物治疗、手术治疗、运动治疗、心理疏导及照料护理等,但最重要且贯穿疾病全过程的手段依然是药物治疗。但现有疗法只能改善症状,不能阻止病情发展,可谓治标不治本。因此,寻找可治疗帕金森病的新药尤为重要。
天然产物是生物为适应环境、竞争拮抗、化学感应、抵御外侵、传递信号等目的产生的小分子化合物。其生物合成诱因复杂、结构与功能事先难料,一直被认为是新药发现的重要源泉。
真菌资源丰富,生物合成能力强,是青霉素等抗感染药物的重要源头,但至今未见真菌产生的抗帕金森等神经退行性疾病的药物。发明人通过广泛筛选,发现Acauliumsp.H-JQSF 等真菌可产结构全新的、能抗此病的系列大环内酯,急需对它们的来源、制备和用途予以专利保护。
发明内容
本发明的目的是为了解决现有棘手医学难题,而提出对新型大环内酯化合物acautalides A-C及其制备方法和应用予以保护。
为了实现上述目的,本发明采用了如下技术方案:本发明的新型大环内酯化合物acautalides A-C,所述的新型大环内酯化合物acautalides A-C的化学结构式如式(I)所示:
本发明所述的新型大环内酯化合物acautalides A-C的制备方法,包括如下步骤:
(1)将真菌Acaulium sp.H-JQSF菌株,采用PDA平板发酵培养;
(2)将步骤(1)所得培养物切块,放入大米等组成的培养基中,30±5℃静置培养25-35 天;
(3)将步骤(2)所得培养基采用乙酸乙酯等有机溶剂萃取,浓缩得到浸粗膏F1;
(3)对浸粗膏F1进行硅胶柱层析,先采用石油醚和乙酸乙酯等溶剂混合物洗脱,再用二氯甲烷和甲醇等溶剂混合物洗脱,共获得14个组分;
(4)再将这些组分,反复用反向硅胶、凝胶柱层析和HPLC分离纯化,制得三种新型大环内酯,命名为acautalides A-C。
进一步地,在步骤(1)中,PDA平板发酵培养的培养条件为26~30℃培养5-7天。
进一步地,在步骤(4)中,HPLC色谱条件为:使用反相色谱柱,流动相A:水,流动相B:乙腈,等度洗脱程序为:流动相A的质量百分比为5%,流动相B的质量百分比为95%,时间为25分钟;HPLC分离纯化过程如下:HPLC半制备反相高效液相色谱:ODS-2Hypersil 柱,流动相为:乙腈和水的体积比为19:1,以2mL/min流速梯度洗脱25分钟;泵的型号可以为Hitachi pump L-7100,紫外灯的型号可以为UV detector L-7400。
本发明所述的Acaulium sp.H-JQSF菌株,其特征在于:真菌Acaulium sp.H-JQSF菌株为无茎真菌属(Acaulium)菌株;保藏名称:Acaulium sp.H-JQSF菌株;保藏编号:CCTCCNo: M2021342;保藏地点:中国典型培养物保藏中心(China Center for Type CultureCollection,简称CCTCC),中国,武汉大学;保藏日期:2021年04月09日。
本发明所述的真菌Acaulium sp.H-JQSF菌株制备的Acaulium sp.H-JQSF菌株菌剂。
进一步地,其活性成分为如下(a)、(b)和(c)中的至少一种:
(a)所述的Acaulium sp.H-JQSF菌株的培养物;
(b)所述的Acaulium sp.H-JQSF菌株细胞的超声裂解上清;
(c)所述的Acaulium sp.H-JQSF菌株细胞的超声裂解沉淀。
本发明所述的三种新型大环内酯化合物在制备帕金森等神经损伤类疾病的治疗药物中的应用。
进一步地,所述的三种新型大环内酯化合物用作研发帕金森神经损伤类疾病药物的先导分子。
进一步地,所述的新型大环内酯化合物能修复多巴胺神经元的损伤。
有益效果:本发明首次从真菌大米发酵产物中,发现了三种新型大环内酯类化合物 acautalides A-C,其线虫动物模型活性数据表明,acautalides A-C能够修复由1-甲基-4-苯基- 吡啶离子(MPP+)引起的多巴胺神经元损伤,其中acautalides A和B的修复效果更显著,可进一步作为用于治疗帕金森等神经损伤疾病类的先导药物。
附图说明
图1-图24为本发明acautalides A-C核磁数据和质谱数据谱图。
图25为本发明acautalides A-C在线虫动物模型活性实验结果图,其中acautalides A和B 修复多巴胺神经元损伤效果更为显著。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中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。
实施例1
本发明的本发明的新型大环内酯化合物acautalides A-C,所述的新型大环内酯化合物 acautalides A-C的化学结构式如式(I)所示:
本发明所述的新型大环内酯化合物acautalides A-C的制备方法,包括如下步骤:
(1)将真菌Acaulium sp.H-JQSF菌株,采用PDA平板发酵培养;PDA平板发酵培养的培养条件为26~30℃培养6天。
(2)将步骤(1)所得培养物切块,放入大米等组成的培养基中,30±5℃静置培养25-35 天;
(3)将步骤(2)所得培养基采用乙酸乙酯等有机溶剂萃取,浓缩得到浸粗膏F1;
(3)对浸粗膏F1进行硅胶柱层析,先采用石油醚和乙酸乙酯等溶剂混合物洗脱,再用二氯甲烷和甲醇等溶剂混合物洗脱,共获得14个组分;
(4)再将这些组分,反复用反向硅胶、凝胶柱层析和HPLC分离纯化,制得三种新型大环内酯,命名为acautalides A-C。HPLC色谱条件为:使用反相色谱柱,流动相A:水,流动相B:乙腈,等度洗脱程序为:流动相A的质量百分比为5%,流动相B的质量百分比为95%,时间为25分钟;HPLC分离纯化过程如下:HPLC半制备反相高效液相色谱:ODS-2Hypersil 柱,流动相为:乙腈和水的体积比为19:1,以2mL/min流速梯度洗脱25分钟;泵的型号可以为Hitachi pump L-7100,紫外灯的型号可以为UV detector L-7400。
本发明所述的Acaulium sp.H-JQSF菌株,真菌Acaulium sp.H-JQSF菌株为无茎真菌属(Acaulium)菌株;保藏名称:Acaulium sp.H-JQSF菌株;保藏编号:CCTCC No:M2021342;保藏地点:中国典型培养物保藏中心(China Center for Type Culture Collection,简称CCTCC),中国,武汉大学;保藏日期:2021年04月09日。
本发明所述的真菌Acaulium sp.H-JQSF菌株制备的Acaulium sp.H-JQSF菌株菌剂。其活性成分为如下(a)、(b)和(c)中的至少一种:
(a)所述的Acaulium sp.H-JQSF菌株的培养物;
(b)所述的Acaulium sp.H-JQSF菌株细胞的超声裂解上清;
(c)所述的Acaulium sp.H-JQSF菌株细胞的超声裂解沉淀。
本发明所述的三种新型大环内酯化合物在制备帕金森等神经损伤类疾病的治疗药物中的应用。
所述的三种新型大环内酯化合物用作研发帕金森神经损伤类疾病药物的先导分子。
所述的新型大环内酯化合物能修复多巴胺神经元的损伤。
实施例2
实施例2与实施例1的区别在于:
本发明所述的新型大环内酯化合物acautalides A-C的制备方法,包括如下步骤:
在步骤(1)中,将真菌Acaulium sp.H-JQSF菌株,采用PDA平板发酵培养;PDA平板发酵培养的培养条件为30℃培养5天。
实施例3
实施例3与实施例1的区别在于:
本发明所述的新型大环内酯化合物acautalides A-C的制备方法,包括如下步骤:
在步骤(1)中,将内生真菌Acaulium sp.H-JQSF菌株,采用PDA平板发酵培养;PDA平板发酵培养的培养条件为30℃培养7天。
试验例1
Acaulium sp.H-JQSF菌株的活化。
将内生真菌Acaulium sp.H-JQSF菌株的冻存干粉涂布于PDA平板培养基(土豆200g、葡萄糖20g、Glucose 4g、琼脂20g、蒸馏水1L,pH7.4~7.6)中,置于30℃恒温箱中培养,得到该真菌。在PDA平板上,培养初期基内菌丝为白色,易产生单个菌落,一周后开始产生大量白色菌丝体。经过形态学和ITS序列鉴定该菌为无茎真菌属Acaulium sp.。该菌名为Acaulium sp.H-JQSF。
试验例2
Acaulium sp.H-JQSF菌株大米培养基发酵
将菌株Acaulium sp.H-JQSF转接至平板PDA培养基上,在30℃恒温箱中进行培养7天。待菌丝体铺满平板后,将其切成小方块,转入大米培养基中,30℃静置培养30天。
试验例3
Acautalides A-C的提取与分离
试验例2中所得的大米培养基加入乙酸乙酯萃取,浓缩得到浸膏F1。对浸膏F1进行正向硅胶柱层析,先用石油醚和乙酸乙酯进行洗脱,再用二氯甲烷和甲醇洗脱,得到14个组分;再将这些组分,分别经反向硅胶、凝胶柱层析和半制备HPLC(色谱柱:Allsphere ODS-2.5mm column),乙腈-水体系,以2mL/min流速,乙腈-水体积比=19:1等度洗脱25min,制得本发明所述acautalide A(25mg)acautalide B(10mg)和acautalide C(6.5mg)。泵的型号可以为 Hitachi pump L-7100,紫外灯的型号可以为UV detector L-7400。
试验例4
Acautalides A-C的结构鉴定。Acautalide A的1H和13C NMR和二维谱图的数据归属如表 1所示:
表1
Acautalide A结构如下:
Acautalide B的1H和13C NMR和二维谱图的数据归属如表2所示:
表2
Acautalide B结构如下:
Acautalide C的1H和13C NMR和二维谱图的数据归属如表3所示:
表3
Acautalide C结构如下:
试验例5
线虫模型动物活性试验
实验材料:BZ555[Pdat-1::GFP]线虫,1-甲基4-苯基吡啶离子(MPP+),LB培养基。
实验方法:
1)线虫培养和MPP+模型建立
将E.coli OP50菌株挑至100mL LB培养基中,37℃培养至OD(λ)600=0.4后将菌液(100μL)涂布在NGM平板上,在室温下过夜后备用。挑取处于产卵期的BZ555[Pdat-1::GFP]线虫若干条放入平板中,培养2天后再使用次氯酸钠和氢氧化钾对线虫进行同步化处理。将L1期的幼虫按每孔30~60条加入到96孔培养板,每孔液体总体积为50μL。实验分组为空白对照组、单独MPP+(浓度为1mM)组、MPP+分别加acautalides A-C(浓度为2μM) 组,共计5组。培养48h测线虫的存活数。
2)活性测定
采用3mmol/L左旋咪唑麻醉的存活线虫放在2%琼脂糖凝胶玻片上,用荧光显微镜观察,计算线虫头部的6条标记绿色荧光的多巴胺能神经元的数量。
实验结果下图25所示:
图25a包括未处理的正常对照组、MPP+处理组、‘MPP++acautalide A’处理组、‘MPP++acautalide B’处理组、‘MPP++acautalide C’处理组。绿色荧光标记蛋白的荧光强度显示:比较acautalides A和B对bz555线虫前多巴胺神经元的保护作用显著。标尺为100μM。图25b 为bz555线虫头部多巴胺神经元计数。所示数据为平均值±SEM。****p<0.0001。每组n= 30-60。
线虫模型动物体内活性实验表明:acautalides A和B修复多巴胺神经元损伤效果较为显著,可进一步作为开发治疗帕金森疾病等神经损伤疾病的先导药物。
本发明所述acautalides A-C的结构是基于它们的质谱、核磁共振谱、化学合成等技术确定的。以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
2.权利要求1所述的新型大环内酯化合物acautalides A-C的制备方法,其特征在于包括如下步骤:
(1)将真菌Acaulium sp.H-JQSF菌株,采用PDA平板发酵培养;
(2)将步骤(1)所得培养物切块,放入大米等组成的培养基中,30±5℃静置培养25-35天;
(3)将步骤(2)所得培养基采用乙酸乙酯等有机溶剂萃取,浓缩得到浸粗膏F1;
(3)对浸粗膏F1进行硅胶柱层析,先采用石油醚和乙酸乙酯等溶剂混合物洗脱,再用二氯甲烷和甲醇等溶剂混合物洗脱,共获得14个组分;
(4)再将这些组分,反复用反向硅胶、凝胶柱层析和HPLC分离纯化,制得三种新型大环内酯,命名为acautalides A-C。
3.根据权利要求2所述的新型大环内酯化合物acautalides A-C的制备方法,其特征在于:在步骤(1)中,PDA平板发酵培养的培养条件为26~30℃培养5-7天。
4.根据权利要求3所述的新型大环内酯化合物acautalides A-C的制备方法,其特征在于:在步骤(4)中,HPLC色谱条件为:使用反相色谱柱,流动相A:水,流动相B:乙腈,等度洗脱程序为:流动相A的质量百分比为5%,流动相B的质量百分比为95%,时间为25分钟;HPLC分离纯化过程如下:HPLC半制备反相高效液相色谱:ODS-2Hypersil柱,流动相为:乙腈和水的体积比为19:1,以2mL/min流速梯度洗脱25分钟;泵的型号可以为Hitachi pump L-7100,紫外灯的型号可以为UV detector L-7400。
5.权利要求2所述的Acaulium sp.H-JQSF菌株,其特征在于:真菌Acaulium sp.H-JQSF菌株为无茎真菌属(Acaulium)菌株;保藏名称:Acaulium sp.H-JQSF菌株;保藏编号:CCTCCNo:M2021342H-JQSF;保藏地点:中国典型培养物保藏中心(China Center for TypeCulture Collection,简称CCTCC),中国,武汉大学;保藏日期:2021年04月09日。
6.权利要求5所述的真菌Acaulium sp.H-JQSF菌株制备的Acaulium sp.H-JQSF菌株菌剂。
7.根据权利要求5所述的Acaulium sp.H-JQSF菌株菌剂,其活性成分为如下(a)、(b)和(c)中的至少一种:
(a)权利要求5所述的Acaulium sp.H-JQSF菌株的培养物;
(b)权利要求5所述的Acaulium sp.H-JQSF菌株细胞的超声裂解上清;
(c)权利要求5所述的Acaulium sp.H-JQSF菌株细胞的超声裂解沉淀。
8.权利要求1至7任一项所述的三种新型大环内酯化合物在制备帕金森等神经损伤类疾病的治疗药物中的应用。
9.权利要求8所述的应用,其特征在于:所述的三种新型大环内酯化合物用作研发帕金森神经损伤类疾病药物的先导分子。
10.根据权利要求9所述的应用,其特征在于:所述的新型大环内酯化合物能修复多巴胺神经元的损伤。
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