CN116836140B - 一类具有抗炎活性的化合物及其制备方法和应用 - Google Patents
一类具有抗炎活性的化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一类具有抗炎活性的化合物或其药学上可接受的盐,或其外消旋混合物、水合物、溶剂合物、前药、对映异构体、非对映异构体、互变异构体,及其在制备用于治疗或预防炎症的药物中的应用,以及两种Wailupemycin类具有抗炎活性的化合物的制备方法。本发明公开的两种Wailupemycin类具有抗炎活性的化合物可由同一株链霉菌通过发酵培养后提纯后得到。
Description
技术领域
本发明涉及微生物药物技术领域,具体涉及一类具有抗炎活性的化合物及其制备方法和应用。
背景技术
海洋生态环境复杂,在海洋生物构建的生态系统中,海洋微生物的存在至关重要,复杂的生存环境促使海洋微生物进化出独特的代谢途径,生产出结构新颖,活性独特的化合物,因此海洋微生物的次级代谢产物研究在新药开发中存在极大的潜力。
Wailupemycin类化合物在抗炎、抗菌、抑制α-糖苷酶等药理研究中具有较好的生物活性。Wailupemycin类化合物首次在海洋来源链霉菌BD-26T的次级代谢产物中分离得到,其中Wailupemycin A对大肠杆菌有良好的抑制活性(Namthip S,et al.Newα-pyrone-containing metabolites from a marine-derived actinomycete.Tetrahedron,1996,52:8073–8080)。之后的研究中,海洋来源链霉菌OUCMDZ-3434的次级代谢产物Wailupemycin H和Wailupemycin I表现出良好的α-糖苷酶抑制活性(Zhengbo C,etal.Newα-glucosidase inhibitors from marine algae-derived Streptomycessp.OUCMDZ-3434.Scientific Reports,2016,6:20004)。
发明内容
本发明对海洋来源的一株链霉菌进行研究,从大米培养基中分离得到2个结构新颖的Wailupemycin类具有抗炎活性的化合物,为先导化合物的开发提供支持。
具有抗炎活性的化合物或其药学上可接受的盐,或其外消旋混合物、水合物、溶剂合物、前药、对映异构体、非对映异构体、互变异构体,所述具有抗炎活性的化合物为具有如下所示结构式的化合物1或化合物2:
本发明又提供了所述的具有抗炎活性的化合物的制备方法,包括步骤:
1)将冻存的链霉菌菌株活化后接种至高氏一号液体培养基中,摇床培养,所得种子液加入到大米固体培养基中,静置培养得到固体发酵液;所述链霉菌菌株为中国典型培养物保藏中心保藏号为CCTCC NO:M2023977的链霉菌;
2)所述固体发酵液经有机溶剂萃取后得到大米萃取液;
3)所述大米萃取液经浓缩后进行分离纯化,获得化合物1或化合物2;
所述分离纯化具体包括步骤:
(a)浓缩后的大米萃取液采用硅胶柱层析的分离方法,通过体积比为6:1、3:1、1:1、1:5、1:10的石油醚-乙酸乙酯体系和5:1、0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得20个馏分,分别记为Fraction A-T;
(b)将硅胶柱层析所得馏分Fraction L通过体积比为100:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得7个馏分Fraction L1-L7;馏分Fraction L3用反相高效液相色谱分离,10μm、21.2mm×250mm的Agilent Pursuit C-18色谱柱进行制备纯化,检测波长210nm,流动相为含0.05vol%三氟乙酸的甲醇体积浓度为40%~60%的甲醇/水体系,以10mL/min梯度洗脱,收集18.7min的色谱峰,回收溶剂,获得化合物1;
(c)将硅胶柱层析所得馏分Fraction F通过体积比为50:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得4个馏分Fraction F1-F4;馏分Fraction F4用反相高效液相色谱分离,10μm、21.2mm×250mm的Agilent Pursuit C-18色谱柱进行制备纯化,检测波长210nm,流动相为含0.05vol%三氟乙酸的甲醇体积浓度为45%~100%的甲醇/水体系,以10mL/min梯度洗脱,收集17.2min的色谱峰,回收溶剂,获得化合物2。
优选的,步骤1)中,所述链霉菌菌株的活化过程包括:将链霉菌菌株划线接种于高氏一号固体培养基中,在27~29℃条件下活化培养3~4天。
进一步优选的,所述高氏一号固体培养基的配制方法为:以培养基1L计,将可溶性淀粉20g、KNO3 1g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4。
优选的,步骤1)中:
所述高氏一号液体培养基的配制方法为:以培养基1L计,将可溶性淀粉20g、KNO31g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4;
所述大米固体培养基的配制方法为:称取25g海盐于1L水中混合均匀,将pH值调至7.2~7.4,配制成海盐水;每个500mL三角瓶中加入40g大米以及60mL海盐水,搅拌均匀,灭菌备用;
所述摇床培养的条件为:27~29℃下,170~190rpm摇床中培养3~4天;
所述种子液加入到大米固体培养基中的量为:10mL/瓶;
所述静置培养的条件为:27~29℃,静置35天。
优选的,步骤2)中,所述有机溶剂为乙酸乙酯。
优选的,步骤3)中,所述浓缩的具体操作为:将所述大米萃取液经减压真空干燥除去溶剂。
本发明还提供了一种药物组合物,包含所述的具有抗炎活性的化合物或其药学上可接受的盐,或其外消旋混合物、水合物、溶剂合物、前药、对映异构体、非对映异构体、互变异构体,以及一种或多种可药用载体、稀释剂、赋形剂。
作为一个总的发明构思,本发明还提供了所述的具有抗炎活性的化合物或其药学上可接受的盐,或其外消旋混合物、水合物、溶剂合物、前药、对映异构体、非对映异构体、互变异构体,或所述的药物组合物在制备用于治疗或预防炎症的药物中的应用。
为进一步测试本发明制备得的具有抗炎活性的化合物的生物活性,采用RAW264.7巨噬细胞进行化合物的抗炎活性评价试验。
试验表明,本发明分离得到的两种化合物具有抗炎活性,因此可用于治疗炎症疾病的药物先导化合物的开发。
优选的,所述炎症为风湿性关节炎、骨质疏松症、骨质溶解、牙周炎等骨骼疾病。
本发明中,所述药学上可接受的盐包括盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐等。
本发明与现有技术相比,有益效果有:
本发明提供了两种Wailupemycin类具有抗炎活性的化合物,均可由同一株链霉菌通过发酵培养后提纯后得到,其结构为天然产物中首次发现,具有结构新颖性,且在抗炎症方面具有效果,可用于治疗炎症疾病的药物先导化合物的开发。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的操作方法,通常按照常规条件,或按照制造厂商所建议的条件。
实验方法与实验设备
化合物的结构确定采用核磁共振(NMR)和/或质谱(MS)。NMR化学位移(δ)以10-6(ppm)为单位给出。NMR的测定采用Bruker AVⅢ600MHz NMR核磁共振波谱仪,测定使用的常规氘代溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)和氘代甲醇(Methanol-d4),以四甲基硅烷(TMS)为内标。质谱(MS)的测定采用液相色谱-质谱联用仪(LC-MS),电离源:电喷雾电离(ESI)。生产商为:Agilent,型号为:Agilent 1260,色谱柱为Eclipse Plus 3.6μm100mm×4.6mm。
薄层层析硅胶板由青岛海洋化工有限公司生产,薄层色谱(TLC)使用的硅胶板厚度0.2-0.25mm;规格50mm×200mm。薄层制备色谱(prep-TLC)使用的硅胶板厚度0.4-0.5mm;规格200mm×200mm。
柱层析硅胶通常使用由青岛海洋化工有限公司生产的硅胶,规格100-200目或300-400目。
高效液相制备色谱采用Agilent Pursuit C-18(10μm,21.2mm×250mm)色谱柱。
菌种来源
本发明菌株为Streptomyces sp.DS-27,从浙江省舟山市岱山县沿海海洋沉积物中分离得到。将海洋沉积物样品涡旋30min,使其充分混匀,用无菌水将样品稀释10倍和100倍,得到两个梯度的泥样稀释液。取200μL泥样稀释液于含40mg/L的萘啶酮酸的高氏一号平板固体培养基上,用涂布棒涂布均匀,做好标记,将培养皿置于28℃恒温培养箱中培养。待平板上长出菌落,观察菌落的形态特征,挑取不同形态的菌株进一步划线纯化,直到有单菌落的产生,最终获得菌株DS-27。
经过16S rDNA序列分析,菌株DS-27的16S rDNA序列(SEQ ID NO:1)含有1476bp,测定结果见序列表,经过与GenBank进行序列比对,菌株属于链霉菌属(Streptomycessp.),保藏于中国典型培养物保藏中心(CCTCC),保藏日期为2023年6月9日,保藏地址为中国湖北省武汉市武昌区八一路299号,菌种保藏号为CCTCC NO:M 2023977。
培养基
高氏一号平板固体培养基:以培养基1L计,将可溶性淀粉20g、KNO3 1g、K2HPO40.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O0.01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4。
高氏一号液体培养基:以培养基1L计,将可溶性淀粉20g、KNO31g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4。
大米固体培养基:称取25g海盐于1L纯净水中混合均匀,将pH值调至7.2~7.4,配制成海盐水,500mL三角瓶中称取40g大米,并加入60mL海盐水。
实施例1:3-Methyl-5-hydroxymethyl-6-isopropyl-4-hydroxy-α-pyrone(即化合物1)的制备与表征
1)菌种种子液
将链霉菌接种于500mL锥形瓶中,每瓶含250mL高氏一号液体培养基,在28℃、180rpm下摇床培养3-4天,得到接种有链霉菌的种子液。
2)化合物的发酵
将步骤1)得到的含有链霉菌的种子液接种至大米培养基(该大米培养基由以下组分制成:大米40g;海盐水60mL),接种体积为10mL,在28℃下静置培养35天,用等体积乙酸乙酯萃取3次,得到发酵产物粗提物。
3)化合物的收集
将步骤2)得到的发酵产物粗提物进行硅胶柱层析,通过体积比为6:1、3:1、1:1、1:5、1:10的石油醚-乙酸乙酯体系和5:1、0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得20个馏分,分别记为Fraction A-T;
将硅胶柱层析所得馏分Fraction L通过体积比为100:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,得到馏分Fraction L3;
Fraction L3用反相高效液相色谱分离(Agilent Pursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为甲醇体积浓度为40~60%甲醇/水体系(含0.05vol%三氟乙酸),以10mL/min梯度洗脱,收集18.7min的色谱峰,回收溶剂,即可得3-Methyl-5-hydroxymethyl-6-isopropyl-4-hydroxy-α-pyrone。
制备得的3-Methyl-5-hydroxymethyl-6-isopropyl-4-hydroxy-α-pyrone分子式根据高分辨质谱HR-ESI-MS推测为C10H14O4([M+H]+199.0964,calculated 199.0965)。
3-Methyl-5-hydroxymethyl-6-isopropyl-4-hydroxy-α-pyrone的核磁共振数据及信号归属如表1所示。
表1(1H NMR 600MHz,13C NMR 100MHz,溶剂CD3OD)
δC | δH(J in Hz) | |
2 | 168.2,C | |
3 | 99.3,C | |
4 | 167.7,C | |
5 | 111.0,C | |
6 | 166.7,C | |
7 | 55.7,CH2 | 4.55,s |
8 | 30.3,CH | 3.21,m |
9 | 20.7,CH3 | 1.23,d(6.8) |
10 | 20.7,CH3 | 1.23,d(6.8) |
11 | 8.7,CH3 | 1.91,s |
实施例2:Wailupemycin Q(即化合物2)的制备与表征
将实施例1中硅胶柱层析所得馏分Fraction F通过体积比为50:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,得到馏分Fraction F4;
Fraction F4用反相高效液相色谱分离(Agilent Pursuit C-18(10μm,21.2mm×250mm)色谱柱进行制备纯化,检测波长210nm),采用的流动相为甲醇体积浓度为45~100%甲醇/水体系(含0.05vol%三氟乙酸),以10mL/min梯度洗脱,收集17.2min的色谱峰,回收溶剂,即可得Wailupemycin Q。
制备得的Wailupemycin Q分子式根据高分辨质谱HR-ESI-MS推测为C18H19O6([M+H]+331.1175,calculated 331.1176)。
Wailupemycin Q的核磁共振数据及信号归属如表2所示。
表2(1H NMR 600MHz,13C NMR 100MHz,溶剂DMSO)
实施例3:化合物的抗炎活性测定
1、测试目的
本实验通过检测化合物对LPS(脂多糖)诱导的RAW 264.7巨噬细胞NO产生的抑制作用,根据NO产生量的变化评价本发明制得的化合物抗炎活性。
2、实验方法
取对数生长期的RAW 264.7细胞,以1.5×105个/孔铺于24孔板培养板,CO2培养箱中培养过夜,取出培养板后每孔加入不同浓度的待测样品,给药3h-4h后加入脂多糖(LPS),同时设置不加药物只加LPS组(模型组),阴性对照组(细胞不加药,不加LPS组)。待LPS作用24小时后取上清,采用NO检测试剂盒检测NO浓度。
其中阳性对照组为总一氧化氮合酶(NOS)抑制剂(L-NMMA)。
3、实验结果
抗炎活性测定结果表明化合物1和化合物2在较低浓度下均可表现出抗炎作用(表3)。
表3化合物对LPS诱导下RAW264.7巨噬细胞NO产生量的抑制作用
化合物 | 浓度 | 抑制率(%) |
化合物1 | 10μM | 11.20 |
化合物2 | 10μM | 18.46 |
L-NMMA | 100μM | 76.72 |
此外应理解,在阅读了本发明的上述描述内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (9)
1.具有抗炎活性的化合物或其药学上可接受的盐,其特征在于,所述具有抗炎活性的化合物为具有如下所示结构式的化合物1或化合物2:
2.根据权利要求1所述的具有抗炎活性的化合物的制备方法,其特征在于,包括步骤:
1)将冻存的链霉菌菌株活化后接种至高氏一号液体培养基中,摇床培养,所得种子液加入到大米固体培养基中,静置培养得到固体发酵液;所述链霉菌菌株为中国典型培养物保藏中心保藏号为CCTCC NO:M2023977的链霉菌;
2)所述固体发酵液经有机溶剂萃取后得到大米萃取液;
3)所述大米萃取液经浓缩后进行分离纯化,获得化合物1或化合物2;
所述分离纯化具体包括步骤:
(a)浓缩后的大米萃取液采用硅胶柱层析的分离方法,通过体积比为6:1、3:1、1:1、1:5、1:10的石油醚-乙酸乙酯体系和5:1、0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得20个馏分,分别记为Fraction A-T;
(b)将硅胶柱层析所得馏分Fraction L通过体积比为100:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得7个馏分Fraction L1-L7;馏分Fraction L3用反相高效液相色谱分离,10μm、21.2mm×250mm的Agilent Pursuit C-18色谱柱进行制备纯化,检测波长210nm,流动相为含0.05vol%三氟乙酸的甲醇体积浓度为40%~60%的甲醇/水体系,以10mL/min梯度洗脱,收集18.7min的色谱峰,回收溶剂,获得化合物1;
(c)将硅胶柱层析所得馏分Fraction F通过体积比为50:1至0:1的二氯甲烷-甲醇体系进行梯度洗脱,收集包含目标化合物的馏分,合并得4个馏分Fraction F1-F4;馏分Fraction F4用反相高效液相色谱分离,10μm、21.2mm×250mm的Agilent Pursuit C-18色谱柱进行制备纯化,检测波长210nm,流动相为含0.05vol%三氟乙酸的甲醇体积浓度为45%~100%的甲醇/水体系,以10mL/min梯度洗脱,收集17.2min的色谱峰,回收溶剂,获得化合物2。
3.根据权利要求2所述的制备方法,其特征在于,步骤1)中,所述链霉菌菌株的活化过程包括:将链霉菌菌株划线接种于高氏一号固体培养基中,在27~29℃条件下活化培养3~4天。
4.根据权利要求3所述的制备方法,其特征在于,所述高氏一号固体培养基的配制方法为:以培养基1L计,将可溶性淀粉20g、KNO3 1g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g、琼脂20g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4。
5.根据权利要求2所述的制备方法,其特征在于,步骤1)中:
所述高氏一号液体培养基的配制方法为:以培养基1L计,将可溶性淀粉20g、KNO3 1g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、NaCl 0.5g、FeSO4·7H2O 0.01g、海盐25g混合后加水至1L,再调节pH值至7.2~7.4;
所述大米固体培养基的配制方法为:称取25g海盐于1L水中混合均匀,将pH值调至7.2~7.4,配制成海盐水;每个500mL三角瓶中加入40g大米以及60mL海盐水,搅拌均匀,灭菌备用;
所述摇床培养的条件为:27~29℃下,170~190rpm摇床中培养3~4天;
所述种子液加入到大米固体培养基中的量为:10mL/瓶;
所述静置培养的条件为:27~29℃,静置35天。
6.根据权利要求2所述的制备方法,其特征在于,步骤2)中,所述有机溶剂为乙酸乙酯。
7.根据权利要求2所述的制备方法,其特征在于,步骤3)中,所述浓缩的具体操作为:将所述大米萃取液经减压真空干燥除去溶剂。
8.一种药物组合物,其特征在于,包含权利要求1所述的具有抗炎活性的化合物或其药学上可接受的盐,以及一种或多种可药用载体、稀释剂、赋形剂。
9.根据权利要求1所述的具有抗炎活性的化合物或其药学上可接受的盐,或根据权利要求8所述的药物组合物在制备用于治疗或预防炎症的药物中的应用。
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