CN106957216A - 一种抗真菌新化合物制备方法和抗真菌用途 - Google Patents
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Abstract
本发明涉及一种抗真菌新化合物制备方法和抗真菌用途。本发明公开了一种以放线菌Streptomyces albolongus为来源分离制备的具有下述结构式I的新化合物,化学名为(1β,4β,4aβ,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol,分子式为C12H22O2。本发明还提供了该化合物的制备方法和应用。本发明所述方法包括:菌株S.albolongus经发酵培养后,离心获得培养液,培养液经过大孔吸附树脂柱吸附,95%乙醇洗脱部分减压浓缩获得粗提物;粗提物经正相硅胶,葡聚糖凝胶LH-20和反相硅胶柱色谱获得结构I所示的单体化合物,采用核磁共振波谱和质谱鉴定其结构为新化合物。体外抗真菌活性研究表明,本发明提供的化合物I对多种致病真菌,包括白色念珠菌、近平滑念珠菌和新型隐球菌具有明显的抑制作用,有望开发成为新的抗真菌药物。
Description
技术领域
本发明涉及具有抗真菌活性的化合物,具体的说,涉及一种从放线菌Streptomyces albolongus发酵液中提取得到的抗真菌活性的新化合物及其在治疗真菌感染疾病中的用途,属于医药技术领域。
背景技术
近年来随着免疫功能低下而被真菌感染的人群数量不断增加,尤其是深部真菌感染发病率的大幅上升,抗真菌药物研究成为热点领域。目前抗真菌药物主要为咪唑类(如酮康唑)和多烯类(如两性霉素B),它们对人体均有严重的毒副作用。同时,临床上耐药病原菌的不断出现,使发现新型高效低毒抗真菌药物成为当务之急。
念珠菌(Candida)又称假丝酵母菌,为条件致病性真菌。在机体免疫功能低下时,可引起皮肤黏膜念珠菌病、阴道念珠菌病等。另外,在深部真菌感染病人中,念珠菌已成为第四位最常见医院内血液感染的致病菌,死亡率高达40%。对人类致病的念珠菌中白色念珠菌(Candida albicans)和近平滑念珠菌(Candida parapsilosis)临床分离率较高。
新型隐球菌(Cryptococcus neoformans)属于隐球菌属,是一种临床重要条件致病真菌,可以引起致死性新型隐球菌性脑膜炎。近年来新型隐球菌感染发生率呈明显上升趋势,突出表现在艾滋病人群中,己成为艾滋病患者死亡的首要原因。
放线菌是药物发现的重要资源,临床治疗和农业生产中应用的抗生素大约其中75%来源于放线菌。本发明实验研究从来自大象(Elephas maximus)粪便中分离得到的放线菌Streptomyces albolongus发酵液中分离得到一种抗真菌活性新化合物。同时,抗真菌实验显示,该化合物对条件致病真菌白色念珠菌、近平滑念珠菌和新型隐球菌显示了较好的抗真菌活性。
发明内容
本发明人从放线菌Streptomyces albolongus发酵液中分离得到一种新化合物I,命名为(1β, 4β, 4aβ, 8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol,并对此进行了深入地研究,证明该化合物可以用于预防和治疗真菌感染性疾病,从而完成了本发明。
本发明的一个目的在于提供一种如结构I所示抗真菌活性新化合物I。
本发明的另一目的在于提供菌株Streptomyces albolongus制备化合物I的方法。
本发明的再一目的在于提供所述的化合物I在制备治疗或预防真菌感染性疾病药物中的应用。
本发明的又一目的在于提供所述的化合物I在制备治疗或预防念珠菌属和隐球菌属真菌感染性疾病药物中的应用。
本发明的又一目的在于提供所述的化合物I在制备治疗或预防条件致病真菌白色念珠菌(C. albicans)、近平滑念珠菌(C. parapsilosis)和新型隐球菌(Cryptococcusneoformans)感染药物中的应用。
为达上述目的,本发明提供了一种化合物I的制备方法,所述方法包括:
(1) Streptomyces albolongus的种子培养:
种子培养基:酵母膏2~10 g, 葡萄糖2~10 g, 麦芽膏2~10 g, 1~5 mL 复合维生素,1~5 mL微量元素,蒸馏水1 L。培养条件:pH 6.5~8.0, 培养温度25~32摄氏度,培养时间24~72小时。
(2) Streptomyces albolongus的发酵培养:
发酵培养基 :大豆粉10 g/L, 蛋白胨2 g/L, 葡萄糖20 g/L, 可溶性淀粉5 g/L, 酵母膏2 g/L, 氯化钠4 g/L, 磷酸氢二钾0.5 g/L, 硫酸镁0.5 g/L, 碳酸钙2 g/L。发酵培养条件:pH 6.5~8.0, 培养温度25~32摄氏度,培养时间96~192小时。
(3)获得粗提物:将步骤(2)获得的发酵液离心,上清液经过大孔树脂柱层析,先用水洗脱除去培养基成分,再用体积为大孔树脂填料体积10-30倍的体积浓度为75-95%的乙醇水溶液洗脱,收集乙醇溶液并进行浓缩,得到粗提物。
(4)单体化合物分离、纯化:上述步骤(3)获得的膏状物,采用正相硅胶柱色谱二氯甲烷-甲醇(50:1~1:1)梯度洗脱,通过薄层检测合并含有化合物I的组分,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并含有化合物I的组分得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,甲醇-水(70: 30)洗脱,得到新化合物I。
根据本发明所述的方法所述的大孔树脂可以为本领域常规的大孔树脂,譬如D101,XAD16等树脂。
(5) 结构鉴定:本发明综合应用高分辨质谱(HRMS)、核磁共振氢谱(1H NMR)、核磁共振碳谱(13C NMR)和二维核磁共振波谱(包括COSY、NOESY、HSQC和HMBC谱)确定了单体化合物结构。
另一方面,本发明还提供了所述方法制备的新化合物I。
再一方面,本发明还提供了所述的新化合物I在制备治疗或预防真菌感染性疾病药物中的应用。
又一方面,本发明还提供所述的化合物I在制备治疗或预防条件致病真菌白色念珠菌(Candida albicans)、近平滑念珠菌(Candida parapsilosis)和新型隐球菌(Cryptococcus neoformans)感染药物中的应用。
综上所述,本发明提供了一种抗真菌新化合物及其制备方法和应用。
附图说明
图1化合物I结构。
图2 化合物I的高分辨质谱。
图3化合物I核磁共振氢谱图1H NMR (600 MHz,DMSO-d6)。
图4化合物I核磁共振碳谱图13C NMR (150 MHz,DMSO-d6)。
图5化合物I的COSY谱图。
图6化合物I的NOESY谱图。
图7化合物I的HSQC谱图。
图8化合物I的HMBC谱图。
具体实施方式
以下通过具体实施例详细说明本发明的实施过程和产生的有益效果,旨在帮助阅读者更好地理解本发明的实质和特点,不作为对本案可实施范围的限定。
实施例1
新化合物I的制备
1.1 Streptomyces albolongus的种子培养。
种子培养基:酵母膏0.4 g, 葡萄糖0.4 g, 麦芽膏0.5 g, 0.1 mL 复合维生素,1.0 mL微量元素,蒸馏水100 mL。培养条件:pH 7.2, 培养温度28摄氏度,培养时间48小时。
1.2 Streptomyces albolongus的发酵培养。
发酵培养基 :大豆粉10 g/L, 蛋白胨2 g/L, 葡萄糖20 g/L, 可溶性淀粉5 g/L,酵母膏2 g/L, 氯化钠4 g/L, 磷酸氢二钾0.5 g/L, 硫酸镁0.5 g/L, 碳酸钙2 g/L, 蒸馏水70 L。培养条件:pH 7.8, 培养温度28摄氏度,培养时间168小时。
1.3提取。
将步骤(2)获得的发酵液离心,上清液经过XAD-16大孔吸附树脂(大孔吸附树脂体积为35 L)柱层析,先用12倍填料体积的20%乙醇洗脱除去培养基成分,再用体积为大孔树脂填料体积12倍的体积浓度为95%的乙醇水溶液洗脱,收集乙醇溶液并进行减压浓缩,得到粗提物。
1.4分离纯化
上述步骤(3)获得的膏状物,甲醇溶解后与100~200目硅胶拌样,采用200~300目硅胶干法装柱,干法上样。分别采用二氯甲烷甲醇(50:1,20:1,10:1,4:1)梯度洗脱,通过薄层检测合并相同组分,得到13个组分(Fr.1~13)。组分Fr.3含有化合物I,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并成6个组分(Fr.3.1~Fr.3.6)。组分Fr.3.1主要含有化合物I,减压蒸干后得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,甲醇-水(70: 30)洗脱,得到新化合物I。
1.5 结构鉴定
化合物I,为白色固体,ESIMS m/z 221 [M+Na]+, 419 [2M+Na]+; HRESIMS m/z221.1513 [M+Na]+ (calcd for C12H22O2Na, 221.1518),提示该化合物分子量为198,分子式为C12H22O2。化合物I碳谱显示12个碳信号,包括2个季碳信号,2个次甲基信号,6个亚甲基信号和2个甲基信号。氢谱中在化学位移δ 0.70 (3H, d, 6.0 Hz)和0.89 (3H, s)处给出2个甲基质子信号,在化学位移δ 3.27(1H, m)处给出含氧次甲基质子信号,在化学位移δ5.38 (1H, d, 4.5 Hz)和4.75 (1H, s)处给出2个活泼氢质子信号,在氢谱中其余的质子信号属于6个亚甲基质子信号。结合上述信息推测化合物I的不饱和度为2,分子中含有2个羟基基团。通过HSQC谱归属了化合物碳和相应氢的信号(见表1),结合化合物的COSY、HMBC和NOESY谱确定了化合物的结构如下:
化学名为:(1β, 4β, 4aβ, 8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol.
表1 化合物I的1H NMR (600 MHz) 和 13C NMR (150 MHz) 数据(DMSO-d6)
实施例2:抗真菌活性实验
化合物I溶于DMSO中,倍比稀释配置成9个不同浓度(22.0, 11.0, 5.5, 2.75, 1.38,0.69, 0.36, 0.18, 0.09 mg/mL)。病原菌条件致病真菌白色念珠菌(Candida albicansATCC MYA-2876)、近平滑念珠菌(Candida parapsilosis ATCC 22019)和新型隐球菌(Cryptococcus neoformans ATCC 208821)分别接种在RPMI-1640培养基 (10.4 g L-1 RPMI-1640, 2 g L-1 NaHCO3, 34.53 g L-1 MOPs, pH 7.0)制成菌悬液,白色念珠菌(C.albicans)和近平滑念珠菌(C. parapsilosis)菌悬液浓度为 2 × 103 cfu/mL,新生隐球菌(C.neoformans)菌悬液浓度为5 × 104 cfu/mL。样品采用RPMI-1640培养基稀释10倍,10 μL的样品和100 μL的菌悬液加到96孔板中,等体积的DMSO溶液作阴性对照,两性霉素B作阳性对照。结果表明化合物I对白色念珠菌和近平滑念珠菌显示较强的抑制活性,MIC值分别为12.5 和3.3 μg/mL, 对新型隐球菌显示较弱的抑菌活性,MIC值为200 μg/mL。阳性对照药两性霉素B对三株菌的MIC值分别为1.0,2.0和2.0 μg/mL。
Claims (5)
1.一种具有抗真菌活性的新化合物I,其特征在于,分子式为C12H22O2,化学名为(1β, 4β, 4aβ, 8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol,结构式如下:
。
2.一种制备权力要求1所述的具有抗真菌活性化合物I的方法,其特征在于包括以下步骤:
(1)菌株Streptomyces albolongus的种子培养:种子培养基:酵母膏2~10 g, 葡萄糖2~10 g, 麦芽膏2~10 g, 1~5 mL 复合维生素,1~5 mL微量元素,蒸馏水1 L;
培养条件:pH 6.5~8.0, 培养温度25~32摄氏度,培养时间24~72小时;
(2)菌株Streptomyces albolongus的发酵培养:发酵培养基:大豆粉10 g/L, 蛋白胨2g/L, 葡萄糖20 g/L, 可溶性淀粉5 g/L, 酵母膏2 g/L, 氯化钠4 g/L, 磷酸氢二钾0.5g/L, 硫酸镁0.5 g/L, 碳酸钙2 g/L;
培养条件:pH 6.5~8.0, 培养温度25~32摄氏度,培养时间96~192小时;
(3)获得粗提物:将步骤(2)获得的发酵液离心,上清液经过大孔树脂柱层析,大孔树脂用量和发酵液按体积比0.5~1:1,先用体积为大孔树脂填料体积10-20倍体积的0~20%乙醇洗脱除去培养基成分,再用体积为大孔树脂填料体积10-20倍体积的浓度为75-95%的乙醇水溶液洗脱,收集乙醇溶液并进行浓缩,得到粗提物;
(4)分离纯化:上述步骤(3)获得的膏状物,采用正相硅胶柱色谱二氯甲烷-甲醇(50:1~1:1)梯度洗脱,通过薄层检测合并含有化合物I的组分,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并含有化合物I的组分得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,甲醇-水(70: 30)洗脱,得到新化合物I。
3.如权利要求1所述的抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途。
4.如权利要求3所述的具有抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途,其中特征在于,所述的真菌为念珠菌属和隐球菌属真菌。
5.如权利要求4所述的具有抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途,其中特征在于,所述的真菌为白色念珠菌(Candida albicans)、近平滑念珠菌(Candida parapsilosis)和新型隐球菌(Cryptococcus neoformans)。
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