CN108276363A - 帚状曲霉次生代谢物及其在制备抗真菌药物中的应用 - Google Patents
帚状曲霉次生代谢物及其在制备抗真菌药物中的应用 Download PDFInfo
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Abstract
本发明提供一种帚状曲霉次生代谢物sphingofungins,其制备方法,及其在制备抗真菌药物中的应用和其在医药中的应用。本发明主要是采用植物化学研究手段,从普洱茶后发酵优势菌帚状曲霉Aspergillus penicilliodes Speg.的发酵菌丝中获得sphingofungins G和H,并按照常规方法制成固体、液体或膏状体剂型。本发明所得抗菌素为天然抗菌素,对人体细胞无毒无害。
Description
技术领域:
本发明属于医药领域,具体地,涉及帚状曲霉次生代谢物sphingofungins的制备及其在抗真菌药物组合物中的应用和其在医药中的应用。
背景技术:
真菌性疾病是人类健康的一大威胁。人类真菌性疾病包括由皮肤癣菌、马拉色菌和白色念珠菌等接触皮肤或粘膜诱发角质层、毛发和甲板产生的浅部真菌感染病变;及由念珠菌、曲霉菌及其它病原真菌诱发的深部真菌感染。
近年来,由于环境质量的下降及肥胖、外用糖皮质激素类药物滥用、糖尿病发病率升高以及不良生活习惯等因素导致临床浅部真菌感染的病例逐年增高,其中体癣和脂溢性皮炎增多尤为明显。此外,由于老年性器官衰退、抗生素的超剂量使用、免疫抑制剂的使用、恶性肿瘤、器官移植及肺结核等疾病引发的深部真菌感染严重危害人类健康,甚至危及生命。
目前常用的抗真菌药主要有多烯类的两性霉素B,三唑类的氟康唑、伊曲康唑、伏立康唑,嘧啶类的氟胞嘧啶和棘白菌素类的卡伯芬净。同一抗菌素的连续使用容易导致病原真菌产生耐药性和抗药性,降低其对侵染性真菌病害的治疗效果,对天然产物中抗真菌活性成分的深度挖掘,丰富抗真菌药物的化学基础将有利于人类健康的发展。
鞘氨酸化合物是一类来源于土壤真菌烟曲霉菌丝次生代谢产生的含氮化合物,通常具有抑制人体病原真菌隐球菌、念珠菌和酵母菌的生长和繁殖,对人类真菌病害具有一定的疗效。
然而,迄今为止,现有技术中未见有源自普洱茶后发酵优势菌帚状曲霉的固体发酵菌丝体的次生代谢物sphingofungin G和H,未见其制备方法报道,未见其在治疗人体浅部真菌感染和深部真菌感染相关的药理活性研究,也未见其在治疗人体浅部真菌感染和深部真菌感染药物及药物制剂中的报道。
发明内容:
本发明的目的旨在提供帚状曲霉次生代谢物sphingofungins G和H的制备方法,以其为活性成分的药物组合物,及其在制备治疗人体浅部真菌感染和深部真菌感染类药物组合物中的应用和其在医药中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
如下结构式所示的化合物sphingofungin G、Sphingofungin H,
所述的化合物sphingofungin G、Sphingofungin H,该两种化合物来源于普洱茶后发酵优势菌帚状曲霉菌丝体的次生代谢物。
所述的上述sphingofungins G和H的制备是采用天然产物的系统分离方法从普洱茶后发酵优势菌株帚状曲霉的固体发酵菌物中分离、纯化和结构鉴定。
所述的普洱茶后发酵优势菌株帚状曲霉固体发酵菌物的制备是指,将分离到的帚状曲霉接种在马铃薯葡萄糖琼脂培养基中,待菌落表面长满孢子,将孢子配制成浓度为3×106/mL的悬浮液,按重量比1∶50接种于马铃薯葡萄糖麦粒培养基中,28℃条件下暗培养20天,即得帚状曲霉固体发酵培养物。
所述的sphingofungins G和H的分离、纯化是指帚状曲霉固体发酵物用3倍体积的氯仿:甲醇体积比为1∶1的溶液冷浸提取3次,每次5天。过滤,滤液减压浓缩除去有机溶剂后溶解于水中,水溶液分别用石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯萃取部分减压浓缩干燥得40.5g,上硅胶层析柱,用氯仿:甲醇体积比为100:0,98:2,95:5,90:10,85:15和80:20的溶液洗脱得6个部分Fractions 1-6;11g的Fraction 3经硅胶柱层析,丙酮-石油醚为0-100%的溶液进行梯度洗脱,进一步细分得7个sub-fractions 1-7,sub-fractions 4经Sephadex LH-20凝胶柱,用甲醇洗脱,洗脱液体浓缩后经半制备高效液相色谱柱,用甲醇:水体积比为65∶35的溶液洗脱,真空浓缩干燥得权利要求1和2所述化合物。
所述的sphingofungins G和H的结构鉴定是指将分离得到的单体化合物进行红外光谱、紫外光谱、高分辨质谱和核磁共振图谱分析,确定结构。
抗菌药物组合物,其含有所述的sphingofungins G和H,单体或混合物作为有效成分,并至少还包含一种药学上可接受的载体。
所述的化合物对红色毛癣菌(Trichophyton rubrum)和新型隐球菌(Cryptococcusneoformans)抗菌活性评价,是指将红色毛癣菌接种在沙保氏固体培养基斜面上,于28℃、黑暗条件下培养7-14d,产生孢子,加入含有0.1%Tween 80的沙保氏液体培养基制备菌悬液,菌悬液静置3-5min后,取上层含孢子以及菌丝片段均质液体至无菌试管,振荡5s,镜检,用沙保氏液体培养基调整菌悬液浓度3×104-4×104CFU·mL-1作为红色毛癣菌的标准菌悬液。将新型隐球菌接种在沙保氏固体斜面培养基中,于28℃、黑暗条件下培养36h,以沙保氏液体培养基稀释至菌含量为0.2×104-1.0×104CFU·mL-1作为新型隐球菌的标准菌悬液。用DMSO将sphingofungins G和H分别溶解为1mg/mL的原液,根据浓度稀释法稀释成14个浓度梯度,各取10μL分别加入96孔微量板中(以DMSO为阴性对照,以两性霉素B为阳性对照),所有孔加入标准菌悬液40μL,每孔总体积为50μL。红色毛癣菌35℃培养72h,新型隐球菌每孔加入0.25%TTC 5μL,继续培养3h,重复3次。用肉眼和倒置显微镜观察培养孔,以未见可见菌生长的最低样品浓度为其MIC,两性霉素B的MIC值判定为没有可见菌生长的最低药物浓度。
所述的sphingofungins G和H在制备预防或治疗人体浅部或深部真菌感染的药物中的应用。
所述的药物组合物在制备预防或治疗人体浅部或深部真菌感染疾病的药物中的应用。
本发明的化合物sphingofungins G和H或其盐可经口或不经过口给药,给药量因药物不同而各有不同,对成人来说,每天1-80mg比较合适。
经口服给药时,首先使化合物与常规的药用辅剂如赋形剂、崩解剂、黏合剂、润滑剂、抗氧化剂、包衣剂、着色剂、芳香剂、表面活性剂等混合,将其制成颗粒剂、胶囊、片剂等形式给药;非经口给药时可以注射液、输液剂、栓剂或涂抹剂等形式给药。制备上述制剂时,可使用常规的制剂技术。
具体实施方式:
下面以本发明的实施例来进一步说明本发明的实质性内容,这些实例仅是对本发明优选方案的说明,而并不以任何方式限制本发明的保护范围。
实施例1:
Sphingofungin G的制备及其抗真菌药物组合物,和其在医药中的应用。
步骤一:sphingofungin G的制备:
帚状曲霉接种在马铃薯葡萄糖琼脂培养基中,待菌落表面长满孢子,将孢子配制成浓度为3×106/mL的悬浮液,按重量比1∶50接种于马铃薯葡萄糖麦粒培养基中,28℃条件下暗培养20天,即得帚状曲霉固体发酵培养样物。
所述的sphingofungin G的分离、纯化是指帚状曲霉固体发酵物用3倍体积的氯仿:甲醇体积比为1∶1的溶液冷浸提取3次,每次5天。过滤,滤液减压浓缩除去有机溶剂后溶解于水中,水溶液分别用石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯萃取部分减压浓缩干燥得40.5g,上硅胶层析柱,用氯仿:甲醇体积比为100:0,98:2,95:5,90:10,85:15和80:20的溶液洗脱得6个部分Fractions 1-6;11g的Fraction 3经硅胶柱层析,丙酮-石油醚为0-100%的溶液进行梯度洗脱,进一步细分得7个sub-fractions 1-7,sub-fractions 4经Sephadex LH-20凝胶柱,用甲醇洗脱,洗脱液体浓缩后经半制备高效液相色谱柱,用甲醇:水体积比为65∶35的溶液洗脱,真空浓缩干燥得单体化合物。
该单体化合物经鉴定为sphingofungin G,即Sphingofungin G,为一新化合物。
其理化数据如下:白色无定型粉末;[α]20 D+22.5(c 0.05,MeOH);UV(MeOH)λmax(logε)201nm(3.66);IR(KBr)νmax 3421,2928,1774,1654,1375,1164和579cm-1;HRTOFMS(m/z434.2514[M+Na]+,calcd 434.2513),分子式C22H37NO6; 1H NMR(CHCl3,600MHz)和13C NMR(CHCl3,600MHz)数据见表1.
表1.化合物1的氢谱和碳谱数据
步骤二:化合物sphingofungin G的抗菌活性评价。
选择红色毛癣菌(Trichophyton rubrum)和新型隐球菌(Cryptococcusneoformans)为抗菌活性评价对象。将红色毛癣菌接种在沙保氏固体培养基斜面上,于28℃、黑暗条件下培养培养7-14d,产生孢子,加入含有0.1%Tween 80的沙保氏液体培养基制备菌悬液,菌悬液静置3-5min后,取上层含孢子以及菌丝片段均质液体至无菌试管,振荡5s,镜检,用沙保氏液体培养基调整菌悬液浓度3×104-4×104CFU·mL-1作为红色毛癣菌的标准菌悬液。将新型隐球菌接种在沙保氏固体斜面培养基中,于28℃、黑暗条件下培养36h,以沙保氏液体培养基稀释至菌含量为0.2×104-1.0×104CFU·mL-1作为新型隐球菌的标准菌悬液。用DMSO将sphingofungins G分别溶解为1mg/mL的原液,根据浓度稀释法稀释成最终浓度为14个浓度梯度,各取10μL分别加入96孔微量板中,使其最终浓度为1.0μg/mL、1.5μg/mL、2.0μg/mL、2.5μg/mL、3.0μg/mL、3.5μg/mL、4.0μg/mL、4.5μg/mL、5.0μg/mL、6.0μg/mL、7.0μg/mL、8.0μg/mL、9.0μg/mL、10.0μg/mL。同时,以DMSO为阴性对照,以两性霉素B为阳性对照,所有孔加如标准菌悬液40μL,每孔总体积为50μL。红色毛癣菌35℃培养72h,新型隐球菌每孔加入0.25%TTC 5μL,继续培养3h,重复3次。用肉眼和倒置显微镜观察培养孔,以未见可见菌生长的最低样品浓度为其MIC,两性霉素B的MIC值判定为没有可见生长的最低药物浓度,实验结果如表2所示。
表2 sphingofungin G对红色毛癣菌和新型隐球菌的MIC值
实施例2
Sphingofungin H的制备及其抗真菌药物组合物,和其在医药中的应用。
步骤一:sphingofungin H的制备
帚状曲霉接种在马铃薯葡萄糖琼脂培养基中,待菌落表面长满孢子,将孢子配制成浓度为3×106/ml的悬浮液,按重量比1∶50接种于马铃薯葡萄糖麦粒培养基中,28℃条件下暗培养20天,即得帚状曲霉固体发酵培养物。
所述的sphingofungin H的分离、纯化是指帚状曲霉固体发酵物用3倍体积的氯仿∶甲醇体积比为1∶1的溶液冷浸提取3次,每次5天。过滤,滤液减压浓缩除去有机溶剂后溶解于水中,水溶液分别用石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯萃取部分减压浓缩干燥得40.5g,上硅胶层析柱,用氯仿:甲醇体积比为100:0,98:2,95:5,90:10,85:15和80:20的溶液洗脱得6个部分Fractions 1-6;11g的Fraction 3经硅胶柱层析,丙酮-石油醚为0-100%的溶液进行梯度洗脱,进一步细分得7个sub-fractions 1-7,sub-fractions 4经SephadexLH-20凝胶柱,用甲醇洗脱,洗脱液体浓缩后经半制备高效液相色谱柱,用甲醇∶水体积比为65∶35的溶液洗脱,真空浓缩干燥得单体化合物。
该单体化合物经鉴定为sphingofungin H,即Sphingofungin H,为一新化合物。
其理化数据如下:白色无定型粉末;[α]20 D+38.8(c 0.10,MeOH);UV(MeOH)λmax(logε)201nm(3.66);IR(KBr)νmax 3421,2928,1774,1654,1375,1164和579cm-1;HRTOFMS(m/z436.2672[M+Na]+,calcd 436.2670),分子式C22H39NO6;1H NMR(CHCl3,600MHz)和13C NMR(CHCl3,600MHz)数据见表3.
表3.化合物2的氢谱和碳谱数据
步骤二:化合物sphingofungin H的抗菌活性评价。
选择红色毛癣菌(Trichophyton rubrum)和新型隐球菌(Cryptococcusneoformans)为抗菌活性评价对象。将红色毛癣菌接种在沙保氏固体培养基斜面上,于28℃、黑暗条件下培养培养7-14d,产生孢子,加入含有0.1%Tween 80的沙保氏液体培养基制备菌悬液,菌悬液静置3-5min后,取上层含孢子以及菌丝片段均质液体至无菌试管,振荡5s,镜检,用沙保氏液体培养基调整菌悬液浓度3×104-4×104CFU·mL-1作为红色毛癣菌的标准菌悬液。将新型隐球菌接种在沙保氏固体斜面培养基中,于28℃、黑暗条件下培养36h,以沙保氏液体培养基稀释至菌含量为0.2×104-1.0×104CFU·mL-1作为新型隐球菌的标准菌悬液。用DMSO将sphingofungins H分别溶解为1mg/mL的原液,根据浓度稀释法稀释成最终浓度为14个浓度梯度,各取10μL分别加入96孔微量板中,使其最终浓度为1.0μg/mL、1.5μg/mL、2.0μg/mL、2.5μg/mL、3.0μg/mL、3.5μg/mL、4.0μg/mL、4.5μg/mL、5.0μg/mL、6.0μg/mL、7.0μg/mL、8.0μg/mL、9.0μg/mL、10.0μg/mL。同时,以DMSO为阴性对照,以两性霉素B为阳性对照,所有孔加如标准菌悬液40μL,每孔总体积为50μL。红色毛癣菌35℃培养72h,新型隐球菌每孔加入0.25%TTC 5μL,继续培养3h,重复3次。用肉眼和倒置显微镜观察培养孔,以未见可见菌生长的最低样品浓度为其MIC,两性霉素B的MIC值判定为没有可见生长的最低药物浓度,实验结果如表4所示。
表4 sphingofungin H对红色毛癣菌和新型隐球菌的MIC值
制剂实施例1:
按实施例1和2的方法,帚状曲霉接种在马铃薯葡萄糖琼脂培养基中,待菌落表面长满孢子,将孢子配制成浓度为3×106/ml的悬浮液,按重量比1∶50接种于马铃薯葡萄糖麦粒培养基中,28℃条件下暗培养20天,即得帚状曲霉固体发酵培养样物。
所述的sphingofungins G和H的分离、纯化:将帚状曲霉固体发酵物用3倍体积的氯仿:甲醇体积比为1∶1的溶液冷浸提取3次,每次5天。过滤,滤液减压浓缩除去有机溶剂后溶解于水中,水溶液分别用石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯萃取部分减压浓缩干燥得40.5g,上硅胶层析柱,用氯仿:甲醇体积比为100:0,98:2,95:5,90:10,85:15和80:20的溶液洗脱得6个部分Fractions 1-6;11g的Fraction 3经硅胶柱层析,丙酮-石油醚为0-100%的溶液进行梯度洗脱,进一步细分得7个sub-fractions 1-7,sub-fractions 4经Sephadex LH-20凝胶柱,用甲醇洗脱,洗脱液体浓缩后经半制备高效液相色谱柱,用甲醇∶水体积比为65∶35的溶液洗脱,真空浓缩干燥得化合物sphingofungins G和H。按常规加注射用水,精滤,灌封灭菌制成注射液。
制剂实施例2:
按实施例1和2的方法先制得sphingofungins G和H,将其溶于无菌注射用水中,搅拌使其充分溶解,用无菌抽滤漏斗过滤,再无菌精滤,分装于2安瓿中,低温冷冻干燥后无菌熔封得粉针剂。
制剂实施例3:
将按实施例1和2的方法所得到sphingofungins G和H与赋形剂重量比为9:1的比例加入赋形剂,制成粉剂。
制剂实施例4:
按实施例1和2的方法先制得sphingofungins G和H,按其与赋形剂重量比为1:5-1:10的比例加入赋形剂,制粒压片。
制剂实施例5:
按实施例1-2的方法先制得sphingofungins G和H,或按常规口服液制法制成口服液。制剂实施例6:
按实施例1和2的方法先制得sphingofungins G和H,按其与赋形剂重量比为5:1的比例加入赋形剂,涂抹剂或清洗剂。
制剂实施例7:
按实施例1和2的方法先制得sphingofungins G和H按其与赋形剂重量比为3:1的比例加入赋形剂,制成涂抹剂或清洗剂。
Claims (7)
1.如下结构式所示的化合物sphingofungin G、Sphingofungin H,
2.如权利要求1所述的化合物sphingofungin G、Sphingofungin H,其特征在于该两种化合物来源于普洱茶后发酵优势菌帚状曲霉菌丝体的次生代谢物。
3.药物组合物,由权利要求1所示的化合物sphingofungin G、Sphingofungin H,和药用载体所组成。
4.权利要求1所述的sphingofungin G、Sphingofungin H的制备方法,其特征在于该方法包括帚状曲霉的固体发酵和化合物的制备,
所述的帚状曲霉的固体发酵是将分离到的帚状曲霉接种在马铃薯葡萄糖琼脂培养基中,待菌落表面长满孢子,用无菌水将孢子配制成浓度为3×106/mL的悬浮液,按重量比1∶50接种于马铃薯葡萄糖麦粒培养基中,28℃条件下暗培养20天,得帚状曲霉固体发酵物;
所述的化合物的制备方法,将帚状曲霉固体发酵物用3倍体积的氯仿∶甲醇体积比为1∶1的溶液冷浸提取3次,每次5天,过滤,滤液减压浓缩除去有机溶剂后溶解于水中,水溶液分别用石油醚、乙酸乙酯和正丁醇萃取,乙酸乙酯萃取部分减压浓缩干燥,上硅胶层析柱,用氯仿∶甲醇体积比为100:0,98:2,95:5,90:10,85:15和80:20的溶液洗脱得6个部分Fractions 1-6;Fraction 3经硅胶柱层析,丙酮-石油醚为0-100%的溶液进行梯度洗脱,进一步细分得7个sub-fractions 1-7,sub-fractions 4经Sephadex LH-20凝胶柱,用甲醇洗脱,洗脱液体浓缩后经半制备高效液相色谱柱,用甲醇∶水体积比为65∶35的溶液洗脱,真空浓缩干燥得化合物sphingofungin G、Sphingofungin H。
5.权利要求1所述的化合物sphingofungin G、Sphingofungin H在制备预防或治疗真菌性疾病的药物中的应用。
6.权利要求1所述的化合物sphingofungin G、Sphingofungin H在制备预防或治疗浅部或深部真菌感染的药物中的应用。
7.权利要求3所述的药物组合物在制备治疗真菌性疾病的药物中的应用。
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JP2002060388A (ja) * | 2000-08-16 | 2002-02-26 | Sankyo Co Ltd | スフィンゴフンジンeの製造方法及びその合成中間体 |
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