CN114934084B - 一种吲哚二酮哌嗪生物碱的制备方法 - Google Patents
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- IRABMPNBAAQRLL-UHFFFAOYSA-N 1h-indole;piperazine-2,3-dione Chemical class O=C1NCCNC1=O.C1=CC=C2NC=CC2=C1 IRABMPNBAAQRLL-UHFFFAOYSA-N 0.000 title claims abstract description 10
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Abstract
本发明公开一种吲哚二酮哌嗪生物碱的制备方法,通过花斑曲霉(Aspergillus versicolor)CCTCC NO:M2022064的发酵培养和发酵产物的提取分离,获得高产率、高纯度吲哚二酮哌嗪生物碱Brevianamide K,使得Brevianamide K产率达到2.7g/kg大米,纯度达到98.61%;该制备方法具有发酵条件简单、菌种易培养、工艺步骤少、生产周期短等优势,具有工业化、规模化生产的潜力。本方法对于解决高纯度Brevianamide K的制备难题,保障后续以Brevianamide K为先导的创新药物研制具有重要意义。
Description
(一)技术领域
本发明属于微生物技术领域,具体涉及一种吲哚二酮哌嗪类生物碱BrevianamideK的制备。
(二)背景技术
二酮哌嗪是由两个氨基酸通过肽键缩合而成的环肽类结构,因其稳定的六元环骨架结构使其成为一个重要的药效团,表现出了抗细菌、抗真菌、抗病毒、抗肿瘤、免疫抑制、神经保护、抗疟疾、抗高血糖等多种生物活性。
Brevianamide K是一种吲哚二酮哌嗪生物碱,呈黄色针状晶体,分子式为C21H21N3O2,相对分子质量为347,熔点157-158℃,其化学结构如式(I)所示。
Brevianamide K具有显著的体外α-葡萄糖苷酶抑制活性(IC50=7.60μM)(YeG.T.et al.,Marine Drugs,2021,19,402),且对结核分枝杆菌酪氨酸磷酸酶B(mPTPB)具有显著的抑制活性(IC50=7.07μM)(佘志刚等.海洋真菌来源的哌嗪类衍生物及其在制备抗结核药物中的应用.公开号:CN104230938A;公开日:2014.12.24),展现出了被开发成为糖尿病和结核病治疗药物的巨大潜力。Brevianamide K于2009年首次从杂色曲霉AspergillusversicolorA.3.4186次级代谢产物中发现。目前,现有文献报道的Brevianamide K制备方法均为曲霉发酵法,包括液体发酵和固体发酵。其中,液体发酵法制备的最高产率为0.383mg/L(Kong X.L.et al.,J.Ocean Univ.China,2014,13,691-695),而固体发酵的最高产率为67.5mg/kg大米(Li G.Y.et al.,Org.Lett.,2009,11,3714-3717.)。
(三)发明内容
本发明的目的是提供一种吲哚二酮哌嗪生物碱的制备方法,该方法另辟蹊径,采用特殊生境真菌——花斑曲霉(Aspergillusversicolor)CCTCC NO:M2022064,通过菌株的发酵培养和发酵产物的提取分离,获得高产率、高纯度Brevianamide K,解决了现有技术中产率低的缺陷。
为实现上述发明目的,本发明采用的技术方案是:
本发明提供一种吲哚二酮哌嗪生物碱(Brevianamide K)的制备方法,所述制备方法为:(1)将花斑曲霉(Aspergillusversicolor)ZJUTE2接种至大米固体培养基,在20-30℃条件下暗培养15-30天,获得大米发酵产物;所述花斑曲霉ZJUTE2,保藏于中国典型培养物保藏中心,保藏日期为2022年1月13日,保藏编号为CCTCC NO:M2022064,地址为中国,武汉,武汉大学;所述大米固体培养基为大米和蒸馏水的混合物,其中蒸馏水用量以大米质量计为1-5mL/g(优选1.35mL/g);(2)将大米发酵产物(优选捣碎)中加入有机溶剂,室温(25-30℃)浸提,提取液浓缩至无液体流出,获得粗提物浸膏;(3)将步骤(2)粗提物浸膏用水悬浮,以有机溶剂萃取,收集有机相,减压浓缩至干,得到萃取物浸膏;(4)将步骤(3)萃取物浸膏用甲醇溶解后进行MCI CHP20P柱层析(优选直径d=4cm,高h=60cm),以体积浓度30-100%甲醇-水为洗脱液,收集体积浓度70-80%的甲醇-水洗脱部位,减压浓缩至干,得浓缩物;(5)将步骤(4)所得浓缩物用甲醇溶解,于室温重结晶,过滤,得晶体,即得到式(Ⅰ)所示Brevianamide K;
优选的,步骤(1)所述花斑曲霉ZJUTE2接种至大米固体培养基前,先进行活化培养,再将活化后的菌悬液以体积浓度0.1-1%(优选1%)的接种量接种大米固体培养基,所述活化是指将花斑曲霉ZJUTE2接种至马铃薯葡萄糖琼脂(PDA)培养基,28℃培养7天,使菌株活化,将活化后的菌株用含体积浓度2%吐温80的无菌水重悬,得到菌悬液;所述菌悬液中菌体浓度为1×105-1×108个/mL,优选1×106个/mL;所述PDA培养基组成为马铃薯200g/L、葡萄糖20g/L、琼脂18g/L,溶剂为蒸馏水,pH自然。
优选的,步骤(1)中发酵培养条件为:28℃条件下暗培养20天。
优选的,步骤(2)中所述有机溶剂为95%乙醇、甲醇或丙酮;所述有机溶剂体积用量以大米固体培养基中大米干重计为2~8mL/g(优选5-6mL/g);所述浸提至少进行3次,每次提取时间为3~5天,每次浸提用的有机溶剂体积用量以大米固体培养基中大米干重计为4mL/g。
优选的,步骤(3)中所述水体积用量以粗提物浸膏重量计为2-5mL/g(优选3.1mL/g);所述有机溶剂为乙酸乙酯,所述有机溶剂与水的体积比为1:0.5-3;萃取3-5次,每次萃取用的有机溶剂与水体积比优选1:1。
优选的,步骤(4)方法为:萃取物浸膏以甲醇溶解后进行MCI CHP20P柱层析,依次以体积比为30:70、40:60、50:50、60:40、70:30、80:20、90:10和100:0的甲醇/水混合溶剂为洗脱剂进行梯度洗脱,每一梯度洗脱2~5个(优选2个)柱体积,流速10-20mL/min(优选15mL/min);收集体积比70:30-80:20的甲醇-水洗脱部位,减压浓缩至干,得浓缩物;所述甲醇体积用量以萃取物浸膏质量计为1-5mL/g,优选2mL/g。
优选的,步骤(5)甲醇体积用量以浓缩物质量计为20-30mL/g(优选26-27mL/g)。
与现有技术相比,本发明有益效果主要体现在:
本发明利用花斑曲霉ZJUTE2的发酵培养,高产率制备高纯度Brevianamide K,使得Brevianamide K产率达到2.7g/kg大米,纯度达到98.61%;该制备方法具有发酵条件简单、菌种易培养、工艺步骤少、生产周期短等优势,具有工业化、规模化生产的潜力。本方法对于解决高纯度Brevianamide K的制备难题,为后续新型抗结核药物的研发提供化学实体具有较大意义。
(四)附图说明
图1是AspergillusversicolorZJUTE2的菌落照片。
图2是Brevianamide K的1H-NMR谱图。
图3是Brevianamide K的13C-NMR谱图。
图4是Brevianamide K的高效液相色谱图。
(五)具体实施方式
本发明结合附图和实施例,对本发明的上述内容做进一步的详细说明,但不应将此理解为本发明上述主题的范围仅限于下述的实例,凡基于本发明上述内容所实现的技术均属于本发明的范围。
本发明所述室温是指25-30℃。所述PDA培养基组成为马铃薯200g/L、葡萄糖20g/L、琼脂18g/L,溶剂为蒸馏水,pH自然。
实施例1、冬虫夏草定殖真菌—花斑曲霉Aspergillus versicolor ZJUTE2高产Brevianamide K培养基筛选
1、菌株活化
取出保存于-80℃冰箱的Aspergillus versicolor ZJUTE2菌株冻存管,于4℃解冻后,在超净台内用无菌棉棒从冻存管蘸取适量孢子悬液,接种至PDA培养基,于28℃恒温培养箱中静置培养7天,进行菌种活化。
活化7天后,用10mL含有体积浓度2%吐温80的无菌水将菌株孢子洗下,置于装有100mL含体积浓度2%吐温80的无菌水中,得到孢子悬液(孢子浓度为1×106个/mL);分别移取100μL孢子悬液,接种至于121℃高温灭菌20min后,冷却的PDB培养基(马铃薯葡萄糖肉汤350mg,蒸馏水10mL)、沙氏培养基(蛋白胨100mg、葡萄糖400mg、蒸馏水10mL)、察氏培养基(硝酸钠30mg、磷酸氢二钾10mg、硫酸镁5mg、氯化钾5mg、硫酸亚铁0.1mg、蔗糖300mg、蒸馏水10mL)的50mL离心管中,于28℃恒温、180rpm的摇床中暗培养7天,获得不同的液体培养基发酵产物;另移取100μL孢子悬液,接种至装有大米培养基(大米4g,蒸馏水10mL,于121℃高温灭菌20min后,冷却)的50mL离心管中,于28℃恒温暗培养7天,获得大米发酵产物。
3、菌悬液萃取
将10mL乙酸乙酯分别加入到液体培养基发酵产物中进行萃取,取乙酸乙酯层减压浓缩至干,得各液体培养基发酵产物乙酸乙酯萃取物,用甲醇溶解后,采用HPLC检测Brevianamid K含量。
大米发酵产物捣碎后,加入10mL的甲醇浸泡1天,过滤,滤液减压浓缩得粗提物浸膏,该粗提物浸膏用10mL蒸馏水悬浮,用10mL乙酸乙酯萃取1次,取乙酸乙酯层减压浓缩至干,得大米培养基发酵产物乙酸乙酯萃取物,用甲醇溶解后,采用HPLC检测Brevianamid K含量。
HPLC检测条件为:仪器:Agilent 1200series;色谱柱:Cosmosil 5C18-PAQ(4.6mmI.D.×250mm);流动相:甲醇-水(50:50﹣80:20,v/v);流速:1mL/min;检测波长:305nm;进样量:20μL;柱温:室温。
HPLC分析结果显示,大米培养基发酵产物乙酸乙酯萃取物中Brevianamid K含量最高。
实施例2、冬虫夏草定殖真菌—花斑曲霉Aspergillus versicolor ZJUTE2的发酵培养
1、菌株活化
取出保存于-80℃冰箱的Aspergillus versicolor ZJUTE2菌株冻存管,于4℃解冻后,在超净台内用无菌棉棒从冻存管蘸取适量孢子悬液,接种至PDA培养基,于28℃恒温培养箱中静置培养7天,进行菌种活化。
2、菌株发酵
活化7天后,用10mL含有体积浓度2%吐温80的无菌水将菌株孢子洗下,置于装有100mL含体积浓度2%吐温80的无菌水中,得到孢子悬液(孢子浓度为1×106个/mL);分别移取1mL孢子悬液,接种至50个装有大米培养基(大米100g,蒸馏水135mL,于121℃高温灭菌20min后,冷却)的1L三角瓶中,于28℃恒温暗培养20天,获得50瓶大米发酵产物。
实施例3、Brevianamide K的提取分离
(1)将实施例2制备的50瓶大米发酵产物混合并捣碎,加入95%乙醇20升,室温浸提4天,过滤,滤饼重复浸提3次(每次加入3升95%乙醇,每次4天),合并提取液并减压浓缩至干,得粗提物浸膏475g。
(2)将步骤(1)粗提物浸膏(475g)用1.5L蒸馏水悬浮,用乙酸乙酯萃取,每次1.5L,共萃取3次,合并乙酸乙酯相,并减压浓缩至干,得到乙酸乙酯萃取物浸膏45g。
(3)将步骤(2)乙酸乙酯萃取物浸膏45g溶解于100mL甲醇后进行MCI CHP20P柱层析(直径d=4cm,高h=60cm),依次以体积比为30:70、40:60、50:50、60:40、70:30、80:20、90:10和100:0的甲醇/水混合溶剂为洗脱剂进行梯度洗脱,每一梯度洗脱1.5L(2个柱体积),流速15mL/min;收集甲醇/水=70:30﹣80:20(v/v)的洗脱部位,合并,减压浓缩至干,得浓缩物22.8g。
(4)往步骤(3)获得的22.8g浓缩物中加入600mL甲醇,于50℃水浴加热至溶解,于室温进行重结晶,过滤,收集黄色针状晶体,即得式(I)所示化合物13.5g,产率达到2.7g/kg大米。
实施例4、Brevianamide K的结构鉴定及纯度检测
1.Brevianamide K的结构鉴定
Brevianamide K:黄色针状晶体。如图2和图3所示,其1H-NMR和13C-NMR数据如下:1H-NMR(600MHz,CDCl3)δH8.48(1H,s,2-NH),7.64(1H,s,18-NH),7.41(1H,d,8.0,H-13),7.34(1H,m,H-16),7.26(1H,s,H-10),7.24(1H,d,7.5,H-15),7.20(1H,t,7.4,H-14),6.29(1H,t,3.1,H-8),6.12(1H,dd,17.4,10.5,H-21),5.27(1H,d,10.5,Ha-22),5.24(1H,d,17.4,Hb-22),4.25(2H,t,9.1,H2-6),2.94(2H,td,9.2,3.1,H2-7),1.58(3H,s,H3-23),1.58(3H,s,H3-24)。13C-NMR(150MHz,CDCl3)δC 155.2(C-4),154.4(C-1),144.4(C-21),144.0(C-19),134.5(C-17),133.9(C-9),126.3(C-12),126.1(C-3),122.5(C-15),121.2(C-14),119.9(C-13),119.0(C-8),113.4(C-16),111.4(C-22),111.3(C-10),103.3(C-11),45.9(C-6),39.3(C-20),28.3(C-7),27.5(C-23),27.5(C-24)。以上波谱数据与文献(Zhang,S.S.et al.Chem.Nat.Compds.2020,56,964-967.)报道的Brevianamide K一致。因此,实施例2制备的晶体即为式(I)所示Brevianamide K。
2.Brevianamide K的纯度检测
1)将实施例3制备所得的Brevianamide K用甲醇溶解,定量稀释配制成浓度为100μg/mL的样品溶液。
2)取样品溶液进行高效液相色谱分析,色谱条件如下。仪器:Agilent1200series;色谱柱:Cosmosil 5C18-PAQ(4.6mm I.D.×250mm);流动相:甲醇-水(50:50﹣80:20,v/v);流速:1mL/min;检测波长:305nm;进样量:20μL;柱温:室温。检测记录色谱图。
3)待测样品的高效液相色谱检测结果如图4所示。Brevianamide K的保留时间为14.527min,峰面积分析其纯度达98.61%。
以上所述的实施例只是本发明的较佳方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (8)
1.一种吲哚二酮哌嗪生物碱的制备方法,其特征在于,所述方法按如下步骤进行:(1)将花斑曲霉(Aspergillus versicolor)ZJUTE2接种至大米固体培养基,在20-30℃条件下暗培养15-30天,获得大米发酵产物;所述花斑曲霉ZJUTE2,保藏于中国典型培养物保藏中心,保藏日期为2022年1月13日,保藏编号为CCTCC NO:M2022064,地址为中国,武汉,武汉大学;所述大米固体培养基为大米和蒸馏水的混合物,其中蒸馏水用量以大米质量计为1-5mL/g;(2)将大米发酵产物中加入有机溶剂,室温浸提,提取液浓缩至无液体流出,获得粗提物浸膏;(3)将步骤(2)粗提物浸膏用水悬浮,以有机溶剂萃取,收集有机相,减压浓缩至干,得到萃取物浸膏;(4)将步骤(3)萃取物浸膏用甲醇溶解后进行MCI CHP20P柱层析,以体积浓度30-100%甲醇-水为洗脱液,收集体积浓度70-80%的甲醇-水洗脱部位,减压浓缩至干,得浓缩物;(5)将步骤(4)所得浓缩物用甲醇溶解,于室温重结晶,过滤,得晶体,即得到式(Ⅰ)所示吲哚二酮哌嗪生物碱;
2.如权利要求1所述的制备方法,其特征在于,步骤(1)所述花斑曲霉ZJUTE2接种至大米固体培养基前,先进行活化培养,再将活化后的菌悬液以体积浓度0.1-1%的接种量接种大米固体培养基,所述活化是指将花斑曲霉ZJUTE2接种至PDA培养基,28℃培养,使菌株活化,将活化后的菌株用含体积浓度2%吐温80的无菌水重悬,得到菌悬液。
3.如权利要求1所述的制备方法,其特征在于,步骤(1)中发酵培养条件为:28℃条件下暗培养20天。
4.如权利要求1所述的制备方法,其特征在于,步骤(2)中所述有机溶剂为95%乙醇、甲醇或丙酮;所述有机溶剂体积用量以大米固体培养基中大米干重计为2~8mL/g。
5.如权利要求1所述的制备方法,其特征在于,步骤(2)所述浸提至少进行3次,每次提取时间为3~5天。
6.如权利要求1所述的制备方法,其特征在于,步骤(3)中所述水体积用量以粗提物浸膏重量计为2-5mL/g;所述有机溶剂为乙酸乙酯,所述有机溶剂与水的体积比为1:0.5-3;萃取3-5次。
7.如权利要求1所述的制备方法,其特征在于,步骤(4)方法为:萃取物浸膏以甲醇溶解后进行MCI CHP20P柱层析,依次以体积比为30:70、40:60、50:50、60:40、70:30、80:20、90:10和100:0的甲醇/水混合溶剂为洗脱剂进行梯度洗脱,每一梯度洗脱2~5个柱体积,流速10-20mL/min;收集体积比70:30-80:20的甲醇-水洗脱部位,减压浓缩至干,得浓缩物。
8.如权利要求1所述的制备方法,其特征在于,步骤(5)甲醇体积用量以浓缩物质量计为20-30mL/g。
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