CN110218759B - 抗胶质瘤活性物质青霉碱醚a的制备和用途 - Google Patents
抗胶质瘤活性物质青霉碱醚a的制备和用途 Download PDFInfo
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Abstract
本发明提供一种抗胶质瘤活性物质青霉碱醚A的制备方法,利用一株海洋灰黄青霉菌株ZZ380,用大米培养基培养灰黄青霉,大大提高了抗胶质瘤活性物质青霉碱醚A的产率,克服了现有方法制备青霉碱醚A产量低的不足。所述青霉碱醚A对人胶质瘤细胞增殖具有显著的抑制活性,显示其抗胶质瘤活性强的特色,可与药学上可接受的载体组成各种剂型的药物,用于治疗胶质瘤。因此,青霉碱醚A在制备抗胶质瘤药物方面具有好的应用前景。
Description
技术领域
本发明属医药领域,涉及一种抗胶质瘤活性物质青霉碱醚A的制备和在制备治疗胶质瘤药物中的用途。
背景技术
胶质瘤是全球最常见和死亡率最高的脑肿瘤。由于胶质瘤在生物学行为上呈现浸润性生长,而且多位于脑的重要功能区,要在保证患者脑组织功能完整的情况下将肿瘤完全切除极为困难,所以药物对胶质瘤的防治显得极为重要。替莫唑胺是目前临床上唯一可单独用于胶质瘤治疗的一线药物(金标药)。但是包括替莫唑胺和传统的第一代抗胶质瘤药物卡莫司汀、洛莫司汀、丙卡巴肼和顺铂等均是细胞毒的烷化剂,具有毒副作用大、耐药性和疗效有限等严重不足,而且,新型靶向药物对胶质瘤的效果也不理想。因此,迫切需要研发新型抗胶质瘤药物。
海洋特殊的生态环境,使海洋微生物具有与陆地微生物不同的新陈代谢途径、生存繁殖方式和适应机制,因而产生许多陆地微生物无法产生的化学结构新颖的次级代谢产物。这些代谢物质是发现新型抗肿瘤活性物质或药物先导化合物的重要资源。
中国专利(申请号:2018113087533)公开了抗耐药菌活性化合物青霉碱醚A的制备及其在制备抗耐甲氧西林金黄色葡萄球菌药物方面的应用,所述的青霉碱醚A是从海洋来源的灰黄青霉(Penicillium griseofulvum)菌株ZZ380在PDB液体培养基中产生的代谢产物。但是,该化合物在PDB液体培养基中的产率很低(0.29μg/mL)。
发明内容
本发明的第一个目的是提供抗胶质瘤活性物质青霉碱醚A的制备,所述的青霉碱醚A的化学结构式为:
所述的青霉碱醚A的制备方法,克服现有方法制备青霉碱醚A产量低的不足,大大提高青霉碱醚A的产量,并通过以下步骤实现:
(1)灰黄青霉(Penicillium griseofulvum)ZZ380的菌种,所述的灰黄青霉的菌株保藏于中国典型培养物保藏中心,分类命名为灰黄青霉(Penicillium griseofulvum)ZZ380,保藏编号CCTCC NO:M 2018344,保藏日2018.06.04,保藏地址:中国-武汉-武汉大学。
(2)灰黄青霉(Penicillium griseofulvum)ZZ380培养物的制备
将灰黄青霉(Penicillium griseofulvum)ZZ380的菌种接种到还有大米培养基的三角烧瓶中,在室温条件下静置培养一定时间,得到含有青霉碱醚A的大米培养物。
所述的三角烧瓶为500毫升;所述的每个三角烧瓶的大米培养基的组成为大米40克和3.5%海盐水60毫升;所述的室温条件为20~30℃;所述的培养时间为25~50天。
(3)青霉碱醚A的提取分离
菌株ZZ380的大米培养物用乙酸乙酯提取得到粗提取物。将该粗提取物先用十八烷基硅烷键合硅胶柱层析分离,分别用50%、70%和90%的甲醇洗脱,得到组分I~III,组分III再用制备型高效液相色谱分离纯化,得到纯化合物青霉碱醚A。
所述柱层析的十八烷基硅烷键合硅胶的用量与上柱的样品量比例是25~55克:1.0克;所述的高效液相分离条件是:岛津LC-20AP高效液相色谱仪,富士C18CT-30色谱柱(280×30 mm,10μm),甲醇和水为流动相(91/9,体积比),检测波长210nm,流速为15.0mL/min。
(4)青霉碱醚A的结构鉴定
青霉碱醚A的结构是根据它的紫外光谱、一维和二维核磁共振谱、高分辨质谱数据和X 射线单晶衍射方法而确定。
本发明的第二个目的是提供所述的抗胶质瘤活性物质青霉碱醚A在制备治疗胶质瘤药物中的应用。所述的青霉碱醚A在抑制人胶质瘤细胞增殖方面的效果非常显著,可与药学上可接受的载体组成各种剂型的药物,用于治疗胶质瘤。
本发明通过研究发现,用大米培养基培养灰黄青霉(Penicillium griseofulvum)ZZ380,大大提高了青霉碱醚A的产率(46.7μg/g),可以克服现有方法制备青霉碱醚A产量低(0.29μg/g) 的不足。本发明还发现,青霉碱醚A对人胶质瘤细胞增殖具有显著的抑制活性,显示其抗胶质瘤活性强的特色。因此,青霉碱醚A在制备抗胶质瘤药物方面具有好的应用前景。总之,本发明利用已知的一株海洋灰黄青霉菌株ZZ380,通过特定培养方法,大大提高了抗胶质瘤活性物质青霉碱醚A的产量,克服了现有方法制备青霉碱醚A的不足,并首次证明了青霉碱醚A显著抑制人胶质瘤细胞增殖的活性,可为胶质瘤的治疗提供一种新的药物分子。因此,所述的青霉碱醚A在制备抗胶质瘤药物方面具有应用潜力。
附图说明
图1是灰黄青霉(Penicillium griseofulvum)ZZ380的菌落图。
图2是青霉碱醚A的紫外光谱图。
图3是青霉碱醚A的高分辨质谱。
图4~6是青霉碱醚A的NMR氢谱。
图7~9是青霉碱醚A的NMR碳谱。
图10是青霉碱醚A的1H-1H COSY谱。
图11是青霉碱醚A的HSQC谱。
图12是青霉碱醚A的HMBC谱。
图13是青霉碱醚A的X单晶衍射获得的结构图。
图14是青霉碱醚A的1H-1H COSY和HMBC相关示意图。
具体实施例
以下结合附图和实施例对本发明作进一步详细描述,但是本发明不限于这些实施例。
实施例1.灰黄青霉(Penicillium griseofulvum)ZZ380在大米培养基中的扩大培养
首先将保存在斜面培养基上的灰黄青霉ZZ380接种至BY固体培养基(马铃薯粉6克、葡萄糖20克、琼脂20克、氯霉素0.1克、海盐35克,水1升)上复苏,再从BY培养皿中挑取单一菌落(附图1)转移至含200mL BY液体培养基(马铃薯粉6克、葡萄糖20克、氯霉素0.1 克、海盐35克,水1升)的500mL三角烧瓶中,置于震荡培养箱(180rpm,28℃)中生长5 天,用作种子液。分别用移液枪吸取6mL种子液均匀加到大米固体培养基表面,将大米培养基(大米40克和3.5%海盐水60毫升,共300瓶)在26℃实验环境下,静置培养30天,得到菌株ZZ380在大米培养基的培养物。本发明所用的菌株ZZ380为灰黄青霉(Penicillium griseofulvum)ZZ380,该菌株已被中国典型培养物保藏中心保藏,保藏编号:CCTCC NO:M 2018344,保藏日:2018.06.04,保藏地址:中国-武汉-武汉大学。
实施例2.青霉碱醚A的提取和分离
将实施例1获得的菌株ZZ380的大米培养基的培养物用乙酸乙酯萃取得到粗提物(15.1 克)。总提物用十八烷基硅烷键合硅胶(1000克)柱层析分离,分别用甲醇和水混合溶剂(50/50、 70/30、90/10,体积比)洗脱,得到三个组分I~III。组分III(2.26g)用半制备型高效液相色谱仪分离(仪器:岛津LC-20AP;色谱柱:富士C18CT-30,280×30mm,10μm;流动相:甲醇/水体系,体积比91/9;检测波长:210nm,流速:15.0mL/min),得到化合物青霉碱醚A(560mg,保留时间41.0min)。
实施例1所述的灰黄青霉培养方法和实施例2所述的青霉碱醚A的提取和分离方法与中国专利(申请号:2018113087533)公开的相应方法不同,主要区别是因为使用不同培养基和培养方法而导致青霉碱醚A产率的提高。本发明用大米固体培养基培养灰黄青霉(Penicillium griseofulvum)ZZ380,可大大提高青霉碱醚A的产率(46.7μg/g),克服现有方法(中国专利: 2018113087533)制备青霉碱醚A产率低(0.29μg/mL)的不足。
附:中国专利CN 2018113087533公开的发酵菌液制备方法。
灰黄青霉(Penicillium griseofulvum)ZZ380发酵菌液的制备:挑取马铃薯葡萄糖琼脂(PDA) 固体斜面培养基上的灰黄青霉(Penicillium griseofulvum)ZZ380,接种到含有250mL马铃薯- 葡萄糖肉汤(PDB,马铃薯100g,葡萄糖10g,海盐35g,水1L)液体培养基的500mL三角烧瓶中。将含有ZZ380菌种的培养液在28℃条件下旋转(180rpm)振摇培养3天后得到菌种液。将5mL菌种液转入到含有250mL的PDB的500mL三角烧瓶中,在28℃条件下静置培养30 天,得到含有活性物质青霉碱醚A的发酵菌液。
青霉碱醚A的提取分离纯化:将上述实验获得的发酵菌液(55.0升)离心得到菌丝体和菌液。菌丝体用甲醇提取三次得到甲醇提取物,菌液用乙酸乙酯萃取得到乙酸乙酯萃取物,合并得到总提物(23.0g)。总提物用ODS(900g)柱层析分离,分别用甲醇和水混合溶剂(40/60,60/40, 80/20,100/0,体积比)洗脱,总共得到组分I~IV。其中组分IV(0.19g)用半制备型高效液相色谱仪分离(仪器:岛津LC-20AP;色谱柱:富士C18CT-30,280×30mm,10μm;流动相:甲醇/水体系,体积比91/9;检测波长:210nm,流速:15.0mL/min),得到化合物青霉碱醚A(16.1mg,保留时间41.0min,产率:0.29μg/mL)。
实施例3.青霉碱醚A的结构鉴定
青霉碱醚A:无色块状晶体;分子式C32H41NO5;比旋光度(c 0.10,MeOH);紫外光谱(附图2)(UV,MeOH)λmax(logε)202(4.20),229(3.66),278(2.67)nm;电子圆二色谱(ECD,10mg/L,MeOH)λmax(Δε)208(-57.22),233(+64.65),286(+34.08)nm;高分辨质谱(HRESIMS)为m/z[M+H]+520.3063(计算值C32H42NO5,520.3063)和[M+Na]+542.2877(计算值C32H41NNaO5,542.2882)。通过分析青霉碱醚A的高分辨质谱(附图3)、NMR氢谱(附图4~6)、NMR碳谱(附图7~9)、1H-1H COSY谱(附图10)、HSQC谱(附图11)、HMBC谱(附图12)和X 射线单晶衍射(附图13),确定了青霉碱醚A的结构,其13C和1H核磁共振信号归属见表一。它的1H-1HCOSY和HMBC相关示意图见附图14。
实施例4.青霉碱醚A的抗胶质瘤活性
人胶质瘤U251细胞用DMEM和10%FBS培养基,而胶质瘤U87MG细胞和正常人胶质细胞HA分别用MEM培养基和AM培养基。所有细胞均在37℃和5%二氧化碳的孵化箱中经过三代培养用于本发明的实验研究。
用磺酰罗丹明B(SRB)法测定青霉碱醚A对肿瘤细胞增殖的抑制活性,阿霉素用于阳性药对照。细胞接种于96孔板中,贴壁24h后加入不同浓度的测试化合物,在孵化箱培养72h 后用SRB染色,用酶标仪测定515nm处的吸收光度值,检测肿瘤细胞的存活率,计算各测试化合物抑制胶质瘤细胞增殖的IC50值。实验结果(表二)表明:青霉碱醚A显著抑制胶质瘤U87MG和U251细胞的增殖,其IC50值1.64~5.50μM,其活性与阳性对照药阿霉素(IC50: 1.90~8.03μM)相当。同时也测定了青霉碱醚A对人正常胶质细胞的细胞毒性(CC50),其CC50值分别为23.28±1.05μM,选择性指数(CC50/IC50)为4.2~14.2。而阳性对照药阿霉素对正常胶质细胞的细胞毒活性的CC50值为8.57±0.76μM,其选择性指数为1.1~4.5。这样,青霉碱醚A与阿霉素比较,,其抗胶质瘤活性相当,但是毒性更低。因此,青霉碱醚A在制备治疗胶质瘤药物方面具有应用前景。
表一、青霉碱醚A的13C和1H NMR数据(溶剂:氘代吡啶)
a说明:δH 7.22的信号与氘代溶剂吡啶的信号重叠。
表二、青霉碱醚A抑制胶质瘤细胞和正常胶质细胞增殖的作用
序列表
<110> 浙江大学
<120> 抗胶质瘤活性物质青霉碱醚A的制备和用途
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Unknown)
<400> 1
cttggtcatt tagaggaagt aa 22
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Unknown)
<400> 2
gctgcgttct tcatcgatgc 20
<210> 3
<211> 558
<212> DNA
<213> 灰黄青霉(Penicillium griseofulvumZZ380)
<400> 3
cttccgtagg gggacctgcg gaaggatcat taccgagtgc gggcccctcg gggcccaacc 60
tcccacccgt gttgcccgaa cctatgttgc ctcggcgggc cccgcgcccg ccgacggccc 120
ccctgaacgc tgtctgaagt tgcagtctga gacctataac gaaattagtt aaaactttca 180
acaacggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga taactaatgt 240
gaattgcaga attcagtgaa tcatcgagtc tttgaacgca cattgcgccc tctggtattc 300
cggagggcat gcctgtccga gcgtcattgc tgccctcaag cccggcttgt gtgttgggcc 360
ccgtcccccc cgccgggggg acgggcccga aaggcagcgg cggcaccgcg tccggtcctc 420
gagcgtatgg ggcttcgtca cccgctctag taggcccggc cggcgccagc cgacccccaa 480
cctttaatta tctcaggttg acctcggatc agagtcaggg atacccgctg aacttaagca 540
tatcaataag cggaggaa 558
Claims (2)
1.一种抗胶质瘤活性物质青霉碱醚A的制备方法,所述的青霉碱醚A的化学结构式为:
其特征在于,通过以下步骤实现:
(1) 取灰黄青霉ZZ380的菌种,所述的灰黄青霉的菌株保藏于中国典型培养物保藏中心-武汉中心,分类命名为Penicillium griseofulvum ZZ380,保藏编号CCTCC NO:M2018344,保藏日2018.06.04;
(2) 灰黄青霉ZZ380培养物的制备
将灰黄青霉ZZ380的菌种接种到装有大米培养基的三角烧瓶中,在室温条件下静置培养一定时间,得到含有青霉碱醚A的大米培养物;其中三角烧瓶装的大米培养基的组成为大米 40 克和3.5%海盐水 60毫升;所述的室温条件为20~30℃;所述的培养时间为25~50天;
(3) 青霉碱醚A的提取分离
菌株ZZ380的大米培养物用乙酸乙酯提取得到粗提取物,将该粗提取物先用十八烷基硅烷键合硅胶柱层析分离,分别用50%、70%和90%的甲醇洗脱,得到组分I~III,组分III再用制备型高效液相色谱分离纯化,得到纯化合物青霉碱醚A;所述柱层析的十八烷基硅烷键合硅胶的用量与上柱的样品量比例是25-55 克:1.0 克;所述的高效液相分离条件是: 岛津LC-20AP高效液相色谱仪,富士C18 CT-30 色谱柱 280 30 mm,10 μm,甲醇和水为流动相,其体积比为91/9,检测波长210 nm,流速为15.0 mL/min;
(4) 青霉碱醚A的结构鉴定
青霉碱醚A的结构是根据它的紫外光谱、一维和二维核磁共振谱、高分辨质谱数据和X射线单晶衍射方法而确定。
2.权利要求1所述方法制备的抗胶质瘤活性物质青霉碱醚A在制备治疗胶质瘤药物中的应用。
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