CN107619803B - 一种暗黄链霉酸和暗黄链霉酮的制备及其医药用途 - Google Patents
一种暗黄链霉酸和暗黄链霉酮的制备及其医药用途 Download PDFInfo
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Abstract
本发明提供的一种极暗黄链霉菌,从海洋动物纵条肌海葵(Haliplanella lineata)中分离获得。分类命名为Streptomycesfulvissimus ZZ406,已被中国典型培养物保藏中心‑武汉中心保藏,保藏编号:CCTCC M 2017435,保藏日:2017年8月3日。利用该极暗黄链霉菌获得的化合物暗黄链霉酸(1)和暗黄链霉酮(2)具有抗胶质瘤活性,可显著地抑制不同脑胶质瘤细胞的增殖和明显降低胶质瘤细胞糖酵解代谢关键酶的蛋白表达水平,具有独特的抗肿瘤作用机制,可在在制备治疗脑胶质瘤药物中的应用。化合物暗黄链霉酸(1)和暗黄链霉酮(2)的结构式如下:
Description
技术领域
本发明属医药领域,涉及从海洋放线菌极暗黄链霉菌(StreptomycesfulvissimusZZ406)中制备抗肿瘤活性化合物暗黄链霉酸和暗黄链霉酮的方法及它们在制备治疗脑胶质瘤药物方面的应用。
背景技术
胶质瘤是脑颅内最常见和死亡率高的脑肿瘤。由于胶质瘤多位于许多脑的重要功能区,使得手术难度大而且不容易将肿瘤全部切除,所以放疗和药物对胶质瘤的治疗更为重要。但是,目前抗胶质瘤药物严重缺乏,主要包括传统的卡氯芥(carmustine)、罗莫司丁(lomustine)、丙卡巴肼(procarbazine)和第二代的替莫唑胺(temozolomide,TMZ)等少数抗胶质瘤药物,其中替莫唑胺是唯一可选择的单独用于胶质瘤治疗的一线药物。而且,包括替莫唑胺在内的现有多数治疗胶质瘤药物都是细胞毒的烷化剂,多有疗效有限、毒副作用大和严重耐药性的不足。因此,临床上迫切需要疗效更好、安全性更高和作用机制独特的新型抗胶质瘤药物。来源于海洋微生物的次生代谢产物是抗肿瘤药或抗肿瘤药先导化合物发现的重要资源。肿瘤细胞代谢重编程为新型抗胶质瘤药物的研究与开发提供了新靶标。
肿瘤细胞的过度糖酵解(Glycolysis)以满足其细胞快速无限制的增殖是肿瘤细胞不同于正常细胞的突出代谢特征之一。肿瘤细胞从微环境中摄取大量葡萄糖在糖酵解环节由胶质瘤细胞特异依赖而高表达的多个关键酶包括己糖激酶2(HK2)、磷酸果糖激酶/果糖2,6-二磷酸酶(PFKFB3)、丙酮酸激酶M2(PKM2)和乳酸脱氢酶5(LDH5)等共同参与产生大量中间产物和终产物乳酸,这些中间产物是肿瘤细胞合成生物大分子的重要起始原料,而乳酸分泌至细胞外可抑制机体免疫细胞对肿瘤细胞的清除能力,可促进肿瘤细胞扩散,高表达的糖酵解关键酶HK2还可提高肿瘤细胞对放疗和替莫唑胺化疗的抗性。不难理解,正是肿瘤细胞高度利用其代谢中间产物合成生物大分子DNA、RNA、蛋白质和生物膜的能力促使其快速无限制地增殖,调控肿瘤细胞糖酵解的特异关键酶可有效抑制肿瘤细胞的增殖。因此,靶向肿瘤代谢特异关键酶的多靶点作用的药物可能具有更好的抗肿瘤疗效。暗黄链霉酸和暗黄链霉酮是从海洋细菌极暗黄链霉菌(StreptomycesfulvissimusZZ406)中分离得到的两个新化合物,它们对多种不同胶质瘤细胞的增殖具有显著的抑制作用,可显著降低胶质瘤细胞糖酵解的重要特异关键酶HK2、PFKFB3、PKM2和LDH5的蛋白表达水平,具有独特的抗肿瘤作用机制,因此,暗黄链霉酸和暗黄链霉酮在制备抗胶质瘤药物方面具有应用前景。
发明内容
本发明的目的是提供一种极暗黄链霉菌,分类命名为StreptomycesfulvissimusZZ406,已被中国典型培养物保藏中心-武汉中心保藏,保藏编号:CCTCC M 2017435,保藏日:2017年8月3日。所述极暗黄链霉菌从海洋动物纵条肌海葵(Haliplanella lineata)中分离,通过以下步骤分离培养而获得:
(1)极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的分离培养
取一定量的新鲜纵条肌海葵(Haliplanella lineata)用灭菌的天然海水清洗三次后匀浆,制成不同浓度的悬浮液。取一定量不同浓度的悬浮液均匀分散到含有固体培养基的培养皿中,在室温条件下培养一定时间后,将不同的菌落分别转移到另一含有固体培养基的培养皿中,在室温条件下继续培养一定时间。最后将生长良好的单一菌落(ZZ406)接种到斜面培养基培养后置4℃冰箱保存备用。
所述的纵条肌海葵(Haliplanella lineata)是从浙江普陀山海滩的岩石缝中获得,天然海水是从浙江舟山市附近的东海海域中获得;所述悬浮液的不同浓度为1×10-4~1×10-1g/mL;所述的样品悬浮液的取样量为100~300μL;所述培养皿所含的固体培养基为高氏琼脂(Gause′s agar)培养基或其它固体培养基;所述的斜面培养基为高氏琼脂培养基或其它固体斜面培养基;所述的室温培养温度为22~28℃;所述的培养时间为5~15天。
(2)极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌种鉴定
上述步骤(1)分离培养所获得的菌株ZZ406用目前实验室普遍使用的16S rDNA序列分析方法鉴定其种类,确定为极暗黄链霉菌,分类命名为StreptomycesfulvissimusZZ406,已被中国典型培养物保藏中心-武汉中心保藏,保藏编号:CCTCC M 2017435。
本发明的第二个目的是提供两个具有抗胶质瘤活性的化合物暗黄链霉酸(1)和暗黄链霉酮(2),所述的暗黄链霉酸和暗黄链霉酮为新化合物,其化学结构式为:
本发明的第三个目的是提供暗黄链霉酸(1)和暗黄链霉酮(2)的制备方法,通过以下步骤实现:
(1)极暗黄链霉菌(Streptomycesfulvissimus ZZ406)发酵菌液的制备
取极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌落接种到含有一定量的液体菌种培养基的大三角烧瓶中,将含有ZZ406菌种的培养液在室温条件下振荡培养一定时间后得到菌种液。最后将菌种液转入含有一定量的液体发酵培养基的大三角烧瓶中,在室温条件下振荡培养一定时间后,得到具有抗肿瘤活性的ZZ406的培养液。
所述的菌种培养基为液体高氏培养基,所述的用量为150-250mL;所述的液体发酵培养基为SC液体培养基(可溶性淀粉10克,干酪素0.3克,硝酸钾2克,硫酸镁0.5克,磷酸氢二钾2克,碳酸钙0.02克,硫酸亚铁0.01克,天然海水1升,pH值7.2);所述的大三角培养瓶为500mL;所述的室温培养温度为22~28℃;所述振荡的转速为160~180rpm;所述的培养时间为5~15天。
(2)暗黄链霉酸和暗黄链霉酮的提取分离纯化
菌株ZZ406的发酵菌丝体用甲醇提取,甲醇提取物再用乙酸乙酯萃取得到乙酸乙酯萃取物。乙酸乙酯萃取物先用正相硅胶柱层析分离,用乙酸乙酯和环己烷混合溶剂梯度洗脱,并用薄层层析分析,得到九个组分(Fr.1~Fr.9)。组分Fr4和组分Fr 8再分别用制备型高效液相色谱(HPLC)仪分离,得到纯化合物暗黄链霉酸(1)和暗黄链霉酮(2)。
所述柱层析的正相硅胶用量与上量的样品量比例是30~50g:1.0g;所述的高效液相分离条件是:创新通恒CXTH-3000高效液相色谱仪,富士C18CT-30色谱柱(280×30mm,10μm),甲醇和水为流动相,检测波长220nm,流速为10.0mL/min。
(3)暗黄链霉酸和暗黄链霉酮的结构鉴定
暗黄链霉酸和暗黄链霉酮的结构是根据它们的一维和二维NMR光谱、高分辨质谱数据和ECD计算等方法而确定的。
本发明的第四个目的是提供暗黄链霉酸和暗黄链霉酮在制备治疗脑胶质瘤药物中的应用。所述的暗黄链霉酸和暗黄链霉酮均可显著抑制多种胶质瘤细胞的增殖,降低胶质瘤细胞糖酵解途径中多个关键酶的蛋白水平表达,具有独特的抗肿瘤作用机制。
所述药物为暗黄链霉酸或暗黄链霉酮单独,或它们两者的组合,或单独或组合与其它活性物质一起,与药学上可接受的载体组成的药物。
本发明提供的极暗黄链霉菌(Streptomycesfulvissimus ZZ406),从海洋动物纵条肌海葵(Haliplanella lineata)中分离获得。研究表明,利用该极暗黄链霉菌获得的化合物暗黄链霉酸(1)和暗黄链霉酮(2)具有抗胶质瘤活性,可显著地抑制不同脑胶质瘤细胞的增殖和明显降低胶质瘤细胞糖酵解代谢关键酶的蛋白表达水平,具有独特的抗肿瘤作用机制,可在在制备治疗脑胶质瘤药物中的应用。
附图说明
图1是极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌落图。
图2是暗黄链霉酸的氢谱。
图3是暗黄链霉酸的碳谱。
图4是暗黄链霉酸的HMBC谱。
图5是暗黄链霉酸主要的HMBC相关示意图。
图6是暗黄链霉酸的高分辨质谱。
图7是暗黄链霉酮的氢谱。
图8是暗黄链霉酮的碳谱。
图9-10是暗黄链霉酮的1H-1HCOSY谱。
图11是暗黄链霉酮的HSQC谱。
图12是暗黄链霉酮的HMBC谱。
图13是暗黄链霉酮主要的1H-1H COSY和HMBC相关示意图。
图14是暗黄链霉酮的高分辨质谱。
图15是暗黄链霉酸和暗黄链霉酮对U87-MG细胞不同代谢关键酶HK2、PFKFB3、PKM2、LDH5、ACO2和CytoC的蛋白表达水平的影响。
具体实施方式
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
1.极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的分离培养
取新鲜纵条肌海葵(Haliplanella lineata)5克,用灭菌的天然海水清洗三次后匀浆,制成浓度为1×10-1g/mL的悬浮液。将1×10-1g/mL的悬浮液依次稀释成浓度为1×10-2、1×10-3和1×10-4g/mL的悬浮液。取各浓度的悬浮液200μL均匀分散到含有高氏琼脂(Gause′s agar)固体培养基的培养皿中,在28℃条件下培养5天后,将不同的菌落分别转移到另一含有高氏琼脂(Gause′sagar)固体培养基的培养皿中,在28℃条件下继续5天。最后将生长良好的单一菌落(ZZ406)接种到高氏琼脂固体斜面培养基培养后置4℃冰箱保存备用。
2.极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌种鉴定
使用16S rDNA序列分析方法鉴定所获得菌株ZZ406的种类。
2.1实验试剂及仪器
PCR试剂:EX Taq酶(TaKaRa),dNTP(TaKaRa),引物(Invitrogen合成),引物序列是:
SEQ ID No:1:AGAGTTTGATCCTGGCTCAG
SEQ ID No:2:GGTTACCTTGTTACGACTT
Marker:DL5000
实验仪器:离心机,电泳仪,PCR仪,ABI 3730XL测序仪。
2.2实验步骤
细菌基因组DNA抽提
电泳检测
PCR扩增
a.PCR反应体系
b.PCR反应条件
c.电泳检测
d.测序:切胶纯化测序
e.分析结果:拼接序列。
2.3实验结果
拼接后的序列为SEQ ID No:3:
TGCAGTCGACGATGAAGCCGCTTCGGTGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAATACTCCTGCCTGCATGGGTGGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCTTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGCCGTGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGA
以上获得的16S rDNA序列与美国NIH的NCBI GenBank数据库比较,其结果表明:菌株ZZ406的16S rDNA序列与GenBank库中的Streptomyces fulvissimus DSM 40593的16SrDNA序列有99%的相似性(登录号:NC 021177.1)。因此,本发明所获得的海洋菌株ZZ406定为极暗黄链霉菌(Streptomycesfulvissimus ZZ406)(附图1)。所获得的极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌株已被中国典型培养物保藏中心-武汉中心保藏,保藏编号:CCTCC M 2017435,保藏日:2017年8月3日。
实施例2 极暗黄链霉菌(Streptomycesfulvissimus ZZ406)发酵菌液的制备
取高氏琼脂固体斜面培养基的极暗黄链霉菌(Streptomycesfulvissimus ZZ406)的菌接种到含有250mL液体高氏培养基的500mL三角烧瓶中,将含有ZZ406菌种的培养液在28℃条件下旋转(180rpm)振摇培养5天后得到菌种液。将5mL菌种液转入到含有250mL的SC液体培养基(可溶性淀粉10克,干酪素0.3克,硝酸钾2克,硫酸镁0.5克,磷酸氢二钾2克,碳酸钙0.02克,硫酸亚铁0.01克,天然海水1升,pH值7.2)的500mL三角烧瓶中,在28℃条件下旋转(180rpm)振摇培养13天,得到具有抗肿瘤活性的ZZ406的发酵菌液。
实施例3
1.暗黄链霉酸和暗黄链霉酮的提取分离纯化
实施例2获得的发酵菌液(70.0升)过滤得到菌丝体。菌丝体用甲醇提取三次得到甲醇提取物。甲醇提取物再用乙酸乙酯萃取得到乙酸乙酯萃取物(8.0克),乙酸乙酯萃取物用硅胶(250克)柱层析分离,用环己烷和乙酸乙酯(4:1到1:1)梯度洗脱,用薄层层析分析各洗脱液,合并含有相同成分的组分,总共得到9个组分(Fr.1~Fr.9)。组分Fr.4(30毫克)用半制备型高效液相色谱仪分离(仪器:创新通恒CXTH-3000;色谱柱:富士C18CT-30,280×30mm,10μm;流动相:甲醇/水,75/25;检测波长:220nm,流速:10.0mL/min),得到化合物暗黄链霉酸(1,9.3mg,tR 31min)。同样,用相同的高效液相色谱分离条件(但不同的流动相:甲醇/水,21/79)分离组分Fr.8(20mg)得到纯化合物暗黄链霉酮(2,8.0mg,tR 35min)。
2.暗黄链霉酸和暗黄链霉酮的结构鉴定
暗黄链霉酸(1):黄色粉末;分子式C16H10O6;紫外吸收UV(MeOH)λmax(logε)217(4.32),279(4.22),411(3.66)nm;高分辨质谱(HRESIMS)为m/z[M-H]-297.0407(计算值C16H9O6297.0399)。根据暗黄链霉酸的1H谱(附图2)、13C谱(附图3)、HMBC谱(附图4、附图5)和高分辨质谱(附图6),确定了暗黄链霉酸的结构,为一个新化合物,其13C和1H NMR信号归属见表一。
表一、暗黄链霉酸的13C和1H NMR数据(溶剂:氘代二甲基亚砜DMSO-d6)
暗黄链霉酮(2):黄色粉末;分子式C16H16O6;[α]D 25-5.6(c 0.20,MeOH);紫外吸收UV(MeOH)λmax(logε)224(4.09),302(3.63)nm;高分辨质谱(HRESIMS)为m/z[M+H]+305.0998(计算值C16H17O6 305.1025)。根据暗黄链霉酮的1H谱(附图7)、13C谱(附图8)、1H-1H COSY谱(附图9、附图10、附图13)、HSQC谱(附图11)、HMBC谱(附图12、附图13)、高分辨质谱(附图14)和ECD谱计算,确定了暗黄链霉酮的结构,为一个新化合物,暗黄链霉酮的13C和1H NMR信号归属见表二。
表二、暗黄链霉酮(2)的13C和1H NMR数据(溶剂:氘代二甲基亚砜DMSO-d6)
实施例4 暗黄链霉酸和暗黄链霉酮的活性
1.暗黄链霉酸和暗黄链霉酮抑制胶质瘤细胞增殖的作用
人脑胶质瘤U251细胞用DMEM和10%FBS培养基,而胶质瘤U87MG细胞、SHG44细胞和正常人胶质细胞HA分别用MEM培养基、RPMI-1640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)法测定暗黄链霉酸和暗黄链霉酮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。实验结果表明:暗黄链霉酸和暗黄链霉酮显著抑制三种不同胶质瘤细胞的增殖,其IC50值为4.7-25.8μM,其中暗黄链霉酸的活性更强,其IC50值为4.7-8.1μM(表三)。同时也测定了暗黄链霉酸和暗黄链霉酮对正常胶质细胞(HA)的细胞毒性,它们的CC50值均大于100μM,暗黄链霉酸的选择性指数(CC50/IC50)为大于12.3到21.3,而暗黄链霉酮的选择性指数(CC50/IC50)为大于3.8到4.6,与阳性对照药阿霉素类似。结果表明:暗黄链霉酸对胶质瘤细胞的选择性作用要高于阳性对照药阿霉素,换言之,暗黄链霉酸对正常胶质细胞的毒性要低于对照药阿霉素(表三)。
表三、暗黄链霉酸和暗黄链霉酮抑制胶质瘤细胞和正常胶质细胞增殖的作用
2.暗黄链霉酸和暗黄链霉酮对胶质瘤细胞代谢酶的影响
蛋白样品的制备:人胶质瘤U87MG细胞用MEM和10%FBS培养基在37℃和5%二氧化碳的孵化箱中培养,经过三代培养的细胞用于本发明的实验研究。细胞(1.5×107)接种于10厘米的培养皿中,贴壁24h后加入暗黄链霉酸(1,30μM)、暗黄链霉酮(2,60μM)、或2-脱氧葡萄糖(2-DG,6mM)共培养48小时后,先用冰冷的PBS缓冲液洗两次,后用冰冷的裂解缓冲液(200μL)裂解15分钟。裂解液经4℃低温高速(11200rpm)离心,上清液为蛋白样品。
蛋白含量的测定:使用BCA试剂盒测定各个蛋白样品的蛋白含量。将试剂盒中试剂A和B以50:1比例配成工作试剂液,用牛血清白蛋白(BSA)将标准品BCA稀释配成不同浓度的BCA标准溶液。取10μL标准溶液或蛋白样品液和200μL工作试剂液混匀后在37℃孵化30分钟.孵化液用酶标仪在562nm波长测定吸收度。以BCA量为横坐标,吸收度为纵坐标绘制标准曲线,计算回归方程。从回归方程计算各蛋白样品液的蛋白含量。
Western Blotting:含等量蛋白(15μg)的各样品用10%十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,将凝胶电泳蛋白图谱转到聚偏氟乙烯膜(PVDF)上,PVDF膜用还有5%去脂牛奶的0.1%TBST在室温下封闭2小时。封闭后的PVDF膜先与测试酶的一抗在4℃孵育过夜,用TBST洗膜;后与HRP标记的二抗在室温孵育2小时。TBST洗膜三次后,用增强化学发光试剂检测免疫反应性,然后显影,水洗,定影,水洗,晾干,观察实验结果。以β-肌动蛋白(β-actin)作为内参对照。
实验结果(附图15)表明:与阴性对照组(没有药物处理的U87MG细胞,CON)比较,暗黄链霉酸(1)和暗黄链霉酮(2)均可显著降低肿瘤细胞糖酵解的关键酶HK2、PFKFB3、PKM2和LDH5的蛋白水平表达。但是,暗黄链霉酸和暗黄链霉酮对三羧酸循环酶顺乌头酸酶2(ACO2)和氧化磷酸化酶细胞色素C(CytoC)的蛋白表达水平影响不明显。CON:U87-MG细胞对照组;CON:阴性对照组;1:暗黄链霉酸处理组;2:暗黄链霉酮处理组;2-DG:2-去氧葡萄糖;HK2:己糖激酶2:PFKFB3:磷酸果糖激酶/果糖2,6-二磷酸酶;PKM2:丙酮酸激酶M2;LDH5:乳酸脱氢酶5;ACO2:顺乌头酸酶2;CytoC:细胞色素C;β-actin:β-肌动蛋白,内部参照)。已知三羧酸循环和氧化磷酸化是正常细胞主导的代谢途径,而ACO2和CytoC分别是三羧酸循环途径和氧化磷酸化途径的关键酶之一。以上结果及分析表明:暗黄链霉酸和暗黄链霉酮两个活性化合物可能是通过选择性影响胶质瘤糖酵解的多个关键酶而产生其抗胶质瘤活性,具有独特的抗肿瘤作用机制。
总之,本发明从海洋动物纵条肌海葵(Haliplanella lineata)中分离到一种海洋放线菌极暗黄链霉菌(Streptomycesfulvissimus ZZ406),该菌可以产生抗胶质瘤活性化合物暗黄链霉酸和暗黄链霉酮。本发明提供了极暗黄链霉菌的分离培养方法、活性化合物暗黄链霉酸和暗黄链霉酮的提取分离纯化方法以及暗黄链霉酸和暗黄链霉酮的抗胶质瘤活性。由于暗黄链霉酸和暗黄链霉酮具有良好的抗胶质瘤活性和独特的调控肿瘤细胞代谢酶的抗肿瘤作用机制,所以,暗黄链霉酸和暗黄链霉酮在制备治疗脑胶质瘤药物方面具有应用前景。
序列表
<110> 浙江大学
<120> 一种暗黄链霉酸和暗黄链霉酮的制备及其医药用途
<160> 3
<170> SIPOSequenceListing 1.0
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<213> 人工设计(Unknown)
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agagtttgat cctggctcag 20
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<212> DNA
<213> 人工设计(Unknown)
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ggttaccttg ttacgactt 19
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<211> 1390
<212> DNA
<213> Streptomyces fulvissimus ZZ406
<400> 3
tgcagtcgac gatgaagccg cttcggtggt ggattagtgg cgaacgggtg agtaacacgt 60
gggcaatctg cccttcactc tgggacaagc cctggaaacg gggtctaata ccggataata 120
ctcctgcctg catgggtggg ggttgaaagc tccggcggtg aaggatgagc ccgcggccta 180
tcagcttgtt ggtggggtaa tggcctacca aggcgacgac gggtagccgg cctgagaggg 240
cgaccggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300
atattgcaca atgggcgaaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg 360
ggttgtaaac ctctttcagc agggaagaag cgcaagtgac ggtacctgca gaagaagcgc 420
cggctaacta cgtgccagca gccgcggtaa tacgtagggc gcaagcgttg tccggaatta 480
ttgggcgtaa agagctcgta ggcggcttgt cacgtcggat gtgaaagccc ggggcttaac 540
cccgggtctg cattcgatac gggctagcta gagtgtggta ggggagatcg gaaattcctg 600
gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg 660
gccattactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720
gtccacgccg taaacgttgg gaactaggtg ttggcgacat tccacgtcgt cggtgccgca 780
gctaacgcat taagttcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840
tgacgggggc ccgcacaagc agcggagcat gtggcttaat tcgacgcaac gcgaagaacc 900
ttaccaaggc ttgacatata ccggaaagca tcagagatgg tgcccccctt gtggtcggta 960
tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020
cgagcgcaac ccttgttctg tgttgccagc atgcctttcg gggtgatggg gactcacagg 1080
agactgccgg ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat 1140
gtcttgggct gcacacgtgc tacaatggcc ggtacaatga gctgcgatgc cgtgaggcgg 1200
agcgaatctc aaaaagccgg tctcagttcg gattggggtc tgcaactcga ccccatgaag 1260
tcggagttgc tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt 1320
acacaccgcc cgtcacgtca cgaaagtcgg taacacccga agccggtggc ccaacccctt 1380
gtgggaggga 1390
Claims (1)
1.一种极暗黄链霉菌(Streptomyces fulvissimus),分类命名为Streptomycesfulvissimus ZZ406,已被中国典型培养物保藏中心-武汉中心保藏,保藏编号: CCTCC NO:M 2017435,保藏日:2017年8月3日。
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