CN111646993B - 海洋抗胶质瘤活性物质异壁咔啉碱c及制备和用途 - Google Patents
海洋抗胶质瘤活性物质异壁咔啉碱c及制备和用途 Download PDFInfo
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Abstract
本发明提供一种海洋抗胶质瘤活性物质异壁咔啉碱C的制备和用途,利用源自海洋沉积物中的蓝灰色异壁放线菌的大米发酵产物制备获得异壁咔啉碱C,蓝灰色异壁放线菌,其分类命名为Actinoalloteichus cyanogriseus ZZ1866,保藏编号:CCTCC M 2020121。所述异壁咔啉碱C显著抑制胶质瘤细胞的增殖,在制备抗胶质瘤药物方面具有应用前景。异壁咔啉碱C的化学结构式为:
Description
技术领域
本发明属医药领域,涉及从海洋来源的蓝灰色异壁放线菌Actinoalloteichuscyanogriseus ZZ1866的代谢产物中获得的具有抗胶质瘤活性的化合物异壁咔啉碱C(Actinoallocarboline C)及其制备方法和在制备抗胶质瘤药物中的应用。
背景技术
脑胶质瘤是一种最常见的原发性颅内肿瘤,具有高侵袭性、易复发等特性。目前,手术切除联合术后放化疗是胶质瘤的标准治疗模式,但患者预后不佳。在过去几十年中,尽管对胶质瘤发展的基本机制的认识有了巨大进展,但胶质瘤的治疗效果仍没有显著改变。胶质瘤无完整包膜,手术难以根除,放疗易因放射抵抗导致肿瘤复发,因此,新型抗胶质瘤药物的筛选对胶质瘤的临床治疗具有重要意义。
海洋放线菌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,其中,放线菌中除链霉菌以外的其它检出率较小的稀有放线菌也能产生许多活性化合物,如庆大霉素、小诺米星、利福霉素等,且近年来从稀有放线菌中得到新化合物的数量也呈上升态势,是药物先导化合物的重要来源。异壁放线菌Actionalloteichus是 1984年发现的稀有放线菌,于2000年经过再分类研究正式建立的一个新属。近年来的报道显示异壁放线菌能产生多种不同类型的天然活性产物,其中包括具有显著抗肿瘤活性的化合物。异壁咔啉碱C(Actinoallocarboline C)是从海洋来源的稀有放线菌蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的代谢产物中分离得到的一个结构新颖的生物碱类化合物,对人胶质瘤U87MG及U251细胞的增殖具有强的抑制作用, 在制备抗胶质瘤药物方面具有应用前景。
发明内容
本发明的第一个目的是提供一个新化合物异壁咔啉碱C(ActinoallocarbolineC), 其化学结构式为:
本发明的第二个目的是提供异壁咔啉碱C的制备方法,通过以下步骤实现:
(1)蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的分离培养
取空气干燥的海底沉积物用灭菌自来水溶解后摇床振荡过夜,将样品混悬液稀释成一定的浓度,取一定量的样品稀释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ZZ1866单一菌落接种到斜面培养基培养后置4℃冰箱保存备用。
所述的海底沉积物是从浙江省舟山市普陀岛附近的海域获得;所述样品稀释液的浓度为1×10-4~1×10-2g/mL;所述振荡的转速为160~180rpm;所述样品稀释液的取样量为200μL;所述培养皿和斜面培养的固体培养基均为高氏琼脂培养基;所述的室温培养温度为20~28℃;所述的培养时间为7~14天。
(2)蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的菌种鉴定
上述步骤(1)分离培养所获得的菌株ZZ1866用目前实验室普遍使用的16S rDNA序列分析方法鉴定其种类,确定为蓝灰色异壁放线菌,分类命名为Actinoalloteichuscyanogriseus ZZ1866,已被中国典型培养物保藏中心-武汉中心保藏,保藏编号:CCTCCNO: M 2020121。
(3)蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866固体发酵产物的制备
将上述步骤(2)获得的蓝灰色异壁放线菌Actinoalloteichus cyanogriseusZZ1866的菌株接种到含有一定量的液体培养基的大三角烧瓶中,将含有ZZ1866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固体大米培养基的大三角烧瓶中,在室温静置培养一定时间后,得到含有抗胶质瘤活性的化合物异壁咔啉碱C的ZZ1866固体发酵产物。
所述的ZZ1866菌株为产生抗肿瘤活性物质异壁咔啉碱C的异壁放线菌;所述的液体培养基为高氏液体培养基(可溶性淀粉20克,硝酸钾1克,七水合硫酸镁0.5克, 磷酸氢二钾0.5克,氯化钠0.5克,硫酸亚铁0.01克,天然海水1升),所述的用量为250mL;所述的固体培养基为大米培养基(大米40克和海水60毫升);所述的大三角培养瓶为500mL;所述的室温培养温度为20~28℃;所述的菌种培养时间为6~10天,大米培养基的培养时间为60天。
(4)异壁咔啉碱C的提取分离纯化
将上述步骤(3)获得的菌株ZZ1866的固体发酵产物用乙酸乙酯提取得到乙酸乙酯提取物。将乙酸乙酯总提取物先用硅胶柱层析分离,用环己烷和乙酸乙酯混合溶剂以及乙酸乙酯和甲醇混合溶剂梯度洗脱,收集洗脱液,用高效液相色谱(HPLC)检测各组分,合并含有相同成分的组分,得到六个组分(A~F)。组分D用十八烷基硅烷键合硅胶 (ODS)柱层析分离,得到组分D1和D2,组分D2再用凝胶Sephadex LH-20柱层析分离,得到三个组分D2a~D2c。最后,组分D2b用高效液相色谱(HPLC)分离纯化得到纯化合物异壁咔啉碱C。
所述的硅胶柱层析的硅胶用量与上柱的样品量比例是10~15g:1.0g;所述的环己烷和乙酸乙酯混合溶剂梯度为10:1、5:1、2:1和1:1(体积比),乙酸乙酯和甲醇混合溶剂梯度为5:1和1:1(体积比);所述的十八烷基硅烷键合硅胶(ODS)柱层析的ODS用量与上柱的样品量比例是20~30g:1.0g;所述凝胶柱Sephadex LH-20柱层析的ODS 用量与上柱的样品量比例是20~30g: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C18 CT-30色谱柱(280×30mm,10μm),甲醇和水为流动相(82/18,体积比),检测波长210nm,流速为10.0mL/min。
(5)异壁咔啉碱C的结构鉴定
异壁咔啉碱C的结构是根据它的紫外光谱、红外光谱、一维和二维核磁共振(NMR)波谱和高分辨质谱(HRESIMS)数据而确定。
本发明的第三个目的是提供异壁咔啉碱C在制备抗胶质瘤药物中的应用。所述的异壁咔啉碱C可显著抑制胶质瘤U87MG和U251细胞的增殖,在制备治疗胶质瘤药物方面具有应用前景。
本发明的有益之处在于:(1)海洋来源的蓝灰色异壁放线菌Actinoalloteichuscyanogriseus ZZ1866在所述的培养条件下可以产生具有抗胶质瘤的生物碱类化合物异壁咔啉碱C;(2)异壁咔啉碱C为一结构新颖的新生物碱类化合物;(3)异壁咔啉碱C 显著抑制胶质瘤细胞U87MG和U251细胞的增殖,可为胶质瘤的治疗提供一种新的候选药物分子。
附图说明
图1是蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的菌落图。
图2是异壁咔啉碱C的高分辨质谱图。
图3~4是异壁咔啉碱C的氢谱。
图5~6是异壁咔啉碱C的碳谱。
图7~8是异壁咔啉碱C的HMBC谱。
图9是异壁咔啉碱C的HMBC相关示意图。
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.蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的分离培养
将从浙江省舟山市普陀岛附近海域获得的海底沉积物在28℃的条件下干燥8天。取干燥的沉积物1克,溶解于9mL的无菌水中,振荡过夜后用灭菌自来水配成浓度为 1×10- 3g/mL的样品溶液。取200μL样品液均匀分散到含有高氏琼脂固体培养基的培养皿中,在28℃的条件下培养7天时间后,将不同的菌落分别转移到另一含有高氏琼脂固体培养基的培养皿中,在28℃的条件下继续培养7天。最后将生长良好的ZZ1866 单一菌落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.蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866的菌种鉴定
使用16S rDNA序列分析方法鉴定所获得菌株ZZ1866的种类。
2.1实验试剂及仪器
PCR试剂:PrimeSTAR Max DNA Polymerase(TaKaRa),引物(Invitrogen合成),引物序列是:
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
2.3实验结果
拼接后的序列为:
GGTTGGGCCACGGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAA GGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTC ACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGAGATTCGCTCCA CCTTGCGATTTCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGACATAA GGGGCATGATGACTTGACCTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCCA TGAGTCCCCACCATTACGTGCTGGCAACATGGAACAAGGGTTGCGCTCGTTGCGGGACTT AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGCACACTGACCTT ACGGAAACCCCATCTCTGAGGCGATCCAGTGCATGTCAAACCCAGGTAAGGTTCTTCGCG TTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGT TTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGAG GACGTGGAAATCCCCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCT AATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGACCCGCC TTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTCCACCGCTACACCAGGAATTCCAGTC TCCCCTACCGAACTCAAGTCTGCCCGTATCGACTGCACGCCCACAGTTAAGCTGTAGGTT TTCACAGTCGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACG CTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGCGCC TACCGTCACTTTCGCTTCTTCGGCGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCC TCACGCGGCGTCGCTGCGTCAGGCTTTCGCCCATTGCGCAATATTCCCCACTGCTGCCTCC CGTAGGAGTCTGGGCCGTATCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTA CCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATC CTGCACCGCCGGAACTTTCCACCCACCCCCATGCGGGGGAAGGTCGTATCCGGTATTAGCCCCGGTTTCCCGAGGTTATCCCAGAGTGCAGGGCAGGTTGCCCACGTGTTACTCACCCGT TCGCCGCTCGTGTACCCCGAAGGGCCTTACCGCTCGACTTGCATGTGTTAAGCACGCCGC CAGCGTTCGTCCTGAGCA(1401bp)。
以上获得的16S rDNA序列与美国美国国立卫生研究院的NCBI GenBank数据库比较,其结果表明:菌株ZZ1866的16S rDNA序列与GenBank库中的蓝灰色异壁放线菌Actinoalloteichus cyanogriseus DSM 40103的16s rDNA序列有100%的相似性(登录号:HG917901.1)。因此,本发明所获得的蓝灰色异壁放线菌ZZ1866的分类命名为Actinoalloteichus cyanogriseus ZZ1866(附图1),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 2020121,保藏日:2020.5.15;保藏地址:中国.武汉.武汉大学。
3.蓝灰色异壁放线菌Actinoalloteichus cyanogriseus ZZ1866发酵产物的制备
取高氏琼脂固体斜面培养基的蓝灰色异壁放线菌Actinoalloteichuscyanogriseus ZZ1866的菌接种到含有250mL液体高氏培养基(可溶性淀粉20克,硝酸钾1克,七水合硫酸镁0.5克,磷酸氢二钾0.5克,氯化钠0.5克,硫酸亚铁0.01克,天然海水1 升)的500mL三角烧瓶中,将含有ZZ1866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菌种液转入到含有大米固体培养基(大米40克和天然海水60毫升)的500mL三角烧瓶中,在28℃条件下静置培养60天,得到含有抗胶质瘤活性物质异壁咔啉碱C的培养物。本实验总计制备了200瓶菌株ZZ1866的大米培养物。
4.异壁咔啉碱C的提取分离纯化
将上述实验获得的200瓶菌株ZZ1866的大米培养物分别用乙酸乙酯提取3次,将乙酸乙酯提取液合并,减压浓缩得到总提取物47.83克。将总提取物先用硅胶(800 克)柱层析分离,用环己烷和乙酸乙酯混合溶剂(10:1,5:1,2:1,1:1,体积比,各1000mL) 以及乙酸乙酯和甲醇混合溶剂(5:1,1:1,体积比,各1000mL)梯度洗脱,每500mL收集为一组分,用高效液相色谱(HPLC)检测各组分,合并含有相同成分的组分,得到六个组分(A~F)。组分D(5克)用十八烷基硅烷键合硅胶(ODS,150克)柱层析分离,分别用1500mL的70%和100%的甲醇洗脱,得到组分D1和D2。组分D2再用凝胶Sephadex LH-20(100g)柱层析分离,以甲醇洗脱得到三个组分D2a~D2c。最后,组分D2b(20mg) 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C18 CT-30, 280×30mm,10μm;流动相:甲醇/水,82/18;检测波长:210nm,流速:10.0mL/min), 得到化合物异壁咔啉碱C(5.9mg,保留时间35min)。
5.异壁咔啉碱C的结构鉴定
异壁咔啉碱C(Actinoallocarboline C)为白色无定形粉末;分子式C24H21N3O3;[α]D 20–42°(c 0.28,CHCl3);紫外光谱:UV(MeOH)λmax(log ε)241(3.82),290(3.71), 352(3.54),369(3.52)nm;红外光谱:IR(MeOH)νmax 2932,1723,1675,1612,1391,1266, 747cm–1;高分辨质谱(附图2):HRESIMS m/z[M+Na]+422.1476(计算值C24H21N3O3Na, 422.1481),[2M+Na]+821.3066(计算值C48H42N6NaO6 821.3064)。通过化合物的1H谱(附图3~4)、13C谱(附图5~6)和HMBC谱(附图7~8)分析,确定了异壁咔啉碱C的化学结构,为一个新化合物,其13C和1H核磁共振信号归属见表一,它的HMBC相关示意图见附图9。
6.异壁咔啉碱C的抗胶质瘤活性
用磺酰罗丹明B(SRB)法测定异壁咔啉碱C对胶质瘤细胞U87MG和U251细胞的增殖的抑制作用,阿霉素用于阳性对照。胶质瘤U87MG和U251细胞分别用含有 10%FBS的MEM和DMEM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。细胞接种于96孔板中,贴壁24h后加入不同浓度的异壁咔啉碱C,药物处理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,计算异壁咔啉碱C抑制肿瘤细胞增殖的IC50值。实验结果表明:异壁咔啉碱C显著抑制胶质瘤细胞的增殖,其抑制胶质瘤U87MG和U251 细胞增殖的IC50值分别为2.28和11.9μM(表二)。
表一、异壁咔啉碱C的13C和1H NMR数据(溶剂:氘代二甲基亚砜DMSO-d6)
No. | <sup>13</sup>C(150MHz) | <sup>1</sup>H(600MHz,J in Hz) | No. | <sup>13</sup>C(150MHz) | <sup>1</sup>H(600MHz,J in Hz) |
1 | 140.8,C | – | 13 | 101.5,C | – |
2 | 111.1,CH | 7.73,d(8.0) | 14 | 22.4,CH<sub>3</sub> | 2.20,s |
3 | 127.4,CH | 7.57,t(8.0) | 15 | 116.1,CH | 5.96,d(9.2) |
4 | 121.2,CH | 7.32,t(8.0) | 16 | 129.0,CH | 6.65,d(9.2) |
5 | 121.8,CH | 8.26,d(8.0) | 17 | 135.1,C | – |
6 | 121.5,C | – | 18 | 128.6,CH | 7.34,d(7.5) |
7 | 123.2,C | – | 19 | 128.2,CH | 7.28,t(7.5) |
8 | 128.7,C | – | 20 | 127.8,CH | 7.22,t(7.5) |
9 | 152.5,C | – | 21 | 128.2,CH | 7.28,t(7.5) |
10 | 127.1,C | – | 22 | 128.6,CH | 7.34,d(7.5) |
11 | 99.8,CH | 7.88,s | 23 | 49.0,CH<sub>3</sub> | 2.94,s |
12 | 156.5,C | – | 24 | 31.4,CH<sub>3</sub> | 4.31,s |
表二、异壁咔啉碱C对胶质瘤细胞增殖的抑制活性(IC50:μM)
以上实验结果说明,异壁咔啉碱C显著地抑制胶质瘤细胞的增殖,在制备治疗抗胶质瘤药物方面具有良好的应用潜力。
序列表
<110> 浙江大学
<120> 海洋抗胶质瘤活性物质异壁咔啉碱C的制备和用途
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Unknow)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Unknow)
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1401
<212> DNA
<213> 蓝灰色异壁放线菌(Actinoalloteichus cyanogriseus ZZ1866)
<400> 3
ggttgggcca cgggcttcgg gtgttaccga ctttcatgac gtgacgggcg gtgtgtacaa 60
ggcccgggaa cgtattcacc gcagcgttgc tgatctgcga ttactagcga ctccgacttc 120
acggggtcga gttgcagacc ccgatccgaa ctgagaccgg ctttaagaga ttcgctccac 180
cttgcgattt cgcagccctc tgtaccggcc attgtagcat gtgtgaagcc ctggacataa 240
ggggcatgat gacttgacct catccccacc ttcctccgag ttgaccccgg cagtctccca 300
tgagtcccca ccattacgtg ctggcaacat ggaacaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga cagccatgca ccacctgcac actgacctta 420
cggaaacccc atctctgagg cgatccagtg catgtcaaac ccaggtaagg ttcttcgcgt 480
tgcatcgaat taatccacat gctccgccgc ttgtgcgggc ccccgtcaat tcctttgagt 540
tttagccttg cggccgtact ccccaggcgg ggcgcttaat gcgttagcta cggcacggag 600
gacgtggaaa tcccccacac ctagcgccca ccgtttacgg cgtggactac cagggtatct 660
aatcctgttc gctccccacg ctttcgctcc tcagcgtcag tatcggccca gagacccgcc 720
ttcgccaccg gtgttcctcc tgatatctgc gcattccacc gctacaccag gaattccagt 780
ctcccctacc gaactcaagt ctgcccgtat cgactgcacg cccacagtta agctgtaggt 840
tttcacagtc gacgcgacaa accgcctacg agctctttac gcccaataat tccggacaac 900
gctcgcaccc tacgtattac cgcggctgct ggcacgtagt tagccggtgc ttcttctgcg 960
cctaccgtca ctttcgcttc ttcggcgctg aaagaggttt acaacccgaa ggccgtcatc 1020
cctcacgcgg cgtcgctgcg tcaggctttc gcccattgcg caatattccc cactgctgcc 1080
tcccgtagga gtctgggccg tatctcagtc ccagtgtggc cggtcgccct ctcaggccgg 1140
ctacccgtcg tcgccttggt aggccattac cccaccaaca agctgatagg ccgcgggccc 1200
atcctgcacc gccggaactt tccacccacc cccatgcggg ggaaggtcgt atccggtatt 1260
agccccggtt tcccgaggtt atcccagagt gcagggcagg ttgcccacgt gttactcacc 1320
cgttcgccgc tcgtgtaccc cgaagggcct taccgctcga cttgcatgtg ttaagcacgc 1380
cgccagcgtt cgtcctgagc a 1401
Claims (3)
1.一种海洋抗胶质瘤活性物质异壁咔啉碱C,其化学结构式为:
通过以下步骤实现:
(1) 蓝灰色异壁放线菌发酵产物的制备
将蓝灰色异壁放线菌的菌株接种到含有液体培养基的大三角烧瓶中,将含有异壁放线菌的菌种培养液在室温条件下振荡培养后得到菌种液,最后将菌种液转入含有大米固体培养基的大三角烧瓶中,特定温度静置培养一定时间后,得到含有活性物质异壁咔啉碱C的培养物;蓝灰色异壁放线菌,其分类命名为Actinoalloteichus cyanogriseus ZZ1866,已被中国典型培养物保藏中心保藏,保藏编号: CCTCC M 2020121,保藏日:2020.5.15;保藏地址:中国.武汉.武汉大学;
(2) 异壁咔啉碱C的提取分离纯化
将上述步骤(1)获得的菌株ZZ1866的固体发酵产物用乙酸乙酯提取得到乙酸乙酯提取物,将乙酸乙酯总提取物先用硅胶柱层析分离,用环己烷和乙酸乙酯混合溶剂以及乙酸乙酯和甲醇混合溶剂梯度洗脱,收集洗脱液,用高效液相色谱检测各组分,合并含有相同成分的组分,得到A-F六个组分,组分D用十八烷基硅烷键合硅胶柱层析分离,得到组分D1和D2,组分D2再用凝胶Sephadex LH-20柱层析分离,得到三个组分D2a-D2c,最后,组分D2b用高效液相色谱分离纯化得到纯化合物异壁咔啉碱C;
(3) 异壁咔啉碱C的结构鉴定
异壁咔啉碱C的结构根据它的紫外光谱、红外光谱、一维和二维核磁共振波谱和高分辨质谱数据确定。
2.根据权利要求1所述的异壁咔啉碱C的制备方法,其特征在于,步骤(2)所述的硅胶柱层析的硅胶用量与上柱的样品量比例是10-15 g: 1.0 g;所述的环己烷和乙酸乙酯混合溶剂梯度体积比为10:1、5:1、2:1和1:1,乙酸乙酯和甲醇混合溶剂梯度体积比为5:1和1:1;所述的十八烷基硅烷键合硅胶柱层析的ODS用量与上柱的样品量比例是20-30 g: 1.0 g;所述凝胶柱Sephadex LH-20柱层析的ODS用量与上柱的样品量比例是20-30 g: 1.0 g;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C18 CT-30 色谱柱28030 mm, 10 μm,甲醇和水为流动相,检测波长210 nm,流速为10.0 mL/min。
3.权利要求1所述的异壁咔啉碱C在制备抗胶质瘤药物中的应用。
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