技术领域：Technical field:
本发明属于生物和医药领域，具体涉及化合物acaromycinA及其制备方法和在制备抗肿瘤药物中的应用。The invention belongs to the field of biology and medicine, and specifically relates to compound acaromycin A, its preparation method and its application in the preparation of antitumor drugs.
背景技术：Background technique:
海洋拥有特殊的环境，尤其是深海海洋环境，栖息于海洋环境的海洋微生物为了适应这些高盐、弱碱性、低营养、缺氧等特殊环境，因此海洋微生物的次级代谢产物具有独特的结构类型和丰富多样的生物活性。这些新化学实体不仅能揭示新的生物合成途径，而且还能启发人们对这些化学实体进行仿生合成和生物活性研究。因此，海洋微生物次生代谢产物具有重要的科研和应用价值，是药物开发的重要资源(SunL,etal.2012)。The ocean has a special environment, especially the deep-sea marine environment. In order to adapt to these special environments such as high salinity, weak alkalinity, low nutrition, and anoxic environment, marine microorganisms inhabit the marine environment, so the secondary metabolites of marine microorganisms have unique structures. types and diverse biological activities. These new chemical entities can not only reveal new biosynthetic pathways, but also inspire people to study biomimetic synthesis and biological activities of these chemical entities. Therefore, marine microbial secondary metabolites have important scientific research and application value, and are important resources for drug development (SunL, et al. 2012).
深海真菌AcaromycesingoldiiFS121于2011年9月从南海(18°44.606′N,119°44.263′E)水深3415米处海底表层沉积物中分离得到。文献查阅显示，到目前为止没有对于Acaromyces属真菌的次生代谢产物的研究，因此有必要对其次级代谢产物进行深入研究。The deep-sea fungus Acaromycesingoldii FS121 was isolated from the seabed surface sediments at a depth of 3415 meters in the South China Sea (18°44.606′N, 119°44.263′E) in September 2011. Literature review shows that so far there is no research on the secondary metabolites of fungi of the genus Acaromyces, so it is necessary to conduct in-depth research on their secondary metabolites.
发明内容：Invention content:
本发明的第一个目的是提供对肿瘤细胞具有抑制活性的化合物acaromycinA。The first object of the present invention is to provide the compound acaromycinA which has inhibitory activity on tumor cells.
本发明通过深海真菌AcaromycesingoldiiFS121的发酵培养、发酵产物的分离纯化和结构鉴定，获得了1个具有抗肿瘤活性的3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10-dione类新化合物，命名为acaromycinA。3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10-dione类化合物具有广泛的生物活性，对革兰氏阳性菌都有一定的抑制活性(GuptaR.B.etal.1989)，能够抑制脂多糖(LPS)刺激下小鼠RAW264.7的巨噬细胞产生一氧化氮(NO)(ByeongMP.etal.2010)，对恶性疟原虫Plasmodiumfalciparum3D7和人胚肺成纤维细胞(MRC-5)有一定的抑制活性(PaulaF.C.etal.2016)。因此，从深海真菌AcaromycesingoldiiFS121中发现的新化合物acaromycinA可为抗肿瘤药物研发提供候选化合物，为开发利用海洋真菌资源提供科学依据。The present invention obtains a 3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10 with anti-tumor activity through the fermentation and cultivation of the deep-sea fungus Acaromycesingoldii FS121, the separation and purification of the fermentation product and the structural identification -dione class of new compounds named acaromycinA. 3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10-dione compounds have a wide range of biological activities, and have certain inhibitory activity against Gram-positive bacteria (GuptaR.B.etal .1989), can inhibit lipopolysaccharide (LPS) stimulation of mouse RAW264.7 macrophages to produce nitric oxide (NO) (ByeongMP.etal.2010), on Plasmodium falciparum3D7 and human embryonic lung fibroblasts ( MRC-5) has certain inhibitory activity (PaulaF.C.etal.2016). Therefore, the new compound acaromycinA discovered from the deep-sea fungus Acaromycesingoldii FS121 can provide candidate compounds for the development of anti-tumor drugs, and provide a scientific basis for the development and utilization of marine fungal resources.
本发明的化合物acaromycinA，其特征在于，结构如式(I)所示：Compound acaromycinA of the present invention, is characterized in that, structure is as shown in formula (I):
本发明的第二个目的是提供化合物acaromycinA的制备方法，其特征在于，所述的化合物acaromycinA是从深海真菌AcaromycesingoldiiFS121的发酵培养物中制备得到。The second object of the present invention is to provide a preparation method of the compound acaromycinA, which is characterized in that the compound acaromycinA is prepared from the fermentation culture of the deep-sea fungus Acaromycesingoldii FS121.
优选，具体步骤如下：Preferably, the specific steps are as follows:
制备AcaromycesingoldiiFS121的发酵培养物，将该发酵培养物的发酵液和菌丝体分离，发酵液用乙酸乙酯萃取，萃取液经蒸馏浓缩后得到浸膏；preparing the fermentation culture of Acaromycesingoldii FS121, separating the fermentation liquid and mycelium of the fermentation culture, extracting the fermentation liquid with ethyl acetate, and obtaining the extract after distillation and concentration of the extract;
将浸膏过正相硅胶柱，先用石油醚-乙酸乙酯从30:1,20:1,10:1,7:1,9:2,3:1,2:1,1:1,1:2v/v梯度洗脱，再用乙酸乙酯洗脱，最后用二氯甲烷:甲醇＝5:1v/v洗脱；收集浸膏被石油醚:乙酸乙酯体积比1:2洗脱的馏分，该馏分再经TLC以石油醚:乙酸乙酯＝1:4v/v展开得Rf＝2.7/3.7的馏分E22，该馏分E22经SephadexLH-20柱，以二氯甲烷:甲醇＝1:1v/v作为洗脱剂，收集TLC以石油醚:乙酸乙酯＝1:2v/v展开得Rf＝1.5/3.5的组分，然后过反相硅胶柱层析，以体积比甲醇:水＝2:8等度洗脱，收集洗脱下来的馏分，上样正相硅胶薄层层析板，以二氯甲烷:甲醇＝10:1为展开剂展开，割板纯化Rf＝1.4/3.3的组分，得到化合物acaromycinA。Pass the extract through a normal-phase silica gel column, first use petroleum ether-ethyl acetate from 30:1, 20:1, 10:1, 7:1, 9:2, 3:1, 2:1, 1:1, 1:2v/v gradient elution, then elution with ethyl acetate, and finally dichloromethane:methanol=5:1v/v elution; the collected extract was eluted with petroleum ether:ethyl acetate volume ratio 1:2 The cut, the cut is developed by TLC with petroleum ether: ethyl acetate=1:4v/v to obtain the cut E22 ofRf =2.7/3.7, the cut E22 is passed through the SephadexLH-20 column, with dichloromethane:methanol=1 : 1v/v is used as eluent, collects TLC to develop the component of Rf=1.5/3.5 with sherwood oil: ethyl acetate=1:2v/v, crosses reverse-phase silica gel column chromatography then, with volume ratio methanol: water = 2:8 isocratic elution, collect the eluted fractions, load the normal phase silica gel thin layer chromatography plate, develop with dichloromethane:methanol=10:1 as the developing solvent, cut the plate purification Rf=1.4/3.3 Components to obtain the compound acaromycinA.
所述的制备AcaromycesingoldiiFS121的发酵培养物优选是将活化的AcaromycesingoldiiFS121菌种接入含质量分数1.5％海盐的马铃薯葡萄糖液体培养基中，28℃，120rpm，培养5d制得种子液，将种子液以体积比10％的接种量接入到含质量分数1.5％海盐的马铃薯葡萄糖液体培养基中，相同条件下培养7d，制得发酵培养物。The fermentation culture for the preparation of AcaromycesingoldiiFS121 is preferably to insert the activated AcaromycesingoldiiFS121 bacterial classification into the potato glucose liquid medium containing 1.5% sea salt in mass fraction, 28 ℃, 120rpm, and cultivate 5d to obtain seed liquid, and the seed liquid is obtained by volume The 10% inoculum was inserted into the potato glucose liquid medium containing 1.5% sea salt by mass fraction, and cultivated under the same conditions for 7 days to obtain a fermentation culture.
本发明的第三个目的是提供AcaromycesingoldiiFS121在制备化合物acaromycinA中的应用。The third object of the present invention is to provide the application of AcaromycesingoldiiFS121 in the preparation of compound acaromycinA.
本发明通过肿瘤细胞生长抑制活性测试发现，化合物acaromycinA对肿瘤细胞株SF-268、MCF-7、NCI-H460和HePG-2均具有良好的抑制活性，对上述4种肿瘤细胞的IC50值分别为7.8±0.5，6.7±1.6，10.0±0.1和7.3±1.8μM，阳性对照药物顺铂对上述四种肿瘤细胞株的IC50值分别为4.1±0.2，2.9±0.5，2.9±0.2和2.5±0.2μM。结果表明：本发明的化合物acaromycinA对肿瘤细胞具有显著的抑制活性，能用于在制备抗肿瘤药物。The present invention finds through the test of tumor cell growth inhibitory activity that the compound acaromycinA has good inhibitory activity on tumor cell lines SF-268, MCF-7, NCI-H460 and HePG-2, and the IC50 values for the above four kinds of tumor cells are respectively 7.8±0.5, 6.7±1.6, 10.0±0.1 and 7.3±1.8μM, and the IC50 values of the positive control drug cisplatin on the above four tumor cell lines were 4.1±0.2, 2.9±0.5, 2.9±0.2 and 2.5±0.2 0.2 μM. The results show that the compound acaromycinA of the present invention has significant inhibitory activity on tumor cells and can be used in the preparation of antitumor drugs.
本发明的第四个目的是提供化合物acaromycinA在制备抗肿瘤药物中的应用。The fourth object of the present invention is to provide the application of the compound acaromycinA in the preparation of antitumor drugs.
所述的抗肿瘤药物优选为抗神经胶质瘤、乳腺癌、非小细胞肺癌或肝癌的药物。The anti-tumor drugs are preferably anti-glioma, breast cancer, non-small cell lung cancer or liver cancer drugs.
本发明的第五个目的是提供一种抗肿瘤药物，其特征在于，含有化合物acaromycinA作为有效成分。The fifth object of the present invention is to provide an antitumor drug characterized in that it contains the compound acaromycinA as an active ingredient.
所述的抗肿瘤药物优选为抗神经胶质瘤、乳腺癌、非小细胞肺癌或肝癌的药物。The anti-tumor drugs are preferably anti-glioma, breast cancer, non-small cell lung cancer or liver cancer drugs.
本发明从深海真菌AcaromycesingoldiiFS121的发酵培养物中分离得到新化合物acaromycinA，该化合物对肿瘤细胞具有良好的抑制活性，可为抗肿瘤新药研发提供候选化合物，为开发利用海洋真菌资源提供科学依据。The invention isolates a new compound acaromycin A from the fermentation culture of the deep-sea fungus Acaromycesingoldii FS121. The compound has good inhibitory activity on tumor cells, can provide candidate compounds for the development of new anti-tumor drugs, and provide scientific basis for the development and utilization of marine fungal resources.
本发明的AcaromycesingoldiiFS121公开于文献：23株海洋真菌的分子鉴定及其抗植物病原真菌和细胞毒活性研究，杨小岚,陈玉婵,李浩华,章卫民2014,0(8):132-137。该菌株本申请人也持有，并保证自申请日起20年内向公众提供。The Acaromycesingoldii FS121 of the present invention is disclosed in the literature: Molecular Identification of 23 Strains of Marine Fungi and Their Anti-phytopathogenic Fungi and Cytotoxic Activities, Yang Xiaolan, Chen Yuchan, Li Haohua, Zhang Weimin 2014, 0(8):132-137. This strain is also held by the applicant and guaranteed to be available to the public within 20 years from the filing date.
附图说明：Description of drawings:
图1是化合物1的1H-NMR谱；Fig. 1 is the1 H-NMR spectrum of compound 1;
图2是化合物1的13C-NMR谱；Fig. 2 is the13 C-NMR spectrum of compound 1;
图3是化合物1的DEPT135谱；Fig. 3 is the DEPT135 spectrum of compound 1;
图4是化合物1的HSQC谱；Fig. 4 is the HSQC spectrum of compound 1;
图5是化合物1的HMBC谱；Fig. 5 is the HMBC spectrum of compound 1;
图6是化合物1的1H-1HCOSY谱；Fig. 6 is the1 H-1 HCOSY spectrum of compound 1;
图7是化合物1的NOESY谱；Fig. 7 is the NOESY spectrum of compound 1;
图8是化合物1的HRESIMS谱；Fig. 8 is the HRESIMS spectrum of compound 1;
图9是化合物1的CD谱。Figure 9 is the CD spectrum of compound 1.
具体实施方式：detailed description:
以下实施例是对本发明的进一步说明，而不是对本发明的限制。The following examples are to further illustrate the present invention, rather than limit the present invention.
实施例1：Example 1:
将活化的深海真菌AcaromycesingoldiiFS121接入含质量分数1.5％海盐的马铃薯葡萄糖液体培养基(每升含有15g海盐、200g马铃薯、葡萄糖20g，余量为水，其配制方法是：将马铃薯洗净去皮，切成大小约为1cm3的小块，称量200g，放入1L水中煮沸20-30分钟，用双层纱布过滤，取滤液，在滤液中加入海盐15g、葡萄糖20g，加水补足1L，灭菌备用。)中，28℃，120rpm，培养5d制得种子液，将种子液以体积比10％的接种量接入到新的含质量分数1.5％海盐的马铃薯葡萄糖液体培养基中，28℃，120rpm下振荡培养7d，制得发酵培养物。The deep-sea fungus Acaromycesingoldii FS121 of activation is inserted into the potato dextrose liquid culture medium containing mass fraction 1.5% sea salt (every liter contains 15g sea salt, 200g potato, 20g of glucose, the remainder is water, and its preparation method is: the potato is washed and peeled, Cut into small pieces about1cm3 in size, weigh 200g, boil in 1L of water for 20-30 minutes, filter with double gauze, take the filtrate, add 15g of sea salt and 20g of glucose to the filtrate, add water to make up 1L, and sterilize Standby.), 28°C, 120rpm, cultured for 5 days to obtain seed liquid, the seed liquid was inserted into a new potato glucose liquid medium containing 1.5% sea salt by volume with an inoculum size of 1.5%, at 28°C, Shake culture at 120 rpm for 7 days to obtain a fermentation culture.
将该发酵培养物的发酵液和菌丝体分离，发酵液用乙酸乙酯萃取，乙酸乙酯萃取液经蒸馏浓缩后得到浸膏。将浸膏过正相硅胶柱，先用石油醚-乙酸乙酯从30:1,20:1,10:1,7:1,9:2,3:1,2:1,1:1,1:2(v/v)梯度洗脱，再用乙酸乙酯洗脱，最后用二氯甲烷:甲醇＝5:1(v/v)洗脱；收集浸膏被石油醚:乙酸乙酯＝1:2(v/v)洗脱的馏分，该馏分再经TLC薄层层析，以石油醚:乙酸乙酯＝1:4(v/v)展开得Rf＝2.7/3.7的馏分E22。The fermentation broth and mycelia of the fermentation culture are separated, the fermentation broth is extracted with ethyl acetate, and the ethyl acetate extract is concentrated by distillation to obtain an extract. Pass the extract through a normal-phase silica gel column, first use petroleum ether-ethyl acetate from 30:1, 20:1, 10:1, 7:1, 9:2, 3:1, 2:1, 1:1, 1:2 (v/v) gradient elution, then elution with ethyl acetate, and finally with dichloromethane:methanol=5:1 (v/v) elution; collect the extract by petroleum ether:ethyl acetate= The fraction eluted at 1:2 (v/v), which was then subjected to TLC thin layer chromatography, developed with petroleum ether: ethyl acetate = 1:4 (v/v) to obtain fraction E22 with Rf =2.7/3.7 .
馏分E22以二氯甲烷:甲醇＝1:1(v/v)洗脱过SephadexLH-20，收集TLC以石油醚:乙酸乙酯＝1:2v/v展开得Rf＝1.5/3.5的组分，然后过反相硅胶柱层析，以体积比甲醇:水＝2:8等度洗脱，收集洗脱下来的馏分，上样正相硅胶薄层层析，以二氯甲烷:甲醇＝10:1(v/v)为展开剂展开，割板纯化Rf＝1.4/3.3的组分，得到化合物acaromycinA(化合物1，2.6mg)。Fraction E22 was eluted with SephadexLH-20 with dichloromethane:methanol=1:1 (v/v), and collected TLC components developed with petroleum ether:ethyl acetate=1:2v/v to obtain Rf=1.5/3.5, Then through reverse-phase silica gel column chromatography, with volume ratio methanol:water=2:8 isocratic elution, collect the fraction that elutes, load normal phase silica gel thin-layer chromatography, with dichloromethane:methanol=10: 1 (v/v) is developing with the developing agent, and the fraction with Rf =1.4/3.3 was purified by cutting plate to obtain the compound acaromycinA (compound 1, 2.6 mg).
化合物1经质谱和核磁共振波谱分析，其结构鉴定如下：Compound 1 was analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy, and its structure was identified as follows:
如图1-9所示，图1是化合物1的1H-NMR谱；图2是化合物1的13C-NMR谱；图3是化合物1的DEPT135谱；图4是化合物1的HSQC谱；图5是化合物1的HMBC谱；图6是化合物1的1H-1HCOSY谱；图7是化合物1的NOESY谱；图8是化合物1的HRESIMS谱；图9是化合物1的CD谱。As shown in Figures 1-9, Figure 1 is the1 H-NMR spectrum of Compound 1; Figure 2 is the13 C-NMR spectrum of Compound 1; Figure 3 is the DEPT135 spectrum of Compound 1; Figure 4 is the HSQC spectrum of Compound 1; Fig. 5 is the HMBC spectrum of compound 1; Fig. 6 is the1 H-1 HCOSY spectrum of compound 1; Fig. 7 is the NOESY spectrum of compound 1; Fig. 8 is the HRESIMS spectrum of compound 1; Fig. 9 is the CD spectrum of compound 1.
化合物1具有以下理化和波谱特性：黄色固体粉末；+102.0(c0.1,MeOH)；UV(MeOH)λmax(logε)194(3.69),204(4.05),286(3.67)nm；CD(c1.8×10-3,MeOH)λ(Δε)474(0),425(-0.25),405(0),370(0.76),314(0.19),287(2.1),257(0),239(-0.67),221(0)nm；IR(KBr)νmax3498,1647,1625,1257,1026,792cm–1。1H和13CNMR数据；HRESIMSm/z[M+Na]+299.0533(calcdfor299.0532)。1H-NMR(500MHz,CDCl3)δ:7.61(1H,d,J＝1.9Hz,H-9),7.60(1H,s,H-6),7.23(1H,dd,J＝5.6,4.0Hz,H-8),4.96(1H,d,J＝4.3,0.9Hz,H-4),4.30(1H,q,J＝6.7,1.0Hz,H-2),4.03(1H,dd,J＝4.3,1.1Hz,H-3),1.65(3H,d,J＝6.7Hz,H-11)。13C-NMR(125MHz,CDCl3)δ:186.5(C-5),183.8(C-10),162.2(C-7),155.2(C-10a),132.1(C-9a),118.8(C-4a),114.1(C-5a),137.2(C-6),124.7(C-8),119.3(C-9),76.2(C-2),66.9(C-3),65.1(C-4),16.7(C-11)。CD谱数据显示该化合物在287、314、370nm显示正的cotton效应，在239、425nm显示负的cotton效应，与(+)-cryptosporin的CD数据一致(BradeandVasella,1989)。因此，可以确定该化合物1在C-2、C-3、C-4均为R构型。经文献检索，该化合物1为新化合物，属于3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10-dione类新化合物，命名为acaromycinA，具体结构如式I中所示。Compound 1 has the following physical, chemical and spectral properties: yellow solid powder; +102.0(c0.1,MeOH); UV(MeOH)λmax (logε)194(3.69),204(4.05),286(3.67)nm; CD(c1.8×10-3 ,MeOH)λ(Δε )474(0),425(-0.25),405(0),370(0.76),314(0.19),287(2.1),257(0),239(-0.67),221(0)nm; IR (KBr)νmax 3498,1647,1625,1257,1026,792cm–1 .1 H and13 CNMR data; HRESIMS m/z [M+Na]+ 299.0533 (calcdfor299.0532).1 H-NMR (500MHz, CDCl3 ) δ: 7.61 (1H, d, J = 1.9 Hz, H-9), 7.60 (1H, s, H-6), 7.23 (1H, dd, J = 5.6, 4.0 Hz,H-8),4.96(1H,d,J＝4.3,0.9Hz,H-4),4.30(1H,q,J＝6.7,1.0Hz,H-2),4.03(1H,dd,J = 4.3, 1.1 Hz, H-3), 1.65 (3H, d, J = 6.7 Hz, H-11).13 C-NMR (125MHz, CDCl3 ) δ: 186.5(C-5), 183.8(C-10), 162.2(C-7), 155.2(C-10a), 132.1(C-9a), 118.8(C -4a), 114.1(C-5a), 137.2(C-6), 124.7(C-8), 119.3(C-9), 76.2(C-2), 66.9(C-3), 65.1(C- 4), 16.7 (C-11). CD spectral data show that the compound exhibits positive cotton effect at 287, 314, and 370nm, and negative cotton effect at 239, 425nm, which is consistent with the CD data of (+)-cryptosporin (Brade and Vasella, 1989). Therefore, it can be confirmed that the compound 1 is in the R configuration at C-2, C-3, and C-4. After literature search, the compound 1 is a new compound, which belongs to the 3,4-dihydro-2H-naphtho-[2,3-b]pyran-5,10-dione class of new compounds, named acaromycinA, and the specific structure is as in formula I shown.
分离得到的化合物acaromycinA的结构式如下式(I)所示：The structural formula of the isolated compound acaromycinA is shown in the following formula (I):
实施例2：Example 2:
采用SRB法(SkehanP.etal.1990)测试化合物acaromycinA对肿瘤细胞的生长抑制活性。The growth inhibitory activity of compound acaromycin A on tumor cells was tested by SRB method (SkehanP. et al. 1990).
所述的肿瘤细胞株为神经胶质瘤细胞SF-268、乳腺癌细胞MCF-7、非小细胞肺癌细胞NCI-H460和肝癌细胞HePG-2。The tumor cell lines are glioma cell SF-268, breast cancer cell MCF-7, non-small cell lung cancer cell NCI-H460 and liver cancer cell HePG-2.
1、实验方法：将本发明制备的化合物acaromycinA用二甲基亚砜(DMSO)溶解得浓度为10mmol/L的母液，再用RPMI-1640培养基稀释至所需浓度，阳性对照药物为顺铂。取对数生长期的NCI-H460、SF-268、MCF-7和HePG-2细胞，用胰酶消化，台盼蓝染色计数，台盼蓝排斥实验检测细胞活力大于95％后，用新鲜RPMI-1640培养基调整细胞浓度为3×104个/mL，细胞接种于96孔板，每孔加入180μL的细胞悬液，并设3个空白孔调零，于37℃、5％CO2培养箱中培养24h。待细胞贴壁后，每孔加入20μL一定浓度的所述化合物溶液，阴性对照加20μLRPMI-1640培养基，以顺铂作阳性对照。置37℃、5％CO2培养箱中培养72h后，加入50μL50％冷三氯醋酸固定细胞，4℃放置1h后用蒸馏水洗涤5次，空气中自然干燥。然后加入由1％冰醋酸配制的浓度为4mg/mL的SRB溶液100μL/孔，室温中染色30min，去上清，用1％冰醋酸洗涤5次。最后加入200μL/孔10mmol/mL的Tris溶液溶解，用酶标仪测定570nm处的吸光值(A)，用以下公式计算药物对细胞生长的抑制率：细胞生长抑制率(％)＝(1-A样品组/A对照组)×100％。1. Experimental method: the compound acaromycinA prepared by the present invention is dissolved with dimethyl sulfoxide (DMSO) to obtain a mother solution with a concentration of 10mmol/L, and then diluted to the required concentration with RPMI-1640 medium, and the positive control drug is cisplatin . Take the NCI-H460, SF-268, MCF-7 and HePG-2 cells in the logarithmic growth phase, digest them with trypsin, and count them with trypan blue staining. After the trypan blue exclusion test shows that the cell viability is greater than 95%, use fresh RPMI Adjust the cell concentration to3 ×104/mL in -1640 medium, inoculate the cells in a 96-well plate, add 180 μL of cell suspension to each well, set 3 blank wells to zero, and culture at 37°C and 5% CO2 Cultivate in the box for 24h. After the cells adhered to the wall, 20 μL of a certain concentration of the compound solution was added to each well, 20 μL of RPMI-1640 medium was added to the negative control, and cisplatin was used as the positive control. After culturing in a 37°C, 5%CO2 incubator for 72 hours, 50 μL of 50% cold trichloroacetic acid was added to fix the cells, placed at 4°C for 1 hour, washed 5 times with distilled water, and dried naturally in the air. Then add 100 μL/well of SRB solution with a concentration of 4 mg/mL prepared by 1% glacial acetic acid, stain at room temperature for 30 min, remove the supernatant, and wash 5 times with 1% glacial acetic acid. Add the Tris solution of 200 μ L/ hole 10mmol/mL to dissolve at last, measure the absorbance value (A) at 570nm place with microplate reader, calculate the inhibitory rate of medicine to cell growth with following formula: Cell growth inhibitory rate (%)=(1- Asample group /Acontrol group )×100%.
2、实验结果：本发明制备的化合物acaromycinA和阳性对照药物顺铂对肿瘤细胞株SF-268、MCF-7、NCI-H460和HePG-2的IC50值见表1。结果表明：本发明的化合物acaromycinA对肿瘤细胞具有显著的抑制活性，因此，本发明的实现为研究与开发新的抗肿瘤药物提供了候选化合物，为开发利用海洋真菌资源提供了科学依据。2. Experimental results: See Table 1 for the IC50 values of the compound acaromycinA prepared by the present invention and the positive control drug cisplatin on tumor cell lines SF-268, MCF-7, NCI-H460 and HePG-2. The results show that the compound acaromycinA of the present invention has significant inhibitory activity on tumor cells. Therefore, the realization of the present invention provides a candidate compound for the research and development of new anti-tumor drugs, and provides a scientific basis for the development and utilization of marine fungal resources.
表1化合物1对肿瘤细胞生长的抑制活性(n＝3)The inhibitory activity of table 1 compound 1 to tumor cell growth ( n=3)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610137725.4ACN105601607B (en) | 2016-03-10 | 2016-03-10 | Compound a caromycin A and preparation method thereof and the application in antineoplastic is prepared |
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| CN116003489A (en)* | 2021-10-29 | 2023-04-25 | 山东国际生物科技园发展有限公司 | Preparation of Atramycin C and application of Atramycin C in drug research and development |
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| CN103012530A (en)* | 2012-12-26 | 2013-04-03 | 广东省微生物研究所 | Preparation method of compound endecaphyllacins B and its application in the preparation of antitumor drugs |
| CN104593443A (en)* | 2014-12-25 | 2015-05-06 | 广东省微生物研究所 | Preparation method of agilawood chromone component |
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| CN107501072B (en)* | 2017-08-15 | 2020-04-07 | 广东省微生物研究所（广东省微生物分析检测中心） | Compound colletotriconeA, preparation method thereof and application thereof in preparing antitumor drugs |
| CN116003489A (en)* | 2021-10-29 | 2023-04-25 | 山东国际生物科技园发展有限公司 | Preparation of Atramycin C and application of Atramycin C in drug research and development |
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