CN112300188B - Compounds myrothecin H and I, and preparation method and application thereof - Google Patents
Compounds myrothecin H and I, and preparation method and application thereof Download PDFInfo
- Publication number
- CN112300188B CN112300188B CN202011127846.3A CN202011127846A CN112300188B CN 112300188 B CN112300188 B CN 112300188B CN 202011127846 A CN202011127846 A CN 202011127846A CN 112300188 B CN112300188 B CN 112300188B
- Authority
- CN
- China
- Prior art keywords
- myrothecin
- compound
- compounds
- volume ratio
- roridum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 241000233866 Fungi Species 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 17
- 230000004151 fermentation Effects 0.000 claims abstract description 17
- 240000002505 Pogostemon cablin Species 0.000 claims abstract description 16
- 235000011751 Pogostemon cablin Nutrition 0.000 claims abstract description 16
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 12
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 9
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 8
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 8
- 208000032612 Glial tumor Diseases 0.000 claims abstract description 8
- 206010018338 Glioma Diseases 0.000 claims abstract description 8
- 206010023774 Large cell lung cancer Diseases 0.000 claims abstract description 8
- 201000007270 liver cancer Diseases 0.000 claims abstract description 8
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 8
- 201000009546 lung large cell carcinoma Diseases 0.000 claims abstract description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 32
- 238000004809 thin layer chromatography Methods 0.000 claims description 20
- 244000061456 Solanum tuberosum Species 0.000 claims description 15
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 15
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 238000009630 liquid culture Methods 0.000 claims description 9
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 6
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- 238000011894 semi-preparative HPLC Methods 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 239000008121 dextrose Substances 0.000 claims 1
- 230000000259 anti-tumor effect Effects 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 abstract 1
- 229930013292 trichothecene Natural products 0.000 abstract 1
- 229940125782 compound 2 Drugs 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 19
- 229940125904 compound 1 Drugs 0.000 description 19
- 238000001228 spectrum Methods 0.000 description 17
- 206010028980 Neoplasm Diseases 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 6
- 230000034994 death Effects 0.000 description 5
- 231100000517 death Toxicity 0.000 description 5
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 238000005100 correlation spectroscopy Methods 0.000 description 4
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
- 229960004316 cisplatin Drugs 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241001529821 Agastache Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241001072961 Pogostemon Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003474 anti-emetic effect Effects 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007822 cytometric assay Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- -1 novel trichothecene compound Chemical class 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
- C07D493/18—Bridged systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses compounds myrothecin H and I, and a preparation method and application thereof. The invention separates myrothecin H and I with anti-tumor activity from the liquid fermentation culture of the Pogostemon cablin endophytic fungus Parastyrtech roridum A697, which are respectively shown as a formula (I) and a formula (II). myrothecin H and I are trichothecene new compounds, and have IC effect on liver cancer cell HepG-2, breast cancer cell MCF-7, glioma cell SF-268 and large cell lung cancer cell NCI-H46050The value range is 0.09-9.72 mu M, and the antitumor activity is relatively obvious. The invention provides a candidate drug for researching and developing novel anti-tumor drugs and provides a scientific basis for developing and utilizing the patchouli endophytic fungi resources.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to compounds myrothecin H and I, and a preparation method and application thereof.
Background
Cancer is one of the major diseases that currently endanger human health. Data released by the international anticancer alliance show that 1270 ten thousand people suffer from cancer and the number of death people is as high as 760 ten thousand in 2008. The number of deaths from cancer worldwide is much greater than the sum of aids, malaria and tuberculosis. If no effective measures are taken, it is expected that 2600 million new cancer cases and 1700 million cancer deaths will occur annually by 2030. The cancer incidence rate is in a rapid rise period in China, the number of cancer diseases is about 260 thousands and 180 thousands of deaths each year. Cancer has become the first cause of death of residents in cities and rural areas in China, and the economic burden and the adverse effect on social development caused by cancer are increasingly highlighted. Among the three major treatments for cancer, drug therapy has been the dominant site.
Endophytic fungi (endophytic fungi) refer to fungi that live in healthy plant tissues at some or all stages of their life history, but do not cause significant disease symptoms to plant tissues (Tan RX and Zou WX, 2001). Endophytic fungi are abundant and diverse in species, are in special environments inside plants, can produce secondary metabolites with various structures, have the structural types far exceeding the range of plant metabolites, are easy to discover compounds with novel structures from the compounds, and have various biological activities, so the endophytic fungi become important resources for discovering new natural active substances and have important application potential in agriculture and medicine industry (executed Qi Yuan et al, 2007).
Herba Agastaches (Pogostemon cablin) is a plant of Pogostemon of Labiatae, is a perennial herb, is a whole herb medicine, and is a clinically common aromatic dampness-resolving traditional Chinese medicine. The stem or leaf of herba Agastaches is used for treating vomiting of pregnancy, stomachache and preventing influenza, and is aromatic, stomach invigorating, antipyretic and antiemetic.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides novel compounds myrothecin H and I which are discovered by patchouli endophytic fungi and have the function of anti-tumor activity, and a preparation method and application thereof.
The first object of the present invention is to provide a compound myrothecin H represented by the formula (I) and a compound myrothecin I represented by the formula (II),
the second object of the present invention is to provide a process for the preparation of compounds myrothecin H and myrothecin I, which are isolated from the fermentation culture of the Pogostemon cablin endophytic fungus Parastyrothecin A697.
Preferably, the preparation method comprises the following steps:
a. preparing a fermentation culture of Pogostemon cablin endophytic fungus Paramythium roridum A697, separating mycelium and fermentation liquor, extracting the fermentation liquor with ethyl acetate, and concentrating the extract to obtain extract;
b. extract process C18Reversed phase column chromatography, eluting with methanol-water as eluent at a volume ratio of 60:40 to 100:0, collecting fraction Fr3 eluted with methanol-water at a volume ratio of 70:30, subjecting Fr3 to gel column chromatography Sephadex LH-20, eluting with chloroform-methanol at a volume ratio of 1:3, collecting fractions, combining by TLC thin layer chromatography, collecting TLC thin layer chromatography, developing with n-hexane and ethyl acetate at a volume ratio of 1:1v/v to obtain fraction Fr3.3 with Rf of 0.3-0.5, subjecting fraction Fr3.3 to silica gel column chromatography, eluting with n-hexane and ethyl acetate at a volume ratio of 5:1, 2:1, 1:1, 1:2, 1:3, collecting fractions, combining by TLC thin layer chromatography, collecting fraction Fr 1.5 with Rf of 0.4-0.5 by TLC thin layer chromatography and developing with n-ethyl acetate at a volume ratio of 1:1v/v, the compounds myrothecin H and myrothecin I were obtained by further semi-preparative HPLC from component Fr 3.1.1.
Preferably, the component Fr3.1.1 is subjected to further semi-preparative HPLC to obtain compounds myrothecin H and myrothecin I by using YMCpack ODS-A/AQ column with mobile phase of acetonitrile/water at volume ratio of 50:50 and flow rate of 3mL/min to obtain compounds myrothecin H, tR12.6min and myrothecin I, tR 13.4min。
Preferably, the step a of preparing the fermentation culture of the pogostemon cablin endophytic fungus Parapyrotech roridum A697 comprises the following steps: selecting mycelium of Pogostemon cablin endophytic fungus Paramythium roridum A697, inoculating to potato glucose liquid culture medium, and culturing at 28 deg.C and 120r/min for 5 days to obtain seed solution; then inoculating the seed solution into a potato glucose liquid culture medium according to the inoculation amount of 10%, and culturing for 7 days at the temperature of 28 ℃ and at the speed of 120r/min to obtain a fermentation culture of Parayrothecium roridum A697;the potato glucose liquid culture medium is prepared by the following method per liter: boiling 200g of potato in 500mL of pure water for 20min, and filtering to obtain potato juice; adding glucose 20g and KH2PO4 3g、MgSO41.5g, vitamin B110 mg, and water to make up to 1000 mL.
The third purpose of the invention is to provide the application of the compound myrothecin H and/or myrothecin I, or the medicinal salt thereof in preparing the antitumor drugs.
Preferably, the anti-tumor drug is a drug for resisting liver cancer, breast cancer, glioma or large cell lung cancer. The fourth object of the present invention is to provide an antitumor agent comprising an effective amount of the compound myrothecin H and/or myrothecin I, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier.
Preferably, the anti-tumor drug is a drug for resisting liver cancer, breast cancer, glioma or large cell lung cancer.
The invention also provides application of the patchouli endophytic fungus Parapyrothecium roridum A697 in preparation of compounds myrothecin H and myrothecin I.
Experiments show that the compounds myrothecin H and I have IC (integrated Circuit) on liver cancer cell HepG-2, breast cancer cell MCF-7, glioma cell SF-268 and large cell lung cancer cell NCI-H46050The value range is 0.09-9.72 mu M, and the positive control substance cis-platinum has IC (integrated Circuit) on the four tumor cell lines50The values were 2.65, 3.02, 3.25 and 2.31. mu.M, respectively. This result shows that: the compounds myrothecin H and I have relatively obvious antitumor activity.
The compounds myrothecin H and I are prepared and separated from the Pogostemon cablin endophytic fungus Parastyrtech rhizodum A697, have antitumor activity, can be used for preparing antitumor drugs, provide candidate compounds for researching and developing new antitumor drugs, and provide scientific basis for developing and utilizing natural active substances of medicinal plant endophytic fungi.
The patchouli endophytic fungus parayrothecium roridum a697 of the present invention is also disclosed in non-patent literature (web page) at 2016, 7, 25.h, with the web addresses: https:// www.ncbi.nlm.nih.gov/nuccore/KU529828.1, which the applicant also owned and guaranteed to be available to the public within 20 years from the filing date.
Drawings
FIG. 1 is a scheme showing that Compound 1(myrothecin H)1H-NMR spectrum;
FIG. 2 is a drawing showing the preparation of Compound 1(myrothecin H)13C-NMR spectrum;
FIG. 3 is a COSY spectrum of Compound 1(myrothecin H);
FIG. 4 is an HSQC spectrum of Compound 1(myrothecin H);
FIG. 5 is an HMBC profile of compound 1(myrothecin H);
FIG. 6 is a NOESY spectrum of Compound 1(myrothecin H);
FIG. 7 is an HR-ESIMS spectrum of Compound 1(myrothecin H);
FIG. 8 is a drawing of Compound 2(myrothecin I)1H-NMR spectrum;
FIG. 9 is of Compound 2(myrothecin I)13C-NMR spectrum;
FIG. 10 is a COSY spectrum of Compound 2(myrothecin I);
FIG. 11 is an HSQC spectrum of Compound 2(myrothecin I);
FIG. 12 is an HMBC profile of compound 2(myrothecin I);
FIG. 13 is a NOESY spectrum of Compound 2(myrothecin I);
FIG. 14 is an HR-ESIMS spectrum of Compound 2(myrothecin I).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
separation, purification and identification of patchouli endophytic fungus A697
The endophytic fungus A697 is obtained by separating from the leaves of a cablin potchouli herb plant collected from Shuizhou of Yangchun city of Guangdong province in 2015 1 month, and has the GenBank gene accession number as follows through ITS sequence analysis and identification: KU529828.1, identified the strain as Paratyrothecium roridum by blast alignment and homology analysis, and named Paratyrothecium roridum A697 (hereinafter referred to as strain A697).
Secondly, liquid fermentation of the strain A697
The culture medium is a potato glucose liquid culture medium, and each liter of culture medium is prepared by the following method: decocting 200g of potato in 500mL of water, boiling for 20min, filtering to obtain potato juice, and adding glucose 20g and KH2PO4 3g、MgSO41.5g, vitamin B110mg, supplementing to 1000mL with water, autoclaving at 121 deg.C for 20min, and cooling for use.
Selecting appropriate amount of strain A697 mycelium, inoculating into potato glucose liquid culture medium, and culturing at 28 deg.C and 120r/min for 5 days to obtain seed solution. Then inoculating the seed solution into a 1000mL triangular flask filled with 500mL potato glucose liquid culture medium according to the inoculation amount of 10 percent of the volume ratio, fermenting for 40L in total, and culturing for 7 days at the temperature of 28 ℃ and at the speed of 120r/min to obtain a liquid fermentation culture of the strain A697.
Preparation of compounds myrothecin H and I
And centrifuging 40L of liquid fermentation culture to obtain fermentation liquor and mycelia, extracting the fermentation liquor for three times by ethyl acetate, combining extraction liquids, distilling the extraction liquids under reduced pressure, and recovering a solvent to obtain 4.7g of concentrated extract.
Extract process C18Reversed phase column chromatography, eluting with methanol-water as eluent at a volume ratio of 60: 40-100: 0, collecting fraction Fr3 (obtained by TLC thin layer chromatography with n-hexane: ethyl acetate at a ratio of 1:1v/v to obtain component Rf at a ratio of 0.3-0.7) eluted at a methanol-water volume ratio of 70:30, subjecting Fr3 to gel column chromatography Sephadex LH-20, and eluting with chloroform-methanol at a volume ratio of 1:3 as eluent to obtain four components Fr 3.1-Fr.3.4. The fraction Fr3.3 (fraction Rf of 0.3-0.5 developed by TLC thin layer chromatography with n-hexane: ethyl acetate of 1:1 v/v) was subjected to silica gel column chromatography with n-hexane-ethyl acetate as eluent, and gradient elution was carried out from the volume ratio of 5:1, 2:1, 1:1, 1:2, 1:3 to give two fractions Fr3.1.1 and Fr3.1.2. Fraction Fr3.1.1 (fraction Rf of 0.4-0.5 developed by TLC thin layer chromatography using n-hexane: ethyl acetate: 1 v/v) was subjected to further semi-preparative HPLC using YMCpack ODS-A/AQ columnThe mobile phase is acetonitrile/water with the volume ratio of 50:50, and the flow rate is 3mL/min, thus obtaining the compound myrothecin H (4.0mg, t)R12.6min) and myrothecin I (5.5mg, t)R13.4min)。
Structure identification of compound myrothecin H and I
1H NMR、13C NMR and HMBC nuclear magnetic resonance spectrograms are measured by a Bruker advanced-500 nuclear magnetic resonance spectrometer, and Tetramethylsilane (TMS) is used as an internal standard; ESI-MS data were measured with VG Autospec-3000 type mass spectrometer; the ultraviolet spectrum was measured with a UV6000 ultraviolet-visible spectrophotometer, Shanghai Yuan analysis Instrument Co., Ltd.
As shown in FIGS. 1 to 12, FIG. 1 is a drawing showing the preparation of Compound 1(myrothecin H)1H-NMR spectrum; FIG. 2 is a drawing showing the preparation of Compound 1(myrothecin H)13C-NMR spectrum; FIG. 3 is a COSY spectrum of Compound 1(myrothecin H); FIG. 4 is an HSQC spectrum of Compound 1(myrothecin H); FIG. 5 is an HMBC profile of compound 1(myrothecin H); FIG. 6 is a NOESY spectrum of Compound 1(myrothecin H); FIG. 7 is an HR-ESIMS spectrum of Compound 1(myrothecin H); FIG. 8 is a drawing of Compound 2(myrothecin I)1H-NMR spectrum; FIG. 9 is of Compound 2(myrothecin I)13C-NMR spectrum; FIG. 10 is a COSY spectrum of Compound 2(myrothecin I); FIG. 11 is an HSQC spectrum of Compound 2(myrothecin I); FIG. 12 is an HMBC profile of compound 2(myrothecin I); FIG. 13 is a NOESY spectrum of Compound 2(myrothecin I); FIG. 14 is an HR-ESIMS spectrum of Compound 2(myrothecin I).
Compound 1(myrothecin H): compound myrothecin H was a white solid (its nuclear magnetic data are shown in table 1); determining the molecular weight of myrothecin H to be 528 according to the ESIMS excimer ion peak; according to HRESIMS [ M + H ]]+m/z528.2440，C15H11O8Calculated value of 528.2432, the molecular formula of the compound was determined to be C29H36O10The unsaturation degree is 12; the hydrogen spectrum and carbon spectrum data have greater similarity with the known compound myrothecin A (Shen, et al.2006,12, 5596-. The structure of compound 1 should be one having a terminal double bond of C-9,a novel trichothecene compound with C-10 and C-12 bridged rings.
Compound 2(myrothecin I) is a white solid (its nuclear magnetic data are shown in table 1); determining the molecular formula of the compound as C according to high resolution mass spectrum29H36O9The unsaturation degree is 12; the 1D and 2D spectra of the compound 2 are analyzed to find that the spectrum of the compound 2 has great similarity with the spectrum of the compound 1, and the main difference is that the C-9 terminal double bond in the compound 1 is an intra-ring double bond in the compound 2. Thus, the structure of compound 2 was determined.
TABLE 1 Nuclear magnetic data (Δ in ppm, J in Hz, CD) for Myrothecin H and I3OD)
Thus, the chemical structural formula of the compound 1(myrothecin H) is shown as a formula (I), and the chemical structural formula of the compound 2(myrothecin I) is shown as a formula (II).
Example 2:
compounds myrothecin H and I were tested for anti-tumor activity using the SRB method [ Skehan P, stopping R, Dominic S.New colorimetric cytometric assay for anti-cancer-drug screening [ J ]. J Natl cancer Inst ].
1. Test reagents: the prepared compounds myrothecin H and I are dissolved in dimethyl sulfoxide (DMSO) to obtain a mother solution with the concentration of 10mg/mL, and then diluted to the required concentration by using RPMI-1640 culture medium. The positive control is cisplatin aqueous solution.
The tumor cell strains used in the experiment are liver cancer cell HepG-2, breast cancer cell MCF-7, glioma cell SF-268 and large cell lung cancer cell NCI-H460.
2. The experimental method comprises the following steps: taking HepG-2, MCF-7, SF-268 and NCI-H460 cells in logarithmic growth phase, digesting with pancreatin, staining and counting with trypan blue, detecting the cells with high activity by trypan blue exclusion experimentAfter 95%, the cell concentration was adjusted to 3X 10 with fresh RPMI-1640 medium4one/mL, cells were seeded in 96-well plates, 180. mu.L of cell suspension was added to each well, and 3 blank wells were set to zero, at 37 ℃ with 5% CO2The incubator is used for 24 h. After the cells adhered, 20. mu.L of solutions of myrothecin H and I with a certain concentration were added to each well, 20. mu.L of RPMI-1640 medium was added for negative control, and cisplatin was used as a positive control. Placing at 37 ℃ and 5% CO2After culturing for 72h in an incubator, 50 μ L of 50% cold trichloroacetic acid was added to fix the cells, the cells were left at 4 ℃ for 1 hour, washed with distilled water 5 times, and air-dried. Then 100. mu.L/well of a 4mg/mL solution of SRB prepared with 1% glacial acetic acid was added, stained at room temperature for 30min, the supernatant removed, washed 5 times with 1% glacial acetic acid and air dried. Finally, 200 mu L/hole 10mmol/mL Tris solution is added, the absorbance (A) at 570nm is measured by a microplate reader, and the inhibition rate of the drug on the cell growth is calculated by the following formula: cell growth inhibition (%) - (1-A)Sample set/AControl group) X 100%, three replicates per sample.
3. The experimental results are as follows: IC of prepared compounds myrothecin H and I on liver cancer cell HepG-2, breast cancer cell MCF-7, glioma cell SF-268 and large cell lung cancer cell NCI-H46050The value range is 0.09-9.72 mu M (Table 2), and the IC of the positive control cisplatin on the four tumor cell lines is50The values were 2.65, 3.02, 3.25 and 2.31. mu.M, respectively. This result shows that: the compounds myrothecin H and I have obvious antitumor activity, so the invention provides a candidate compound for researching and developing new antitumor drugs and provides a scientific basis for developing and utilizing natural active substances of endophytic fungi.
TABLE 2 inhibitory Effect of compounds myrothecin H and I on tumor cells
aValues are expressed as the mean±SD。
Claims (6)
1. A compound myrothecin H shown in a formula (I) or a compound myrothecin I shown in a formula (II),
2. a process for the preparation of the compounds myrothecin H and myrothecin I as claimed in claim 1, comprising the steps of:
a. preparing a fermentation culture of Pogostemon cablin endophytic fungus Paramythium roridum A697, separating mycelium and fermentation liquor, extracting the fermentation liquor with ethyl acetate, and concentrating the extract to obtain extract;
b. extract process C18Reversed phase column chromatography, eluting with methanol-water as eluent at a volume ratio of 60:40 to 100:0, collecting fraction Fr3 eluted with methanol-water at a volume ratio of 70:30, subjecting Fr3 to gel column chromatography Sephadex LH-20, eluting with chloroform-methanol at a volume ratio of 1:3, collecting fractions, combining by TLC thin layer chromatography, collecting TLC thin layer chromatography, developing with n-hexane and ethyl acetate at a volume ratio of 1:1v/v to obtain fraction Fr3.3 with Rf of 0.3-0.5, subjecting fraction Fr3.3 to silica gel column chromatography, eluting with n-hexane and ethyl acetate at a volume ratio of 5:1, 2:1, 1:1, 1:2, 1:3, collecting fractions, combining by TLC thin layer chromatography, collecting fraction Fr 1.5 with Rf of 0.4-0.5 by TLC thin layer chromatography and developing with n-ethyl acetate at a volume ratio of 1:1v/v, component Fr3.1.1 was subjected to further semi-preparative HPLC to give compounds myrothecin H and myrothecin I;
the preparation of the fermentation culture of the Pogostemon cablin endophytic fungus Parastyrtech roridum A697 in the step a comprises the following steps: selecting mycelium of Pogostemon cablin endophytic fungus Paramythium roridum A697, inoculating to potato glucose liquid culture medium, and culturing at 28 deg.C and 120r/min for 5 days to obtain seed solution; then inoculating the seed solution into a potato glucose liquid culture medium according to the inoculation amount of 10%, and culturing for 7 days at the temperature of 28 ℃ and at the speed of 120r/min to obtain a fermentation culture of Parayrothecium roridum A697; the above-mentionedThe potato dextrose liquid culture medium is prepared by the following method per liter: boiling 200g of potato in 500mL of pure water for 20min, and filtering to obtain potato juice; adding glucose 20g and KH2PO4 3g、MgSO41.5g, vitamin B110 mg, and water to make up to 1000 mL.
3. The process according to claim 2, wherein the fraction Fr3.1.1 is subjected to further semi-preparative HPLC to obtain the compounds myrothecin H and myrothecin I by using YMCpack ODS-A/AQ column with A mobile phase of acetonitrile/water in A volume ratio of 50:50 and A flow rate of 3mL/min to obtain the compounds myrothecin H, tR12.6min and myrothecin I, tR 13.4min。
4. The use of the compound myrothecin H and/or myrothecin I or a pharmaceutically acceptable salt thereof as claimed in claim 1 for the preparation of an anti-neoplastic drug against liver cancer, breast cancer, glioma or large cell lung cancer.
5. An antitumor agent comprising an effective amount of the compound myrothecin H and/or myrothecin I or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier, wherein the antitumor agent is an agent against liver cancer, breast cancer, glioma or large cell lung cancer.
6. Use of the patchouli endophytic fungus Parapyrothecium roridum A697 for the preparation of the compounds myrothecin H and myrothecin I according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011127846.3A CN112300188B (en) | 2020-10-19 | 2020-10-19 | Compounds myrothecin H and I, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011127846.3A CN112300188B (en) | 2020-10-19 | 2020-10-19 | Compounds myrothecin H and I, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112300188A CN112300188A (en) | 2021-02-02 |
| CN112300188B true CN112300188B (en) | 2022-03-22 |
Family
ID=74328215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011127846.3A Active CN112300188B (en) | 2020-10-19 | 2020-10-19 | Compounds myrothecin H and I, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112300188B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684035B (en) * | 2019-09-03 | 2021-07-30 | 郑州轻工业学院 | Trichothecene compound and application thereof |
-
2020
- 2020-10-19 CN CN202011127846.3A patent/CN112300188B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112300188A (en) | 2021-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112592350B (en) | Polyketide lithocarpin E-G and preparation method and application thereof | |
| CN109336873B (en) | Compound lithocarolsA-F, preparation method thereof and application thereof in preparation of antitumor drugs | |
| CN110642827B (en) | Compounds of photosteroids A and B, preparation method thereof and application thereof in preparing antitumor drugs | |
| CN106631775A (en) | Compound cytosporaphenone A and preparation method thereof and applications in preparing anti-tumor drugs | |
| CN109232513B (en) | Compound litocarpinols, preparation method thereof and application thereof in preparation of antitumor drugs | |
| CN111285758B (en) | Preparation method of compound trieffulsols C-E and application of compound trieffulsols C-E in preparation of anti-inflammatory drugs | |
| CN108912086B (en) | Active flavonoid compound and preparation method and application thereof | |
| CN111004251B (en) | Marine-derived heteroterpene compounds I and II, preparation method and application thereof in preparation of antitumor drugs | |
| CN108570025B (en) | Oxygen-containing five-membered ring pimarane diterpenoid compound, preparation method and application thereof | |
| CN109776561B (en) | Compounds Cytorhizins B and C, preparation method thereof and application thereof in preparation of antitumor drugs | |
| CN107501072B (en) | Compound colletotriconeA, preparation method thereof and application thereof in preparing antitumor drugs | |
| CN112300188B (en) | Compounds myrothecin H and I, and preparation method and application thereof | |
| CN114213428B (en) | Indole alkaloid compound and preparation method and application thereof | |
| CN111217706A (en) | Marine polyketide hypoxone A, preparation method thereof and application thereof in preparing anti-inflammatory drugs | |
| CN111808088B (en) | Compounds tersaphilone B and E, preparation method thereof and application thereof in preparing antitumor drugs | |
| CN102746995B (en) | Preparation method for isochromophilone VIII and application of same in preparation of antineoplastic drugs | |
| CN113149820B (en) | Monocyclic hetero-terpene structural compound, preparation method and application thereof | |
| CN115536645A (en) | Compound Phonolide B, preparation method thereof and application thereof in antibacterial drugs | |
| CN106279092B (en) | A kind of double 1,4-benzoquinone class compounds and its extracting method | |
| CN109369399B (en) | 1, 3-O-diferulate-2-methoxy propylene glycol and preparation method and application thereof | |
| CN112500374B (en) | Compound tenellone K, preparation method thereof and application thereof in preparing antitumor drugs | |
| CN115181083B (en) | Preparation method of compound Cyophiobiolins A-B and application of compound Cyophiobiolins A-B in preparation of anti-inflammatory drugs | |
| CN112079897A (en) | Two steroid compounds and preparation method and application thereof | |
| CN118063376A (en) | Preparation method and application of compound pararorine A | |
| CN115650854B (en) | Integrin derivative, its preparation method and application in alpha-glucosidase inhibiting medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Address before: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |