CN109942658A - A kind of miscellaneous terpene compound and the preparation method and application thereof and anti-tumor drug - Google Patents
A kind of miscellaneous terpene compound and the preparation method and application thereof and anti-tumor drug Download PDFInfo
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- CN109942658A CN109942658A CN201910354207.1A CN201910354207A CN109942658A CN 109942658 A CN109942658 A CN 109942658A CN 201910354207 A CN201910354207 A CN 201910354207A CN 109942658 A CN109942658 A CN 109942658A
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- terpene compound
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- miscellaneous terpene
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Abstract
The structural formula of miscellaneous terpene compound is as follows in the present invention；
Description
Technical field
The invention belongs to the application fields of marine microorganism drugs, and in particular to miscellaneous terpene compound and preparation method thereof with answer
With and anti-tumor drug.
Background technique
The complexity of disease and variability can not be ignored the importance of new drug development, and natural products is the weight of drug
Want source.Microorganism be easy to cultivate and scale amplification and environmental-friendly feature become the important of Natural products research
Object.The particularity of marine environment gives marine microorganism source natural medicine source again plus the progress of Marine Microorganisms acquiring technology
The research band of compound unprecedented opportunities.Marine fungi in marine microorganism is the abundant of active secondary metabolites
Source.
Proliferation, survival and the differentiation of tumour cell are influenced by a variety of heredity and molecular changes.A plurality of types of chemical combination
Anti-tumor active substance was had been found that in object, such as alkaloid compound, steroid compound, flavone compound, Anthraquinones
Compound etc..
The efficient anti-cancer drugs with novel mechanism of action is found from marine fungi secondary metabolite or derivatives thereof
It is an important directions of cancer drug development, the natural products pair of specific structure type is found to have using marine fungi as raw material
The research and development of Yu Haiyang's drug are of great significance.For this purpose, the present invention has found two kinds of new miscellaneous terpene compounds by raw material of Penicillium notatum,
It is with stronger anti-tumor activity.
Summary of the invention
The present invention obtains a new class of miscellaneous terpene compound by making full use of Deep-Sea Microorganisms resources advantage.
The structural formula of miscellaneous terpene compound is as follows in the present invention；
The present invention also provides a kind of preparation methods of miscellaneous terpene compound, comprising the following steps:
S1, Penicillium notatum is subjected to fermented and cultured, obtain tunning；
S2, tunning extracted, obtain extract liquor；Extract liquor obtains miscellaneous terpene compound after isolating and purifying.
In one of specific embodiment of the invention, Penicillium notatum was obtained at PDA plate culture 3 days in step S1
Mycelium, cultivation temperature are 25 DEG C；Mycelium is inoculated into static culture 30 days in the conical flask of 1L, cultivation temperature 28 again
DEG C, there are 80g rice and 120mL water in conical flask.
At least one of the miscellaneous terpene compound of the present invention and its esters are used for the preparation of anti-tumor drug, and the anti-of preparation swells
Tumor medicine is inhibited to tumour cell.
Specifically, anti-tumor drug is for treating in bladder cancer, lung cancer, the cancer of the esophagus, cancer of pancreas, liver cancer and cervical carcinoma
It is at least one.
A kind of anti-tumor drug, at least one of miscellaneous terpene compound and its salt including effective dose and pharmacy
Upper acceptable carrier.
In terms of existing technologies, the invention has the following advantages:
The miscellaneous terpene compound of the present invention ferments generation by Penicillium notatum in specific culture medium can with anti-tumor activity
It is used to prepare anti-tumor drug, the present invention provides new compound source for research and development anti-tumor drug, utilizes ocean abundant
Microorganism prepares miscellaneous terpene compound as anti-tumor active substance as fermentation raw material fermentation, drug production cost is reduced, to cancer
Cell has apparent inhibiting effect, has a good application prospect.
Specific embodiment
Invention is further described in detail With reference to embodiment.
Penicillium notatum Penicillium allii-sativi is preserved in Chinese Sea Microbiological Culture Collection administrative center, protects
Hiding number is MCCC 3A00580.
The preparation of miscellaneous terpene compound
(1) Penicillium notatum is cultivated 3 days on PDA plate in 25 DEG C.Then fresh mycelium is inoculated into 50 1L's
In conical flask at 28 DEG C static culture, have 80g rice and 120mL distilled water in every bottle, cultivate 30 days.
(2) fermentation material that step (1) obtains is extracted with ethyl acetate three times.It is evaporated off under reduced pressure organic solvent, is had
Machine extract (120g).These extracts are extracted using methanol and with petroleum ether.Methanol layer is concentrated, obtains degreasing extraction
Object (60.4g).
(3) the degreasing extract that step (2) obtains is separated using silica gel column chromatography, with methylene chloride-methanol body
System carries out gradient elution, obtains 8 fractions (Fr.1~Fr.8).
(4) the fraction Fr.3 (5.5g) for having bioactivity for obtaining step (3) is separated using ODS column chromatography, is made
Gradient elution is carried out with Water-Methanol System, then is eluted on silica gel column chromatography using petroleum ether-acetone (5:1), is changed
It closes object 1 (5.0mg).
(5) another bioactivity fraction Fr.5 (6.7g) for obtaining step (3) crosses ODS chromatographic column and Sephadex LH-
20 gel columns, are eluted using methanol.It is finally purified by preparative HPLC using water-methanol gradient (20% → 80%),
Obtain compound 2 (3.5mg).
(6) 1D the and 2D NMR spectra of compound 1 and 2 obtained in above-mentioned steps and high resolution mass spectrum analysis are carried out
Structural Identification:
Compound 1 is colorless oil.Determine that its molecular formula is C in high resolution mass spectrum according to it28H40O7, degree of unsaturation
It is 9.1H and13C NMR data (table 1) 28 carbon signals of display, including eight methyl, a methoxyl group, four methylene, four
Methine (ethylene linkage) and 11 quaternary carbons (two ketone, two carboxyls, an olefinic carbon).Remove 1 methoxyl group [δH 3.54
(3H,s)；δC51.8q] and an acetyl group [δH2.03(3H,s)；δC21.1q, 170.0s] outside, remaining showing of signal
It closes 1 structural formula of object and also contains 25 carbon.Due to two ketone groups, two carboxyls and an ethylenic moieties account for 5 degrees of unsaturation, because
This compound 1 is four ring skeletons.
In COSY spectrum, according to the related of H-3/H-2/H-1, H-5/H-6/H-7 and H-9/H-11/H-21, three are obtained
Segment.In HMBC spectrum, according to Me-18, Me-20, Me-22-Me-25, H-3 are related to H-3''s, and three above segment is connected
Four ring skeletons are connected into, and acetyl group and methoxyl group are being connected on C-3 and C-19 respectively.The flat of compound 1 is thereby determined that
Face structure.
1 compound 1 of table and compound 21H (400MHz) and13C (100MHz) NMR data
The relative configuration of compound 1 is mainly composed by NOESY and is determined.It is related to H-9/Me-25/H-7a according to H-5, H-9 with
16-OH is related, and Me-25 is related to H-2a through 3-OAc, and H-2b is related to Me-24, and H-7b is through Me-22 and Me-23/3'-OMe
It is related.Therefore, H-5/H-9/Me-25/3-OAc/16-OH is in same plane, it is determined as beta comfiguration, with Me-23/H-3/Me-24/
Me-22/Me-18 is opposite.
Electronic circular dichroism (ECD) spectrogram that its absolute configuration is measured by comparison theoretical calculation and experiment, discovery (3S,
5S, 8S, 9S, 10R, 13R, 14R, 16R) -1 calculating map and experiment chromatogram accordance.The absolute structure of compound 1 is thereby determined that
Type, and named are as follows: androsterone A (andrastone A).
Compound 2 is colorless oil, and molecular formula is determined as C according to high resolution mass spectrum28H36O9。1H and13C NMR data
(table 1) and known compound citreohybriddione A are closely similar.It the difference is that only C-16, Me-18 is respectively toward low
Field has been displaced 4.7 and 5.7ppm, thus it is speculated that and the hydroxyl of C-16 should be beta comfiguration in compound 2, rather than
α configuration in citreohybriddione A.NOESY spectrum in, 3'-Me is related to Me-18, so that it is determined that in 2 C-16 hydroxyl
Base is beta comfiguration.By comparing experiment and the calculated value of its ECD, the final structure for determining compound 2 is ent-16 β-
Hydroxycitreohybriddione A, name are as follows: androsterone B (andrastone B).
The anti-tumor activity of miscellaneous terpene compound is investigated
The present embodiment select 7 plants of tumour cells, including HepG2 (liver cancer), A549 (lung cancer), BIU-87 (bladder cancer),
BEL-7402 (liver cancer), ECA-109 (cancer of the esophagus), Hela-S3 (cervical carcinoma) and PANC-1 (cancer of pancreas).
By detection compound sample to the inhibiting rate of these tumour cells, so that detection compound 1 and 2 is antitumor
Activity.
The present embodiment is divided into following 3 groups:
(1) negative control group of compound is not added in cell culture fluid；
(2) cell is free of, but has the blank control group of equivalent culture solution；
(3) Penicillium notatum fermented cpds experimental group: gained Penicillium notatum fermented cpds in embodiment 1；
Present embodiment is using following operation:
(1) with 2000~5000, every hole cell inoculation to 96 orifice plates, every 200 μ L of pore volume.
(2) 37 DEG C, 5%CO2Incubator in cultivate for 24 hours, then be added various concentration compound continue to cultivate.
(3) after 72h, every hole adds 10 μ L 3- (4,5)-dimethylthiahiazo (- z-y1) -3,5-
Diphenytetrazol iumromide (MTT), 37 DEG C are protected from light 3h.After supernatant is abandoned in careful suction, 150 μ L are added in every hole
DMSO vibrates 10 minutes, dissolves crystal sufficiently.
(4) microplate reader measures 570nm absorbance value, then calculates inhibiting rate according to public formula (I).
Public formula (I):
(5)IC50Indicate compound concentration when inhibiting rate is 50%.
The IC of compound 1 and 250Data are as shown in table 2, and compound 1 and compound 2 are to HepG2, A549, BIU-87, BEL-
7402, ECA-109, Hela-S3 and PANC-1 have stronger inhibiting effect, show stronger anti-tumor activity, and compound 1 is right
The growth of hepatoma Hep G 2 cells has significant inhibiting effect.
Inhibiting effect (the IC of 2 compound 1 of table and 2 pairs of compound different tumour cells50, μM)
The corresponding salt of compound 1, the corresponding salt of compound 2 equally have anti-tumor activity.
It will be in the compound 1 of effective dose, the corresponding salt of compound 1, compound 2 and the corresponding salt of compound 2
It is at least one to be mixed with pharmaceutically acceptable carrier with the drug for inhibiting growth of tumour cell.
Only several embodiments of the present invention are expressed for above embodiments, and the description thereof is more specific and detailed, but can not
Therefore it is construed as limiting the scope of the patent.It should be pointed out that for those of ordinary skill in the art,
Under the premise of not departing from present inventive concept, various modifications and improvements can be made, and these are all within the scope of protection of the present invention.
Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (6)
1. a kind of miscellaneous terpene compound, which is characterized in that the structural formula of the miscellaneous terpene compound is as follows；
2. a kind of preparation method of miscellaneous terpene compound as described in claim 1, which comprises the following steps:
S1, Penicillium notatum is subjected to fermented and cultured, obtain tunning；
S2, tunning extracted, obtain extract liquor；Extract liquor obtains miscellaneous terpene compound after isolating and purifying.
3. the preparation method of miscellaneous terpene compound as claimed in claim 2, which is characterized in that step S1It is middle by Penicillium notatum in PDA plate
Culture obtains mycelium for 3 days, and cultivation temperature is 25 DEG C；Mycelium is inoculated into static culture 30 days in the conical flask of 1L again,
Cultivation temperature is 28 DEG C, there is 80g rice and 120mL water in conical flask.
4. a kind of application of miscellaneous terpene compound as described in claim 1, which is characterized in that in the miscellaneous terpene compound and its esters
At least one preparation for anti-tumor drug.
5. the application of miscellaneous terpene compound according to claim 4, which is characterized in that the anti-tumor drug is for treating wing
At least one of Guang cancer, lung cancer, the cancer of the esophagus, cancer of pancreas, liver cancer and cervical carcinoma.
6. a kind of anti-tumor drug, which is characterized in that including effective dose as described in claim 1 miscellaneous terpene compound and its
At least one of salt, and pharmaceutically acceptable carrier.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111217878A (en) * | 2020-02-21 | 2020-06-02 | 自然资源部第三海洋研究所 | Andrastone compounds, preparation method thereof and application thereof in preparing antiallergic drugs |
| WO2022057223A1 (en) * | 2020-09-21 | 2022-03-24 | 自然资源部第三海洋研究所 | Benzothiazole meroterpenoid compound and derivative thereof, and preparation method therefor and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134574A (en) * | 2018-09-28 | 2019-01-04 | 国家海洋局第三海洋研究所 | Steroidal compounds and the preparation method and application thereof and anti-tumor drug |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134574A (en) * | 2018-09-28 | 2019-01-04 | 国家海洋局第三海洋研究所 | Steroidal compounds and the preparation method and application thereof and anti-tumor drug |
Non-Patent Citations (2)
| Title |
|---|
| KOSEMURA, SEIJI1 ET AL: "Meroterpenoids from Penicillium citreo-viride B. IFO 4692 and 6200 hybrid", 《TETRAHEDRON》 * |
| YUDAI MATSUDA ET AL: ""Reconstituted biosynthesis of fungal meroterpenoid andrastin A"", 《TETRAHEDRON》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111217878A (en) * | 2020-02-21 | 2020-06-02 | 自然资源部第三海洋研究所 | Andrastone compounds, preparation method thereof and application thereof in preparing antiallergic drugs |
| WO2022057223A1 (en) * | 2020-09-21 | 2022-03-24 | 自然资源部第三海洋研究所 | Benzothiazole meroterpenoid compound and derivative thereof, and preparation method therefor and use thereof |
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| CN109942658B (en) | 2020-08-14 |
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