CN101928270A - Isobenzofuranone compound, preparation method thereof and use thereof - Google Patents
Isobenzofuranone compound, preparation method thereof and use thereof Download PDFInfo
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- CN101928270A CN101928270A CN 201010222125 CN201010222125A CN101928270A CN 101928270 A CN101928270 A CN 101928270A CN 201010222125 CN201010222125 CN 201010222125 CN 201010222125 A CN201010222125 A CN 201010222125A CN 101928270 A CN101928270 A CN 101928270A
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Abstract
The invention discloses a novel isobenzofuranone compound of a general chemical structural formula (I), a preparation method thereof and use thereof. In the formula, 1, 3, 4a, 4, 5, 6, 7, 7a, 8 and 9 represent C1, C3, C4a, C4, C5, C6, C7, C7a, C8 and C9 respectively; the isobenzofuranone compound is extracted from fungi of which the strain classification is cephalosporium AL031 and the strain collection number is CCTCC No.M2010140. The compound is subjected to antioxidant activity and antitumor activity screening. Experiment results show that the compound has high antioxidant activity and antitumor activity and a promising medicinal application prospect.
Description
Technical field
The present invention relates to a kind of natural drug, more particularly, relate to a kind of new isobenzofuran ketone compound that obtains that from Cephalosporium AL031 secondary fungus metabolite, separates.Simultaneously, the invention still further relates to the preparation method of this compound and the application in pharmacy field.
Background technology
The existing fungi of occurring in nature nearly 250,000 kinds, kind surplus China has 100,000 at least has been reported about 8000 kinds, wherein traditional drugs with and the fungi of test with drug effect reach 400 surplus kind.It is various that fungus metabolite has structure type, the characteristics that physiologically active is various.Thereby, follow the tracks of activity, the fungi chemical ingredients is studied, can be the bioactive natural product of seeking new texture an excellent research direction is provided.
The mould AL031 bacterial strain of cephalo be from separation screening on the arrow bamboo of the firm mountain of Yunnan Province sorrow to a kind of filamentous fungus strain, be accredited as Moniliaceae, Cephalosporium (Cephalosporium Corde) fungi through professor Yang Farong of biology department of Yunnan University.According to the research of people such as Bi Yunmei to this secondary fungus metabolite, the compound that is separated to from this secondary fungus metabolite finds to have broad-spectrum antibacterial activity.Therefore, this genus secondary fungus metabolite is done further research, tool has very important significance.
Summary of the invention
The objective of the invention is to by the chemical ingredients of Cephalosporium fungi AL031 secondary metabolite is furtherd investigate, thereby a kind of new compound that pharmaceutical use is arranged is provided.
Another object of the present invention provides a kind of preparation method of described new compound.
Further aim of the present invention provides the purposes of described new compound aspect the anti-oxidant and antitumor drug of preparation.
Purpose of the present invention is achieved by following technical proposals.
* except as otherwise noted, the percentage ratio that is adopted among the present invention is mass percent.
A. the present invention has isolated a kind of new compound from Cephalosporium fungi AL031 secondary metabolite, and this compound is represented with following structural formula:
This compound is named as 4,6-dihydroxyl-5-methoxyl group-7-methyl-isobenzofuranone.
Wherein, Me-represents methyl, MeO one expression methoxyl group.
B. the invention provides a kind of preparation method of described isobenzofuranone, this method adopts following step:
(1) with rice, Semen Maydis powder, bagasse, wheat bran are raw material, by 12: 2: 3: the sterilization of 3 (W/W/W/W) mixed, cooling;
(2) strain classification called after Cephalosporium AL031 fungi is inoculated in the mixture,, fermented 7 days at 37 ℃ of bottom fermentations; The deposit number of its bacterial strain is CCTCC №: M2010140;
(3) carry 7 days with industrial ethyl acetate lucifuge cold soaking, united extraction liquid, filtration, concentrating under reduced pressure extracting solution get ethyl acetate medicinal extract, keep in Dark Place;
(4) with heavily steaming acetic acid ethyl dissolution extraction 5 times, the combining extraction liquid concentrating under reduced pressure becomes medicinal extract to medicinal extract once more, medicinal extract with acetone solution after with 200～300 order silica gel mixed samples;
(5) 100～200 purpose silica gel carry out rough segmentation with wet method dress post, adopt with pure chloroform give moving phase earlier, add methyl alcohol then, add high polarity gradually, and up to chloroform: the volume ratio of methyl alcohol is 9: 1; Gradient elution is collected the elutriant of various piece; 20: 1 (volume ratio) part of elutriant silica gel, gel filtration chromatography is repeatedly separated, and promptly obtains required compound.
C. this compound has been carried out the anti-oxidant and antitumor activity screening of DPPH, compound exhibits goes out anti-oxidant preferably and anti-tumor activity.Its half to DPPH free radical, HL-60 cell, M49 lung carcinoma cell is removed concentration IC
50Measurement result is respectively 28.7mg/L, 12.5mg/L, 23mg/L.
Beneficial effect of the present invention:
1. Cephalosporium AL031 fungi remains in the laboratory, can ferment in batches, and raw material sources are simple; And The compounds of this invention content in this secondary fungus metabolite is higher, obtains easily;
2. adopted lucifuge operation and conventional column chromatography preparation method, the compound operating process is simple, the The compounds of this invention purity height that is obtained, and suitability for industrialized production subsequently is easy to realize;
3. described compound shows stronger anti-oxidant, anti-tumor activity, can be new compound or lead compound that medicine industry provides pharmaceutical use, is indicating well prospect in medicine.
Description of drawings
Fig. 1 is The compounds of this invention high resolution mass spectrum (ESI-MS);
Fig. 2 be The compounds of this invention proton nmr spectra (
1H NMR);
Fig. 3 be The compounds of this invention carbon-13 nmr spectra (
13C NMR);
Fig. 4 is the DEPT spectrum of The compounds of this invention;
Fig. 5 is the relevant spectrum of the HMBC of The compounds of this invention;
Fig. 6 is the relevant spectrum of the HMBC of The compounds of this invention.
The explanation of preservation biomaterial
Fungal bacterial strain involved in the present invention has been deposited in Chinese typical culture collection center on June 9th, 2010; This centre address: China. Wuhan. Wuhan University.This strain classification called after: Cephalosporium AL031 (Cephalosporium Corda AL031), deposit number is CCTCC:M2010140.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with drawings and Examples.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
Embodiment 1
---the preparation 1 of compound
With rice, Semen Maydis powder, bagasse, wheat bran is raw material, by 12: 2: 3: the sterilization of 3 (W/W/W/W) mixed, cooling.Cephalosporium AL031 fungi is inoculated in the mixture, at 37 ℃ of bottom fermentations, ferments 7 days; Carry 7 days with industrial ethyl acetate lucifuge cold soaking, extract 1 time, extracting liquid filtering, concentrating under reduced pressure get ethyl acetate medicinal extract, keep in Dark Place; Medicinal extract with heavily steaming acetic acid ethyl dissolution extraction, extracts 5 times once more, and the combining extraction liquid concentrating under reduced pressure becomes medicinal extract, medicinal extract with acetone solution after with 200～300 order silica gel mixed samples; 100～200 purpose silica gel carry out rough segmentation with wet method dress post, adopt pure chloroform give moving phase earlier, add methyl alcohol then, add high polarity gradually, and up to chloroform: the volume ratio of methyl alcohol is 9: 1; Collect the elutriant of various piece; 20: 1 (volume ratio) part of elutriant silica gel, gel filtration chromatography is repeatedly separated, and promptly obtains required compound.
Embodiment 2
---the preparation 2 of compound
Repeat the process of embodiment 1, but following difference is arranged: carry 5 days with industrial ethyl acetate lucifuge cold soaking, extract 2 times, united extraction liquid, filtration, concentrating under reduced pressure get ethyl acetate medicinal extract and keep in Dark Place.
Embodiment 3
---the preparation 3 of compound
Repeat the process of embodiment 1, but following difference is arranged: carry 6 days with industrial ethyl acetate lucifuge cold soaking, extract 2 times, united extraction liquid, filtration, concentrating under reduced pressure get ethyl acetate medicinal extract and keep in Dark Place.
Embodiment 4
---the preparation 4 of compound
Repeat the process of embodiment 1, but following difference is arranged: medicinal extract is with heavily steaming ethyl acetate extraction 3 times, and the combining extraction liquid concentrating under reduced pressure becomes medicinal extract, medicinal extract with acetone solution after with 200～300 order silica gel mixed samples; 100～200 purpose silica gel carry out rough segmentation with dry column-packing, and use chloroform: methyl alcohol is gradient elution by a certain percentage, collect the elutriant of various piece; 20: 1 parts of elutriant silica gel, gel filtration chromatography are repeatedly separated, and promptly obtain required compound.
Embodiment 5
---the structure to The compounds of this invention is identified
Compound 1 is colourless needle, 253 ℃ of mp; ESI-MS provides quasi-molecular ion peak m/z:211.0628[M+H]
+(calculated value 211.0562), in conjunction with
1H NMR,
13C NMR infers that molecular formula is C
10H
10O
5, degree of unsaturation is 6.λmax(MeOH)：217nm、255nm。In the infrared spectra (KBr) 3365,3164cm
-1Show that having a plurality of hydroxyls exists.1685cm
-1Show that having carbonyl in the molecule exists, 1611,1515cm
-1Show in the molecule and may have phenyl ring.From
13C NMR (CD
3OD, 100MHz) in as can be seen compound 2 methyl are arranged, 1 methylene radical and 7 quaternary carbons.δ
C172.7 (C) wait signal to determine the existence of ester group, exist and can define phenyl ring according to 6 remaining quaternary carbon signals in the low field.Can infer that in conjunction with the degree of unsaturation of this compound it belongs to the isobenzofuran class.
1H NMR (CD
3There is a methyl [δ in the compound in OD in 400MHz) as can be seen
H=2.42, (3H, s)], a methoxyl group [δ
H=3.87, (3H, s)].Compose data according to the molecular formula of above compound and the carbon spectrum data and the hydrogen of appearance, and can infer to also have 2 OH to exist in the molecule in conjunction with IR.H-3 and C-1 as can be seen from HMBC spectrum, C-4, C-4a, C-7a is relevant, H-8 and C-6, C-7a is relevant.According to HMBC spectrum, the H in the methoxyl group is relevant with C on 5, releases then that methoxyl group is connected on 5 in the compound 1.By the spectrogram analysis-by-synthesis, provide the structure of compound, and its NMR data have been carried out belonging to (seeing Table1.1).。
Table?1.1
1H?NMR，
13C?NMR?and?HMBC?data?for?compound?1
Embodiment 6
Compound with oxidation resistance is active one by one detects
Anti-oxidant activity is represented with the size of removing DPPH free radical ability; 6.5 * 10
-5MolL
-1DPPH solution is prepared with dehydrated alcohol, and the sample dissolve with methanol is a primary dcreening operation concentration with 0.1mg/mL, measures the activity that it removes free radical DPPH.In test tube, add 2.5mL 6.5 * 10 successively
-5MolL
-1DPPH solution and 1.5mL methyl alcohol, cumulative volume are 4.0mL, behind the mixing 20min, measure A (517nm) in the 1cm cuvette, are designated as A
0Add 2.5mL 6.5 * 10
-5MolL
-1DPPH solution and 1.5mL sample to be tested solution, measured value is designated as As; Add 2.5mL methyl alcohol and 1.5mL sample to be tested solution, measured value is designated as Ar.Sample is calculated as follows free radical DPPH clearance rate:
Calculate half and remove concentration IC
50, IC
50Measurement result is 28.7mg/L, shows that compound of the present invention has anti-oxidant activity preferably.
Embodiment 7
Antitumor activity of compound detects one by one
Adopt the increment restraining effect of improvement mtt assay assessing compound to tumour cell
Get standby tumour cell suspension inoculation in 96 well culture plates, 90 μ L/ holes, the given the test agent of adding different concns behind the continuation cultivation 24h, 10 μ L/ holes, making its final concentration is 100,10,1,0.1,0.01 μ g/mL, each concentration is all established 3 multiple holes.The positive contrast of cis-platinum (DDP), the negative contrast of equal-volume RPMI1640 substratum.Corresponding D MSO concentration is the solvent contrast when establishing compound 100,10 μ g/mL simultaneously, to eliminate the influence of DMSO cell growth.Continue to place 37 ℃ after the dosing, 5%CO
2Incubator is cultivated 72h, and every hole adds MTT (5mg/mL) 20 μ L, continues to cultivate 4h, and every hole adds three liquid [10%SDS-5% isopropylcarbinol-0.012mol/L HCl (W/V/V)], 100 μ L Rong Xie Jia Za.Under the 570nm wavelength, measure the OD value in each hole with microplate reader.
Calculate cell increment inhibiting rate by following formula:
Inhibiting rate (%)=(OD value control wells-OD value dosing holes)/OD value control wells * 100%
Data statistic analysis: adopt Microsoft Excel 2003 to calculate inhibiting rate, adopt the logit method, SPSS11.5 software package calculation of half inhibitory concentration IC
50
Calculation of half inhibitory concentration IC
50, to HL-60 cell, M49 lung carcinoma cell IC
50Measurement result is 12.5mg/L, and 23mg/L shows that compound of the present invention has better antitumor activity.
The above only is preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of being done within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. compound with following structural formula:
Wherein, 1,3,4a, 4,5,6,7,7a, 8,9 represent C1, C3, C4a, C4, C5, C6, C7, C7a, C8, C9 respectively.
2. the preparation method of the described compound of claim 1 is characterized in that: this method adopts following steps:
(1) with rice, Semen Maydis powder, bagasse, wheat bran are raw material, by 12: 2: 3: 3 mass ratio mixed sterilization, cooling;
(2) fungi with strain classification called after Cephalosporium AL031 is inoculated in the mixed culture medium, at 37 ℃ of bottom fermentations, ferments 7 days; The deposit number of its bacterial strain is CCTCC №: M2010140;
(3) the solid fermentation product is carried 7 days with industrial ethyl acetate lucifuge cold soaking, and united extraction liquid, filtration, concentrating under reduced pressure extracting solution get ethyl acetate medicinal extract, keep in Dark Place;
(4) medicinal extract is once more with heavily steaming ethyl acetate solvent extraction 5 times, combining extraction liquid, decompression is condensed into medicinal extract once more, medicinal extract with acetone solution after with 200～300 order silica gel mixed samples;
(5) 100～200 purpose silica gel carry out rough segmentation with wet method dress post, adopt pure chloroform give moving phase earlier, add methyl alcohol then, add high polarity gradually, and up to chloroform: the volume ratio of methyl alcohol is 9: 1; Gradient elution is collected the elutriant of various piece; 20: 1 parts of elutriant silica gel, gel filtration chromatography are repeatedly separated, and promptly obtain required compound.
3. the described compound of claim 1 is as the application of antioxidant.
4. the application of the described compound of claim 1 in the preparation antitumor drug.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617529A (en) * | 2012-02-27 | 2012-08-01 | 云南民族大学 | Application for isobenzofuran type compounds in marine biofouling prevention and preparation method thereof |
| CN103772237A (en) * | 2014-01-24 | 2014-05-07 | 先尼科化工（上海）有限公司 | Method for swapping out ammonia with strong base in tetrachloro o-nitrile ammonium benzoate production process |
| CN106008422A (en) * | 2016-06-27 | 2016-10-12 | 云南中烟工业有限责任公司 | Benzo-lactone compound, preparation method of benzo-lactone compound and application of benzo-lactone compound in preparing anti-cancer medicine |
| CN106883243A (en) * | 2017-02-14 | 2017-06-23 | 云南民族大学 | It is a kind of with the isobenzofuran class compound and its tobacco purposes of removing free radical effect in the root of kudzu vine |
| WO2019153954A1 (en) * | 2018-02-12 | 2019-08-15 | 广州自远生物科技有限公司 | Multi-substituted benzene compound having biological activity, preparation method therefor, and application thereof |
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| CN109055233B (en) * | 2018-08-08 | 2021-08-17 | 黑龙江大学 | Fungus for producing dimethoxy isobenzofuran-1-one and method for preparing dimethoxy isobenzofuran-1-one by fungus |
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| US20060009431A1 (en) * | 2003-03-13 | 2006-01-12 | Nitromed, Inc. | Nitrosated and nitrosylated compounds, compositions and methods use |
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| US20060009431A1 (en) * | 2003-03-13 | 2006-01-12 | Nitromed, Inc. | Nitrosated and nitrosylated compounds, compositions and methods use |
Non-Patent Citations (2)
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| 《中草药》 20011231 毕韵梅等 头孢霉AL 031 真菌次生代谢产物的挥发性成分 第32卷, 第12期 2 * |
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Cited By (12)
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| CN102617529A (en) * | 2012-02-27 | 2012-08-01 | 云南民族大学 | Application for isobenzofuran type compounds in marine biofouling prevention and preparation method thereof |
| CN102617529B (en) * | 2012-02-27 | 2014-12-03 | 云南民族大学 | Application for isobenzofuran type compounds in marine biofouling prevention and preparation method thereof |
| CN103772237A (en) * | 2014-01-24 | 2014-05-07 | 先尼科化工（上海）有限公司 | Method for swapping out ammonia with strong base in tetrachloro o-nitrile ammonium benzoate production process |
| CN106008422A (en) * | 2016-06-27 | 2016-10-12 | 云南中烟工业有限责任公司 | Benzo-lactone compound, preparation method of benzo-lactone compound and application of benzo-lactone compound in preparing anti-cancer medicine |
| CN106008422B (en) * | 2016-06-27 | 2018-05-25 | 云南中烟工业有限责任公司 | A kind of benzo lactone compound, its preparation method and the application in anticancer drug is prepared |
| CN106883243A (en) * | 2017-02-14 | 2017-06-23 | 云南民族大学 | It is a kind of with the isobenzofuran class compound and its tobacco purposes of removing free radical effect in the root of kudzu vine |
| CN106883243B (en) * | 2017-02-14 | 2018-11-27 | 云南民族大学 | It is a kind of with the isobenzofuran class compound and its tobacco purposes of removing free radical effect in pueraria lobata |
| WO2019153954A1 (en) * | 2018-02-12 | 2019-08-15 | 广州自远生物科技有限公司 | Multi-substituted benzene compound having biological activity, preparation method therefor, and application thereof |
| CN110143858A (en) * | 2018-02-12 | 2019-08-20 | 华南理工大学 | Biologically active Multi substituted benzenes compound and its preparation method and application |
| EP3753931A4 (en) * | 2018-02-12 | 2021-05-12 | Genifarm Laboratories Inc | Multi-substituted benzene compound having biological activity, preparation method therefor, and application thereof |
| CN110143858B (en) * | 2018-02-12 | 2022-04-29 | 华南理工大学 | Polysubstituted benzene compound with bioactivity and preparation method and application thereof |
| US11376228B2 (en) | 2018-02-12 | 2022-07-05 | Genifarm Laboratories Inc | Polysubstituted benzene, preparation method thereof, and method of using the same |
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