CN104230874B - Preparation method of isocoumarin compound and application of isocoumarin compound as antibacterial agent - Google Patents
Preparation method of isocoumarin compound and application of isocoumarin compound as antibacterial agent Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
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- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The invention relates to a preparation method of an isocoumarin compound and application of the isocoumarin compound as an antibacterial agent. The preparation method comprises the following steps of: firstly carrying out strain culture on a marine fungus, namely Eurotium sp. (HM991283), in a strain culture medium; then carrying out fermentation culture on the marine fungus in a fermentation culture medium; after fermentation liquor and thalli are separated, extracting the fermentation liquor by using ethyl acetate, and carrying out vacuum concentration an extraction solution to obtain a fermentation liquor extract; lixiviating the thalli by using methanol, and carrying out vacuum concentration to obtain a thalli extract; merging the fermentation liquor extract and the thalli extract, and carrying out chromatographic separation to obtain the isocoumarin compound. The antibacterial agent is characterized in that the antibacterial agent taking the isocoumarin compound or salt thereof as an effective component is used for preventing or treating diseases caused by staphylococcus epidermidis, staphylococcus aureus, bacillus cereus, vibrio parahaemolyticus, vibrio anguillarum or escherichia coli.
Description
Technical field
The present invention relates to a kind of preparation method of isocoumarin class compound and the application as antibacterial.
Background technology
In recent years, due to the unreasonable use of antibacterials, the drug resistance of antibacterial has become as severe asking of exception
Topic.World Health Organization (WHO) (who) points out, the appearance of 2010 " superbacteria " shows the progress at full speed of bacterial drug resistance, to people
Class life and health brings great threat.In order to reduce the generation of bacterial drug resistance, it is badly in need of the new quick antibacterials of height of exploitation, to carry
The effectiveness of high clinical application.It is new that the conditions such as ocean high salt, high pressure, low illumination enable Marine microorganism to produce a large amount of structures
The unique secondary metabolite of grain husk, activity, many of which compound has antibacterial activity, for finding potential antibacterials guide
Compound provides important sources.Cephalosporin (cephalosporin) is clinically wide variety of one kind after penicillin
Antibiotic, the source that it is originally found exactly Marine microorganism secondary metabolite.In recent years, with marine natural pharmaceutical chemistry
Development, increasing antibacterial active compounds constantly separate from Marine microorganism and obtain, and carry for finding antibacterial agent
Supply material base.Using the method for artificial culture fermentation, obtain the secondary generation of important antibacterial activity from Marine microorganism
Thank to product, there is the features such as environmental friendliness, sustainable development, medicine source in energy effectively solving drug discovery process etc. is key to ask
Topic, therefore has unique advantage.
Content of the invention
It is an object of the invention to provide a kind of preparation method of the isocoumarin class compound from marine fungi with
As the application of antibacterial, it can meet the demand of prior art.Culture presevation information: depositary institution's title: China is micro-
Biological inoculum preservation administration committee common micro-organisms center；Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica；Preservation date: on August 12nd, 2013；Deposit number: cgmcc7991 Classification And Nomenclature:
eurotium sp..
A kind of isocoumarin class compound or its salt of formula i structure:
Wherein r1、r2Be each independently h,OrAnd r1、r2In have one and be
h；r3、r4It is each independently h, och3, and r3、r4In have one be h.
Formula i compound is selected from following compounds:
The present invention provides a kind of preparation method of formula i compound it is characterised in that comprising the following steps: first in spawn culture
In base, spawn culture is carried out to marine fungi eurotium sp. (hm991283), trueer to above-mentioned ocean in the fermentation medium
Bacterium carries out fermentation culture；After fermentation liquid is separated with thalline, fermentation liquid is extracted with ethyl acetate 2-6 time, extract concentrating under reduced pressure
Obtain fermentation liquid extractum；Thalline is extracted 2-6 time with methanol, is concentrated under reduced pressure to give thalline extractum；Merge fermentation liquid extractum and thalline
Extractum, carries out chromatographic isolation, obtains formula i compound, in wherein said bacterium culture medium contain glucose, yeast extract, peptone,
Agar, coarse sea salt, water, contain glucose, yeast extract, peptone, coarse sea salt, water in fermentation medium；Described chromatographic isolation
Separate for normal phase silica gel column chromatography and separate with efficient liquid phase chiral chromatogram.
In above-mentioned preparation method bacterium culture medium preferably comprise glucose 0.1%-10.0% (percentage by weight, similarly hereinafter),
Yeast extract 0.01%-4%, peptone 0.01%-4%, agar 0.1%-6.0%, coarse sea salt 0.05%-10%, remaining is water；
Cultivation temperature is preferably 5-45 DEG C；Incubation time is preferably 3-10 days；Fermentation medium preferably comprises glucose 0.1%-
10.0% (percentage by weight, similarly hereinafter), yeast extract 0.01%-4%, peptone 0.01%-4%, coarse sea salt 0.05%-10%,
Remaining is water；Cultivation temperature is preferably 5-45 DEG C；Incubation time is preferably 5-50 days；During described normal phase silica gel column chromatography separates
Using fixing phase be preferably 200～300 mesh silica gel, mobile phase be preferably 10%-60% (percent by volume) ethyl acetate-
Petroleum ether mixed solvent；The chromatographic column that efficient liquid phase chiral chromatogram adopts in separating is preferably tbb chiral chromatographic column
(kromasil, 5 μm, 150 × 4.6mm), mobile phase is preferably the isopropanol-normal hexane mixing of 5%-15% (percent by volume)
Solvent.
The present invention provides a kind of antibacterial it is characterised in that using formula i compound or its salt as effective ingredient, for preventing
And/or treatment by staphylococcus epidermidiss (staphylococcus epidermidis), staphylococcus aureuses (s.aureus),
Bacillus cercuses (bacillus cereus), vibrio parahaemolytious (vibrio parahaemolyticus), Vibrio anguillarum
Or the disease that causes of escherichia coli (escherichia coli) (v.anguillarum).
The formula i compound or its salt of the present invention is notable to the antibacterial activity of above pathogenic strain, can be used for developing antibacterial,
And raw material can carry out large-scale production by fungi fermentation, be not subject to resource limit, therefore have a extensive future.
Specific embodiment
For the ease of a further understanding of the present invention, examples provided below has done more detailed description to it.But
Be these embodiments only for be better understood from invention and not for limiting the scope of the present invention or implementation principle, the reality of the present invention
The mode of applying is not limited to herein below.
Embodiment 1
(1) culture of marine fungi eurotium sp. (hm991283) strain
The culture medium used by spawn culture of funguses eurotium sp. (hm991283) contains glucose 1.0% (weight
Percentage ratio, similarly hereinafter), yeast extract 0.1%, peptone 0.2%, agar 1.0%, coarse sea salt 3%, remaining is water, makes during use
Test tube slant, fungal bacterial strain is cultivated 5 days at 28 DEG C.
(2) fermentation of marine fungi eurotium sp. (hm991283)
The fermentation medium used by fermentation culture of funguses eurotium sp. (hm991283) is containing glucose 1.0%
(percentage by weight, similarly hereinafter), yeast extract 0.1%, peptone 0.2%, coarse sea salt 3.0%, remaining is water, and fungal bacterial strain is in 25 DEG C
Culture 30 days.
(3) separation and Extraction of formula i compound
Take step (2) gained fermented product 100l, after fermentation liquid is separated with thalline, fermentation liquid is extracted with ethyl acetate
5 times, extract is concentrated under reduced pressure to give fermentation liquid extractum；Thalline is extracted 5 times with methanol, is concentrated under reduced pressure to give thalline extractum；Merge
Fermentation liquid extractum and thalline extractum, first carry out normal phase silica gel column chromatography and separate, fixing phase: 200～300 mesh silica gel, mobile phase:
The ethyl acetate-light petrol mixed solvent of 10%-60% (percent by volume), separates and obtains 6 raceme mixtures, then
Carry out efficient liquid phase chiral chromatogram respectively to separate, fixing phase: tbb chiral chromatographic column (kromasil, 5 μm, 150 × 4.6mm), stream
The isopropanol of dynamic phase: 5%-15% (percent by volume)-normal hexane mixed solvent, separates and obtains (the change of 12 pale yellow oil
Compound 1-12), as formula i compound.
The structural identification data of gained formula i compound:
Compound 1,2Pale yellow oil；[α]25 d1:+59；
2:-59；Cd1: λmax(δ s) 260 (- 18.3), 340 (+2.8) nm；2: λmax(δ ε) 260 (+18.3), 340 (- 2.8) nm；1、2
Nuclear magnetic data:1h nmr(cdcl3, 600mhz) and δ: 10.85 (1h, s, 2-oh), 6.94 (1h, s, h-4), 6.10 (1h, brs, 5-
Oh), 5.27 (1h, m, h-2 "), 4.69 (1h, d, j=2.4hz, h-1 '), 4.43 (1h, ddd, j=8.4,5.4,2.4hz, h-
2 '), 3.35 (3h, s, 1 '-och3), 3.32 (2h, d, j=7.2hz, h-1 "), 1.96 (1h, m, ha-3 '), 1.86 (1h, m, hb-
3 '), 1.74 (3h, s, h-4 "), 1.68 (3h, s, h-5 "), 1.55 (1h, m, ha-4 '), 1.43 (1h, m, hb-4 '), 1.32
(4h, m, h-5 ', h-6 '), 0.89 (3h, t, j=6.6hz, h-7 ').13c nmr(cdcl3, 150mhz) and δ: 169.1 (c, c-7),
153.6 (c, c-2), 145.5 (c, c-5), 133.4 (c, c-3 "), 131.3 (c, c-3), 123.8 (ch, c-4), 120.1 (ch,
C-2 "), 118.4 (c, c-6), 106.0 (c, c-1), 81.2 (ch, c-2 '), 68.3 (ch, c 1 '), 55.9 (ch3, 1 '-
och3), 30.8 (ch2, c-5 '), 29.3 (ch2, c-3 '), 27.0 (ch2, c-1 "), 25.0 (ch3, c-4 "), 24.1 (ch2, c-
4 '), 21.7 (ch2, c-6 '), 17.1 (ch3, c-5 "), 13.2 (ch3, c-7 ') and .hresimsm/z347.1860 [m h]-
(calcd for c20h27o5, 347.1853).
Compound 3,4Pale yellow oil；[α]25 d3:+38；
4:-38；Cd3: λmax(δ ε) 260 (- 15.2), 340 (+2.0) nm；4: λmax(δ ε) 260 (+15.2), 340 (- 2.0) nm；3、4
Nuclear magnetic data:1h nmr(cdcl3, 600mhz) and δ: 10.89 (1h, s, 2-oh), 6.93 (1h, s, h-4), 5.83 (1h, brs, 5-
Oh), 5.49 (1h, m, h-6 '), 5.42 (1h, m, h-5 '), 5.28 (1h, m, h-2 "), 4.71 (1h, d, j=2.4hz, h-1 '),
4.47 (1h, ddd, j=7.8,5.4,2.4hz, h-2 '), 3.36 (3h, s, 1 '-och3), 3.32 (2h, d, j=7.2hz, h-1 "),
2.17 2.25 (2h, m, h-4 '), 2.06 (1h, m, ha-3 '), 1.86 (1h, m, hb-3 '), 1.75 (3h, s, h-4 "), 1.69
(3h, s, h-5 "), 1.64 (3h, t, j=6.0hz, h-7 ').13c nmr(cdcl3, 150mhz) and δ: 169.6 (c, c-7), 154.4
(c, c-2), 145.2 (c, c-5), 134.1 (c, c-3 "), 132.1 (c, c-3), 129.6 (ch, c-5 '), 126.6 (ch, c-
6 '), 124.5 (ch, c-4), 120.9 (ch, c-2 "), 119.0 (c, c-6), 106.9 (c, c-1), 80.8 (ch, c-2 '), 69.4
(ch, c-1 '), 56.6 (ch3, 1 '-och3), 29.8 (ch2, c-3 '), 27.8 (ch2, c-1 "), 25.8 (ch3, c-4 "), 27.9
(ch2, c-4 '), 18.0 (ch3, c-7 '), 17.8 (ch3, c-5 ") .hresimsm/z345.1706 [m h]-(calcd for
c20h25o5, 345.1697).
Compound 5,6Pale yellow oil；[α]25 d5:+13；
6:-13；Hresimsm/z345.1698 [m h]-(calcd for c20h25o5, 345.1697).
Compound 7,8Pale yellow oil；[α]25 d7:-58；
8:+58；Cd7: λmax(δ ε) 260 (+18.2), 340 (- 2.2) nm；8: λmax(δ ε) 260 (- 18.2), 340 (+2.2) nm；7、8
Nuclear magnetic data:1h nmr(cdcl3, 600mhz) δ: 10.95 (1h, s, 2 oh), 6.93 (1h, s, h-4), 5.82 (1h, brs,
5 oh), 5.28 (1h, m, h-2 "), 4.76 (1h, ddd, j=7.8,4.8,3.6hz, h-2 '), 4.65 (1h, d, j=3.6hz, h-
1 '), 3.39 (3h, s, 1 '-och3), 3.33 (2h, d, j=7.2hz, h-1 "), 1.75 (3h, s, h-4 "), 1.69 (3h, s, h-
5 "), 1.59 (1h, m, ha-3 '), 1.54 (1h, m, hb-3 '), 1.53 (1h, m, ha-4 '), 1.41 (1h, m, hb-4 '), 1.28
(2h, m, h-6 '), 1.27 (2h, m, h-5 '), 0.87 (3h, t, j=6.6hz, h-7 ').13c nmr(cdcl3, 150mhz) and δ:
167.8 (c, c-7), 153.6 (c, c-2), 145.1 (c, c-5), 133.4 (c, c-3 "), 131.5 (c, c-3), 123.8 (ch, c-
4), 120.0 (ch, c-2 "), 116.7 (c, c-6), 105.8 (c, c-1), 81.4 (ch, c-2 '), 70.4 (ch, c-1 '), 55.3
(ch3, 1 '-och3), 31.2 (ch2, c-5 '), 30.6 (ch2, c-3 '), 26.9 (ch2, c-1 "), 25.0 (ch3, c-4 "), 24.2
(ch2, c-4 '), 21.6 (ch2, c-6 '), 17.0 (ch3, c-5 "), 13.1 (ch3, c-7 ') and .hresims m/z347.1860
[m—h]-(calcd for c20h27o5, 347.1853).
Compound 9,10Pale yellow oil；[α]25 d9:-
29；10:+29；Cd9: λmax230 (+5.8), 258 (+10.5), 340 (- 1.5) nm；(δ ε) 10: λmax(δ ε) 230 (- 5.8),
258 (- 10.5), 340 (+1.5) nm；9th, 10 nuclear magnetic data:1h nmr(cdcl3, 600mhz) and δ: 10.96 (1h, s, 2-oh),
6.93 (1h, s, h-4), 5.63 (1h, brs, 5-oh), 5.50 (1h, m, h-6 '), 5.38 (1h, m, h-5 '), 5.28 (1h, m, h-
2 "), 4.77 (1h, ddd, j=8.4,4.8,3.6hz, h-2 '), 4.65 (1h, d, j=3.6hz, h-1 '), 3.39 (3h, s, 1 '-
och3), 3.33 (2h, d, j=7.2hz, h-1 "), 2.18 (2h, m, h-4 '), 1.76 (3h, s, h-4 "), 1.70 (3h, s, h-
5 "), 1.68 (2h, m, h-3 '), 1.64 (3h, dd, j=6.6,1.2hz, h-7 ').13c nmr(cdcl3, 150mhz) and δ: 167.8
(c, c-7), 153.9 (c, c-2), 145.0 (c, c-5), 133.5 (c, c-3 "), 131.7 (c, c-3), 128.4 (ch, c-5 '),
126.2 (ch, c-6 '), 124.0 (ch, c-4), 120.1 (ch, c-2 "), 116.8 (c, c-6), 105.9 (c, c-1), 80.4
(ch, c-2 '), 70.9 (ch, c-1 '), 55.4 (ch3, 1 '-och3), 31.2 (ch2, c-3 '), 27.4 (ch2, c-4 '), 27.0
(ch2, c-1 "), 25.2 (ch3, c-4 "), 17.2 (ch3, c-5 "), 17.1 (ch3, c-7 ') and .hresims m/z345.1706
[m—h]-(calcd for c20h25o5, 345.1697).
Compound 11,12Pale yellow oil；[α]25 d11:-
18；12:+18；Cd11: λmax(δ ε) 232 (+10.6), 255 (+7.5), 340 (- 1.8) nm；12: λmax(δε)232(-
10.6), 255 (- 7.5), 340 (+1.8) nm；11st, 12 nuclear magnetic data:1h nmr(cdcl3, 600mhz) and δ: 10.91 (1h, s, 2-
Oh), 6.93 (1h, s, h-4), 6.28 (1h, brs, 5-oh), 5.94 (1h, m, h-4 '), 5.74 (1h, m, h-3 '), 5.29 (1h,
M, h-2 "), 4.87 (1h, d, j=3.6hz, h-1 '), 4.47 (1h, dd, j=8.4,3.6hz, h-2 '), 3.44 (3h, s, 1 '-
och3), 3.32 (2h, d, j=7.2hz, h-1 "), 2.08 (2h, m, h-5 '), 1.76 (3h, s, h-4 "), 1.69 (3h, s, h-
5 "), 1.42 (2h, m, h-6 '), 0.89 (3h, t, j=7.8hz, h-7 ').13c nmr(cdcl3, 150mhz) and δ: 168.3 (c, c-
7), 153.6 (c, c-2), 145.0 (c, c-5), 137.8 (ch, c-4 '), 133.4 (c, c-3 "), 131.4 (c, c-3), 124.2
(ch, c-4), 122.3 (ch, c-3 '), 120.1 (ch, c-2 "), 116.9 (c, c-6), 105.4 (c, c-1), 80.2 (ch, c-
2 '), 71.6 (ch, c-1 '), 56.1 (ch3, 1 '-och3), 33.6 (ch2, c-5 '), 26.9 (ch2, c-1 "), 25.1 (ch3, c-
4 "), 21.2 (ch2, c-6 '), 17.0 (ch3, c-5 "), 12.8 (ch3, c-7 ') and .hresims m/z345.1698 [m h]-
(calcd for c20h25o5, 345.1697).
Embodiment 2
(1) culture of marine fungi eurotium sp. (hm991283) strain
Bacterium culture medium contains glucose 0.1%-10.0% (percentage by weight, similarly hereinafter), yeast extract 0.01%-4%, egg
White peptone 0.01%-4%, agar 0.1%-6.0%, coarse sea salt 0.05%-10%, remaining is water；Cultivation temperature is 5-45 DEG C；Training
The foster time is 3 10 days.
(2) fermentation of marine fungi eurotium sp. (hm991283)
Fermentation medium contains glucose 0.1%-10.0% (percentage by weight, similarly hereinafter), yeast extract 0.01%-4%, egg
White peptone 0.01%-4%, coarse sea salt 0.05%-10%, remaining is water；Cultivation temperature is 5-45 DEG C；Incubation time is 5-50 days.
(3) separation and Extraction of formula i compound
Taking the fermented product 50l of step (2) gained, after fermentation liquid is separated with thalline, fermentation liquid is extracted with ethyl acetate 2～
6 times, extract is concentrated under reduced pressure to give fermentation liquid extractum；Thalline is extracted 2～6 times with methanol, is concentrated under reduced pressure to give thalline extractum；Close
And fermentation liquid extractum and thalline extractum, first carry out normal phase silica gel column chromatography and separate, fixing phase: 200～300 mesh silica gel, mobile phase:
The ethyl acetate-light petrol mixed solvent of 10%-60% (percent by volume), separates and obtains 6 raceme mixtures, then
Carry out efficient liquid phase chiral chromatogram respectively to separate, fixing phase: tbb chiral chromatographic column (kromasil, 5 μm, 150 × 4.6mm), stream
The isopropanol of dynamic phase: 5%-15% (percent by volume)-normal hexane mixed solvent, separates and obtains (the change of 12 pale yellow oil
Compound 1-12), as formula i compound.The structural identification data of its Chinese style i compound is consistent with corresponding data in embodiment 1.
Other spawn culture of not particularly pointing out in embodiment 12, fermentation condition, and normal phase silica gel column chromatography separate,
Other experimental operating conditions such as efficient liquid phase chiral chromatogram separation are the conventional experimental operating conditions in this area, the skill of this area
Art personnel can reasonably be selected according to actual needs.
Embodiment 3
In bacterium culture medium, spawn culture is carried out to marine fungi eurotium sp. (hm991283), then in fermentation training
In foster base, fermentation culture being carried out to above-mentioned marine fungi, after fermentation liquid is separated with thalline, fermentation liquid is extracted with ethyl acetate 2～
6 times, extract is concentrated under reduced pressure to give fermentation liquid extractum；Thalline is extracted 2～6 times with methanol, is concentrated under reduced pressure to give thalline extractum；Close
And fermentation liquid extractum and thalline extractum, obtain 12 pale yellow oil (compound 1-12) after carrying out chromatographic isolation and be formula i
Compound, its structural identification data is consistent with corresponding data in embodiment 1.In wherein said bacterium culture medium contain glucose,
Yeast extract, peptone, agar, coarse sea salt, water, in described fermentation medium contain glucose, yeast extract, peptone, coarse sea salt,
Water；Described chromatographic isolation separates for normal phase silica gel column chromatography and separates with efficient liquid phase chiral chromatogram.
Widely it is applied to the method preparing formula i compound in order to explore, bacterium culture medium in the present embodiment,
The interpolation of each composition in fermentation medium, is all added in ratio conventional in this area or adds in any proportion, during chromatographic isolation
The selection of the specification of used silica gel, the model of chiral chromatographic column and eluting solvent, is the conventional selection of this area.Experiment knot
Fruit shows, the above-mentioned conventional preparation method selecting, and all can obtain 12 pale yellow oil invented, i.e. formula i compound, its knot
Structure confirmation data is consistent with corresponding data in embodiment 1, only exists small difference in terms of purity and yield for the individual compound
Different.
The result of embodiment 1-3 shows, according to the conventional spawn culture in this area, fermentation condition, the purification on normal-phase silica gel of routine
Pillar layer separation, the detached condition of efficient liquid phase chiral chromatogram are trained to marine fungi eurotium sp. (hm991283)
Support, ferment, isolate and purify, all can obtain the compound of formula i structure.The preparation method of formula i compound, preferably
Method described in embodiment 1-2.
Embodiment 4
The antibacterial activity test of formula i compound
(1) formula i compound is according to literature method (pierce c.g.；uppuluri p.；teistan a.r.；
wormley jr.f.l.；mowat e.；ramage g.；Lopez-ribot j.l.nat.protoc.2008,3,1494-
1500), test is to 3 plants of gram positive bacterias: staphylococcus epidermidiss (staphylococcus epidermidis), golden yellow Portugal
Grape coccus (s.aureus), Bacillus cercuses (bacillus cereus), and 3 plants of gram negative bacterias: vibrio parahaemolytious
(vibrio parahaemolyticus), Vibrio anguillarum (v.anguillarum), escherichia coli (escherichia coli)
Antibacterial activity.
(2) antibacterial activity of formula i compound
Formula i compound is to staphylococcus epidermidiss (staphylococcus epidermidis), golden yellow Fructus Vitis viniferae
Coccus (s.aureus), Bacillus cercuses (bacillus cereus), vibrio parahaemolytious (vibrio
Parahaemolyticus), Vibrio anguillarum (v.anguillarum) or escherichia coli (escherichia coli) have significantly
Antibacterial activity, all not higher than 6.25 μm of its minimal inhibitory concentration (mic).Wherein 1,2 pairs of staphylococcus epidermidiss of compound
(s.epidermidis) antibacterial activity is the strongest, and its minimal inhibitory concentration (mic) is 0.39 μm, is that positive control cyclome third is husky
4 times of star (ciprofloxacin), as shown in table 1.The activity knot of the compounds of this invention 1-3,5,7,8,10,12 is listed in table 1
Really.
The inhibitory activity to antibacterial for the segment boundses i compound of table 1 present invention
The present invention provides a kind of antibacterial it is characterised in that containing formula i coumarin kind compound or its salt as effective one-tenth
Point, for preventing or treating by staphylococcus epidermidiss (staphylococcus epidermidis), staphylococcus aureuses
(s.aureus), Bacillus cercuses (bacillus cereus), vibrio parahaemolytious (vibrio parahaemolyticus),
What Vibrio anguillarum (v.anguillarum) or escherichia coli (escherichia coli) caused has related disorders, and raw material can lead to
Cross fungi fermentation and carry out large-scale production, be not subject to resource limit, therefore have a extensive future.
Claims (6)
1. a kind of preparation method of the isocoumarin class compound of formula i structure is it is characterised in that comprise the following steps: first in strain
In culture medium, spawn culture is carried out to marine fungi eurotium sp. (hm991283), more in the fermentation medium to above-mentioned sea
Foreign funguses carry out fermentation culture, and after fermentation liquid is separated with thalline, fermentation liquid is extracted with ethyl acetate 2～6 times, and extract reduces pressure
It is concentrated to give fermentation liquid extractum；Thalline is extracted 2～6 times with methanol, is concentrated under reduced pressure to give thalline extractum；Merge fermentation liquid extractum and
Thalline extractum, carries out chromatographic isolation, obtains formula i compound；Its Chinese style i compound structure is:r1、
r2Be each independently h, OrAnd r1、r2In have one be h；r3、r4Independently of one another
For h, och3, and r3、r4In have one be h；Glucose, yeast extract, peptone, fine jade is contained in wherein said bacterium culture medium
Fat, coarse sea salt, water；Glucose, yeast extract, peptone, coarse sea salt, water is contained in described fermentation medium；Described chromatograph
It is separated into normal phase silica gel column chromatography separation to separate with efficient liquid phase chiral chromatogram.
2. preparation method as claimed in claim 1 is it is characterised in that described bacterium culture medium contains glucose 0.1%-
10.0%th, yeast extract 0.01%-4%, peptone 0.01%-4%, agar 0.1%-6.0%, coarse sea salt 0.05%-10%, its
Yu Weishui, above-mentioned percentage composition is all weight percentage；Cultivation temperature is 5-45 DEG C；Incubation time is 3-10 days.
3. preparation method as claimed in claim 1 is it is characterised in that described fermentation medium contains glucose 0.1%-
10.0%th, yeast extract 0.01%-4%, peptone 0.01%-4%, coarse sea salt 0.05%-10%, remaining is water, above-mentioned percentage
Content is all weight percentage；Cultivation temperature is 5-45 DEG C；Incubation time is 5-50 days.
4. preparation method as claimed in claim 1 is it is characterised in that consolidating of adopting in separating of described normal phase silica gel column chromatography
Fixed is mutually 200～300 mesh silica gel, and mobile phase is the ethyl acetate-light petrol mixed solvent of 10%-60%；Described efficient liquid
The chromatographic column that phase chiral chromatogram adopts in separating is tbb chiral chromatographic column kromasil, 5 μm, 150 × 4.6mm, and mobile phase is
The isopropanol of 5%-15%-normal hexane mixed solvent；Above-mentioned mixed solvent percentage ratio is percent by volume.
5. a kind of antibacterial is it is characterised in that include the formula i compound or its salt of claim 1 preparation as effective ingredient.
6. the formula i compound or its salt of claim 1 preparation in preparation prevention and/or is treated by staphylococcus epidermidiss, golden yellow
Application in the medicine of the disease that staphylococcuses, Bacillus cercuses, vibrio parahaemolytious, Vibrio anguillarum or escherichia coli cause.
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| Bioactive chemical constituents of two endophytic fungi;H. Hussain et al.;《Biochemical Systematics and Ecology》;20071231;第35卷;898-900 * |
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