CN108794368A - A kind of alkaloid compound and preparation method and application with various bacteriostatic activity - Google Patents
A kind of alkaloid compound and preparation method and application with various bacteriostatic activity Download PDFInfo
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Abstract
The invention discloses a kind of alkaloid compounds and preparation method and application with various bacteriostatic activity.The present invention is from the isolated alkaloid compound of a structure novel of wart spore bacterium V.GIFhornensis FIM06-0025, with preferable antibacterial activity, therefore can be used for preparing antibacterials.
Description
Technical field：
The invention belongs to natural product fields, and in particular to a kind of alkaloid compound and preparation method and application.
Background technology：
Since nineteen twenty-nine finds medical antibiotics penicillin, finds farm antibiotics blasticidin-S S within 1958, full generation so far
Boundary has found that more than 4000 plant antibiotic, played an important role in terms of person poultry disease's prevention and control and agricultural pest prevention,
The progress of human civilization and science and technology is pushed.
But the use of a large amount of broad-spectrum antibiotics, the evolution of pathogenic bacteria is accelerated, pathogen especially bacterial pathogens
Drug resistance enhances increasingly, and the effect of making existing drug declines.In addition, the type of multi-drug resistant bacteria is also increasing.Drug resistance is removed from office certainly
Blue negative bacillus develops to gram-positive bacteria, and community infection is developed to by nosocomial infection, bacterial resistance problem be 21 century most
One of serious public health problem, the emergence of drug-resistant bacteria and multidrug resistance bacterium and fast propagation, make bacterium infection
Prevent, treat and be faced with stern challenge with control.Enterobacteriaceae lactobacteriaceae of the outstanding behaviours in Carbapenem-resistant class antibiotic
With non-fermented sugar bacterium, wherein especially the tightest with the drug resistance of Klebsiella Pneumoniae, Acinetobacter bauamnnii, pseudomonas aeruginosa etc.
Weight, clinical trials difficult.Although scholar has carried out a large amount of research work, in past 30 years, Zhi oxazolidine ketones
It goes through to list with two class resisting gram-positive bacteria medicine of cyclic lipopeptide, the drug of anti-Gram-negative bacteria is less, and clinical application is existing
Shape still allows of no optimist.Face bacterial drug resistance constantly upgrades, antibacterials curative effect reduces, it is clinical without medicine can with etc. intractable ask
Topic, the work for researching and developing novel drug-resistance bacteria medicine are extremely urgent.
It is the world that active compound is found from Secondary Metabolites of Microorganisms as lead compound Development of New Drugs
One of the effective way that pharmacy worker generally acknowledges, microbial resources provide infinite source for innovation drug research.Microorganism is rich
Rich various secondary metabolite provides a large amount of extremely precious mode configurations and medicine for chemicals new drug research and exploitation
Object precursor small molecule is the important substance basis of drug discovery source treatment and Continuous Innovation, the research to entire original new drug
With decisive significance.
Actinomyces are the main sources of microbial medicine, wherein based on streptomyces and some rare actinomycetes category.But
From after the seventies, the probability of noval chemical compound is found from streptomycete with regard to fewer and fewer, repetitive rate is higher and higher.Wart spore bacterium, promise
The few actinomyces (being commonly called as rare actinomycete) of these types for obtaining at present such as Cattell bacterium are by growing interest.Rare actinomycete
The reactive compound for containing a large amount of structure novels in secondary metabolite, improves the probability for obtaining targeted activity compound.
Wart spore bacterium (Verrucosispora) belongs to Actinomycetal Micromonosporaceae (Micromonosporaceae).1998
Year Rheims etc., scientists were one by one from ocean mud sample, mangrove after earliest isolated wart spore bacterium in the mud of marsh
The category bacterial strain is separated in woods, ascidian and sponge.Wart spore bacterium contains extremely abundant natural products resource.Just become micro- life
One important bacterium source of object drug.Such as 2004, is isolated from the deposit of sea of Japan 289m depths and belong to Micromonosporaceae
Verrucosispora maris AB 18-032, the bacterial strain can generate the active polyketides deep-sea anti-MRSA, VRSA
Plain (Abyssomicin C), the compound have new bacteriostasis target spot, have actively meaning to finding new and effective antibiotic
Justice.Bull and Stach has found that it is included at least by the genome analysis to verrucosispora maris AB 18-032
The gene cluster of 20 natural products synthesis, this demonstrates wart spore bacterium not only together with the research conclusion of Nett etc. and can synthesize anti-
The active material of bacterium, and more native compounds can be synthesized.2008, divide from the deposit of the 250 meters of depths in the Norwegian Sea
From to verrucosispora sp.MG-37, Fuan class compound (proximicins) of the bacterial strain synthesizing antitumor.
2010, Dai etc. isolated verrucosispora sediminis MS426 from the deposit of the 3602 meters of depths in the South SeaT, and
From the strain isolation to 8 Cyclic dipeptides (cyclo-dipetides) and two kinds of Nocardamin thunder scholar's object (nocardamine-
Like), some of which has antibacterium and fungi activity.2010, Shirai etc. was from bacterial strain verrucosispora
Two new diterpene-kind compounds with anti-prostate cancer are isolated in gifhornensis YM28-088
(gifhornennolones A and B).In addition, being also separated to antitumoral compounds thiochondrilline C from wart spore bacterium
With structure novels, the compound of good activity such as Harrucomicin C.Structure novel or new role are screened from wart spore bacterium
The antibacterials of mechanism have realistic feasibility to cope with Multidrug resistant bacteria.
Invention content：
The first purpose of the invention is to provide a kind of alkaloid compounds with bacteriostatic activity.
The alkaloid compound, shown in structure such as formula (1)：
Second object of the present invention is to provide the preparation method of above-mentioned alkaloid compound, which is characterized in that described
Alkaloid compound be from the fermentation culture of wart spore bacterium Verrucosisporagifhornensis FIM06-0025
Isolated.
It is preferred that being as follows：
The fermentation culture of wart spore bacterium V.gifhornensis FIM06-0025 is isolated into mycelium and supernatant, bacterium
Filament volume ratio 1:1 methanol/acetone extraction, extracting solution concentration removal methanol and acetone, obtains medicinal extract 1, by medicinal extract 1 and upper
Clear liquid is extracted with ethyl acetate after merging, and obtains medicinal extract 2 after extract liquor concentration, medicinal extract 2 uses first through C18 reversed-phase silica gel column chromatographies
Alcohol/water volume ratio is 30:100,50:100,70:100 carry out gradient elution for eluant, eluent, collect methanol/water wash-out concentration and are
70:100 component, obtains component A-1, and component A-1 is through silica gel column chromatography, with chloroform/methanol from volume ratio 9:1~1:1 carries out
Gradient elution, fraction collection eluent, is detected through TLC, and solvent is chloroform/methanol volume ratio 10:1, iodine is color developing agent, is merged
Same composition collects the component 3 that TLC detection Rf values are 0.55, then through Sephadex LH-20 column chromatographies, with methanol/acetonitrile 1:
1, v:V is eluant, eluent, and elution fraction is detected through TLC, and solvent is chloroform/methanol volume ratio 10:1, it is 0.57 to collect Rf values
Component obtains monomeric compound FW252.
The fermentation culture of the wart spore bacterium V.gifhornensis FIM06-0025 is by wart spore bacterium
V.gifhornensis FIM06-0025 are inoculated into fermentation medium, fermented acquisition；
Every liter of the fermentation medium contains soluble starch 20.0g, K2HPO40.5g, KNO35.0g, MgSO4·7H2O
0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0g, surplus are seawater, pH 7.2~7.5.
The fermented acquisition, fermentation condition are：It 26 DEG C, is cultivated under the conditions of 240rpm.
Third object of the present invention is to provide application of the above-mentioned alkaloid compound in preparing antibacterials.
Fourth object of the present invention is to provide a kind of antibacterials, which is characterized in that contains above-mentioned alkaloids chemical combination
Object is as active constituent.
The antibacterials are anti-Gram-negative bacteria helicobacter pylori, pseudomonas aeruginosa, Acinetobacter bauamnnii, lung
Scorching klebsiella and Escherichia coli and gram-positive bacteria staphylococcus aureus, Candida albicans and enterococcus faecium medicine
Object.
Fifth object of the present invention is to provide wart spore bacterium Verrucosispora gifhornensis FIM06-0025,
Preserving number is：CGMCC No.15242
Sixth object of the present invention is to provide wart spore bacterium V.gifhornensis FIM06-0025 to prepare above-mentioned biology
Application in alkaloid compound.
The present invention is from the isolated alkaloid of a structure novel of wart spore bacterium V.gifhornensis FIM06-0025
Class compound with preferable antibacterial activity, therefore can be used for preparing antibacterials.
The Verrucosispora gifhornensis FIM06-0025 of the present invention were preserved on 01 18th, 2018
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, preserving number are：CGMCC No.15242.
Description of the drawings：
Fig. 1 is single bacterium colony forms of the bacterial strain FIM06-0025 in nutrient agar；
Fig. 2 is the scanning electron microscope (SEM) photograph of bacterial strain FIM06-0025；
Fig. 3 is the HRESI-TOF-MS spectrums of compound FW252；
Fig. 4 is the IR spectrums of compound FW252；
Fig. 5 is compound FW2521H-NMR is composed；
Fig. 6 is compound FW2521H-1H COSY spectrums；
Fig. 7 is the HMBC spectrums of compound FW252；
Fig. 8 is compound FW25213C-NMR is composed；
Fig. 9 is the hsqc spectrum of compound FW252；
Figure 10 is the NOESY spectrums of compound FW252；
Figure 11 is the chemical constitution of compound FW252 and main1H-1H COSY are related to HMBC
Specific implementation mode：
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1：
The present inventor obtains a bacterial strain from the sponge of the Fujian East Sea, is named as：FIM06-0025, taxology feature is such as
Under：
1.1, the physicochemical property of bacterial strain
Single bacterium colony forms (Fig. 1) of the bacterial strain FIM06-0025 in nutrient agar.From bacterial strain FIM06-0025's
As it can be seen that substrate mycelium physically well develops, branch has every 0.4 μm of diameter, single spore is born in short sporophore electron-microscope scanning figure
On, spore has handle, surface to have verruca (Fig. 2).
Under aseptic condition, bacterial strain FIM06-0025 is inoculated into oese on ISP1-ISP7 agar slant culture-mediums,
32 DEG C of 2~5d of culture, observe and record the cultural characteristics (table 1) of FIM06-0025 in different medium.
Nutrient agar trains culture medium (g/L)：Beef extract 3.0, peptone 10.0, yeast extract 2.0, sodium chloride 5.0, agar
12.0, distilled water 1000mL, pH is 7.2-7.5 before sterilizing.
The cultural characteristics of 1 FIM06-0025 of table in different medium
Note：+++ indicate well-grown；++ indicate that growth is general；+ indicate that growth is poor；Expression is not grown；Color and color value
With reference to ISCC-NBC standards.
ISP1 culture mediums (g/L)：Tryptone 0.5, yeast extract 0.3, agar 2, distilled water 1L, pH7.2~7.5 before sterilizing.
ISP2 culture mediums (g/L)：Yeast extract 0.4, malt extract 1, glucose 0.4, agar 2, distilled water 1L, before sterilizing
pH7.3。
ISP3 culture mediums (g/L)：Oatmeal 2, agar 2, micro- 1mL, agar 2, distilled water 1L, pH7.4 before sterilizing.
ISP4 culture mediums (g/L)：Soluble starch 10.0, K2HPO41.0, MgSO41.0, NaCl 1.0, (NH4)2SO42.0, CaCO32.0, micro- 1mL, agar 2, distilled water 1L, pH 7.2. before sterilizing
ISP5 culture mediums (g/L)：L- asparagines 1.0, K2HPO41.0, glycerine 10.0, agar 20.0, micro- 1mL, fine jade
Fat 2, distilled water 1L, pH 7.2-7.4 before sterilizing.
ISP6 culture mediums (g/L)：Peptone-iron-agar 36.0, yeast extract 1.0, agar 2, distilled water 1L, pH 7.2- before sterilizing
7.4.Peptone yeast extract iron agar.
ISP7 culture mediums (g/L)：Glycerine 1.5, l-tyrosine 0.05, L- asparagines 0.1, K2HPO40.05, MgSO40.05,
NaCl 0.05, micro- 1mL/L, agar 1.5, distilled water 1L, pH 7.2-7.4 before sterilizing.
Trace element solution (g/L)：FeSO4·7H2O 0.1, MnCl4H2O 0.1, ZnSO4·7H2O 0.1, distilled water
100mL。
Utilization intensity of the microorganism to this carbon source is characterized with growth rate of microorganism under the conditions of single carbon source.Choosing
It is tested with ISP9 basal mediums.21 kinds of carbon sources, including melibiose, raffinose, D-MANNOSE, L- xyloses, inositol, bigcatkin willow
Element, L- rhamnoses, dulcitol, sorbierite, dithiothreitol (DTT), D- galactolipins, sweet and pure, aesculin, erythrite, cellobiose, Ah
Eastern sugar alcohol, melezitose, trehalose, L-arabinose, D-Glucose and chitin.Each carbon source adds 0.8g/L, to be not added with carbon
Source as a contrast, each handles 3 repetitions.Under aseptic condition, by bacterial strain FIM06-0025 with oese be inoculated into it is above-mentioned containing
On the ISP9 base culture base ware tablets of different carbon source, 32 DEG C are inverted culture, and different incubation time record results are simultaneously same empty
It is white to compare, analyze the result (table 2) of utilization of carbon source.
ISP9 basal mediums (g/L)：K2HPO45.65 KH2PO42.38, (NH4)2SO42.64 MgSO4·7H2O 1.0,
Agar 12.0, carbon source 1.0, micro- 1mL, distilled water 1000mL, pH 7.5.Trace element is prepared：CuSO4·5H2O
0.64g, FeSO4·7H2O 0.11g, ZnSO4·7H2O 0.15g, MnCl2·4H2O 0.79g, distilled water 100mL.
2 bacterial strain FIM06-0025 utilization of carbon source situations of table
Note：+ indicate to be fully utilized；± indicate partly to utilize；Expression cannot utilize completely.
1.2 bacterial strain 16S rRNA identifications
FIM06-0025 the bacterium solutions 5mL, 8000r/min for taking culture to exponential phase centrifuge 15min, and incline supernatant,
Collect thalline.Using CTAB/NaCl methods extraction phage gene group DNA.Steps are as follows：1.35mLTE solution (pH is added in thalline
8.0) it, suspends, 10% lauryl sodium sulfate of 0.3mL (SDS) and 150 μ L 100mg/ml lysozymes and 150 μ L is added
100mg/mL Proteinase Ks, mixing, 60 DEG C of water-bath 1h add 0.25mL 5mol/L NaCl and 0.2m L CTAB/NaCl solution, and 65
DEG C water-bath 10min, is respectively extracted 3 times with isometric phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol, 10000r/min centrifugations
0.6 times of volume isopropanol is added in 10min, Aspirate supernatant, is positioned over -20 DEG C and precipitates DNA, and DNA is dissolved in 50 μ L TE, and -20
It DEG C saves backup.Utilize the genomic DNA integrality of 1% agarose gel electrophoresis detection extracting.Using 16S rRNA genes
Universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') are to carrying
The DNA taken carries out PCR amplification.Supreme marine growth Engineering Co., Ltd is sent to be sequenced the DNA product after amplification, 16S
RRNA sequences are as shown in SEQ ID NO.1.The 16S rRNA sequences included with the GenBank databases of BL AST softwares and NCBI
It is compared, Blast searches for homologous sequence.Bacterial strain FIM06-0025 and wart born of the same parents bacterium Verrucosispora
gifhornensis DSM 44337TThe similitudes of 16S rRNA gene orders be 99%, the Physiology and biochemistry of comprehensive bacterial strain with
And Molecular Identification is as a result, determine that bacterial strain FIM06-0025 is wart born of the same parents bacterium Verrucosispora.It is by the Strain Designation：Wart born of the same parents bacterium
Verrucosispora gifhornensis FIM06-0025 were preserved in Chinese microorganism strain on 01 18th, 2018
Preservation administration committee common micro-organisms center (CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number
For：CGMCC No.15242.
The fermentation of 1.3 bacterial strains
Take a platinum loop bacterial strain Verrucosispora gifhornensis FIM06-0025 Gao Shi asparagine agar slants
Culture accesses in seed culture medium, and 26 DEG C, 2~3d of shaking table culture culture, obtains V.gifhornensis under the conditions of 240rpm
The seed culture fluid of FIM06-0025.By the seed culture fluid of bacterial strain FIM06-0025 with the inoculum concentration of volume fraction 5~10%
It is inoculated into fermentation medium, 26 DEG C, 4~5d of shaking table culture culture, obtains V.gifhornensis under the conditions of 240rpm
The fermentation culture of FIM06-0025.
Gao Shi asparagine agar slant culture-mediums：Soluble starch 20.0g, asparagine 0.5g, sea salt 16.5g, NaCl
0.5g, KNO31.0g, K2HPO40.5g, MgSO4·7H2O 0.5g, CaCO31.0g, agar 12.0g, seawater 1000mL, natural pH
Value, preparation method is to be uniformly mixed each component by its content, 121 DEG C of high-temperature sterilization 30min, spare.
The preparation of seed culture medium：Soluble starch 15.0g, yeast powder 5.0g, peptone 5.0g, glucose 5.0g,
K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, (NH4)2SO40.5g, CaCO31.0g, 1L seawater are prepared, and are prepared
Method is to be uniformly mixed each component by its content, pH7.5 before sterilizing.121 DEG C of high-temperature sterilization 30min, it is spare.
The preparation of fermentation medium：Soluble starch 20.0g, K2HPO40.5g, KNO35.0g, MgSO4·7H2O 0.5g,
NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0g, seawater 1000mL, preparation method are by each component by its content
It is uniformly mixed, pH 7.2~7.5.121 DEG C of high-temperature sterilization 30min, it is spare.
The preparation of 1.4 compounds
By the fermentation culture (50L) of above-mentioned ocean V.gifhornensis FIM06-0025 under the conditions of 4500rpm from
The mycelium and supernatant that gains in depth of comprehension arrive, the methanol/acetone (1 of 1.5~2.0 times of volumes of mycelium：1, v:V) ultrasonic extraction 2 times,
Ultrasonic temperature is less than 40 DEG C, ultrasonic time 30min.Extracting solution obtains medicinal extract in less than 40 DEG C reduced pressure removal methanol and acetone
1 (210g), medicinal extract 1 is merged with supernatant, and the ethyl acetate of 1.5~2.0 times of volumes of amalgamation liquid extracts 2 times, ethyl acetate
Removal ethyl acetate is concentrated under reduced pressure less than 40 DEG C and obtains medicinal extract 2 (169g) for extract liquor.Medicinal extract 2 is used through C18 reversed-phase silica gel column chromatographies
Methanol/water volume ratio is 30:100,50:100,70:100 elute for eluent gradient, and it is 70 to merge methanol/water wash-out concentration:
100 component obtains component A-1 (110mg).100~200 mesh silica gel mixed samples of component A-1, after dry column-packing, with chloroform/first
Alcohol is from volume ratio 9:1~1:1 carries out gradient elution, and fraction collector is collected eluent, detected through TLC, and solvent is chloroform/first
Alcohol volume ratio 10:1, iodine is color developing agent, merges same composition, obtain five kinds of components (Rf values are respectively 0.27,0.39,0.55,
0.67 and 0.75), component 3 (30mg, TLC detect the component that Rf values are 0.55) is again through Sephadex LH-20 column chromatographies, with first
Alcohol/acetonitrile 1:1, v:V is eluant, eluent, and elution fraction is detected through TLC, and solvent is chloroform/methanol volume ratio 10:1, collect Rf values
For 0.57 component, monomeric compound FW252 (6.5mg) is obtained.
1.5, Structural Identification
The data tests such as mass spectrum, ultraviolet spectra, infrared spectrum and nuclear magnetic resonance are carried out to compound FW252, so that it is determined that
The structure of compound.
Compound FW252：Pale pink powder.(c=1.0, MeOH)；UV(MeOH),λmax:241nm,
301nm；HR-TOF-MS(m/z 208.1035[M+H]+) (calcd for 208.0974) (Fig. 3), molecular formula C11H13NO3,
It is 6 to calculate its degree of unsaturation.Infrared absorption spectrum (IR) is in 3053,1619,1490 and 1582cm-1Place has absorption explanation containing virtue
Fragrant benzene radicals, 1642cm-1The strong absworption peak at place illustrates the presence (Fig. 4) for having amide group.
1HNMR (Fig. 5) shows 1 methyl proton [δH1.70 (d, J=6.5Hz, 3H)] signal, 2 methene protons
[δH3.94 (dd, J=12.0,3.5Hz, 1H)), 3.81 (dd, J=12.0,3.0Hz, 1H)] signal, 2 methine protons letter
Number [δH4.27 (m, 1H), 5.38 (m, 1H)], the Hydrogen Proton [δ of 2-OHH(12.3,4.9)] signal and 4 phenyl protons [δH7.95 (dd, J=8.5,2.0Hz, 1H), 7.12 (ddd, J=8.5,8.5,2.0Hz, 1H), 7.72 (ddd, J=8.5,8.0,
1.0Hz, 1H) and 7.16 (dd, J=8.0,1.0Hz, 1H)] signal.It can be seen that from the coupling constant of 4 phenyl protons signals
Phenyl ring containing 1 ortho position substitution in compound,1H-1H COSY and HMBC wave spectrums further demonstrate above-mentioned conclusion (Fig. 6, figure
7)。13C-NMR and DEPT135 (DMSO-d6, 125MHz) (Fig. 8, Fig. 9) show compound contain 11 carbon signals, including 1
A methyl (δC20.2), 1 methylene (δC61.6), 6 methine (δC 140.0、132.3、121.7、118.0、107.8、
67.7) and 3 quaternary carbon (δ 84.9 andC170.5,161.9 and 107.8).The compound that nuclear magnetic data shows contains 1 ortho position substitution
Phenyl ring and 1 four carbon long-chain fragment structure (CH3-C10-C8-C7), 8-H (δ 3.90), 10-H (δ in HMBC spectrums
4.68) with 9-C (δC164.4) segment CH known to the correlation of amide carbon3-C10-C8-C7With amide group by being formed with N atoms
Three-membered ring structures are attached, moreover, pass through 2-H (δHIt 7.60) can will be upper with the coupled relation of the amide carbon of 9-C (δ 170.5)
Structure fragment is stated with benzene radicals to be connected.In summary it analyzes, and combines HSQC (Figure 10) and HMBC spectrograms, obtain compound
Whole carbon signals and the hydrogen signal ownership (table 4) of FW252.It is last true according to degree of unsaturation, molecular weight and NOESY spectrums (Figure 11)
Shown in the chemical constitution such as formula (2) for having determined compound FW252, chemical name is：(2-(hydroxymethyl)-3-(2-
(hydroxymethyl)-3-methylaziridin-1-yl)(2-hydroxyphenyl)methanone.Through SciFinder
With the database retrievals such as INSPEC, the compound of same structure is found no, thus determines that FW252 is a new construction
Alkaloid compound.The chemical constitution of FW252 and main1H-1H COSY are related to HMBC such as Figure 11.
3 compound FW252's of table1H NMR(in Methanol-d4, 400MHz) and13C-NMR(in Methanol-d4,
100MHz) data
1.6, antibacterial activity is tested
Using micro broth dilution method, using Cefotaxime Sodium as positive reference substance, the minimum of test compound FW252 is antibacterial
Concentration (minimum inhibitory concentration, MIC).Test bacterium bag includes Gram-negative bacteria：Helicobacter pylori
(H.pylori) ATCC 43504, Klebsiella Pneumoniae (K.pneumoniae) ATCC 4352, Escherichia coli (E.coli) ATCC
25922, pseudomonas aeruginosa (P.aeruginosa) ATCC 27853 and Acinetobacter bauamnnii (A.baumanniiin) ATCC
17978 and gram-positive bacteria：Staphylococcus aureus (S.aureus) ATCC 25923, Candida albicans
(C.albicans) ATCC 90028 and enterococcus faecium (E.faecium) ATCC 35667 (table 5).
Concrete operation step is as follows：
1) MH broth bouillons are prepared：Weigh MH broth bouillon 21.0g, be added in 1L distilled water, heating boil to
It is completely dissolved, is sub-packed in test tube, 121 DEG C of high pressure sterilization 15min are spare.
2) test bacterium culture early period：Under aseptic condition, test bacterium is inoculated into 100mL MH broth bouillons, is placed in 35
DEG C incubator culture 12h is spare.
3) prepared by storage liquid：Appropriate sample to be tested and positive reference substance are weighed respectively, and positive reference substance is sterile water-soluble with 1mL
Solution, sample to be tested 1mL methanol dissolve.Respectively the initial concentration of storage liquid should be more than 1000 μ g/mL.
4) preparation of bacterium solution is tested：Under aseptic condition, above-mentioned cultured test bacterium is corrected to 0.5 wheat with MH meat soups
1 is pressed after family name's unit turbidity standard:20 ratio is diluted, and bacterial concentration is about 5 × 10 at this time6CFU/mL, it is spare.
5) diluting stock solutions and inoculation test bacterium：Sterile working takes sterile 96 orifice plate one.Except 160 μ are added in the first hole
Outside L MH meat soups, 100 μ L of MH meat soups are added per hole in remaining, and 40 μ L mixings of sample to be tested stoste are added in the 1st hole, then draw
100 μ L to the 2nd hole are drawing 100 μ L to the 3rd hole mixing after mixing, and so continuous doubling dilution is to the 3rd hole reciprocal, and from falling
It always draws 100 μ L and discards in the 3rd hole of number.2nd hole reciprocal is the growth control of not drug containing, and last 1 hole is nonvaccinated control.So
It is added 10 μ L of the above-mentioned test bacterium inoculum prepared in every hole afterwards, the bacterial concentration for keeping every hole final is about 5 ×
105CFU/mL。
6) it is incubated：96 orifice plates for being inoculated with test bacterium are covered into lid, sets in 35 DEG C of biochemical cultivation cases and cultivates 16-20h, remember
Record result.
7) MIC terminals interpretation：It is the sample to this kind that bacterial growth can be with the naked eye completely inhibited seen in 96 orifice plates
The minimum inhibitory concentration of bacterium.
The results are shown in Table 4, and compound FW252 has wide spectrum inhibitory activity to Gram-negative bacteria and gram-positive bacteria
(MIC value is 3.4-200 μ gmL-1), it is especially blue to Gram-negative bacteria helicobacter pylori and Friedlander's bacillus and leather
Family name's positive bacterium S. aureus has stronger inhibitory activity.
4 bacteriostatic test measurement result of table
Sequence table
<110>Fujian Microorganism Inst.
<120>A kind of alkaloid compound and preparation method and application with various bacteriostatic activity
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1397
<212> DNA
<213>Wart spore bacterium FIM06-0025 (Verrucosispora gifhornensis FIM06-0025)
<400> 1
tgcttacaca tgcaagtcga gcggaaggcc cttcggggta ctcgagcggc gaacgggtga 60
gtaacacgtg agcaacctgc cctaggcttt gggataaccc tcggaaacgg gggctaatac 120
cgaatattca ctcatgggcg catgtttgtg ggtggaaagt ttttcggctt gggatgggct 180
cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg ggtagccggc 240
ctgagagggc gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300
cagtggggaa tattgcacaa tgggcggaag cctgatgcag cgacgccgcg tgagggatga 360
cggccttcgg gttgtaaacc tctttcagca gggacgaagc gcaagtgacg gtacctgcag 420
aagaagcgcc ggccaactac gtgccagcag ccgcggtaag acgtagggcg cgagcgttgt 480
ccggatttat tgggcgtaaa gagctcgtag gcggcttgtc gcgtcgactg tgaaaacccg 540
tggctcaact gcgggcctgc agtcgatacg ggcaggctag agttcggtag gggagactgg 600
aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg 660
gtctctgggc cgatactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 720
ccctggtagt ccacgctgta aacgttgggc gctaggtgtg gggggcctct ccggttctct 780
gtgccgcagc taacgcatta agcgccccgc ctggggagta cggccgcaag gctaaaactc 840
aaaggaattg acgggggccc gcacaagcgg cggagcatgc ggattaattc gatgcaacgc 900
gaagaacctt acctgggttt gacatcgccg gaaatcctgc agagatgtgg ggtccttcgg 960
ggccggtgac aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1020
cccgcaacga gcgcaaccct cgttcgatgt tgccagcgcg ttatggcggg gactcatcga 1080
agactgccgg ggtcaactcg gaggaaggtg gggatgacgt caagtcatca tgccccttat 1140
gtccagggct tcacgcatgc tacaatggcc ggtacaatgg gctgcgatac cgtgaggtgg 1200
agcgaatccc aaaaagccgg tctcagttcg gatcggggtc tgcaactcga ccccgtgaag 1260
tcggagtcgc tagtaatcgc agatcagcaa cgctgcggtg aatacgttcc cgggccttgt 1320
acacaccgcc cgtcacgtca cgaaagtcgg caacacccga agccggtggc ccaacccttg 1380
tggagggagc cgtcgaa 1397
Claims (10)
1. alkaloid compound or its salt, shown in structure such as formula (1)：
2. a kind of preparation method of alkaloid compound described in claim 1, which is characterized in that the alkaloids
It is isolated from the fermentation culture of wart spore bacterium Verrucosispora gifhornensis FIM06-0025 to close object.
3. preparation method according to claim 2, which is characterized in that be as follows：
The fermentation culture of wart spore bacterium V.gifhornensis FIM06-0025 is isolated into mycelium and supernatant, mycelium
With volume ratio 1:1 methanol/acetone extraction, extracting solution concentration removal methanol and acetone, obtain medicinal extract 1, by medicinal extract 1 and supernatant
It is extracted with ethyl acetate after merging, obtains medicinal extract 2 after extract liquor concentration, medicinal extract 2 uses methanol/water through C18 reversed-phase silica gel column chromatographies
Volume ratio is 30:100,50:100,70:100,70:90 carry out gradient elution for eluant, eluent, collect methanol/water wash-out concentration and are
70:100 component, obtains component A-1, and component A-1 is through silica gel column chromatography, with chloroform/methanol from volume ratio 9:1~1:1 carries out
Gradient elution, fraction collection eluent, is detected through TLC, and solvent is chloroform/methanol volume ratio 10:1, iodine is color developing agent, is merged
Same composition collects the component 3 that TLC detection Rf values are 0.55, then through Sephadex LH-20 column chromatographies, with methanol/acetonitrile 1:
1, v:V is eluant, eluent, and elution fraction is detected through TLC, and solvent is chloroform/methanol volume ratio 10:1, it is 0.57 to collect Rf values
Component obtains alkaloid compound described in claim 1.
4. preparation method according to claim 3, which is characterized in that the wart spore bacterium V.gifhornensis
The fermentation culture of FIM06-0025 is that wart spore bacterium V.gifhornensis FIM06-0025 are inoculated into fermentation medium,
Fermented acquisition；
Every liter of the fermentation medium contains soluble starch 20.0g, K2HPO40.5g, KNO35.0g, MgSO4·7H2O
0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0g, surplus are seawater, pH 7.2~7.5.
5. the application of alkaloid compound described in claim 1 or its salt in preparing antibacterials.
6. application according to claim 6, which is characterized in that the antibacterials are anti-helicobacter pylori, verdigris vacation
Monad, Acinetobacter bauamnnii, Klebsiella Pneumoniae, Escherichia coli, staphylococcus aureus, Candida albicans or enterococcus faecium
Drug.
7. a kind of antibacterials, which is characterized in that contain alkaloid compound described in claim 1 or its salt as activity
Ingredient.
8. antibacterials according to claim 8, which is characterized in that the antibacterials are anti-helicobacter pylori, copper
Green pseudomonad, Acinetobacter bauamnnii, Klebsiella Pneumoniae, Escherichia coli, staphylococcus aureus, Candida albicans or dung intestines
The drug of coccus.
9. wart spore bacterium Verrucosispora gifhornensis FIM06-0025, preserving number are：CGMCC No.15242.
10. the wart spore bacterium V.gifhornensis FIM06-0025 described in claim 9 are preparing life described in claim 1
Application in object alkaloid compound.
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Cited By (3)
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| CN109280034A (en) * | 2018-08-01 | 2019-01-29 | 福建省微生物研究所 | A kind of Benzoxazepin class compound and the preparation method and application thereof with bacteriostatic activity |
| CN110092758A (en) * | 2019-03-27 | 2019-08-06 | 福建省微生物研究所 | A kind of new bio alkali cpd and fermentation prepare the wart spore bacterial strain of the compound |
| CN111793015A (en) * | 2020-07-14 | 2020-10-20 | 遵义医科大学 | Nitrogen-containing three-membered ring compound streptomycin A and separation method and antibacterial application thereof |
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| CN103408503A (en) * | 2013-08-21 | 2013-11-27 | 中国科学院南海海洋研究所 | Preparation method of alkaloid compounds, and application of alkaloid compounds in preparing medicines for inhibiting calcium ion channels |
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| CN103408503A (en) * | 2013-08-21 | 2013-11-27 | 中国科学院南海海洋研究所 | Preparation method of alkaloid compounds, and application of alkaloid compounds in preparing medicines for inhibiting calcium ion channels |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109280034A (en) * | 2018-08-01 | 2019-01-29 | 福建省微生物研究所 | A kind of Benzoxazepin class compound and the preparation method and application thereof with bacteriostatic activity |
| CN109280034B (en) * | 2018-08-01 | 2021-06-15 | 福建圣维生物科技有限公司 | Benzoxazepine compound with antibacterial activity and preparation method and application thereof |
| CN110092758A (en) * | 2019-03-27 | 2019-08-06 | 福建省微生物研究所 | A kind of new bio alkali cpd and fermentation prepare the wart spore bacterial strain of the compound |
| CN110092758B (en) * | 2019-03-27 | 2021-06-18 | 福建省微生物研究所 | Novel alkaloid compound and wart spore strain for preparing compound by fermentation |
| CN111793015A (en) * | 2020-07-14 | 2020-10-20 | 遵义医科大学 | Nitrogen-containing three-membered ring compound streptomycin A and separation method and antibacterial application thereof |
| CN111793015B (en) * | 2020-07-14 | 2021-08-27 | 遵义医科大学 | Nitrogen-containing three-membered ring compound streptomycin A and separation method and antibacterial application thereof |
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