CN108794368B - Alkaloid compound with diverse antibacterial activities and preparation method and application thereof - Google Patents

Abstract

The invention discloses an alkaloid compound with diverse antibacterial activities, and a preparation method and application thereof. The alkaloid compound with a novel structure is separated from verruca vulgaris V.gifhornnensis FIM06-0025, has good antibacterial activity, and can be used for preparing antibacterial drugs.

Description

Alkaloid compound with diverse antibacterial activities and preparation method and application thereof

The technical field is as follows:

the invention belongs to the field of natural products, and particularly relates to an alkaloid compound, and a preparation method and application thereof.

Background art:

since the discovery of medical antibiotic penicillin in 1929 and the discovery of agricultural antibiotic blasticidin S in 1958, more than 4000 antibiotics have been discovered all over the world so far, play an important role in the prevention and control of human and animal diseases and the prevention and control of agricultural pests, and promote the progress of human civilization and scientific technology.

However, the use of a large number of broad-spectrum antibiotics accelerates the evolution of pathogenic bacteria, and the drug resistance of pathogens, particularly bacterial pathogens, is increasing day by day, so that the curative effect of the existing drugs is reduced. In addition, the number of types of multi-drug resistant bacteria is increasing. Drug resistance develops from gram-negative bacilli to gram-positive bacteria, and from nosocomial infection to extramural infection, the problem of bacterial drug resistance is one of the most serious public health problems in the 21 st century, and the continuous emergence and rapid propagation of drug-resistant bacteria and multi-drug-resistant bacteria make prevention, treatment and control of bacterial infection face serious challenges. The antibacterial agent is highlighted in the carbapenem antibiotic-resistant Enterobacteriaceae bacteria and non-fermentation sugar bacteria, particularly the drug resistance of Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the like is the most serious, and the clinical treatment is difficult. Although researchers have conducted a great deal of research, only two classes of gram-positive antibacterial agents, namely oxazolidinones and cyclic lipopeptides, have been approved for the market in the last 30 years, and the number of gram-negative antibacterial agents is less, so that the current clinical application is still not optimistic. The work of developing novel anti-drug-resistant bacteria medicaments is urgent in the face of the troublesome problems of continuous upgrade of the drug resistance of bacteria, reduction of the curative effect of antibacterial medicaments, no clinical medicament availability and the like.

The method for searching active compounds from microbial secondary metabolites as lead compounds to create new drugs is one of the accepted effective ways of pharmaceutical workers in the world, and microbial resources provide an infinite source for innovative drug research. The abundant and diverse secondary metabolites of the microorganisms provide a large number of precious mode structures and drug precursor micromolecules for the research and development of new chemical drugs, are important material bases for the innovation of drug discovery sources and continuous innovation, and have decisive significance for the research of the whole innovative drugs.

Actinomycetes are the main sources of microbial drugs, and are mainly Streptomyces and some rare actinomycetes. However, since the 70 s, the chances of finding new compounds from streptomyces became less and more high. These currently available actinomycetes (commonly called rare actinomycetes) of a small variety, such as verruca, nocardia, and the like, have been attracting increasing attention. A large number of active compounds with novel structures are contained in secondary metabolites of rare actinomycetes, so that the probability of obtaining target active compounds is improved.

Verruculopsis (Verrucosispora) belonging to the order ActinomycetalesMonosporaceae (Micromonosporaceae). After the earliest isolation of wart fungi from marsh sludge in 1998, Rheims et al, scientists continued to isolate this genus of strain from marine muds, mangroves, ascidians and sponges. Wart spore fungus contains abundant natural product resources. Is becoming an important bacterial source for microbial drugs. In 2004, verrucosispora maris AB 18-032 belonging to Micromonosporaceae was isolated from 289m deep sediments in the Japanese sea, and the strain can produce polyketide abysomicin C with MRSA and VRSA activity, and the compound has a new antibacterial effect target and has positive significance for searching novel high-efficiency antibiotics. Bull and Stach found that they contained at least 20 gene clusters synthesized by natural products through genome analysis of verrucosispora maris AB 18-032, together with the findings of Net et al, demonstrated that wart could synthesize not only antibacterial active substances but also more natural compounds. In 2008 verrucosispora sp.MG-37 isolated from sediments 250 m deep in the Norwegian sea, this strain synthesized anti-tumor Folan compounds (proximicins). In 2010, Dai et al isolated verrucosispora sediminis MS426 from sediments in the south sea at 3602 m depth^TAnd 8 cyclic-dipeptides (cyclo-dipeptides) and two nocardiam leishmanials (nocardamine-like) were isolated from the strain, some of which had antibacterial and antifungal activity. In 2010, Shirai et al isolated two new diterpenoids (gifhornenolones A and B) with anti-prostate cancer from the strain verrucosispora gifhornensis YM 28-088. In addition, compounds with novel structures and excellent activity, such as antineoplastic compounds thiochromdriline C and Harrucomicin C, are also separated from the verruca. The antibacterial agent with a novel structure or a novel action mechanism is screened from the verruca acuminata, so that the method has practical feasibility for coping with multi-drug resistant bacteria.

The invention content is as follows:

the first purpose of the invention is to provide an alkaloid compound with bacteriostatic activity.

The structure of the alkaloid compound is shown in the formula (1):

the second object of the present invention is to provide the method for producing the alkaloid compounds, wherein the alkaloid compounds are isolated from a fermentation broth of Verrucosissisporagifhornensis FIM06-0025, which is a fungus of Verrucosissisporus.

Preferably, the specific steps are as follows:

separating mycelium and supernatant from a fermentation culture solution of verruca vulgaris V.gifhornensis FIM06-0025, extracting the mycelium by using methanol/acetone with the volume ratio of 1:1, concentrating an extracting solution to remove methanol and acetone to obtain an extract 1, combining the extract 1 and the supernatant, extracting by using ethyl acetate, concentrating an extracting solution to obtain an extract 2, performing C18 reverse phase silica gel column chromatography on the extract 2, performing gradient elution by using eluent with the methanol/water volume ratio of 30:100, 50:100 and 70:100, collecting components with the methanol/water elution concentration of 70:100 to obtain a component A-1, performing silica gel column chromatography on the component A-1, performing gradient elution by using chloroform/methanol from the volume ratio of 9:1 to 1:1, collecting eluents in parts, performing TLC detection, using a developing agent with the chloroform/methanol volume ratio of 10:1, using iodine as a color developing agent, combining the same components, collecting a component 3 with Rf value of 0.55 by TLC detection, performing Sephadex LH-20 column chromatography, detecting the eluted fraction by TLC with methanol/acetonitrile 1:1 and v: v as eluent and chloroform/methanol volume ratio of 10:1 as a developing solvent, and collecting a component with Rf value of 0.57 to obtain a monomeric compound FW 252.

The fermentation culture solution of the verruca fungus V.gifhornensis FIM06-0025 is obtained by inoculating the verruca fungus V.gifhornensis FIM06-0025 into a fermentation culture medium and fermenting;

the fermentation medium contains 20.0g of soluble starch per liter, K₂HPO₄0.5g，KNO₃5.0g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g, the balance being seawater, pH 7.2-7.5.

The fermentation conditions are as follows: the culture was carried out at 26 ℃ and 240 rpm.

The third purpose of the invention is to provide the application of the alkaloid compound in the preparation of antibacterial drugs.

A fourth object of the present invention is to provide an antibacterial agent characterized by containing the above alkaloid compound as an active ingredient.

The antibacterial drug is a drug for resisting gram negative bacteria helicobacter pylori, pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, escherichia coli, gram positive bacteria staphylococcus aureus, candida albicans and enterococcus faecium.

The fifth purpose of the invention is to provide Verrucosispora gifhornensis FIM06-0025 with the preservation number as follows: CGMCC No.15242

The sixth purpose of the invention is to provide the application of verruca v. gifhornrensis FIM06-0025 in preparing the alkaloid compounds.

The alkaloid compound with a novel structure is separated from verruca vulgaris V.gifhornnensis FIM06-0025, has good antibacterial activity, and can be used for preparing antibacterial drugs.

The Verrucosispora gifhornensis FIM06-0025 is preserved in China general microbiological culture Collection center (CGMCC) in 2018, month 01 and day 18, and the address is as follows: the Beijing West Lu No.1 Hospital No. 3 of Chaoyang district, the preservation number is: CGMCC No. 15242.

Description of the drawings:

FIG. 1 is the single colony morphology of strain FIM06-0025 in nutrient agar medium;

FIG. 2 is a scanning electron micrograph of strain FIM 06-0025;

FIG. 3 is an HRESI-TOF-MS spectrum of Compound FW 252;

fig. 4 is an IR spectrum of compound FW 252;

FIG. 5 is of compound FW252¹H-NMR spectrum;

FIG. 6 is of compound FW252¹H-¹H COSY spectrum;

fig. 7 is an HMBC spectrum of compound FW 252;

FIG. 8 is of compound FW252¹³C-NMR spectrum;

fig. 9 is an HSQC spectrum of compound FW 252;

FIG. 10 is a NOESY spectrum of compound FW 252;

FIG. 11 shows the chemical structure and essential of Compound FW252¹H-¹H COSY and HMBC correlation

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

the inventor obtains a strain from east China sea sponge of Fujian province, and the strain is named as: FIM06-0025, which has the taxonomic characteristics as follows:

1.1 physicochemical Properties of the Strain

Single colony morphology of strain FIM06-0025 in nutrient agar medium (FIG. 1). As seen from the electron microscope scanning of the strain FIM06-0025, the intrabasal mycelium developed well, the branches were separated, the diameter was 0.4 μm, the single spore grew on the brachycephalus, the spore had a handle, and the surface had wart-like prominences (FIG. 2).

Under the aseptic condition, the strain FIM06-0025 is inoculated to ISP 1-ISP 7 agar slant culture medium by using an inoculating loop, the culture is carried out for 2-5 days at 32 ℃, and the culture characteristics of the FIM06-0025 in different culture media are observed and recorded (Table 1).

Nutrient agar culture medium (g/L): 3.0 parts of beef extract, 10.0 parts of peptone, 2.0 parts of yeast extract, 5.0 parts of sodium chloride, 12.0 parts of agar and 1000mL of distilled water, wherein the pH value before sterilization is 7.2-7.5.

TABLE 1 cultivation characteristics of FIM06-0025 in different media

Note: , + + + + indicates good growth; + indicates general growth; + represents a growth difference; -means no growth; color and color values were referenced to the ISCC-NBC standard.

ISP1 medium (g/L): 0.5 parts of tryptone, 0.3 parts of yeast extract, 2 parts of agar, 1L of distilled water and the pH value of the mixture before sterilization is 7.2-7.5.

ISP2 medium (g/L): 0.4 parts of yeast extract, 1 part of malt extract, 0.4 part of glucose, 2 parts of agar and 1L of distilled water, and the pH value is 7.3 before sterilization.

ISP3 medium (g/L): oat flour 2, agar 2, trace elements 1mL, agar 2, distilled water 1L, pH7.4 before sterilization.

ISP4 medium (g/L): soluble starch 10.0, K₂HPO₄1.0，MgSO₄1.0，NaCl 1.0，(NH₄)₂SO₄2.0，CaCO₃2.0, 1mL of trace elements, 2 parts of agar, 1L of distilled water, and pH7.2 before sterilization.

ISP5 medium (g/L): L-Aspartame 1.0, K₂HPO₄1.0, 10.0 glycerol, 20.0 agar, 1mL microelement, 2 agar, 1L distilled water, and pH 7.2-7.4 before sterilization.

ISP6 medium (g/L): peptone-iron-agar 36.0, yeast extract 1.0, agar 2, distilled water 1L, pH 7.2-7.4 before sterilization. Peptone yeast extract iron agar.

ISP7 medium (g/L): glycerol 1.5, L-tyrosine 0.05, L-aspartyl 0.1, K₂HPO₄0.05，MgSO₄0.05, NaCl 0.05, trace elements 1mL/L, agar 1.5, distilled water 1L, and pH 7.2-7.4 before sterilization.

Microelement solution (g/L): FeSO₄·7H₂O 0.1，MnCl·4H₂O 0.1，ZnSO₄·7H₂O0.1, distilled water 100 mL.

The strength of the carbon source utilization by the microorganism is characterized by the growth rate of the microorganism under the condition of a single carbon source. ISP9 basic medium is selected for testing. 21 carbon sources including melibiose, raffinose, D-mannose, L-xylose, inositol, saligenin, L-rhamnose, dulcitol, sorbitol, dithiothreitol, D-galactose, dulcitol, esculin, erythritol, cellobiose, adonitol, melezitose, trehalose, L-arabinose, D-glucose and chitin. 0.8g/L of each carbon source was added, 3 replicates per treatment, with no carbon source added as a control. Under aseptic conditions, the strain FIM06-0025 was inoculated with an inoculating loop onto ISP9 minimal medium petri dish plates containing different carbon sources, cultured upside down at 32 ℃, the results were recorded at different culture times and compared with a blank, and the results of carbon source utilization were analyzed (Table 2).

ISP9 radicalBasal medium (g/L): k₂HPO₄5.65，KH₂PO₄2.38，(NH₄)₂SO₄2.64，MgSO₄·7H₂O1.0, agar 12.0, carbon source 1.0, trace elements 1mL, distilled water 1000mL, pH 7.5. Preparing trace elements: CuSO₄·5H₂O 0.64g，FeSO₄·7H₂O 0.11g，ZnSO₄·7H₂O 0.15g，MnCl₂·4H₂O0.79 g, distilled water 100 mL.

TABLE 2 carbon source utilization of strain FIM06-0025

Note: + indicates that it is fully available; ± means partially available; -means completely unusable.

1.2 identification of 16S rRNA of Strain

5mL of FIM06-0025 bacterial liquid cultured to logarithmic phase is taken, centrifuged for 15min at 8000r/min, supernatant is decanted, and thalli are collected. Extracting thallus genome DNA by a CTAB/NaCl method. The method comprises the following steps: adding 1.35mL of LTE solution (pH 8.0) into the thalli, suspending, adding 0.3mL of 10% Sodium Dodecyl Sulfate (SDS), 150 mu L of 100mg/mL lysozyme and 150 mu L of 100mg/mL proteinase K, uniformly mixing, carrying out water bath at 60 ℃ for 1h, adding 0.25mL of 5mol/L NaCl and 0.2m L of CTAB/NaCl solution, carrying out water bath at 65 ℃ for 10min, extracting for 3 times by using equal volumes of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol respectively, centrifuging for 10min at 10000r/min, sucking supernatant, adding 0.6 times of volume of isopropanol, placing at-20 ℃ for precipitating DNA, dissolving the DNA in 50 mu L of TE, and storing at-20 ℃ for later use. The integrity of the extracted genomic DNA was checked by electrophoresis on a 1% agarose gel. The extracted DNA was subjected to PCR amplification using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the 16S rRNA gene. Sending the amplified DNA product to Shanghai bioengineering Co., Ltd for sequencing, wherein the 16S rRN isThe sequence A is shown in SEQ ID NO. 1. BL AST software is used for carrying out comparison analysis with 16S rRNA sequences recorded in a GenBank database of NCBI, and Blast searches homologous sequences. Strain FIM06-0025 and Verrucosispora gifhornensis DSM 44337^TThe similarity of the 16S rRNA gene sequence is 99 percent, and the strain FIM06-0025 is determined to be verruca verrucosa Verrucosissispora by integrating physiological and biochemical and molecular identification results of the strain. This strain was named: verruca verrucosa Verrucosispora gifhornnensis FIM06-0025, which was deposited at 18.01.2018 in the china general microbiological culture collection center (CGMCC), address: the Beijing West Lu No.1 Hospital No. 3 of Chaoyang district, the preservation number is: CGMCC No. 15242.

1.3 fermentation of the Strain

Inoculating a platinum loop strain Verrucosispora gifhornensis FIM06-0025 Gaoshan aspartyl agar slant culture into a seed culture medium, and performing shake culture at 26 ℃ and 240rpm for 2-3 days to obtain a seed culture solution of V.gifhornensis FIM 06-0025. Inoculating the seed culture solution of the strain FIM06-0025 into a fermentation culture medium in an inoculation amount of 5-10% by volume fraction, and performing shake culture at 26 ℃ for 4-5 days at 240rpm to obtain the fermentation culture solution of V.gifhornensis FIM 06-0025.

Aspartyl agar slant culture medium: 20.0g of soluble starch, 0.5g of aspartyl, 16.5g of sea salt, 0.5g of NaCl and KNO₃1.0g，K₂HPO₄0.5g，MgSO₄·7H₂O 0.5g，CaCO₃1.0g, agar 12.0g, seawater 1000mL, natural pH value, and its preparation method comprises mixing the components according to their content, and sterilizing at 121 deg.C for 30 min.

Preparation of seed culture medium: 15.0g of soluble starch, 5.0g of yeast powder, 5.0g of peptone, 5.0g of glucose and K₂HPO₄0.5g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，(NH₄)₂SO₄0.5g，CaCO₃1.0g and 1L of seawater, and the preparation method comprises the steps of uniformly mixing the components according to the content, and the pH value is 7.5 before sterilization. Sterilizing at 121 deg.C for 30 min.

Preparation of a fermentation medium: soluble starch 20.0g，K₂HPO₄0.5g，KNO₃5.0g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g and 1000mL of seawater, and the preparation method comprises the step of uniformly mixing the components according to the content, wherein the pH value is 7.2-7.5. Sterilizing at 121 deg.C for 30 min.

1.4 preparation of the Compounds

Centrifuging the fermentation culture solution (50L) of the marine V.gifhornensis FIM06-0025 at 4500rpm to obtain mycelium and supernatant, and ultrasonically extracting the mycelium for 2 times by using methanol/acetone (1: 1, v: v) with the volume of 1.5-2.0 times, wherein the ultrasonic temperature is lower than 40 ℃, and the ultrasonic time is 30 min. Concentrating the extract at a temperature lower than 40 ℃ under reduced pressure to remove methanol and acetone to obtain extract 1(210g), mixing the extract 1 with the supernatant, extracting the combined solution with ethyl acetate with the volume of 1.5-2.0 times of that of the combined solution for 2 times, and concentrating the ethyl acetate extract at a temperature lower than 40 ℃ under reduced pressure to remove ethyl acetate to obtain extract 2(169 g). Subjecting extract 2 to C18 reverse phase silica gel column chromatography, gradient eluting with methanol/water at volume ratio of 30:100, 50:100, and 70:100 as eluent, and mixing with the component with methanol/water elution concentration of 70:100 to obtain component A-1(110 mg). Mixing the component A-1 with 100-200 mesh silica gel, performing dry column loading, performing gradient elution with chloroform/methanol from a volume ratio of 9: 1-1: 1, collecting eluate in a fraction collector, performing TLC detection, using a developing agent of chloroform/methanol in a volume ratio of 10:1, using iodine as a color developing agent, combining the same components to obtain five components (Rf values are 0.27, 0.39, 0.55, 0.67 and 0.75 respectively), performing Sephadex LH-20 column chromatography on the component 3(30mg, component with Rf value of 0.55 detected by TLC), performing TLC detection on the eluate with methanol/acetonitrile in a volume ratio of 1:1 and v: v as an eluent, and collecting a component with Rf value of 0.57 to obtain a monomer compound FW252(6.5 mg).

1.5, structural identification

The compound FW252 is subjected to data tests such as mass spectrometry, ultraviolet spectrometry, infrared spectrometry, nuclear magnetic resonance and the like, so as to determine the structure of the compound.

Compound FW 252: 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 C₁₁H₁₃NO₃The unsaturation was calculated to be 6. Infrared absorption spectra (IR) at 3053, 1619, 1490 and 1582cm^-1The absorption at (B) indicates that the aromatic benzene ring group is contained at 1642cm^-1The strong absorption peak at (a) indicates the presence of an amide group (fig. 4).

¹HNMR (FIG. 5) showed 1 methyl proton [ delta ]_H1.70(d,J＝6.5Hz,3H)]Signal, 2 methylene protons [ delta ]_H3.94(dd,J＝12.0,3.5Hz,1H))、3.81(dd,J＝12.0,3.0Hz,1H)]Signal, 2 methine proton signal [ delta ]_H4.27(m,1H)、5.38(m,1H)]Hydrogen protons of 2-OH [ delta ]_H(12.3，4.9)]Signal and 4 phenyl protons [ delta ]_H7.95(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)]A signal. From the coupling constants of 4 phenyl proton signals, it can be seen that the compound contains 1 ortho-substituted benzene ring,¹H-¹the H COSY and HMBC spectra further confirmed the above conclusions (fig. 6, fig. 7).¹³C-NMR and DEPT135 (DMSO-d)₆125MHz) (fig. 8, fig. 9) shows that the compound contains 11 carbon signals, including 1 methyl group (δ)_C20.2),1 methylene (. delta.) group_C61.6), 6 methines (. delta.)_C140.0, 132.3, 121.7, 118.0, 107.8, 84.9, and 67.7) and 3 quaternary carbons (δ_C170.5, 161.9, and 107.8). Nuclear magnetic data indicate that the compound contains 1 ortho-substituted benzene ring and 1 long-chain fragment structure of four Carbons (CH)₃-C₁₀-C₈-C₇) 8-H (. delta.3.90), 10-H (. delta.4.68) and 9-C (. delta.4.68) in HMBC spectra_C164.4) correlation of amide carbons to known fragment CH₃-C₁₀-C₈-C₇The amide group is linked by a three-membered ring structure with the N atom, and then, by 2-H (. delta.) (_H7.60) coupling to the amide carbon of 9-C (. delta.170.5) can link the above-mentioned structural fragment to a benzene ring group. Combining the above analyses, and combining HSQC (FIG. 10) and HMBC spectra, the total carbon signal and hydrogen of compound FW252 were obtainedSignal attribution (table 4). Finally, the chemical structure of the compound FW252 is determined as shown in the formula (2) according to the unsaturation degree, the molecular weight and the NOESY spectrum (figure 11), and the chemical name is: (2- (hydroxymethyl) -3- (2- (hydroxymethyl) -3-methylazidin-1-yl) (2-hydroxyphenyl) methyl one. by database search of Scifinder and INSPEC, no compound having the same structure as the 2- (hydroxymethyl) -3-methylazidin-1-yl is found, so that FW252 is determined to be an alkaloid compound having a new structure, and the chemical structure and main components of FW252 are¹H-¹H COSY and HMBC are related as shown in FIG. 11.

TABLE 3 of compound FW252¹H NMR(in Methanol-d₄400MHz) and¹³C-NMR(in Methanol-d₄100MHz) data

1.6 antimicrobial Activity test

Compound FW252 was tested for Minimum Inhibitory Concentration (MIC) using a broth dilution method with cefotaxime sodium as a positive control. Test bacteria include 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, acinetobacter baumannii (a.baumannii) 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).

The specific operation steps are as follows:

1) MH broth culture medium preparation: weighing MH broth culture medium 21.0g, adding into 1L distilled water, heating and boiling to dissolve completely, subpackaging in test tube, and autoclaving at 121 deg.C for 15min for use.

2) Early culture of test bacteria: the test bacteria were inoculated into 100mL MH broth under aseptic conditions and cultured in an incubator at 35 ℃ for 12 hours for use.

3) Preparation of a stock solution: weighing a proper amount of a sample to be detected and a positive control respectively, dissolving the positive control by using 1mL of sterile water, and dissolving the sample to be detected by using 1mL of methanol. The initial concentration of each stock solution should be greater than 1000. mu.g/mL.

4) Preparation of test bacterium solution: the cultured test bacteria are diluted with MH broth to 0.5 McLee unit turbidity standard at a ratio of 1:20 under aseptic condition to obtain a bacterial solution with a concentration of about 5 × 10⁶CFU/mL, spare.

5) Dilution of stock and inoculation of test bacteria: sterile procedures were performed, taking one sterile 96-well plate. Adding MH broth 100 μ L into each well except the first well, adding sample stock solution 40 μ L into the 1 st well, mixing, sucking 100 μ L to the 2 nd well, mixing, sucking 100 μ L to the 3 rd well, diluting in multiple times to the 3 rd last well, and discarding 100 μ L from the 3 rd last well. The penultimate 2 wells are growth controls without drug and the last 1 well is an ungaccinated control. Then, 10. mu.L of the prepared inoculum of the test bacteria was added to each well to give a final bacterial liquid concentration of about 5X 10 per well⁵CFU/mL。

6) And (3) incubation: covering the 96-well plate inoculated with the test bacteria with a cover, placing the plate in a biochemical incubator at 35 ℃ for culturing for 16-20h, and recording the result.

7) MIC endpoint interpretation: the lowest inhibitory concentration of the sample against the species of bacteria that completely inhibited bacterial growth seen with the naked eye in 96-well plates.

As a result, the compound FW252 showed broad-spectrum inhibitory activities (MIC value 3.4-200. mu.g. multidot.mL) against gram-negative and gram-positive bacteria as shown in Table 4^-1) Especially has strong inhibitory activity to gram negative bacteria helicobacter pylori and Klebsiella pneumoniae and gram positive bacteria staphylococcus aureus.

TABLE 4 results of the bacteriostatic test

Sequence listing

<110> institute of microorganisms of Fujian province

<120> alkaloid compound with diverse antibacterial activities, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1397

<212> DNA

<213> Verrucaria 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 (6)

1. A preparation method of an alkaloid compound is characterized by comprising the following steps:

separating mycelium and supernatant from a fermentation culture solution of verruca vulgaris V.gifhornensis FIM06-0025, extracting the mycelium with methanol/acetone in a volume ratio of 1:1, concentrating an extracting solution to remove methanol and acetone to obtain an extract 1, combining the extract 1 and the supernatant, extracting with ethyl acetate, concentrating the extracting solution to obtain an extract 2, subjecting the extract 2 to C18 reverse phase silica gel column chromatography, performing gradient elution with an eluent of methanol/water in a volume ratio of 30:100, 50:100, 70:100 and 70:90 to obtain a component A-1, subjecting the component A-1 to silica gel column chromatography, performing gradient elution with chloroform/methanol in a volume ratio of 9: 1-1: 1, collecting eluates in parts, detecting by TLC, using a developing agent of chloroform/methanol in a volume ratio of 10:1, using iodine as a color developing agent, combining the same components, collecting component 3 with Rf value of 0.55 by TLC detection, performing Sephadex LH-20 column chromatography, eluting with methanol/acetonitrile at volume ratio of 1:1 as eluent, detecting eluate with TLC with chloroform/methanol at volume ratio of 10:1 as developing solvent, and collecting component with Rf value of 0.57 to obtain alkaloid compound;

the structure of the alkaloid compound is shown in the formula (1):

2. the preparation method according to claim 1, wherein the fermentation culture solution of verruca v.gifhornensis FIM06-0025 is obtained by inoculating verruca v.gifhornensis FIM06-0025 into a fermentation culture medium and fermenting;

the fermentation medium contains 20.0g of soluble starch per liter, K₂HPO₄ 0.5g，KNO₃ 5.0g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g, the balance being seawater, pH 7.2-7.5.

3. The application of alkaloid compounds or salts thereof in preparing antibacterial drugs;

the structure of the alkaloid compound is shown in the formula (1):

4. the use according to claim 3, wherein said antibacterial agent is an agent against helicobacter pylori, pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, escherichia coli, staphylococcus aureus, candida albicans or enterococcus faecium.

5. Verrucaria Verrucosispora gifhornensis FIM06-0025 with the preservation number: CGMCC No. 15242.

6. Use of the verruca v.gifhornensis FIM06-0025 according to claim 5 for the preparation of alkaloid compounds;

the structure of the alkaloid compound is shown in the formula (1):

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