An antibacterial active compound, its preparation method and application

Abstract

The application belongs to the technical field of natural products and medicines, and particularly relates to an alkaloid compound with antibacterial activity and a preparation method and application thereof. The structure of the alkaloid compound is shown as formula (1). The alkaloid compound is a new compound, which is separated from a fermentation product of actinomycetes. Antibacterial activity experiments show that the compound has obvious inhibitory activity on the formation of a biofilm. Meanwhile, the method for preparing the compound is simple and has good repeatability, and therefore can be better applied in the preparation of antibacterial drugs.

Description

Technical Field

[0001] This invention relates to the field of natural products and pharmaceutical technology, and in particular to a compound with antibacterial activity, its preparation method, and its application. Background Technology

[0002] Antibiotic resistance has become one of the most serious global threats to human health in the 21st century. Currently, China ranks first in the world in antibiotic consumption, accounting for approximately 50% of global usage. It is estimated that without effective measures, by 2050, China will experience approximately one million deaths annually due to antibiotic resistance, resulting in a cumulative economic loss of US$20 trillion. The overuse of antibiotics exacerbates bacterial resistance, and the continued rise in antibiotic resistance and the shortage of effective antibiotics necessitate the development of new antibiotics.

[0003] Microbial secondary metabolites are an important source of antibiotics, and more than half of microbial antibiotics are obtained from Streptomyces. It has been reported that Streptomyces rod-shaped bacteria can produce secondary metabolites with unique structures and diverse biological activities; for example, lactoquinacrine A exhibits significant inhibitory activity against Gram-positive strains. To enrich the compound resources and their diverse activities, this application isolated a Streptomyces sp. ZSA65 strain from marine sediment samples. Antibacterial activity was observed in the rice solid culture medium fermentation product of this strain, which showed antibacterial activity and inhibited biofilm formation by Pseudomonas aeruginosa. Through the isolation and purification of the active components, we obtained a novel antibacterial natural product. Summary of the Invention

[0004] This invention marks the first time an alkaloid compound has been isolated and purified from the fermentation products of actinomycetes. The chemical structure of the new compound was determined by analyzing the 1H, 1C, COSY, HMQC, HMBC, and NOSEY spectra of the alkaloid. A series of studies have revealed that this new compound exhibits significant biofilm inhibitory activity and can be used in the preparation of antibiotic drugs, showing great promise for development and application.

[0005] In a first aspect, the present invention provides a compound as shown in formula (1), which is an alkaloid with significant biomembrane inhibitory activity and can be used to prepare antibiotic drugs, showing great promise for development and application. Its structural formula (1) is shown below.

[0006]

[0007] Secondly, the present invention provides a method for preparing the above-mentioned alkaloid compound, which includes the following steps:

[0008] 1) Take Streptomyces sp. ZSA65 and inoculate it onto Gao's No. 1 solid medium for activation. Inoculate a single colony of the activated strain into Gao's No. 1 liquid medium and culture with shaking to obtain seed liquid. Inoculate the obtained seed liquid into rice medium and culture statically to obtain fermentation product.

[0009] 2) The fermentation product was extracted with an organic solvent, and the organic solvent was removed by vacuum distillation using a rotary evaporator to obtain a crude extract.

[0010] 3) The obtained crude extract was separated by silica gel column chromatography, the eluent was collected, and each component was detected by thin-layer chromatography. The components containing antibacterial activity were combined.

[0011] 4) The antibacterial active components were separated and purified by preparative high performance liquid chromatography to obtain the antibacterial active compound alkaloid (1).

[0012] Preferably, the Streptomyces sp. ZSA65 mentioned in step 1) is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2024611.

[0013] Preferably, the formula of Gao's No. 1 solid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 20g agar, 25g sea salt, and 1L water.

[0014] Preferably, the formula of Gao's No. 1 liquid culture medium used in step 1) is as follows: 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 25g sea salt, and 1L water.

[0015] Preferably, in step 1), the rice culture medium is prepared from rice and sea salt water, and the weight-to-volume ratio of the rice to the sea salt water is 40g:60mL; the sea salt water is formulated as 2.5g of sea salt per 100mL of water.

[0016] Preferably, the organic solvent in step 2) is an ester-based organic solvent.

[0017] Preferably, the silica gel column chromatography conditions in step 3) are as follows: column chromatography is performed using eluents with volume ratios of dichloromethane to methanol of 1:0, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1, and the fractions are combined by TLC to obtain 13 fractions, namely Fr.A to Fr.M.

[0018] Preferably, in step 4), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit C18 column (21.2 mm × 250 mm × 10 μm), using a gradient elution system of methanol-acid-water with a volume percentage of 8–95%, wherein the volume ratio of trifluoroacetic acid to pure water in the acid water is 0.05:100, the flow rate is 10 mL / min, the detection wavelength is 210 nm, and the fraction collected has a retention time of 19.1 min.

[0019] Thirdly, the present invention provides a pharmaceutical composition comprising the compound of formula (1) of the present invention, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.

[0020] Fourthly, the present invention provides the use of compounds represented by the structure of formula (1) above, their tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof, in the preparation of antibiotic medicaments.

[0021] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their pharmaceutical salts with pharmaceutically acceptable excipients.

[0022] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0023] The beneficial effects of this invention are as follows: This invention extracts and separates a new compound from actinomycete rice fermentation products, and uses a variety of spectroscopic analysis methods to determine that it is an alkaloid compound; through antibacterial activity testing of the obtained compound, it was found that the above-mentioned alkaloid compound has significant inhibitory activity against Pseudomonas aeruginosa biofilm. This invention provides a candidate compound for the development of new antibacterial drugs. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 The compound described in formula (1) 1 H-NMR (600MHz, CD3OD) spectrum;

[0026] Figure 2 The compound described in formula (1) 13 C-NMR (150MHz, CD3OD) spectrum;

[0027] Figure 3 The HSQC (Heteronuclear Singular Qantum Correlation) spectrum of the compound described in formula (1) is shown below.

[0028] Figure 4 HMBC of the compound described in formula (1) 1 H detected heteronuclear multiple bondcorrelation, 1 Heteronuclear multicarbon correlation spectrum of H;

[0029] Figure 5 The compound described in formula (1) 1 H- 1 H COSY (Correlation spectroscopy) spectrum;

[0030] Figure 6 The NOESY (Nuclear Overhauser Effect Spectroscopy) spectrum of the compound described in formula (1);

[0031] Figure 7 The HR-ESI-MS spectrum of the compound described in formula (1);

[0032] Figure 8 The effect of the compound described in formula (1) on biofilm formation

[0033] strain preservation

[0034] Classification and nomenclature: Streptomyces sp.; Strain number: ZSA65

[0035] Preservation Institution: China Center for Type Culture Collection

[0036] Abbreviation for depository institution: CCTCC

[0037] Address: Wuhan University Collection Center

[0038] Deposit date: April 2, 2024

[0039] Collection Center Registration Number: CCTCC NO: M 2024611 Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention are further described below with reference to specific examples. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the examples can be easily obtained from commercial companies unless otherwise specified.

[0042] Source of strain:

[0043] The marine-derived strain Streptomyces sp. ZSA65 used in this application was isolated from sediment samples collected in Daishan, Zhejiang Province, China.

[0044] culture medium

[0045] Gao's No. 1 solid culture medium: Mix 20.0g soluble starch; 1.0g KNO3; 0.5g KH2PO4; 0.5g MgSO4; 0.5g NaCl; 0.01g FeSO4·7H2O; 25.0g sea salt; and 20.0g agar, dissolve in distilled water, bring the volume to 1L, and sterilize in an autoclave at 121℃ for 30min.

[0046] Gao's No. 1 liquid culture medium: Mix 20.0g soluble starch, 1.0g KNO3, 0.5g KH2PO4, 0.5g MgSO4, 0.5g NaCl, 0.01g FeSO4·7H2O, and 25.0g sea salt, dissolve in distilled water, bring the volume to 1L, and sterilize in a high-pressure steam autoclave at 121℃ for 30min.

[0047] Rice culture medium: Weigh 25g of sea salt and mix it evenly in 1L of purified water. Adjust the pH to 7.2-7.4 to prepare sea salt solution. Weigh 40g of rice into a 500mL Erlenmeyer flask, add 60mL of the above-prepared sea salt solution, and sterilize in a high-pressure steam sterilizer at 121℃ for 30min.

[0048] Example 1: Preparation of the compound shown in formula (1)

[0049] (1) Fermentation culture of strain Streptomyces sp. ZSA65: 1) Take Streptomycess sp. ZSA65 (preservation number CCTCC NO: M 2024611, China Center for Type Culture Collection), inoculate it onto Gao's No. 1 solid medium, and incubate it in a 28℃ incubator for 10 days for activation culture; 2) Inoculate a single colony of Streptomycess sp. ZSA65 activated in step 1) into a 500mL Erlenmeyer flask containing 250mL Gao's No. 1 liquid medium, and incubate it at 28℃ with shaking at 180rpm for 4 days to obtain seed liquid; 3) Take 5mL of the seed liquid from step 2) into a 500mL Erlenmeyer flask containing 40g of sterile rice solid medium, and incubate it in a 28℃ incubator for 70 days to obtain fermentation product.

[0050] (2) Extraction: The fermentation product was extracted by soaking in ethyl acetate for 24 hours, with the amount of organic solvent being 1.5 times the volume of the fermentation product. The soaking was repeated three times. The extracts were combined and concentrated under reduced pressure to obtain a crude extract of 10.6 g.

[0051] (3) Separation: After dissolving the crude extract in dichloromethane, the sample was first subjected to gradient elution by silica gel column chromatography with dichloromethane-methanol volume ratios of 1:0, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1. The samples were then combined by TLC, and the antibacterial components were combined to obtain 13 components, namely Fr.A to Fr.M. The antibacterial component Fr.E was separated by preparative high performance liquid chromatography. The Agilent Pursuit C18 column (21.2 mm × 250 mm × 10 μm) was used for gradient elution in a methanol-acid water system with a volume percentage of 8-95% for 40 min. The volume ratio of trifluoroacetic acid to pure water in the acid water was 0.05:100. The flow rate was 10 mL / min. The fraction with a retention time of 19.1 min was collected to obtain 1.9 mg of the compound (1).

[0052] (4) Structural characterization: The compounds obtained in this example were subjected to high-resolution mass spectrometry and nuclear magnetic resonance analysis to determine the structure of the compounds, which is shown below:

[0053]

[0054] The physicochemical constants and spectral data of the obtained compounds are as follows:

[0055] The resulting compound was a pale yellow oil, [α]D 20 = –69.3 (c = 0.12, methanol), HRESIMS m / z 182.0807 [M+H] +(calcd for m / z 182.0812), m / z 385.1355[2M+Na] + (calcd for m / z385.1370). 1 H NMR (600MHz) and 13 The C NMR (150MHz) data are shown in Table 1. 1 The HNMR (600MHz, CD3OD) spectrum is shown below. Figure 1 , 13 The C-NMR (150MHz, CD3OD) spectrum is shown below. Figure 2 HSQC spectrum can be found Figure 3 HMBC spectrum can be found Figure 4 , 1 H- 1 See the H COSY spectrum. Figure 5 See NOESY spectrum Figure 6 HR-ESI-MS spectra can be found Figure 7 .

[0056] Table 1. Compounds 1 H (600MHz) and 13 C(150MHz) data

[0057]

[0058] Example 2: Evaluation of the anti-biofilm activity of the present invention using a Pseudomonas aeruginosa biofilm inhibition model.

[0059] Pseudomonas aeruginosa was inoculated into liquid culture medium and cultured overnight. The bacterial suspension, diluted with M63 restriction medium, was mixed with the test sample and placed in a 96-well plate. The plate was then incubated at 37°C for 16 hours. The bacterial concentration (OD600) in each well was measured using a microplate reader. The culture medium was then removed from the plate and the plate was washed with deionized water. 160 μL of 0.1% crystal violet was added to the plate to stain the biofilm for 10-15 minutes. The plate was washed 3-4 times with sterile water to remove unbound staining agent and then dried. 160 μL of acetic acid (30%) was added to the plate to extract the crystal violet, and the absorbance (Abs595) was measured using a microplate reader. The biofilm formation amount was expressed as Abs595 / OD600.

[0060] See results Figure 8 The results showed that the compound had significant inhibitory activity against biofilm formation, indicating that the compound of formula (1) had strong anti-biofilm activity.

[0061] The embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the terms used herein are descriptive and explanatory, not limiting. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from its scope and spirit. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A compound having antibacterial activity, characterized in that, The compound has a molecular formula of C9H 11 NO3, and a structure as shown in formula (1):

2. A process for the preparation of the antibacterially active compounds as claimed in claim 1, characterized in that: The fermentation product is prepared by fermentation of Streptomyces sp. ZSA65, which is preserved in China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: M 2024611.

3. The process for the preparation of an antibacterially active compound according to claim 2, characterized in that The method comprises the following steps: 1) After activation of Streptomyces sp. ZSA65, a seed solution is prepared, and the seed solution is inoculated into a culture medium for fermentation culture to obtain a fermentation product; 2) The fermentation product is extracted with an organic solvent, and a crude extract is obtained by concentration; 3) The crude extract obtained in step 2) is separated by silica gel column chromatography, and the eluate is collected, and each component is detected by thin layer chromatography (TLC), and the components containing antibacterial activity are combined; 4) The components containing antibacterial activity are separated and purified by preparative high performance liquid chromatography to obtain an antibacterial compound (1).

4. The production method according to claim 3, characterized by, The fermentation conditions in step 1) are as follows: the temperature is 25-30°C, and the time is 50-70 days.

5. The preparation method according to claim 3, characterized in that, The culture medium is a rice medium prepared from rice and sea salt water, and the weight / volume ratio of the rice and the sea salt water is 40g:60mL; the formula of the sea salt water is 2.5g of sea salt in 100mL of water.

6. The preparation method according to claim 3, characterized in that, In step 2), the organic solvent is an ester organic solvent.

7. The preparation method according to claim 3, characterized in that, In step 3), the silica gel column chromatography conditions are as follows: dichloromethane-methanol with a volume ratio of 1:0, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1 are used as eluents for column chromatography, and 13 components, i.e., Fr. A-Fr. M, are obtained by TLC combination.

8. The method of claim 3, wherein In step 4), the preparative high performance liquid chromatography method is as follows: an Agilent pursuit C18 column 21.2mm×250mm×10μm is used, methanol-acid water with a volume percentage of 8-95% is used for gradient elution, the volume ratio of trifluoroacetic acid to pure water in the acid water is 0.05:100, the flow rate is 10mL / min, the detection wavelength is 210nm, and the component with a retention time of 19.1min is collected.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition contains the compound represented by formula (1) or a pharmaceutically acceptable salt thereof as claimed in claim 1, and one or more pharmaceutically acceptable excipients.

10. Use of the compound represented by formula (1) or a pharmaceutically acceptable salt thereof as claimed in claim 1, or the pharmaceutical composition as claimed in claim 9 in the preparation of an antibiotic drug.

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