Preparation method and application of compound with anti-biofilm activity
By isolating and purifying compound (I) from marine sediments, the problem of antibiotic resistance to biofilm infections has been solved, achieving effective biofilm inhibition and showing promise for development into an antibiotic drug.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG OCEAN UNIV
- Filing Date
- 2024-01-25
- Publication Date
- 2026-05-01
AI Technical Summary
Existing antibiotics are ineffective in treating persistent infections caused by bacterial biofilms, as bacteria exhibit strong drug resistance within biofilms.
Nocardiopsis sp ZHD001 strain was isolated from sediments collected from the waters surrounding Zhoushan. Compound (I) was obtained by fermentation, extraction and chromatography, and its chemical structure was determined, verifying that it has significant biofilm inhibitory activity.
Compound (I) significantly inhibits biofilm formation and has the potential to be developed into an antibiotic drug, thus solving the problem of resistance to biofilm infections by existing antibiotics.
Smart Images
Figure CN121949259A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemical technology, specifically to a compound with anti-biofilm activity, its preparation method, and its application. Background Technology
[0002] Infections caused by bacterial biofilms seriously impact public health and are one of the main causes of bacterial resistance. Bacterial biofilms are often associated with intractable infections that are difficult to eradicate. Because bacteria within the biofilm exhibit strong drug resistance, traditional antibiotics are ineffective. Therefore, there is an urgent need to discover compounds with anti-biofilm activity to combat infections caused by bacterial biofilms. Summary of the Invention
[0003] This invention is the first to isolate Nocardiopsis sp ZHD001 from sediments collected from the waters surrounding Zhoushan, and further isolates it to obtain the fermentation product compound (I). This invention analyzes the proton, carbon, HSQC, and HMBC spectra of compound (I). 1 H- 1 The chemical structure of this compound was determined by HCl COSY and NOSEY spectroscopy. Activity assays showed that compound (I) exhibits significant biofilm inhibitory activity, making it suitable for the preparation of antibiotic drugs and demonstrating promising development and application prospects.
[0004] On the one hand, the present invention provides a compound as shown in formula (I), which has significant biofilm inhibitory activity and can be used to prepare antibiotic drugs, and has good development and application prospects.
[0005]
[0006] Furthermore, the compound of formula (I) is a pale yellow oily substance, soluble in methanol, and high-resolution mass spectrometry yields a quasi-ion peak at m / z 326.1968 [M+H]. + The molecular formula was determined to be C 17 H 27 NO5. Specific NMR data are shown in Table 1 below. 1 The H NMR spectrum is shown in [reference]. Figure 1 , 13 The C NMR 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 NOSEY's score. Figure 6 .
[0007] Table 1
[0008]
[0009]
[0010] On the other hand, the present invention provides a method for preparing a natural active compound, which includes the following steps:
[0011] 1) Take Nocardiopsis sp ZHD001, inoculate it onto Gao's No. 1 solid medium for activation, take a single colony from the plate and inoculate it into Gao's No. 1 liquid medium, shake and culture to obtain seed liquid; inoculate the obtained seed liquid into rice medium, let it stand and culture to obtain fermentation product.
[0012] 2) The fermentation product was extracted with ethyl acetate. The ethyl acetate extract was then removed by vacuum distillation using a rotary evaporator to obtain a concentrated solution. The concentrated solution was then separated by silica gel column chromatography to obtain the separated solution.
[0013] 3) The separation solution was separated and purified using a preparative high performance liquid chromatograph to obtain the target compound (I).
[0014] Preferably, the Nocardiopsis sp ZHD001 strain in step 1) has the accession number CCTCC NO:M2022921.
[0015] 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.
[0016] 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.
[0017] Preferably, the rice culture medium in step 1) 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 25g of sea salt per 1L of water.
[0018] Preferably, the volume ratio of fermentation broth to ethyl acetate in step 2) is 1:1.
[0019] Preferably, the silica gel column chromatography conditions in step 2) are as follows: the eluent used is CH2Cl2:CH3OH, and the gradient is 100:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1.
[0020] Preferably, in step 3), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. A methanol-water + 0.05% TFA system (volume percentage 10%–100%) is used for gradient elution at 10 mL / min for 40 min, and the eluent is collected after 31–33 min. After evaporating the eluent to dryness, a mixed component was obtained. The component was further separated using the instrument described above, with a detection wavelength of 210 nm. A gradient elution system of 55%–65% (v / v) methanol-water + 0.05% TFA was used, and the eluent was collected at a rate of 10 mL / min for 100 min. The eluent was collected at 19–23 min. After evaporating the eluent to dryness, a mixed component was obtained. The component was further separated using the instrument described above, with a detection wavelength of 210 nm. An isocratic elution system of 30% (v / v) acetonitrile-water + 0.05% TFA was used, and the eluent was collected at a rate of 10 mL / min for 50 min. The eluent was collected at 17.4–20 min.
[0021] On the other hand, the present invention provides a pharmaceutical composition comprising the compound having formula (I) of the present invention, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.
[0022] The present invention further provides the use of compounds of the structure shown in formula (I), their tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof, in the preparation of antibiotic medicaments.
[0023] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their pharmaceutical salts with pharmaceutically acceptable excipients.
[0024] 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. Attached Figure Description
[0025] Figure 1 For compound (I) 1 HNMR spectrum (methanol-d4).
[0026] Figure 2 For compound (I) 13C10 NMR spectrum (methanol-d4).
[0027] Figure 3 The image shows the HSQC spectrum (methanol-d4) of compound (I).
[0028] Figure 4 The image shows the HMBC spectrum (methanol-d4) of compound (I).
[0029] Figure 5 For compound (I) 1 H- 1 H COSY spectrum (methanol-d4).
[0030] Figure 6 The NOSEY spectrum (methanol-d4) of compound (I)
[0031] Figure 7 This refers to the anti-biofilm activity of compound (I). Accession number: CCTCC NO: M 2022921 Preservation Institution Code: CCTCC - China Center for Type Culture Collection Date of preservation: June 20, 2022 Classification and naming: Nocardiopsis sp.ZHD001 Survival status: Survival Address of the depository: Wuhan, China Detailed Implementation
[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.
[0033] Example 1: A method for preparing a natural active compound (I)
[0034] The strain Nocardiopsis sp ZHD001 was activated by inoculating it onto Gao's No. 1 solid medium. Single colonies from the plates were then inoculated into Gao's No. 1 liquid medium to obtain seed culture. The Gao's No. 1 solid medium consisted of: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 25g sea salt, and 20g agar powder (2%), diluted to 1L with water and adjusted to pH 7.2. The Gao's No. 1 liquid medium consisted of: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, and 25g sea salt, diluted to 1L with water and adjusted to pH 7.2.
[0035] The obtained seed culture was inoculated into rice culture medium, statically cultured, and the fermentation broth was obtained by extraction. The rice culture medium was prepared by rice and sea salt water, with a weight-to-volume ratio of 40g:60ml. The sea salt water formula was 25g of sea salt per 1L of water. The seed culture inoculation amount was 5ml of seed culture water per 40g of rice culture medium. The static culture conditions were 28℃ for 60 days. The extraction conditions were ethyl acetate soaking for 24 hours, repeated 3 times.
[0036] The obtained fermentation broth was concentrated, separated, and purified to obtain a compound with the structural formula shown in formula (1). The separation and purification steps included, in sequence, silica gel column chromatography, TCL analysis, and high-performance preparative liquid chromatography (HPLC) purification. The silica gel column chromatography conditions were as follows: the eluent used was CH2Cl2:CH3OH, with a gradient of 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1. TCL analysis was performed, and the fractions were combined to obtain 13 components (AM). The target product was found in component K. The HPLC conditions were as follows: HPLC separation and purification were performed using an Agilent Pursuit C18 column (21.2 × 250 mm, 10 μm), with a detection wavelength of 210 nm. A methanol-water + 0.05% TFA system (volume percentage 10%–100%) was used, with gradient elution at 10 mL / min for 40 min. The eluent was collected for 31–33 min. After evaporating the eluent to dryness, a mixed fraction was obtained. This fraction was further separated using the aforementioned instrument at a detection wavelength of 210 nm. A gradient elution system of 55%–65% (v / v) methanol-water + 0.05% TFA was used, eluted at 10 mL / min for 100 min, and the eluent was collected for 19–23 min. After evaporating the eluent to dryness, a mixed fraction was obtained. This fraction was further separated using the aforementioned instrument at a detection wavelength of 210 nm. An isocratic elution system of 30% (v / v) acetonitrile-water + 0.05% TFA was used, eluted at 10 mL / min for 50 min, and the eluent was collected for 17.4–20 min. Compound (I) 3.5 mg was obtained. This compound was a pale yellow oily substance soluble in methanol. High-resolution mass spectrometry showed a quasi-ion peak at m / z 326.1959 [M+H]. + The molecular formula was determined to be C. 17 H 27 NO5. Specific NMR data are shown in Table 1. 1 The H NMR spectrum is shown in [reference]. Figure 1 , 13 The C NMR 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 .
[0037] Example 2 uses a Pseudomonas aeruginosa biofilm inhibition activity model to evaluate the anti-biofilm activity of the present invention.
[0038] A single colony of *Pseudomonas aeruginosa* (PA01) was inoculated into LB broth and cultured overnight. The diluted bacterial suspension, obtained by dilution with M63 restriction medium, was then mixed with the test sample and placed in a 96-well plate. The plate was incubated statically at 37°C for 12-16 hours. The bacterial concentration (OD) in each well was measured using a microplate reader. 600 After removing the culture medium from the plate and rinsing with deionized water, the surface was dried. 160 μl of 0.1% crystal violet was added to each well to stain the biofilm for 10 min. Unbound staining was washed away with sterile water, and the surface was dried again. 160 μl of 30% acetic acid was added to each well to dissolve the crystal violet, and the absorbance was measured using a microplate reader. 595 The relative biofilm formation rate is expressed as Abs595 / OD600, with DMSO as the control group.
[0039] See results Figure 7 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.
[0040] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.
Claims
1. A compound, characterized in that: The compound has the structure of formula (I), is a pale yellow oil, soluble in methanol, and high-resolution mass spectrometry shows a quasi-ion peak at m / z 326.1959 [M+H]. + The molecular formula was determined to be C. 17 H 27 NO 5。 2. The method for preparing the compound of formula (I) as described in claim 1, characterized in that, The preparation method includes the following steps: 1) Take Streptomyces sp. ZSA65, inoculate it onto Gao's No. 1 solid medium for activation, take a single colony from the plate and inoculate it into Gao's No. 1 liquid medium, shake and culture to obtain seed liquid; inoculate the obtained seed liquid into rice medium, let it stand and culture to obtain fermentation product. 2) The fermentation product was extracted with ethyl acetate. The ethyl acetate extract was then removed by vacuum distillation using a rotary evaporator to obtain a concentrated solution. The concentrated solution was separated by silica gel column chromatography, and the eluent was collected. Similar components were detected and combined by thin-layer chromatography. 3) The component containing the target product was separated and purified by preparative high performance liquid chromatography to obtain compound (I).
3. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The Nocardiopsis sp ZHD001 strain in step 1) has the accession number CCTCC NO:M2022921.
4. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The formula ratio 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; the formula ratio 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.
5. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... In step 1), the rice culture medium is prepared from rice and sea salt water, with a weight-to-volume ratio of 40g:60ml for rice and sea salt water. The sea salt water is formulated as 25g of sea salt per 1L of water.
6. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The volume ratio of fermentation broth to ethyl acetate in step 2) is 1:
1.
7. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... The silica gel column chromatography conditions in step 2) are as follows: the eluent used is CH2Cl2:CH3OH, and the gradient is 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:
1.
8. The method for preparing the compound of formula (I) as described in claim 2, characterized in that... In step 3), the high-performance liquid chromatography (HPLC) separation and purification column is an Agilent Pursuit column. A C18 column (21.2 × 250 mm, 10 μm) was used with a detection wavelength of 210 nm. A gradient elution system of 10%–100% (v / v) methanol-water + 0.05% TFA was used at 10 mL / min for 40 min, collecting the eluent for 31–33 min. The eluent was then evaporated to dryness to obtain a mixed fraction. This fraction was further separated using the same instrument at a detection wavelength of 210 nm. A gradient elution system of 55%–65% (v / v) methanol-water + 0.05% TFA was used at 10 mL / min for 100 min, collecting the eluent for 19–23 min. The eluent was then evaporated to dryness to obtain a mixed fraction. This fraction was further separated using the same instrument at a detection wavelength of 210 nm. An isocratic elution system of 30% (v / v) acetonitrile-water + 0.05% TFA was used at 10 mL / min for 50 min, collecting the eluent for 17.4–20 min.
9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a compound of formula (I) as claimed in claim 1, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.
10. The use of the compound represented by formula (I) as claimed in claim 1, its tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or the use of the pharmaceutical composition as claimed in claim 9 in the preparation of an antibiotic medicament.