A lipopolysaccharide compound and its preparation method and application

Abstract

The present invention discloses a lipopolysaccharide compound and a preparation method and application thereof. The lipopolysaccharide compound is separated from the fermentation product of Pseudomonas aeruginosa J025. The lipopolysaccharide compound of the scheme of the present invention exhibits good antibacterial activity against fluconazole-resistant strains of Pseudomonas aeruginosa. The lipopolysaccharide compound can be used to prepare antibacterial drugs and has antibacterial clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and in particular relates to a lipopolysaccharide compound and a preparation method and application thereof. Background Art

[0002] Wikstroemia nutans Champ.ex Benth. is a species of the genus Wikstroemia in the family Thymelaeaceae. It is widely distributed in tropical and subtropical areas in southern my country. It is a commonly used Chinese herbal medicine. It is cold and bitter in nature. The whole plant is poisonous. It is used in folk medicine to dispel wind, disperse blood, and relieve pain. Ethnomedicine shows that the plant has certain antibacterial and anti-inflammatory activities, but so far, there has been no research report on the endophytic fungi of Wikstroemia nutans and its secondary metabolites. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a composition.

[0004] The invention also provides a preparation method of the composition.

[0005] The invention also provides a preparation method of the lipopolysaccharide compound.

[0006] The present invention also provides the application of the composition.

[0007] In one aspect of the present invention, a composition is provided, comprising at least one lipopolysaccharide compound having the following structural formula (I)-(IV):

[0008]

[0009]

[0010] According to the second aspect of the present invention, a method for preparing the above-mentioned composition is proposed, the method comprising the following steps: directly fermenting Pestalotiopsis sp.J025 to obtain it, wherein Pestalotiopsis sp.J025 was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 22, 2022, with a collection number of GDM CC NO:62822.

[0011] According to the third aspect of the present invention, a method for preparing a lipopolysaccharide compound is proposed, which comprises the following steps: directly fermenting Pestalotiopsis sp. J025 to obtain a fermentation product; extracting the fermentation product to obtain an extract; and separating and purifying the extract.

[0012] In some embodiments of the present invention, the method comprises the following steps: fermenting the above-mentioned Polytrichomonas spp. J025 to obtain a fermentation product; extracting the fermentation product to obtain an extract; and separating and purifying the extract.

[0013] In some embodiments of the present invention, the fermentation culture conditions are fermentation at a rotation speed of 100-150 r / min and 25-35° C. for 6-15 days.

[0014] In some embodiments of the present invention, the fermentation time is 8 to 12 days.

[0015] In some embodiments of the present invention, the culture medium used in the fermentation includes a culture medium suitable for fungal culture.

[0016] In some embodiments of the present invention, the fermentation includes a bacterial activation step, and the activation adopts a solid PDA medium.

[0017] In some embodiments of the present invention, the culture medium used in the fermentation includes a culture medium suitable for fungal culture.

[0018] In some embodiments of the present invention, the liquid culture medium used in the fermentation is PDB liquid culture medium.

[0019] In some embodiments of the present invention, the extraction step includes solid-liquid separation of the fermentation product, collecting the liquid phase, adding the liquid phase to the organic phase for extraction, collecting the extract, and removing the organic phase from the extract to obtain the extract.

[0020] In some embodiments of the present invention, the organic phase is ethyl acetate, dichloromethane or chloroform.

[0021] In some embodiments of the present invention, solid-liquid separation is performed by filtration.

[0022] In some embodiments of the present invention, the added volume ratio of the liquid phase to the organic phase used for extraction is 1:1.

[0023] In some embodiments of the present invention, the method of removing the organic phase is concentration under reduced pressure.

[0024] In some embodiments of the present invention, the separation and purification comprises the steps of subjecting the extract to gel resin and preparative liquid chromatography separation and purification in sequence.

[0025] In some embodiments of the present invention, the step of separation and purification of the gel resin includes adding the extract to the gel resin, eluting with a first eluent to obtain a crude extract, separating the crude extract using thin layer chromatography technology, and collecting to obtain a first component A and a first component B.

[0026] In some embodiments of the present invention, the first eluent is a solution of dichloromethane and methanol in a volume ratio of (40-60): (60-40).

[0027] In some embodiments of the present invention, the first eluent is a solution of dichloromethane and methanol in a volume ratio of 1:1.

[0028] In some embodiments of the present invention, the first component A is separated by preparative liquid chromatography using a second eluent to obtain a second component C with a retention time of 7.5-8.5 min and a second component D with a retention time of 14-16 min.

[0029] In some embodiments of the present invention, the first component A is separated by preparative liquid chromatography using a second eluent to obtain a second component C with a retention time of 8.0 min and a second component D with a retention time of 15 min.

[0030] In some embodiments of the present invention, the second eluent is a 50-100% methanol aqueous solution.

[0031] In some embodiments of the present invention, the second eluent further comprises 0.05-0.5% of acid.

[0032] In some embodiments of the present invention, the second component C is separated by preparative liquid chromatography using a third eluent to obtain a compound with a structure shown in formula (III) and a compound with a structure shown in formula (IV).

[0033] In some embodiments of the present invention, the third eluent is a 70-90% methanol aqueous solution.

[0034] In some embodiments of the present invention, the second component D is separated by preparative liquid chromatography using a fourth eluent to obtain a compound having a structure as shown in formula (I).

[0035] In some embodiments of the present invention, the fourth eluent is 80-95% methanol in water.

[0036] In some embodiments of the present invention, the first component B is separated by preparative liquid chromatography using a fifth eluent to obtain a third component.

[0037] In some embodiments of the present invention, the fifth eluent is a 50-100% methanol aqueous solution.

[0038] In some embodiments of the present invention, the fifth eluent further comprises 0.05-0.5% of acid.

[0039] In some embodiments of the present invention, the third component is separated by preparative liquid chromatography using a sixth eluent to obtain a compound having a structure as shown in formula (II).

[0040] In some embodiments of the present invention, the sixth eluent is a 40-50% methanol aqueous solution.

[0041] In the fourth aspect of the present invention, the use of the above-mentioned Polytrichomonas spp. J025, the product or the above-mentioned lipopolysaccharide compound in the preparation of antibacterial drugs is proposed.

[0042] In some embodiments of the present invention, the antibacterial drug is used to inhibit Candida albicans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Staphylococcus albus, Micrococcus luteus, Bacillus cereus or Salmonella, etc.

[0043] In some embodiments of the present invention, the Candida albicans includes a fluconazole-resistant strain of Candida albicans.

[0044] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention isolates new spirocyclic lipopolysaccharide compounds from Pestalotiopsis sp. J025, and the new compounds exhibit good antibacterial activity against fluconazole-resistant strains of Pseudomonas aeruginosa. The MIC value is between 0.5 and 4 μM, and the compounds can be used to prepare antibacterial drugs. The compounds have the potential for clinical application in antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0046] Figure 1 This is a morphological diagram of the bacteria in Example 1 of the present invention;

[0047] Figure 2 is the HR-ESI-MS spectrum of the lipopolysaccharide compound with the structure shown in Formula I in Example 2 of the present invention;

[0048] Figure 3 The lipopolysaccharide compound of the structure shown in Formula I in Example 2 of the present invention 1 H-NMR spectrum;

[0049] Figure 4 The lipopolysaccharide compound of the structure shown in Formula I in Example 2 of the present invention 13 C-NMR spectrum;

[0050] Figure 5 is the HR-ESI-MS spectrum of the lipopolysaccharide compound with the structure represented by Formula II in Example 2 of the present invention;

[0051] Figure 6The lipopolysaccharide compound of formula II in Example 2 of the present invention 1 H-NMR spectrum;

[0052] Figure 7 The lipopolysaccharide compound of formula II in Example 2 of the present invention 13 C-NMR spectrum;

[0053] Figure 8 is the HR-ESI-MS spectrum of the lipopolysaccharide compound with the structure represented by Formula III in Example 2 of the present invention;

[0054] Fig. 9 The lipopolysaccharide compound of formula III in Example 2 of the present invention 1 H-NMR spectrum;

[0055] Fig.10 The lipopolysaccharide compound of formula III in Example 2 of the present invention 13 C-NMR spectrum;

[0056] Fig.11 is the HR-ESI-MS spectrum of the lipopolysaccharide compound with the structure represented by Formula IV in Example 2 of the present invention;

[0057] Fig.12 The lipopolysaccharide compound of formula IV in Example 2 of the present invention 1 H-NMR spectrum;

[0058] Fig.13 The lipopolysaccharide compound of formula IV in Example 2 of the present invention 13 C-NMR spectrum. DETAILED DESCRIPTION

[0059] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0060] Example 1 Obtaining Pestalotiopsis sp. J025

[0061] 1. Isolation of Pestalotiopsis sp. J025

[0062] The specific method for isolating the Pestalotiopsis sp.J025 strain is as follows: After washing the fresh rhizomes of the fine-axis wormwood, soak them in a 70% ethanol solution for 1 minute, then rinse them with sterile water 3 times, and dry the water with sterile paper. Use sterilized scissors to cut the rhizomes of the fine-axis wormwood into small pieces and place them on the PDA culture medium, and culture them in a constant temperature water-insulated incubator at 30°C for 5-10 days. Regularly observe the growth of the culture medium. When the hyphae grown from the plant tissue on the plate are observed to stretch around, transfer the hyphae of each colony to a new PDA culture medium for streaking culture. After 3-5 days, observe whether the hyphae are colonies of a single morphology, and further isolate and identify the hyphae. After isolating a single colony, transfer the PDA culture medium with hyphae to a cryovial containing 1mL20% glycerol and store it at -80°C. And it was identified as the Pestalotiopsis sp.J025 by ITS sequence.

[0063] 2. Identification of Pestalotiopsis sp. J025 strain

[0064] (1) Culture characteristics, microscopic examination and morphological features

[0065] The isolated Pestalotiopsis sp. J025 strain was cultured on PDA medium. Figure 1 shown.

[0066] (2) 16s rDNA gene sequence identification

[0067] Pestalotiopsis sp.J025 was identified by ITS sequence, and the strain ITS sequence was: TGAGTTTCTACT (SEQ ID NO.1).

[0068] By submitting the ITS sequence amplification data of the screened fungi to GenBank and using NCBI for homology comparison, similar sequence information was obtained, and finally Pestalotiopsis sp.J025 was determined to be Pseudomonas aeruginosa.

[0069] Based on the above identification results, it was determined that the Pestalotiopsis sp.J025 strain was a Pseudomonas aeruginosa, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short, address: Guangdong Provincial Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on September 22, 2022, with the deposit number GDMCC NO:62822.

[0070] Example 2 Isolation of Compounds

[0071] This example prepares a lipopolysaccharide compound, which is isolated from the fermentation broth of Pestalotiopsis sp. J025. The specific process is as follows:

[0072] (1) Pestalotiopsis sp. J025 was inoculated into PDA solid culture medium and cultured at 30°C for 3 to 7 days before use for fermentation inoculation.

[0073] (2) Fermentation culture: 1.5 L of PDB liquid culture medium was added to each 2 L × 16 conical flask, sterilized and cooled to room temperature, and then inoculated in a clean bench. After the inoculation, the conical flask was placed in a constant temperature shaker (30° C., 150 rpm) and cultured for 10 days to obtain a fermentation broth.

[0074] (3) Filtering the fermentation broth obtained in step (2), adding ethyl acetate to the filtrate for extraction, with an extraction volume ratio of 1:1, repeating the extraction three times, and concentrating the ethyl acetate extract to obtain an organic extract of the secondary metabolites of the endophytic fungus J025 of the fine-spined flower.

[0075] (4) The obtained extract was separated by LH-20 dextran gel, and the mobile phase was dichloromethane:methanol = 1:1 (v:v). After separation by thin layer chromatography, the crude extract was divided into 5 sections: F1-F5. Further separation was carried out by preparative liquid chromatography column Waters XBridge Prep C18 (250×19mm, 5μm), and the mobile phase was methanol-water. F2 component 350mg, the mobile phase was 50–100% (MeOH-H 2 O+0.1% COOH) in a ratio gradient elution to obtain components F2-3 (retention time 8.0 min) and F2-5 (retention time 15.0 min). After further purification by preparative liquid phase, F2-3 was purified by 85% MeOH-H 2 O isocratic elution to obtain 3.0 mg of a monomer compound having a structure represented by Formula III (retention time 9.3 min) and 3.0 mg of a monomer compound having a structure represented by Formula IV; F2-5 was purified by 90% MeOH-H 2 O isocratic elution to obtain 5.0 mg of the monomer compound having the structure shown in Formula I (retention time 8.5 min). F3 component 1.4 g, mobile phase 50-100% (MeOH-H 2 O+0.1% COOH) in a ratio gradient elution to obtain components F3-1 (retention time 10.8 min) and F3-2 (retention time 14.1 min). 2 O isocratic elution was performed to obtain 3.0 mg of a monomer compound having the structure represented by Formula II (retention time 7.6 min).

[0076] (4) Detection of compounds

[0077] The proton nuclear magnetic resonance (NMR) spectrometer was used to measure the 1 H-NMR), 13 C NMR ( 13 C-NMR) was used to detect the lipopolysaccharide compound of formula I, and the test results were as follows Figure 1-3 As shown. Figure 1 As can be seen from the figure, the high resolution mass spectrometry (HR-ESIMS) shows that the quasi-molecular ion peak of the molecule is 831.3802 [MH] - m / z, calculated value: 831.3808[MH] - m / z, the molecular formula is estimated to be C 43 H 60 O 16 There are 14 unsaturated carbons and 43 carbon signals in the structure, including a pair of overlapping signals (δ C 73.9). It includes 7 quaternary carbons (including two carbonyl carbons), 12 methylene signals, 22 methine signals, and two methyl signals.

[0078]

[0079] The proton nuclear magnetic resonance (NMR) spectrometer was used to measure the 1 H-NMR), 13 C NMR ( 13 C-NMR) was used to detect the lipopolysaccharide compound of formula II, and the test results were as follows Figure 4-6 As shown in the figure, it can be seen that the high-resolution mass spectrometry (HR-ESIMS) shows that the quasi-molecular ion peak of the molecule is 651.2288 [MH] - m / z, calculated value: 651.2294 [MH] - m / z, the molecular formula is estimated to be C 31 H 40 O 15 The lipopolysaccharide compound of formula II has 12 unsaturations, 31 carbon signals, including 6 quaternary carbons (including 1 carbonyl carbon), 4 methylene signals, 20 methine signals, and 1 methyl signal.

[0080]

[0081] Proton nuclear magnetic resonance (NMR) was measured using HR-ESIM and 600 MHz NMR spectrometer. 1 H-NMR), 13 C NMR ( 13 C-NMR) was used to detect the lipopolysaccharide compound of formula III, and the test results were as follows Figure 7-9 As shown in the figure, it can be seen that the quasi-molecular ion peak given by HR-ESIM is 803.3495 [MH] - m / z, calculated value is m / z803.3496[MH] - , its molecular formula is estimated to be C 41 H 56 O 16 There are 14 unsaturated carbons and 41 carbon signals in the structure, including a pair of overlapping signals (δ C 73.9), including 7 quaternary carbons (including two carbonyl carbons), 10 methylene signals, 22 methine signals, and two methyl signals.

[0082]

[0083] Proton nuclear magnetic resonance (NMR) was measured using HR-ESIM and 600 MHz NMR spectrometer. 1 H-NMR), 13 C NMR ( 13 C-NMR) was used to detect the lipopolysaccharide compound of formula IV, and the test results were as follows Figure 10-12 As shown in the figure, it can be seen that the quasi-molecular ion peak given by HR-ESIM is 847.3756 [MH] - m / z, calculated value: 847.3758 [MH] - m / z, the molecular formula is estimated to be C 43 H 59 O 18 , with 14 unsaturations. There are 41 carbon signals in the structure, including 7 quaternary carbons (including two carbonyl carbons), 12 methylene signals, 22 methine signals, and two methyl signals.

[0084]

[0085] Example 3 Application of lipopolysaccharide compounds in inhibiting the proliferation of fluconazole-resistant strains of Bacillus albicans

[0086] This example provides the use of the four lipopolysaccharide compounds prepared in Example 2 in inhibiting the proliferation of fluconazole-resistant strains of Bacillus thuringiensis. The specific experimental steps are as follows:

[0087] Take a 96-well culture plate, dilute the samples (the four lipopolysaccharide compounds prepared in Example 2) to a concentration of 100 μM; add the fluconazole-resistant strain of Bacillus albicans to each well, and the final concentration is 1×10 5 CFU / mL; culture at 37℃ for 24h, and measure the OD value at 625nm with a microplate reader. The experiment also set up a blank control of the culture medium (replacing the lipopolysaccharide compound with an equal amount of sterile water), a control of fluconazole-resistant strains of Alpinia aviculare, and amphotericin B control (positive control).

[0088] MIC 50 (50% minimum inhibitory concentration) was calculated according to the Reed & Muench method.

[0089] Table 1

[0090]

[0091] The experimental results are shown in Table 1. It can be seen from the table that compounds Ⅰ and Ⅲ have strong inhibitory effects on fluconazole-resistant strains of P. albicans. Further testing of the MIC of compounds Ⅰ and Ⅲ 50 , experimental data show that the MIC of compound Ⅰ 50 =0.566±0.003μM, MIC of compound III 50 =3.795±0.022 μM. Therefore, among the four lipopolysaccharide compounds prepared by the scheme of the present invention, compound I has the best effect.

[0092] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A composition, characterized in that The composition comprises at least one lipopolysaccharide compound having the structural formula (I)-(IV) as follows:

2. A method for preparing the composition according to claim 1, characterized in that: The method comprises the following steps: directly fermenting Pestalotiopsis sp. J025, wherein Pestalotiopsis sp. J025 was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 28, 2022, with a deposit number of GDMCC NO: 62822.

3. A method for preparing a lipopolysaccharide compound as claimed in claim 1, characterized in that: The method comprises the following steps: directly fermenting Pestalotiopsis sp. J025 to obtain a fermentation product; extracting the fermentation product to obtain an extract; and separating and purifying the extract.

4. The preparation method according to claim 3, characterized in that: The fermentation culture conditions are as follows: fermentation at a rotation speed of 100-150 r / min and a temperature of 25-35° C. for 6-15 days.

5. The preparation method according to claim 3, characterized in that: The culture medium used in the fermentation includes a culture medium suitable for fungal culture.

6. The preparation method according to claim 5, characterized in that: The culture medium used in the fermentation is PDB liquid culture medium.

7. The preparation method according to claim 3, characterized in that: The extraction step comprises separating the fermentation product into solid and liquid, collecting the liquid phase, adding the liquid phase into the organic phase for extraction, collecting the extract, and removing the organic phase from the extract to obtain the extract.

8. The preparation method according to claim 7, characterized in that: The organic phase is ethyl acetate, dichloromethane or chloroform.

9. The preparation method according to claim 3, characterized in that: The separation and purification comprises the steps of subjecting the extract to gel resin and preparative liquid chromatography separation and purification in sequence.

10. The preparation method according to claim 9, characterized in that: The separation and purification step comprises adding the extract to a gel resin, eluting with a first eluent, obtaining a crude extract after eluting with the first eluent, separating the crude extract with a thin layer chromatography technique, and collecting to obtain a first component A and a first component B; subjecting the first component A to preparative liquid chromatography with a second eluent to elution to obtain a second component C with a retention time of 7.5-8.5 min and a second component D with a retention time of 14-16 min; subjecting the second component C to preparative liquid chromatography with a third eluent to elution to obtain a compound with a structure shown in formula (III) and a compound with a structure shown in formula (IV); The second component D is separated by preparative liquid chromatography using a fourth eluent to obtain a compound with a structure shown in formula (I); the first component B is separated by preparative liquid chromatography using a fifth eluent to obtain a third component; the third component is separated by preparative liquid chromatography using a sixth eluent to obtain a compound with a structure shown in formula (II).

11. The preparation method according to claim 10, characterized in that: The first eluent is a dichloromethane and methanol solution in a volume ratio of (40-60): (60-40), the second eluent is a 50-100% methanol aqueous solution, the third eluent is a 70%-90% methanol aqueous solution, the fourth eluent is an 80-95% methanol aqueous solution, the fifth eluent is a 50-100% methanol aqueous solution, and the sixth eluent is a 40-50% methanol aqueous solution.

12. Use of the composition according to claim 1 in preparing an antibacterial product.

13. The use according to claim 12, characterized in that: The product is food, medicine or daily chemical product.

14. The use according to claim 12, characterized in that: The antibacterial product is used for inhibiting Candida albicans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Staphylococcus albus, Micrococcus luteus, Bacillus cereus or Salmonella.

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