A glycolipid compound, and a preparation method and application thereof

Abstract

The application discloses a glycolipid compound and a preparation method and application thereof, and belongs to the technical fields of marine organisms and medicines. Pseudogymnoascus The glycolipid compound comprises a compound shown in formula I, stereoisomers of the compound or pharmaceutically acceptable salts of the compound. The compound shown in formula I is extracted and separated from a strain of sp.OUCMDZ-4032 from an Antarctic marine sediment source, and experiments prove that the compound in combination with antibiotics (such as ciprofloxacin) greatly enhances the bacteriostatic activity of the antibiotics on gram-negative bacteria, and can be used for developing an application of an antibiotic auxiliary agent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of marine biology and medicine, and particularly relates to a glycolipid compound extracted and separated from a microbial fermentation culture from Antarctic marine sediments and application of the glycolipid compound as a biological surfactant and in combination with ciprofloxacin to enhance the activity of the ciprofloxacin in inhibiting gram-negative bacteria. BACKGROUND

[0002] The Antarctic region is long-term exposed to low temperature, drought (cold desert), frequent freeze-thaw cycles, strong wind, high sublimation and evaporation, lack of nutrients and strong ultraviolet radiation. Therefore, microorganisms that have long-term survival in such an extreme environment may have evolved various strategies in metabolism and physiology to adapt to such a harsh environment, forming special physiological metabolic characteristics and chemical defense mechanisms. In particular, psychrophilic fungi have become an important source for discovering new bioactive secondary metabolites. Therefore, the research on psychrophilic fungi and bioactive secondary metabolites has important significance in the fields of biotechnology and pharmaceuticals.

[0003] Multidrug resistance (MDR) is one of the most serious global public health threats in this century. Among them, most of the antibiotic-resistant bacteria are gram-negative pathogens. Due to its unique structure, gram-negative bacteria have stronger drug resistance than gram-positive bacteria, causing serious drug-resistant infections worldwide. However, the development of new antibiotics to overcome drug resistance has been stagnant for decades, and in order to solve the emergence of antibiotic resistance and multidrug-resistant strains, one of the current methods is to use biological surfactants in combination with antibiotics to improve their efficacy. In recent years, nanoparticles have attracted attention as synergistic antibacterial antibiotic adjuvants. However, there are relatively few related natural products. Therefore, it is desirable to develop new adjuvants to restore the drug sensitivity of super drug-resistant bacteria.

[0004] Glycolipid compounds are composed of carbohydrate molecules and fatty acids, and are characterized by high structural diversity and the ability to reduce surface tension and interfacial tension at the interface, thus having biological surfactant activity. Recently, there has been an increase in the attention paid to biological surfactants because of their unique properties, such as low toxicity, high biodegradability, high foaming capacity, antibacterial activity, and selectivity and specificity at extreme temperatures, pH values, and salinity. These properties are considered to be more advantageous than other chemical surfactants, and therefore glycolipid compounds are considered to be a good alternative that can be applied in the environment, oil, food, and pharmaceutical industries, such as glycolipids as food additives and food preservatives, and the development of antibiotic adjuvants for clinical antibacterial applications. SUMMARY

[0005] A first object of the present application is to provide a glycolipid compound isolated from a microorganism derived from Antarctic marine sediments, including but not limited to the glycolipid compound shown in Formula I and all stereoisomers thereof or a pharmaceutically acceptable salt thereof:

[0006] (I).

[0007] The positions of Formula I compounds that can change in configuration include C-6 and C-12 stereoisomers, cis-trans isomers of double bonds, and the configuration of the sugar.

[0008] The glycolipid compound shown in Formula I is extracted, isolated and purified from a strain of sp. OUCMDZ-4032 derived from Antarctic marine sediments. Pseudogymnoascus

[0009] The strain of sp. Pseudogymnoascus OUCMDZ-4032 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 7, 2021, and has the accession number CGMCC No. 21946. A second object of the present application is to provide a preparation method for the glycolipid compound shown in Formula I, which is extracted and isolated from the fermentation culture of the strain of sp. OUCMDZ-4032.

[0010] Pseudogymnoascus The specific preparation method for the compound shown in Formula I includes the following steps:

[0011] (1) Take the strain of sp. OUCMDZ-4032 for fermentation culture, break the culture, extract with ethyl acetate, filter the extract, and then perform vacuum concentration extraction to obtain a total crude extract;

[0012] (2) Perform normal pressure silica gel column chromatography on the obtained total crude extract, use petroleum ether / dichloromethane and dichloromethane / methanol as eluents for gradient elution, vacuum concentrate the eluted components, and then perform LC-MS analysis to divide them into 12 components Fr. 1-Fr. 12; Pseudogymnoascus

[0013] (3) Perform pressure reverse phase silica gel column chromatography on component Fr. 10, use methanol / water as eluent, and separate to obtain 15 components Fr. 10-1-Fr. 10-15;

[0014]

[0015] ​​​​(4) purifying components Fr. 10-14 by HPLC to obtain the compound of formula I.

[0016] The elution gradient in step (2) is petroleum ether / dichloromethane=100:0, 50:1, 10:1, 1:1, 0:100; dichloromethane / methanol=100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:100.

[0017] The volume ratio of methanol / water in step (3) is 10:90-0:100.

[0018] A third object of the present application is to provide the use of the compound of formula I in combination with antibiotics (such as ciprofloxacin) to enhance the inhibitory activity of antibiotics against gram-negative bacteria.

[0019] Technical effects of the present application: the glycolipid compound of formula I is obtained by extraction and separation from the fermentation culture of the fungus from the Antarctic marine sediments; the compound in combination with antibiotics can greatly enhance the antibacterial activity of antibiotics against gram-negative bacteria, and can be used for the development of antibiotic adjuvants. The present application uses 96-well plate method to determine the minimal inhibitory concentration (MIC) of the compound of formula I on 5 strains of bacteria, on this basis, the checkerboard method is used to evaluate the antibacterial activity of the compound of formula I in combination with ciprofloxacin on 3 strains of gram-negative bacteria, and the results show that: 32 μg / mL of the compound of formula I in combination with ciprofloxacin can effectively enhance the antibacterial activity of ciprofloxacin, and can reduce the MIC of ciprofloxacin against Pseudomonas aeruginosa, Escherichia coli and Paratyphi by 1024, 256 and 256 times, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is Pseudogymnoascus Strain morphology chart of sp. OUCMDZ-4032.

[0021] Figure 2 is the nuclear magnetic resonance hydrogen spectrum chart (DMSO- d 6 , 400 MHz) of the compound of formula I.

[0022] Figure 3 is the nuclear magnetic resonance carbon spectrum chart (DMSO- d 6 , 100 MHz) of the compound of formula I.

[0023] Figure 4 is the LR-ESI-MS analysis chart (positive ion mode, [M+H] + ) of the compound of formula I.

[0024] Figure 5 The figure shows the experimental results of water droplet collapse of compound I. Detailed Implementation

[0025] The following specific embodiments are further illustrations of the present invention, but not limitations thereof.

[0026] Unless otherwise specified, the experimental methods in the following specific embodiments are conventional methods; unless otherwise specified, the experimental reagents used in the following embodiments are commercially available products.

[0027] Example 1: Isolation, purification and identification of strains

[0028] 1. Sample Collection

[0029] The samples were collected from Antarctic marine sediments. After collection, they were immediately placed in sterile sealed bags, frozen, and transported to the laboratory, where they were stored at -20°C.

[0030] 2. Sample processing

[0031] The gradient dilution method was used to separate marine sediment samples. 1 g of sample was weighed in a sterile environment, firstly surface sterilized with 75% ethanol, then rinsed repeatedly with sterile seawater, and finally ground into a homogenate in a mortar. The supernatant was then diluted with sterile seawater to three gradients: 0.1 g / mL, 0.01 g / mL, and 0.001 g / mL.

[0032] 3. Isolation and purification of strains

[0033] Microorganisms were isolated and cultured using PDA medium (natural pH). First, 0.2 mL of the medium was aspirated using a sterile pipette.

[0034] Sample solutions of different dilution ratios were added dropwise to PDA solid medium (200g cooked potato, 20g glucose, 15-20g agar, natural pH per liter of water), and then spread evenly using a sterile spreader. After the bacterial solution had penetrated the medium, it was placed in an incubator at 28 ℃ and inverted for 4-7 days. The strain was purified using the streak plating method. The growth of the bacterial strain on the plate was observed. When a single colony appeared, streak plating was performed. The specific method for streak plating is as follows: the inoculation loop was ignited with the outer flame of an alcohol lamp. After complete cooling, a single colony was scraped from the plate, and the inoculation loop was used to streak the agar plate in a zigzag pattern. This operation should be performed in a sterile area near the alcohol lamp flame. After inoculation, the strain number, name, and date were noted on the bottom of the petri dish, and then it was placed in an incubator at 28 ℃ and inverted for further incubation. If the strain was not purified by the first streak, a second streak was performed until the strain was completely purified.

[0035] 4. Preservation method of strains:

[0036] Preservation on slant: the purified strain was inoculated on PDA medium by zigzag line method, and then stored in a refrigerator at 4 ℃ after the strain grew maturely, and three parallels were set for each strain.

[0037] Freeze preservation in glycerol tube: the purified strain was freeze preserved by using PDA liquid medium (20% glycerol was added) as freeze storage solution. 1 mL of the freeze storage solution was taken in a freeze storage tube and sterilized. After cooling, an appropriate amount of mycelium of the strain on the agar plate was scraped into the freeze storage tube, and then stored in a refrigerator at -80 ℃.

[0038] 5. Identification of strains

[0039] The strains were identified by combining morphological and molecular biological methods. The external phenotypic characteristics of the strains were photographed and archived, and the morphological photos of the strains are shown in FIG. 1. The strain has the following morphological characteristics: the aerial mycelium is short, and the mycelium is white; the micro-morphology of the insert under a 400-fold microscope shows that the aerial mycelium is broken into spores, and the spores are spherical and smooth. The ITS DNA sequence of the strain was amplified by using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The total DNA of the strain was extracted as a template, and then subjected to PCR amplification. The length of the ITS DNA sequence of the strain is 521 bp (as shown in the sequence table SEQ No. 1). According to the ITS sequence comparison, the strain is identified as Aspergillus sp. OUCMDZ-4032, and the preservation number is CGMCC No. 21946. Figure 1 Pseudogymnoascus

[0040] Example 2: Separation, purification and structural identification of the compound of formula I

[0041] 1. Preparation of total crude extract

[0042] An appropriate amount of mycelium of the target strain was inoculated into fungal 3# culture medium, and then fermented in a shaker at 16 ℃ and 180 r / min for 5 days to obtain a seed liquid of the strain. The seed liquid was inoculated into a fermentation medium, i.e., rice solid culture medium (80 g of rice + 120 mL of seawater), at a proportion of 5% by volume, and then fermented. A total of 200 bags were fermented. The fermentation was carried out at 16 ℃ in a constant-temperature shaker, and then the strain culture was obtained after fermentation for 30 days.

[0043] ​​The fungus 3# culture medium comprises: 200 grams of boiled potatoes, 10 grams of monosodium glutamate, 20 grams of mannitol, 20 grams of maltose, 10 grams of glucose, 0.3 grams of MgSO4·7H2O, 3 grams of yeast extract, sea water, natural pH, per liter of water.

[0044] The fermentation broth was centrifuged at 4,000 rpm for 10 min, and the supernatant was collected and concentrated to 1 mL by rotary evaporation. Pseudogymnoascus sp. The culture of OUCMDZ-4032 was broken, and 3 times the volume of ethyl acetate was added to extract the fermentation broth. The extraction was repeated three times, each time for 0.5 h of ultrasonic extraction. The ethyl acetate extract was filtered and vacuum concentrated to obtain about 75 g of total crude oil.

[0045] 2. Compound separation and purification

[0046] The total crude oil obtained by fermentation of the strain was separated by normal phase silica gel reduced pressure column chromatography. The elution gradient was set as petroleum ether / dichloromethane 100:0, 50:1, 10:1, 1:1, 0:100; dichloromethane / methanol 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:100, respectively. The elution components were vacuum concentrated and analyzed by LC-MS to obtain 12 components Fr.1-12.

[0047] Component Fr. 10 was separated by reversed phase silica gel pressure column chromatography (methanol: water 10:90-0:100) to obtain 15 components (Fr. 10-1-Fr. 10-15). Component Fr. 10-14 was purified by HPLC (25 min ACN-H2O, 0 min: 10% ACN+90% H2O, 15 min 100% ACN, 20 min-25 min 10% ACN+90% H2O) to obtain a compound of formula I (18.6 mg, t R = 16.5 min).

[0048] 3. Compound structure identification

[0049] The structure of the compound separated and purified by the present application was determined by nuclear magnetic resonance spectroscopy (NMR), ultraviolet spectroscopy (UV), high resolution mass spectrometry (HRESIMS), and other spectroscopic techniques, and was shown as formula I.

[0050] The compound of formula I is characterized by a yellow-brown oily solid; α ] 28 D +89.28( c 1, MeOH), ECD (0.80 mM, MeOH) λmax (Δ ε ) 231 (+15.56), 231(-18.38) nm; UV (MeOH) λ max (log ε ) 230(2.83), 271 (3.13) nm; IR (KBr) ν max 3365, 2951, 2930, 2869, 1681, 1556,1456, 1383,1205, 1147, 1078, 993 cm -1 ; 1 H and 13 CNMR (DMSO, dimethyl sulfoxide -d 6 ) are shown in Table 1; positive high resolution mass spectrum HRESIMS: m / z 629.3515 [M + H] + (C 32 H 53 O 12 calcd 629.3532), molecular formula is C 32 H 52 O 12 . See Figures 2-4 .

[0051] Table 1 Nuclear magnetic resonance data table of compound of formula I 1 H 400 MHz, 13 C 100 MHz, NMR, DMSO -d 6 , TMS, δ ppm)

[0052]

[0053] Example 3: Surface activity test of compound of formula I

[0054] The compound of Formula I consists of a hydrophobic fatty chain and a hydrophilic glycosyl group, suggesting that it may possess good surface activity. A droplet collapse assay was used to qualitatively test the surface activity of Compound I. Ultrapure water was stained with 0.01% crystal violet, and 10 μL droplets were prepared and placed on a paraffin-sealed film. Stained water and water stained with 10% MeOH were used as negative controls. These droplets had only a small contact point with the hydrophobic surface and formed a spherical shape. Stained water containing 10% MeOH and 0.1% Tween 20 was used as a positive control. Due to its low water tension, the droplets collapsed and diffused onto the hydrophobic surface. Compound I was tested at a concentration of 1 mM in stained water containing 10% MeOH; the results are shown below. Figure 5 The degree of collapse of the droplets after the addition of compound I was comparable to, or even slightly greater than, that of the positive control group. This indicates that compound I possesses good biosurface activity.

[0055] Example 4: Evaluation of the antibacterial activity of compound I

[0056] 1. Evaluation of antibacterial activity

[0057] The effects of Formula I compound on five strains of Pseudomonas aeruginosa were initially evaluated using the 96-well plate method. Pseudomonas aeruginosa ATCC10145), Escherichia coli ( Escherichia coli ATCC11775), Salmonella paratyphi ( Salmonella paratyphi ATCC 9150), Bacillus subtilis ( Bacillus subtilis ATCC 6051), Methicillin-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus The antibacterial activity of ATCC43300 was assessed using ciprofloxacin as a positive control and bacterial LB medium as a negative control. The initial concentration of compound I was 64 μg / mL. After incubation, the antibacterial activity was preliminarily determined by visually observing whether the bottom of the 96-well plate was clear. The results of the antibacterial activity are shown in Table 2.

[0058] Table 2 Initial Screening of Antibacterial Activity of Compounds of Formula I

[0059]

[0060] As shown in Table 2, compound I has weak inhibitory activity against Bacillus subtilis (MIC value of 32 μg / mL), but no inhibitory activity against the other four strains (MIC > 64 μg / mL), indicating that compound I itself does not have good antibacterial activity.

[0061] Example 5: Evaluation of the antibacterial activity of compound I in combination with ciprofloxacin

[0062] 1. Checkerboard method for determining bacteriostatic activity

[0063] The checkerboard method was used to evaluate the bacteriostatic activity of the combination of the compound of formula I and ciprofloxacin on 3 strains of gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, and Paratyphi).

[0064] Referring to the CLSI M27-A (yeast broth dilution method for antifungal drug susceptibility testing) scheme, the drug stock solution was diluted with LB liquid medium (0.5% yeast extract, 0.5% NaCl, 1% tryptone) to a 2-fold concentration range of ciprofloxacin 2-0.001953125 μg / mL (Pseudomonas aeruginosa), 0.125-4.8828125×10 -4 μg / mL (Escherichia coli), and 0.25-0.0009765625 μg / mL (Paratyphi). The final concentration of the compound of formula I was 64-1 μg / mL.

[0065] The strains were picked from LB plates and inoculated into LB liquid medium, and the strains were activated at 28°C and 180 rpm for 12 h.

[0066] The operation was performed as follows: 50 μL of the ciprofloxacin dilution was added to columns 1-12 of the 96-well plate in order of high concentration to low concentration; 50 μL of the compound of formula I dilution was added to rows B-G of the 96-well plate in order of low concentration to high concentration. 100 μL of the bacterial suspension was added to each well, and blank controls (200 μL of LB medium) and growth controls (100 μL of LB medium and 100 μL of the bacterial suspension) were additionally added. The 96-well plate was incubated at 28°C for 24 h, and the results were read by visually observing the clear wells. The experiment was repeated 3 times.

[0067] 2. Method for evaluating the effect of the combination of two drugs

[0068] The FICI method was used to evaluate the results, which is a non-parametric model based on the Loewe additivity (LA) theory, and the formula is as follows:

[0069]

[0070] FICl is the fractional bacteriostatic combination index, wherein MIC A and MIC B are the MIC values when the two drugs are used alone, MIC A in combination and MIC B in combinationMIC value of the two drugs in combination. When the MIC value is higher than the highest limit of detection, the FICI is calculated as twice the highest limit concentration. According to Example 3, since the compound of Formula I has no inhibitory activity against the three strains of gram-negative bacteria, the MIC is defined as 128 μg / mL. The FICI method is the most commonly used model for explaining the interaction of antibacterial drugs, but there are still various standards for interpreting the FICI value in different documents. In this application, the standard of "FICI < 0.5 is synergistic, 0.5 < FICI < 4 is no interaction, and FICI > 4 is antagonistic" is used to determine the experimental results.

[0071] FICI: Partial Inhibitory Combination Index is calculated as:

[0072] FICI (Pseudomonas aeruginosa) = MIC A in combination / MIC A +MIC B in combination / MIC B = 0.001953125 / 2 + 32 / 128 = 9.765625 x 10-4+ 0.25 < 0.5, so it is determined that the combination has inhibitory effect.

[0073] FICI (Escherichia coli) = MIC A in combination / MIC A +MIC B in combination / MIC B = 4.8828125 x 10-4 / 0.125 + 32 / 128 = 3.90625 x 10-3+ 0.25 < 0.5, so it is determined that the combination has inhibitory effect.

[0074] FICI (Paratyphi) = MIC A in combination / MIC A +MIC B in combination / MIC B = 0.0009765625 / 0.25 + 32 / 128 = 3.90625 x 10-3+ 0.25 < 0.5, so it is determined that the combination has inhibitory effect.

[0075] The evaluation results are shown in Table 3, and the combination of 32 μg / mL of the compound of Formula I and ciprofloxacin can reduce the MIC of Pseudomonas aeruginosa, Escherichia coli, and Paratyphi by 1024, 256, and 256 times, respectively.

[0076] Table 3 Evaluation of the synergistic antibacterial activity of the combination of the compound of Formula I and ciprofloxacin against three strains of gram-negative bacteria by FICI

[0077]

[0078] The results of this example can lead to the conclusion that 32 μg / mL of the compound of formula I combined with ciprofloxacin can effectively improve the synergistic bacteriostatic effect, and the MIC of Pseudomonas aeruginosa, Escherichia coli and Paratyphi can be reduced by 1024, 256 and 256 times, respectively.

Claims

1. A glycolipid compound as shown in Formula I, or a pharmaceutically acceptable salt thereof: Formula I (Ⅰ)。 2. The method for preparing glycolipid according to claim 1, wherein, The compound of formula I is extracted and separated from a fermentation culture of a strain of sp. OUCMDZ-4032 Pseudogymnoascus sp. OUCMDZ-4032 strain, which was preserved in the China General Microbiological Culture Collection Center on April 7, 2021, with a preservation number of CGMCC No. 21946. Pseudogymnoascus The compound of formula I is extracted and separated from a fermentation culture of a strain of sp. OUCMDZ-4032 Pseudogymnoascus sp. OUCMDZ-4032 strain, which was preserved in the China General Microbiological Culture Collection Center on April 7, 2021, with a preservation number of CGMCC No. 21946. Pseudogymnoascus 3. The method for preparing glycolipid compounds as described in claim 2, characterized in that, comprising the following steps: (1) Take Pseudogymnoascus The strain OUCMDZ-4032 was fermented, the culture was broken, and the culture was extracted with ethyl acetate. After the extract was filtered and vacuum concentrated, a total crude extract was obtained. (2) The obtained total crude extract was subjected to normal silica gel column chromatography under reduced pressure, and gradient elution was performed using petroleum ether / dichloromethane and dichloromethane / methanol as eluents. The eluted components were vacuum concentrated and subjected to LC-MS analysis, and were divided into 12 components Fr.1-Fr.12; (3) Component Fr.10 was subjected to reversed-phase silica gel column chromatography under pressure, and 15 components Fr.10-1-Fr.10-15 were separated using methanol / water as eluents. (4) Component Fr. 10-14 was purified by HPLC to obtain the compound of Formula I.

4. The method for preparing glycolipid according to claim 3, wherein The elution gradient in step (2) was petroleum ether / dichloromethane = 100:0, 50:1, 10:1, 1:1, 0:100; dichloromethane / methanol = 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:100, respectively.

5. The method for preparing glycolipid compounds as described in claim 3, characterized in that, The volume ratio of methanol / water in step (3) was 10:90-0:

100.

6. Use of the glycolipid compound of claim 1, or a pharmaceutically acceptable salt thereof, for preparing a medicament for enhancing the inhibitory activity of ciprofloxacin on Escherichia coli, Pseudomonas aeruginosa, and Paratyphi.

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