Active glycolipid compound and preparation method and application thereof

Abstract

The present application relates to the technical field of microorganism and new medicine and pesticide, and particularly relates to a new glycolipid active compound containing sugar, and further discloses a preparation method and application of the glycolipid active compound containing sugar.The glycolipid active compound containing sugar FW-6-2 has cytotoxic activity on staphylococcus aureus and micrococcus luteus in bacteria through activity testing, and not only a new glycolipid active compound containing sugar is found, but also a lead compound for research and development of a new bacterium inhibiting medicine is provided.

Description

Technical Field

[0001] This invention relates to the fields of microbial and novel pharmaceutical pesticides, specifically to a novel sugar-containing glycerol ester active compound, and further discloses its preparation method and application. Background Technology

[0002] Micromonospora, belonging to the family Micromonosporaceae in the order Actinobacteria, has a wide range of strains, originating from both freshwater lakes and marine environments. According to Talukdar et al., as of 2016, nearly 700 antibiotics originated from metabolites of the genus Micromonospora. Therefore, the genus Micromonospora, a rare actinomycete, represents an important repository of bioactive secondary metabolites (Qi et al., 2020).

[0003] In 2000, Librada M. Canedo et al. discovered a novel spirocyclic macrolide compound, IB-96212, from the marine micromonospora sp. and verified its antibacterial activity, particularly against Micrococcus luteus, where the MIC value reached 0.4 ug / ml. It also exhibited strong cytotoxic activity against various tumor cell lines, especially the P388 cell line. 50 The activity reached 0.1 ng / ml, significantly higher than the reference standards paclitaxel (200 ng / ml) and etoposide (100 ng / ml), and even doxorubicin (20 ng / ml). Furthermore, compared to oligomycin compounds, the IB-96212 class of compounds, in addition to the characteristic spirocyclic structure, possesses an additional sugar ring, L-rhodinose (L-roseose). Currently, only this single sugar ring has been discovered in this class of compounds, and its antibacterial mechanism and biosynthetic mechanism have not yet been reported.

[0004] For example, Chinese patent CN114907367A discloses a method for preparing the natural macrocyclic lactone compound FW-Z using fermentation of *Micromonas marineus*, and verifies that the compound has antifungal properties similar to oligomycins, exhibiting cytotoxic activity against *Aspergillus niger*. Another example is Chinese patent CN115504990A, which discloses a method for preparing the natural sugar-spirocyclic macrocyclic lactone compound FW-5-39 using fermentation of *Micromonas marineus*. This sugar-spirocyclic macrocyclic lactone compound FW-5-39 exhibits antifungal properties similar to oligomycins, and shows cytotoxic activity against *Aspergillus niger* and *Candida albicans*.

[0005] Therefore, the field looks forward to developing more compounds with antibacterial activity, which is of positive significance for the research and development of novel antibiotic drugs. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a novel active glyceroglycolipid compound FW-6-2, which has cytotoxic activity against Staphylococcus aureus and Micrococcus luteus among bacteria;

[0007] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned novel active glyceroglycolipid compound FW-6-2;

[0008] The third technical problem to be solved by the present invention is to provide the use of the above-mentioned novel active glyceroglycolipid compound FW-6-2 in the preparation of novel antibiotic drugs.

[0009] To solve the above-mentioned technical problems, the present invention provides an active glycerol glycolipid compound, characterized in that the compound is designated FW-6-2 and has the structure shown in formula (Ⅰ):

[0010]

[0011] The present invention also discloses a method for preparing the active glycerol glycolipid compound by fermentation, comprising the step of inoculating marine micromonospora strain FIMYZ51 into a suitable fermentation medium for fermentation culture;

[0012] The marine micromonospora strain FIMYZ51, classified as Micromonospora sp., was deposited at the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, with accession number CGMCC No. 24067.

[0013] Specifically, the method for preparing the active glycerol glycolipid compound by fermentation includes the following steps:

[0014] (1) Seed culture: Micromonospora sp. FIMYZ51 preserved on slant was inoculated into liquid seed culture medium and cultured at constant temperature. Seed culture was collected for later use.

[0015] (2) Fermentation broth culture: The seed liquid is transferred to a fermentation medium for constant temperature culture to obtain a fermentation broth containing the desired active glycerol glycolipid compounds.

[0016] Specifically, in the method for preparing the active glycerol glycolipid compound by fermentation, in step (1):

[0017] The seed culture medium comprises: 1-2 wt% soluble starch, 0.3-0.8 wt% glucose, 0.3-0.8 wt% soybean flour, 0.3-0.8 wt% yeast extract, 0.03-0.08 wt% MgSO4·7H2O, 0.03-0.08 wt% NaCl, 0.03-0.08 wt% (NH4)2SO4, 0.05-0.15 wt% CaCO3, and pH 6.0-8.5.

[0018] Preferably, the seed culture medium comprises: 1.5% soluble starch, 0.5% glucose, 0.53% soybean flour, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.05% NaCl, 0.05% (NH4)2SO4, 0.1% CaCO3, and pH 6.0-8.5.

[0019] The constant temperature incubation step is 25-35℃, and the incubation time is 1-3 days.

[0020] Specifically, in the method for preparing the active glycerol glycolipid compound by fermentation, in step (2):

[0021] The fermentation medium comprises: 3-5 wt% soluble starch, 0.3-0.8 wt% glucose, 2-3 wt% corn steep liquor powder, 0.3-0.8 wt% yeast powder, 0.03-0.08 wt% MgSO4·7H2O, 0.03-0.08 wt% K2HPO4, 0.05-0.15 wt% CaCO3, and pH 6.0-8.5.

[0022] Preferably, the fermentation medium comprises: 4% soluble starch, 0.5% glucose, 2.5% corn steep liquor powder, 0.5% soybean oil, 0.5% yeast powder, 0.05% MgSO4·7H2O, 0.05% K2HPO4, 0.1% CaCO3, and pH 6.0-8.5.

[0023] The constant temperature culture step is conducted at 25-35℃ for 3-6 days.

[0024] Specifically, the method for preparing the active glyceroglycolipid compound by fermentation further includes the steps of extracting and purifying the active glyceroglycolipid compound, specifically including:

[0025] (3) Extraction: The collected fermentation broth was added to XAD-16 resin for adsorption, filtered, and the filter cake was desorbed, recovered, and concentrated to obtain crude extract A;

[0026] (4) Purification: The crude extract A was subjected to C18 reversed-phase column chromatography with a gradient elution of methanol:water. The eluent was collected in fractions by high performance liquid chromatography. 40-80% of the eluent fractions were collected and subjected to a preparative C18 reversed-phase high performance liquid chromatography gradient elution. 50% of the fractions were collected to obtain the desired active glycerol glycolipid compound FW6-2 in pure form.

[0027] Specifically, in the method for preparing the active glycerol glycolipid compound by fermentation, in step (3), the volume ratio of the XAD-16 resin to the fermentation broth is 1:10-1:30.

[0028] The desorption step is 100% ethanol desorption.

[0029] Specifically, in the method for preparing the active glycerol glycolipid compound by fermentation, in step (4):

[0030] In the C18 reversed-phase column chromatography step, gradient elution is performed by controlling the volume ratio of methanol to water to be 30%-100%.

[0031] In the preparative C18 reversed-phase high-performance liquid chromatography step, gradient elution is performed using acetonitrile-water at a volume ratio of 40%-75%.

[0032] This invention also discloses the use of the active glyceroglycolipid compound in the preparation of antibacterial agents for medical, veterinary, or agricultural purposes for non-therapeutic purposes;

[0033] Preferably, the antibacterial agent includes Staphylococcus aureus inhibitors and Micrococcus luteus inhibitors.

[0034] This invention also discloses the use of a marine micromonospora strain FIMYZ51 for fermentation to prepare the active glycerol glycolipid compound;

[0035] The marine micromonospora strain FIMYZ51, classified as Micromonospora sp., was deposited at the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, with accession number CGMCC No. 24067.

[0036] This invention utilizes a marine micromonospora sp. FIMYZ51 strain with antifungal activity, isolated and screened from marine actinomycetes, to ferment and prepare the compound FW-6-2. Compound FW6-2, which exhibits inhibitory activity against Staphylococcus aureus and Micrococcus luteus, can be extracted and isolated from the fermentation broth of Micromonospora sp. FIMYZ51. The compound is then purified through extraction from the fermentation broth to obtain a pure product. Both fermentation and extraction efficiencies are quite ideal.

[0037] The active glyceroglycolipid compound FW-6-2 described in this invention has been shown to have cytotoxic activity against Staphylococcus aureus and Micrococcus luteus in activity tests. This discovery not only reveals a new type of glyceroglycolipid compound, but also provides a lead compound for the research and development of new antibacterial drugs. Attached Figure Description

[0038] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0039] Figure 1 This is the high-resolution mass spectrum of the active glyceroglycolipid compound FW-6-2 in this invention;

[0040] Figure 2 It is the active glyceroglycolipid compound FW-6-2 in this invention. 1 H nuclear magnetic resonance image 1 H spectrum;

[0041] Figure 3 It is the active glyceroglycolipid compound FW-6-2 in this invention. 13 C NMR image 13 C spectrum;

[0042] Figure 4 It is the active glyceroglycolipid compound FW-6-2 in this invention. 1 H- 1 HCOSY related graphs;

[0043] Figure 5 This is the HSQC spectrum of the active glyceroglycolipid compound FW-6-2 in this invention;

[0044] Figure 6 This is the HMBC correlation spectrum of the active glyceroglycolipid compound FW-6-2 in this invention;

[0045] Figure 7 These are the antibacterial test results of the active glycerol glycolipid compound FW-6-2 in this invention, wherein (A) is Staphylococcus aureus and (B) is Cladosporium flavum. Detailed Implementation

[0046] In the following embodiments of the present invention, the active glycerol glycolipid compound FW-6-2 was prepared by fermentation using a screened Micromonospora sp. FIMYZ51.

[0047] The Micromonospora sp. strain FIMYZ51, classified as Micromonospora sp., was deposited on December 9, 2021, at the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 24067, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0048] The present invention describes the extraction and isolation of an active glyceroglycolipid compound FW-6-2, which exhibits cytotoxic activity against Staphylococcus aureus and Micrococcus luteus, from the fermentation broth of Micromonospora sp. FIMYZ51.

[0049] Example 1: Obtaining the active glycerol glycolipid compound FW-6-2

[0050] The preserved Micromonospora sp. FIMYZ51 was inoculated onto starch-asparagine agar slant culture and then inoculated into liquid seed culture medium. After culturing at 30℃ for 2 days, the resulting seed liquid was mixed with artificial fermentation medium at a volume ratio of 1:10 and placed at 30℃ for shaking culture for 5 days. The fermentation product was then collected.

[0051] The liquid seed culture medium consists of: 1.5% soluble starch, 0.5% glucose, 0.53% soybean flour, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.05% NaCl, 0.05% (NH4)2SO4, 0.1% CaCO3, prepared with tap water, and a pH of 6.0-8.5.

[0052] The fermentation medium consists of: 4% soluble starch, 0.5% glucose, 2.5% corn steep liquor powder, 0.5% soybean oil, 0.5% yeast powder, 0.05% MgSO4·7H2O, 0.05% K2HPO4, 0.1% CaCO3, prepared with tap water, and a pH of 6.0-8.5.

[0053] The collected fermentation products were subjected to solid-liquid separation, and the fermentation broth and mycelium were collected separately. The fermentation broth was adsorbed with resin XAD-16, and after adsorption, the filter cake was obtained by centrifugation or filtration. The filter cake was soaked in 100% industrial ethanol overnight, and the solvent layer was collected and concentrated under reduced pressure to obtain crude extract A.

[0054] The crude extract A obtained above was subjected to chromatography using a C18 reversed-phase column with gradient elution of methanol:water (30%-100% v / v) (30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%). Detection was performed using high-performance liquid chromatography (HPLC) (Agilent SB-C18 column, 4.6×250 mm) at a detection wavelength of 200 nm. The eluent containing 40%-80% methanol was collected. Preparative gradient elution was then performed using a preparative Agilent SB-C18 (20×250 mm) reversed-phase HPLC (acetonitrile-water, 40%-75% v / v). The compound FW6-2, exhibiting the desired antibacterial activity, was obtained in its pure form in the acetonitrile v / v segment.

[0055] Structural analysis of compound FW-6-2 in Example 2

[0056] In this embodiment, the structure of the above-mentioned active glyceroglycolipid compound FW-6-2 was identified by MS and NMR techniques.

[0057] The compound FW-6-2 was identified as a pale yellow amorphous solid with the molecular formula: C2 33 H 58 O 14 Its degree of unsaturation is 5, and its molecular weight is 678.38; high-resolution mass spectrometry measurement value: m / z [M+Na] + =701.3707, [M+NH4] + = 696.4159, theoretical value m / z 701.3719 [M+Na] + .

[0058] The compound is soluble in organic solvents such as methanol, acetone, acetonitrile, chloroform, ethyl acetate and dimethyl sulfoxide, but insoluble in water.

[0059] The compound FW-6-2 11H NMR spectrum (DMSO-d6, 600MHz): δ 5.35 (ddd, J = 11.1, 7.0, 1.6Hz, 1H), 5.33 (dd, J = 6.9, 1.3Hz, 1H), 5.31 (dd, J = 6.9, 1.3Hz, 1H), 5.29 (ddd, J = 11.1, 7.0, 1.6Hz, 1H), 4.68 (d, J = 3.6Hz, 1H), 4.58 (s, 1H), 4.51 (s, 1H), 4.39 (s, 1H), 4.09 (t, J =3.7Hz,1H),4.04(dd,J=11.2,4.0Hz,1H),3.97(dd,J=11.2,6.6Hz,1H),3.81(dq,J=11.0,6.0Hz,1H),3.70(d,J=3 .9Hz,1H),3.68(d,J=5.6Hz,1H),3.66(d,J=5.6Hz,1H),3.60(m,1H),3.59(dd,J=5.9,2.5Hz,1H),3.57(d,J=5.8Hz, 1H),3.55(d,J=3.9Hz,1H),3.53(d,J=6.3Hz,1H),3.51(d,J=7.0Hz,1H),3.49(d,J=4.5Hz,1H),3.43(dd,J=5.8,4. 5Hz,1H),3.42(dd,J=5.8,3.2Hz,1H),3.33(dd,J=2.5Hz,1H),3.29(d,J=2.1Hz,2H),3.16(s,1H),3.12(s,1H),2.73 (dd,J=7.5,6.1Hz,2H),2.29(t,J=7.5Hz,2H),2.01(q,J=6.8Hz,4H),1.50(q,J=7.2Hz,2H),1.31(t,J=6.9Hz,3H), 1.28(dd,J=4.1,1.8Hz,2H),1.27–1.26(m,6H),1.25(d,J=1.6Hz,2H),1.24(d,J=4.1Hz,2H),0.85(t,J=7.0Hz,3H).

[0060] The compound FW-6-2 13C-NMR spectrum (DMSO-D6, 150MHz): δ173.0,129.8,127.8,104.0,99.5,73.1,72.9,71.3,70.5,70.4,69.6,68.8,68.4,68.1,67.4,66.5,65.5,60.6,33.4,30.9,29.0,28.7,28.6,28.5,28.5,26.6,26.6,25.2,24.4,22.0,13.9.

[0061] In addition, this embodiment also measured multiple NMR spectra of the compound FW-6-2, as shown in the figures below. Figures 1-6 This determined the assignment of all carbon and hydrogen atoms in the compound and its chemical structure, identifying it as a novel sugar-containing glycerol ester compound, FW-6-2. 1 H and 13 The structural characterization information, such as the C(DMSO-6) affiliation, is shown in the table below.

[0062] FW-6-2 NMR data

[0063]

[0064]

[0065] In summary, the structural formula of compound FW-6-2 obtained by extraction and purification in this invention is as follows:

[0066]

[0067] Example 3: Bioactivity assay of compound FW-6-2

[0068] In this embodiment, an in vitro bacterial inhibition test was conducted on the active glyceroglycolipid compound FW-6-2. The results showed that it has the effect of inhibiting the growth of Staphylococcus aureus and Micrococcus luteus.

[0069] In this embodiment, the inhibitory activity of compound FW-6-2 against bacteria and fungi was determined using a paper-agar disk diffusion assay.

[0070] First, mix Escherichia coli, Staphylococcus aureus, Micrococcus occulta, and Bacillus subtilis at a concentration of 10... 7 Colony density of CFU / ml on inverted MH plates; Candida albicans and Aspergillus niger at 10⁻⁶ / ml. 5Colony density (CFU / ml) on a Sabouraud dextrose agar plate; dissolve the obtained compound FW-6-2 in methanol solution, and take 8 μl of the sample to be tested on a 6 mm diameter circular filter paper. Place the filter paper containing the sample on an agar plate containing the above concentrations of test bacteria (Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Candida albicans, and Aspergillus niger). Simultaneously, use dimethyl sulfoxide solution as a negative control. Incubate at 28-35℃ for 24-48 hours. Observe and record the diameter of the inhibition zone; a larger inhibition zone diameter indicates stronger antibacterial activity of the strain. Results are attached. Figure 7 As shown.

[0071] like Figure 7 The experimental results show that the compound FW-6-2 obtained in this invention exhibits inhibitory activity against Staphylococcus aureus (A) and Staphylococcus faecalis (B), with inhibition zone diameters of 7-8 mm and 18-22 mm, respectively. Therefore, compound FW-6-2 shows promise as a lead compound for antibacterial activity.

[0072] In summary, the in vitro antibacterial activity assay of this sugar-containing glyceride demonstrates its antibacterial activity, thus providing a lead compound for the research and development of new antibacterial drugs.

[0073] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An active glycolipid compound, characterized by, The compound is recorded as FW-6-2, and has the structure shown in the following formula (I): 。 2. A process for the fermentative production of the active glycolipid compound according to claim 1, characterized in that, The method comprises the following steps: (1) Seed liquid culture: the marine Micromonospora preserved on a slant is inoculated into a liquid seed culture medium for constant temperature culture, and a seed liquid is collected for standby use; The marine Micromonospora is a Micromonospora strain FIMYZ51, which is classified as Micromonospora sp. and was preserved in the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, with a preservation number of CGMCC No. 24067. Micromonospora sp. and was preserved in the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, with a preservation number of CGMCC No. 24067. The components of the seed culture medium include: soluble starch 1-2 wt%, glucose 0.3-0.8 wt%, soybean powder 0.3-0.8 wt%, yeast extract 0.3-0.8 wt%, MgSO4·7H2O 0.03-0.08 wt%, NaCl 0.03-0.08 wt%, (NH4)2SO4 0.03-0.08 wt%, CaCO3 0.05-0.15 wt%, and pH 6.0-8.5; The temperature of the constant temperature culture step is 25-35°C, and the culture time is 1-3 days; (2) Fermentation liquid culture: the seed liquid is transferred to a fermentation culture medium for constant temperature culture, so as to obtain a fermentation liquid containing the active glycolipid compound; The components of the fermentation culture medium include: soluble starch 4 wt%, glucose 0.5 wt%, corn syrup dry powder 2.5 wt%, soybean oil 0.5 wt%, yeast powder 0.5 wt%, MgSO4·7H2O 0.05 wt%, K2HPO4 0.05 wt%, CaCO3 0.1 wt%, and pH 6.0-8.5; The temperature of the constant temperature culture step is 25-35°C, and the culture time is 3-6 days.

3. The method of claim 2, wherein the active glycolipid compound is produced by fermentation. In the step (1), the components of the seed culture medium include: soluble starch 1.5 wt%, glucose 0.5 wt%, soybean powder 0.53 wt%, yeast extract 0.5 wt%, MgSO4·7H2O 0.05 wt%, NaCl 0.05 wt%, (NH4)2SO4 0.05 wt%, CaCO3 0.1 wt%, and pH 6.0-8.

5.

4. The process for the fermentative production of said active glycolipid compound according to claim 2 or 3, characterized in that, The method further comprises the steps of extracting and purifying the active glycolipid compound, specifically comprising: (3) Extraction: the collected fermentation liquid is added to XAD-16 resin for adsorption, filtration, desorption recovery and concentration of the filter cake, so as to obtain a crude extract A; (4) Purification: the crude extract A is subjected to C18 reverse phase column chromatography, gradient elution is performed with methanol: water in a volume ratio of 30%-100%, high performance liquid chromatography detection is performed, and the eluate is collected in sections; the eluate component in a section with a methanol volume ratio of 40-80% is collected, gradient elution is performed with acetonitrile-water in which the acetonitrile volume ratio is 40-75% by means of preparative C18 reverse phase high pressure liquid chromatography, and the component in a section with an acetonitrile volume ratio of 50% is collected, so as to obtain the pure active glycolipid compound FW6-2.

5. The method of claim 4, wherein the active glycolipid compound is produced by fermentation. In the step (3), the volume ratio of the XAD-16 resin to the fermentation liquid is 1:10-1:30; The desorption step is 100% ethanol desorption.

6. Use of the active glycolipid compound in claim 1 for preparing a medical, veterinary or agricultural antibacterial preparation for non-therapeutic purposes. The antibacterial agent is a Staphylococcus aureus inhibitor and a Micrococcus luteus inhibitor.

7. Use of Micromonospora strain FIMYZ51 for the fermentative preparation of the active glycolipid compound according to claim 1. The marine Micromonospora strain FIMYZ51, which is classified as Micromonospora sp., was preserved in the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, and the preservation number is CGMCC No. 24067. Micromonospora sp., was preserved in the Institute of Microbiology, Chinese Academy of Sciences on December 9, 2021, and the preservation number is CGMCC No. 24067.

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