CN115974773B - Compound produced by sugarcane endophytic fungi and application thereof in bacteriostasis and antioxidation - Google Patents
Compound produced by sugarcane endophytic fungi and application thereof in bacteriostasis and antioxidation Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pyridine Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a compound produced by sugarcane endophytic fungi and application thereof in bacteriostasis and antioxidation. The invention adopts modern spectrum technology to carry out structure identification on the separated monomer compound, deduces the molecular structure of the compound, and names the compound as 5-butenylpyridine-2-formic acid, and experimental results prove that the 5-butenylpyridine-2-formic acid not only has antibacterial effect on various food-borne pathogenic bacteria, but also has certain scavenging capacity on ABTS free radicals and oxygen free radicals (ORAC), and can be used for preparing antibacterial drugs and antioxidant preparations, so that the compound has good application prospect.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a compound produced by sugarcane endophytic fungi and application thereof in bacteriostasis and antioxidation.
Background
Research on endophytic fungi has been focused on research and development and utilization of plant endophytic fungi since the 21 st century. The plant resources in China are rich and various, and the endophyte resource library is utilized, so that low-toxicity, high-efficiency and low-cost anticancer drugs, antibacterial drugs, pesticides, antioxidants, plant hormones, immunosuppressants and the like in the metabolites are discovered, which has very important significance.
Recent researches indicate that endophytic fungi are important resource libraries of various bioactive components with novel structures, and can produce bioactive components identical/similar to symbiotic plants. Compared with the preparation of the compound by plant extraction, the endophytic fungi has obvious advantages such as high growth speed of thalli, easy culture, convenient downstream processing, cost saving, green sustainable property and the like. At present, various endophytic fungi polyphenols have been reported, such as ginger endophytic fungi (Aspergillusaustroafricanus), palm root endophytic fungi (PENICILLI mu M citrin mu M TDPEF), and the like. These studies suggest that sugarcane endophytic fungi might be a new direction for preparing various biologically active ingredients with novel structures.
Recent studies have shown that a variety of active substances (antiviral, antibacterial, etc.) have been isolated from plant endophytic fungi, some of which have specific biological activities. Research on secondary metabolic substances generated by endophytic fungi in plants shows that the effect of the secondary metabolic substances approximates to parasitic plants, wherein a part of the secondary metabolic substances have medicinal potential, and a part of the secondary metabolic substances have activities for inhibiting the growth of various common pathogenic bacteria causing animal and plant diseases to different degrees after the antibacterial experiments, so that the secondary metabolic substances can be used for researching and developing microbial preparations for preventing and controlling diseases.
Endophytic fungi screened from different tissue parts of the food-borne sugarcane are selected for morphological and molecular biological identification, and the antibacterial activity of compounds produced by the endophytic fungi is researched. The precious natural medicine resources with high activity in sugarcane endophytic fungi are developed, so that the application contribution to the human healthy development is expected.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a compound produced by sugarcane endophytic fungi and application thereof in bacteriostasis and antioxidation so as to overcome the technical problems.
The invention is realized in the following way:
the inventors first isolated and purified a novel material from Fusari mu M proliferat. Mu.M 15, which was identified as 5-butenylpyridine-2-carboxylic acid. Proved by researches, the 5-butenyl pyridine-2-formic acid has antibacterial activity and oxidation resistance.
In a first aspect, the invention provides a compound having the formula C 10H11NO2, designated 5-butenylpyridine-2-carboxylic acid, and having the formula:
among them, a sugarcane endophytic fungus for producing the above-mentioned compounds is named Fusari mu M proliferat mu M15, and the preservation number is GDMCC No:62048, collection of microorganism strains from Guangdong province, no.5 building, no. 59, road 100, university, martyr, for 2021, 11, 10 days.
The preparation method for producing the compound comprises the following steps:
(1) Activating endophytic fungi of the sugarcane to obtain fermentation seed liquid of the endophytic fungi of the sugarcane;
(2) Inoculating the sugarcane endophytic fungi fermentation seed liquid into a sugarcane endophytic fungi liquid fermentation medium for fermentation, and collecting fermentation liquid;
(3) Centrifuging, freezing and freeze-drying at low temperature, and collecting fermentation products;
(4) Leaching the crude fermentation product by using an alcohol solution, precipitating by using alcohol, centrifuging to obtain precipitate permeability, and drying to obtain the crude fermentation product;
(5) And re-dissolving the crude fermentation product by using an eluent, removing proteins and pigments, and eluting a target product (5-butenylpyridine-2-carboxylic acid) by using a semi-prepared liquid phase.
In some embodiments, the sugarcane endophytic fungus is Fusari μ M proliferat μΜ 15.
In some embodiments, the medium for activating the sugarcane endophytic fungi is PDB medium, the activation temperature is 20-30 ℃, and the activation time is 2-7d.
In some embodiments, the sugarcane endophytic fungus liquid fermentation medium is a PBD medium.
In some embodiments, the volume ratio of the sugarcane endophyte seed liquid to the sugarcane endophyte liquid fermentation medium is 1:5-15.
In some embodiments, the conditions under which the sugarcane endophyte fermentation seed liquid is fermented in the sugarcane endophyte liquid fermentation medium are: the fermentation time is 1-10d, and the fermentation temperature is 20-30 ℃.
In some embodiments, the alcoholic solution comprises a methanol solution.
In some embodiments, the methanol solution has a volume concentration of 30-70%.
In some embodiments, the eluent comprises a methanol solution.
In some embodiments, the concentration of methanol solution in the eluent is 30-70% by volume.
In some embodiments, the conditions for semi-preparative liquid phase elution are:
The mobile phase is acetic acid aqueous solution and methanol;
The chromatographic column is a C18 chromatographic column;
the elution procedure is isocratic elution;
the flow rate was 1mL/min.
In a second aspect, the invention also provides application of the compound in preparation of antibacterial medicines.
According to the invention, the oxford cup method is used for verifying the antibacterial effect of 5-butenyl pyridine-2-carboxylic acid on various food-borne pathogenic bacteria, and experimental results show that the antibacterial circle range of 5-butenyl pyridine-2-carboxylic acid on tested strains is 25.2-30.0mm under the mass concentration of 100mg/L, so that the 5-butenyl pyridine-2-carboxylic acid has good antibacterial effect on staphylococcus aureus, escherichia coli, salmonella, listeria monocytogenes, staphylococcus lentus, bacillus subtilis, bacillus cereus and micrococcus luteus.
In a third aspect, the invention also provides application of the compound in preparation of an antioxidant preparation.
The determination result of the ABTS free radical and oxygen free radical (ORAC) scavenging ability of the 5-butenylpyridine-2-carboxylic acid shows that the 5-butenylpyridine-2-carboxylic acid has a certain ABTS free radical scavenging ability and increases with the increase of the concentration of the 5-butenylpyridine-2-carboxylic acid; meanwhile, 5-butenylpyridine-2-carboxylic acid has stronger oxygen free radical scavenging capability, which indicates that the 5-butenylpyridine-2-carboxylic acid can be used as a purely natural antioxidant component and can be used for replacing common but toxic synthetic antioxidants (such as 2, 6-di-tert-butyl-4-methylphenol, BHT and butyl hydroxy anisole, BHA).
In a fourth aspect, the invention also provides a biological agent comprising a compound as described above.
In some embodiments, the biological agents include biological agents that inhibit pathogenic bacteria and biological agents that are resistant to oxidation.
The invention has the following beneficial effects:
The invention provides a new compound from sugarcane endophytic fungi, and the compound is named as 5-butenylpyridine-2-formic acid through identification, and experimental results prove that the 5-butenylpyridine-2-formic acid has a bacteriostatic effect on various food-borne pathogenic bacteria, has a certain scavenging capacity on ABTS free radicals and oxygen free radicals (ORAC), can be used for preparing bacteriostatic medicaments and antioxidation preparations, and has good application prospects.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chart of the 1H spectrum of 5-butenylpyridine-2-carboxylic acid of the present invention.
FIG. 2 is a graph of 13C of 5-butenylpyridine-2-carboxylic acid of the present invention.
FIG. 3 is a schematic diagram of the correlation of COSY, HMBC and HSQC of 5-butenylpyridine-2-carboxylic acid of the present invention.
FIG. 4 shows the results of an antioxidant experiment of 5-butenylpyridine-2-carboxylic acid of example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The preparation method of the high-activity sugarcane endophytic fungus fermentation product-5-butenylpyridine-2-carboxylic acid comprises the following specific processes:
(1) The sugarcane endophytic fungus Fusari mu M proliferat mu M15 strain is activated and cultured for 5 days at 25 ℃ in PDB culture medium with pH of 6.5, and strain fermentation liquor seed liquor is obtained.
(2) Inoculating 10% (v/v) sugarcane endophytic fungus fermentation seed liquid into a sugarcane endophytic fungus liquid fermentation medium, and fermenting at room temperature of 25 ℃ for 7-10d; after the fermentation is completed, collecting fermentation liquor.
(3) Centrifuging at low temperature, lyophilizing the fermentation broth, leaching with 30-70% methanol at room temperature for 2-8 hr, repeating the steps for 3 times, mixing the extractive solutions, concentrating under reduced pressure, precipitating with 50% methanol at room temperature, centrifuging at 10000rpm for 20min, dialyzing with ultrapure water for 4 times, concentrating under reduced pressure, and lyophilizing to obtain crude fermentation product.
(4) Obtaining fermentation seed liquid with the yield of the crude fermentation product of 2.68 g/mL; dissolving the obtained crude fermentation product sample into 1mg/mL crude fermentation product solution by using 50% methanol as an eluent, removing proteins and pigments by using H103 macroporous adsorption resin, eluting by using 50% methanol, and collecting eluent. Concentrating under reduced pressure to obtain crude fermentation product with deproteinized and pigment removed; and (3) dissolving the crude fermentation product by using a mobile phase, and performing semi-preparation liquid phase elution to obtain a single component, namely 5-butenyl pyridine-2-formic acid.
Wherein, the semi-preparative liquid phase elution uses an irite P3500 semi-preparative liquid chromatography system, and the mobile phase (v/v) is: 60 (HPLC grade aqueous trifluoroacetic acid): 40 (methanol), column: 10 μm,250×4.6mm C18 column (octadecyl bonded silica gel column liquid phase column), flow rate: 1mL/min, the treatment time was 30min.
(5) The pure 5-butenyl pyridine-2-carboxylic acid is taken and subjected to low-temperature freeze drying, and the yield of the 5-butenyl pyridine-2-carboxylic acid is found to be about 26.8 mg/L.
Example 2
The pure 5-butenyl pyridine-2-carboxylic acid obtained in the example 1 is sent to a detection mechanism for substance structure identification and analysis, and the obtained product is determined to be 5-butenyl pyridine-2-carboxylic acid (shown in the figures 1,2 and 3), and the detection mechanism is a scientific compass scientific research service platform. The following formula is the structural formula of 5-butenylpyridine-2-carboxylic acid prepared in example 1:
Example 3
The antibacterial effect of 5-butenylpyridine-2-carboxylic acid on various food-borne pathogenic bacteria is tested by using the oxford cup method, and the specific operation steps are as follows:
(1) 0.2mL of the indicated bacteria solution (bacteria concentration: 1X 10 7 CFU/mL) was aspirated in a sterile super clean bench and spread on LB solid medium.
(2) Under aseptic conditions, sterilized oxford cups (oxford cup dimensions: outer diameter 8mm, inner diameter 6mm, height 10 mm) were placed vertically on the surface of LB solid medium and gently pressurized. 200. Mu.L of 5-butenylpyridine-2-carboxylic acid was added to the oxford cup and incubated at 37℃for 24 hours.
The bacteriostasis test was repeated 5 times in total. Wherein the indicator bacteria include Staphylococcus aureus, escherichia coli, salmonella, listeria monocytogenes, staphylococcus slowly, bacillus subtilis, bacillus cereus, and Micrococcus luteus.
The test results are shown in table 1:
TABLE 1 diameter of inhibition zone for different test strains
| Strain name | Diameter of inhibition zone (mm) |
| Staphylococcus aureus | 28.17±0.12 |
| Coli bacterium | 31.97±0.47 |
| Salmonella bacteria | 22.30±0.46 |
| Listeria monocytogenes | 21.67±0.31 |
| Staphylococcus slow | 25.57±0.35 |
| Bacillus subtilis | 21.13±0.21 |
| Bacillus cereus | 20.33±0.25 |
| Micrococcus luteus | 18.52±0.28 |
As can be seen from Table 1, 5-butenylpyridine-2-carboxylic acid has good antibacterial effect against Staphylococcus aureus, escherichia coli, salmonella, listeria monocytogenes, staphylococcus slowly, bacillus subtilis, bacillus cereus and Micrococcus luteus, and the diameters of the antibacterial zone are 28.17+ -0.12, 31.97+ -0.47, 22.30+ -0.46, 21.67+ -0.31, 25.57+ -0.35, 21.13 + -0.21, 20.33+ -0.25 and 18.52+ -0.28 mm, respectively.
Example 4
This example is a determination of ABTS free radical clearing ability of 5-butenylpyridine-2-carboxylic acid, and is performed as follows:
(1) ABTS radical stock solution preparation: preparing mixed solution of ABTS and K 2S2O8 with the concentration of 14mM and 4.9mM respectively by adopting ultrapure water, and standing for 16 hours at room temperature in a dark place;
(2) ABTS clearance ability assay: before the experiment, 50% (v/v) ethanol solution is adopted to dilute the ABTS stock solution until the absorbance at 734nm is 0.7+/-0.1, namely the ABTS free radical measuring solution. Adding 0.1mL of 5-butenylpyridine-2-carboxylic acid sample solution to 2.9mL of ABTS assay solution, after 30s oscillation, measuring the absorbance Ai of the reaction at 734nm for 20 min; 2.9ml of 50% ethanol solution was selected as a blank, 0.1ml of 5-butenylpyridine-2-carboxylic acid sample was added thereto, and the absorbance A0 was measured under the same conditions, and the positive control was vitamin C (Vc).
(3) ABTS radical scavenging ability (R) calculation; r= (1-Ai/A0) ×100%
The results of the ABTS radical scavenging ability of 5-butenylpyridine-2-carboxylic acid on ABTS radical scavenging ability are shown in fig. 4B, and compared with ABTS radical scavenging ability of a (control group Vc) in fig. 4, 5-butenylpyridine-2-carboxylic acid has a certain ABTS radical scavenging ability, but is significantly weaker than Vc. ABTS scavenging ability of 5-butenylpyridine-2-carboxylic acid showed dose dependence, all increased with increasing 5-butenylpyridine-2-carboxylic acid concentration.
Example 5
This example is a measurement of the oxygen radical scavenging capacity (ORAC) of 5-butenylpyridine-2-carboxylic acid, and is performed as follows:
(1) Preparing a solution: phosphate Buffer (PBS) of 75mM pH7.4 was prepared, and 10-200. Mu.M Trolox (water-soluble vitamin E) standard solution, 70mM FL (sodium fluorescein, sodi. Mu.M fluorescein, FL) solution, 12.8mM AAPH (2, 2-azo-bis- (2-amidinopropane) dihydrogen chloride, 2-azobis (2-amidinopropane) dihydrochloride, AAPH) solution were prepared from the prepared PBS, and the solution was ready for use. A0.2 mg/mL solution of 5-butenylpyridine-2-carboxylic acid was prepared.
(2) ORAC clearance ability assay: mu.L of antioxidant (Trolox, 5-butenylpyridine-2-carboxylic acid, GSH), 20. Mu.L of PBS buffer and 20. Mu.L of FL solution were added separately to 96-well plates, incubated at 37℃for 15min, then 140. Mu.L of AAPH solution was added rapidly, and the plates were shaken for 10min to ensure adequate mixing of the solutions, read 1 time every 2min, 3 replicates/sample.
(3) ORAC cleanup capability calculation: the Net area of the fluorescence extinction curve (Net AUC) was calculated according to the following formula
NetAUC＝AUCsample-AUCblank
Wherein, f 0-initial absorbance, fi-interval 2min to measure absorbance, AUCsample-fluorescence extinction curve area, AUCblank curve area without antioxidant. Then, the standard curves were plotted as Net AUC and Trolox: y=0.333x+4.175 (R 2 = 0.9905). ORAC clearance of polysaccharide 5-butenylpyridine-2-carboxylic acid and FP was calculated as mu Mol Trolox/g based on the above standard curve, and the positive control group was GSH (glutathione).
The ORAC free radical scavenging ability of 5-butenylpyridine-2-carboxylic acid is shown as 4C, and as a common antioxidant, the ORAC value of the control GSH is 1343.1 + -26.5 μm Trolox/g, while the ORAC value of 5-butenylpyridine-2-carboxylic acid is 625.27 + -22.37 μm Trolox/g, which corresponds to 1/2 of the ORAC value of GSH.
Reactive oxygen radicals can directly or indirectly cause oxidative damage to DNA and proteins within the cells of an organism, thereby causing other collective diseases. Thus, the determination of the ORAC value is of great importance for the evaluation of the oxidation resistance of biologically active ingredients. The above results indicate that 5-butenylpyridine-2-carboxylic acid has relatively strong oxygen radical scavenging ability. This is similar to the ABTS radical scavenging ability evaluation, i.e., 5-butenylpyridine-2-carboxylic acid can be used as a purely natural antioxidant ingredient to replace those commonly used but toxic synthetic antioxidants (e.g., 2, 6-di-tert-butyl-4-methylphenol, BHT and butylated hydroxyanisole, BHA).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The application of a compound in preparing an antioxidant preparation is characterized in that the molecular formula is C 10H11NO2, and the molecular formula is named as 5-butenylpyridine-2-carboxylic acid, and the structural formula is as follows:。
2. The use according to claim 1, wherein the compound is produced by a sugarcane endophytic fungus, named Fusari μ M proliferat μΜ 15, accession No. GDMCC No:62048.
3. The use according to claim 2, wherein the antioxidant formulation has the ability to scavenge free radicals including ABTS free radicals and oxygen free radicals.
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