Kenone fatty acid derivative and salt thereof, preparation method and natural anti-inflammatory active substance thereof

Abstract

The invention provides ketene fatty acid derivatives, salts thereof, a preparation method and natural anti-inflammatory active substances thereof, and belongs to the field of medicine and health and food industry. The ketene fatty acid derivative is (E)-14-oxoctadec-12-enoic acid with a structural formula I, and the structural formula I is # imgabs0. The ketene fatty acid derivative provided by the invention has relatively good in-vitro anti-inflammatory activity, can be used as an active component of an anti-inflammatory drug, and has relatively good practical application.

Description

Technical Field

[0001] The invention relates to the fields of medicine, health and food industry, and relates to an ketene fatty acid derivative and a salt thereof, a preparation method and a natural anti-inflammatory active substance thereof. Background Art

[0002] Inflammation: It is what people usually call "inflammation". It is a defensive reaction of the body to stimulation, which manifests as redness, swelling, heat, pain and dysfunction. Inflammation can be infectious inflammation caused by infection or non-infectious inflammation not caused by infection. In the process of inflammation, a series of local reactions centered on the vascular system limit and eliminate damaging factors, and also promote the healing of damaged tissues. The exudation of fluid can dilute toxins, engulf and transport necrotic tissues to facilitate regeneration and repair, so that pathogenic factors are confined to the site of inflammation and do not spread throughout the body. Therefore, inflammation is a natural local reaction mainly based on defense, which is generally beneficial to the body. It can be imagined that without inflammatory response, bacterial infection cannot be controlled, the damage can never be healed, and it can cause serious harm to the body. However, in some cases, inflammation is potentially harmful. Inflammatory response is the basis for the onset of some diseases. For example, severe hypersensitivity reaction can threaten the patient's life when the inflammation is too severe. In addition, inflammation in special parts or organs can cause serious consequences. For example, inflammation of the brain or the brain can compress the life center, inflammation of the vocal cords can block the larynx and cause suffocation, and severe myocarditis can affect heart function. At this time, anti-inflammatory drugs should be used to suppress the inflammatory response.

[0003] Anti-inflammatory drugs are drugs used to treat inflammation caused by tissue damage. There are two major categories of anti-inflammatory drugs: one is steroidal anti-inflammatory drugs, and the other is non-steroidal anti-inflammatory drugs, which are antipyretic, analgesic and anti-inflammatory drugs such as aspirin in medical practice. However, the anti-inflammatory drugs currently used in clinical practice still have certain toxic and side effects: 1. There are certain side effects on the gastrointestinal tract, mainly manifested in 1) direct damage to the mucosal surface, 2) inhibition of systemic prostaglandin synthesis, and 3) anti-platelet aggregation effects; 2. There are certain toxic and side effects on the kidneys; 3. Effects on the central nervous system, common central nervous system symptoms include drowsiness, confusion, and depression. Summary of the invention

[0004] The purpose of the present invention is to at least partially solve the above-mentioned technical problems, and aims to provide a new type of ketene fatty acid derivatives and their salts, a preparation method and natural anti-inflammatory active substances. According to in vitro activity results, the ketene fatty acid derivatives have good anti-inflammatory activity in vitro.

[0005] The present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, a ketene fatty acid derivative is provided, wherein the ketene fatty acid derivative is (E)-14-oxooctadec-12-enoic acid having a structural formula I, wherein the structural formula I is:

[0007]

[0008] In the second aspect of the present invention, a salt that can be formed by a ketene fatty acid derivative is provided, wherein the salt that can be formed by a ketene fatty acid derivative is formed by the above-mentioned ketene fatty acid derivative.

[0009] In a third aspect of the present invention, a method for preparing an ketene fatty acid derivative, wherein the ketene fatty acid derivative is the above-mentioned ketene fatty acid derivative, comprises:

[0010] 1) Preliminary extraction of actinomycetes: The actinomycete culture was extracted with an organic solvent and concentrated to dryness under reduced pressure at room temperature to obtain a crude extract;

[0011] 2) Normal phase open chromatography column purification: add the actinomycete extract obtained in step 1) to a chromatography column filled with normal phase silica gel, and sequentially use a petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and a petroleum ether-ethyl acetate solvent with a volume ratio of 0:100 to elute the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:10;

[0012] 3) Purification by reverse phase medium and low pressure chromatography column: dissolve the fraction obtained in step 2) in an appropriate amount of the first solvent, add to a chromatography column filled with reverse phase silica gel, and sequentially elute with a methanol solution having a volume concentration of 40%, a methanol solution having a volume concentration of 50%, a methanol solution having a volume concentration of 60%, a methanol solution having a volume concentration of 70%, a methanol solution having a volume concentration of 80%, a methanol solution having a volume concentration of 90%, and a methanol solution having a volume concentration of 100%, and take the fraction eluted with the methanol solution having a volume concentration of 80%;

[0013] 4) Normal phase HPLC purification: The fraction obtained in step 3) is separated and purified by normal phase HPLC to obtain enone fatty acid derivatives, the structure of which is shown in Formula 1.

[0014] In some embodiments of the present invention, the actinomycete is Streptomyces sundarbansensis.

[0015] In some embodiments of the present invention, in step 1), the organic solvent is ethyl acetate.

[0016] In some embodiments of the present invention, in step 4), the elution system used is a n-hexane-isopropanol system with a volume ratio of 95:5.

[0017] In the third aspect of the present invention, a natural anti-inflammatory active substance is provided, wherein the natural anti-inflammatory drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned enoketone fatty acid derivative or a salt thereof.

[0018] In some embodiments of the present invention, the pharmaceutically acceptable carrier is selected from any one of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant, or any combination thereof.

[0019] The ketene fatty acid derivatives and their salts, preparation methods and natural anti-inflammatory active substances according to the embodiments of the present invention have at least one of the following advantages:

[0020] Compared with the prior art, the embodiments of the present invention provide a new use of ketene fatty acid derivatives. In vitro experiments have confirmed that the ketene fatty acid derivatives provided by the present invention have good in vitro anti-inflammatory activity, can be used as active ingredients of anti-inflammatory agents, and have a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments in conjunction with the accompanying drawings, in which:

[0022] Figure 1 This is a hydrogen spectrum data diagram of the compound obtained in Example 1 of the present invention;

[0023] Figure 2 The carbon spectrum data of the compound obtained in Example 1 of the present invention;

[0024] Figure 3 The two-dimensional nuclear magnetic resonance data (COSY) of the compound obtained in Example 1 of the present invention;

[0025] Figure 4 This is a two-dimensional nuclear magnetic resonance data diagram (HSQC) of the compound obtained in Example 1 of the present invention;

[0026] Figure 5 This is a two-dimensional nuclear magnetic resonance data diagram (HMBC) of the compound obtained in Example 1 of the present invention;

[0027] Figure 6This is a high resolution mass spectrum (HRESIMS) data diagram of the compound obtained in Example 1 of the present invention;

[0028] Figure 7 This is a data diagram of the in vitro anti-inflammatory activity of the compound obtained in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further specifically described below by examples and in conjunction with the accompanying drawings. In the specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation of the present invention.

[0030] In order to further illustrate the present invention, the ketene fatty acid derivatives and the preparation method thereof provided by the present invention are described in detail below in conjunction with examples.

[0031] In one embodiment of the present invention, a ketene fatty acid derivative is provided, wherein the ketene fatty acid derivative is (E)-14-oxooctadec-12-enoic acid having a structural formula I, wherein the structural formula I is:

[0032]

[0033] In another embodiment of the present invention, a salt that can be formed by a ketene fatty acid derivative is provided. The salt that can be formed by a ketene fatty acid derivative is formed by the ketene fatty acid derivative according to claim 1.

[0034] In another embodiment of the present invention, a method for preparing a ketene fatty acid derivative is provided, wherein the ketene fatty acid derivative is the ketene fatty acid derivative described above, and the preparation method comprises:

[0035] 1) Preliminary extraction of actinomycetes: The actinomycete culture was extracted with an organic solvent and concentrated to dryness under reduced pressure at room temperature to obtain a crude extract;

[0036] 2) Normal phase open chromatography column purification: add the actinomycete extract obtained in step 1) to a chromatography column filled with normal phase silica gel, and sequentially use a petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and a petroleum ether-ethyl acetate solvent with a volume ratio of 0:100 to elute the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:10;

[0037] 3) Purification by reverse phase medium and low pressure chromatography column: dissolve the fraction obtained in step 2) in an appropriate amount of the first solvent, add to a chromatography column filled with reverse phase silica gel, and sequentially elute with a methanol solution having a volume concentration of 40%, a methanol solution having a volume concentration of 50%, a methanol solution having a volume concentration of 60%, a methanol solution having a volume concentration of 70%, a methanol solution having a volume concentration of 80%, a methanol solution having a volume concentration of 90%, and a methanol solution having a volume concentration of 100%, and take the fraction eluted with the methanol solution having a volume concentration of 80%;

[0038] 4) Normal phase HPLC purification: The fraction obtained in step 3) is separated and purified by normal phase HPLC to obtain enone fatty acid derivatives, the structure of which is shown in Formula 1.

[0039] Specifically, the actinomycete is Streptomyces sundarbansensis.

[0040] In step 1), the organic solvent is ethyl acetate.

[0041] In step 4), the elution system used is a n-hexane-isopropanol system with a volume ratio of 95:5.

[0042] In one embodiment of the present invention, a natural anti-inflammatory active substance is provided, wherein the natural anti-inflammatory drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned enoketone fatty acid derivative or a salt thereof.

[0043] The pharmaceutically acceptable carrier is selected from any one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants, or any combination thereof.

[0044] Example 1. Preparation of the compound of formula I

[0045] The present invention adopts the steps of extraction and purification under conventional laboratory culture conditions to prepare the compound of the present invention, wherein the microorganism used is the actinomycete Streptomyces sundarbansensis.

[0046] The process for a specific compound is as follows:

[0047] 1) Cultivation

[0048] The actinomycetes were cultured in the laboratory using a modified Gottfried medium at 26°C and 120 rpm for 21 days. The formula of the Gottfried medium is: 20 grams of soluble starch, 0.5 grams of sodium chloride, 0.01 grams of ferrous sulfate, 1.0 grams of potassium nitrate, 0.5 grams of magnesium sulfate and 0.5 grams of potassium dihydrogen phosphate per liter of water.

[0049] 2) Extraction

[0050] The actinomycete culture was extracted with an equal volume of ethyl acetate, and the ethyl acetate layer was taken and concentrated to dryness under reduced pressure to obtain 10.3 g of ethyl acetate extract.

[0051] 3) Normal phase silica gel column chromatography separation

[0052] The ethyl acetate extract was loaded onto silica gel and added to a chromatographic column containing 200 g of normal phase silica gel (200-300 mesh). Gradient elution was performed using a petroleum ether-ethyl acetate system (100:5-0:100). The eluted components were collected, and the petroleum ether-ethyl acetate component with a volume ratio of 100:10 was the main collected fraction.

[0053] 4) Reverse phase silica gel column purification

[0054] The selected fraction was dissolved in an appropriate amount of methanol, added to a chromatographic column containing 120 g of reverse phase silica gel (120 angstroms, 30-50 mesh), and gradient eluted with a methanol-water system (40%-100% methanol). The eluted components were collected, of which 80% were the main collected fractions.

[0055] 5) Normal phase HPLC purification

[0056] The selected fractions were analyzed and purified by normal phase HPLC. The analysis conditions were as follows: caprisilsilical column (5 μm, 10×250 mm, ), the elution system is n-hexane-isopropanol isocratic elution, the specific conditions are: n-hexane-isopropanol (95:5, V / V), the flow rate is 1.0 mL / min. The detection wavelength is 203 nm, and the injection volume is 20 μL. The secondary metabolite with the structural formula I is obtained, that is, ketene fatty acid derivatives.

[0057] 6) Structural Identification of Monomer Compound 1

[0058] The monomer compound 1 was tested by nuclear magnetic resonance data, and the measured spectra were 1 H NMR spectrum (H spectrum, as attached Figure 1 As shown, it mainly provides relevant information about the hydrogen element in the compound). 13 C NMR spectrum (carbon spectrum, as attached Figure 2As shown in the figure, it mainly provides relevant information about the carbon element in the compound), isonuclear shift correlation spectrum (COSY, as shown in the figure Figure 3 As shown, it mainly provides information related to hydrogen-hydrogen coupling in compounds) heteronuclear single quantum relationship spectrum (HSQC, as shown in the attached Figure 4 It mainly provides direct information about hydrogen and carbon in compounds) and heteronuclear multi-correlation spectrum (HMBC, as shown in the attached Figure 5 As shown, it mainly provides information related to hydrogen-carbon long-range in the compound, and its purpose is to determine the structure of the compound by analyzing the spectrum. The instrument selected is Varian INOVA500 MHz nuclear magnetic resonance instrument, and the test reagent selected is deuterated methanol.

[0059] The monomer compound I was subjected to high resolution mass spectrometry (HRESIMS, as shown in the attached Figure 6 The molecular weight and molecular formula of the compound were determined by using a Finnigan LCQDECA mass spectrometer.

[0060] Compound 1, (E)-14-oxooctadec-12-enoic acid, colorless needle crystals; H NMR and C NMR data, see Table 1; High resolution ESI mass spectrometry data: (HRESIMS) m / z 295.2281 [MH] - ,319.2252[M+Na] + .

[0061]

[0062]

[0063] The above results show that the structure of the obtained compound is as shown in Formula 1.

[0064] Example 2: Anti-inflammatory activity of compound 1 of the present invention

[0065] (1) Experimental Materials

[0066] Instruments and reagents: Cell line: RAW264.7 mouse macrophage cell line, provided by Professor Su Mingzhi of the Chinese Academy of Sciences; complete culture medium (DMEM+fetal bovine serum+double antibody); MTT reagent (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide); Griess kit (nitrate reduction test reagent).

[0067] Bio-Rad 680 microplate reader, Shimadzu UV-2401PC spectrophotometer.

[0068] Test sample: Secondary metabolite I of Streptomyces sundarbansensis, the compound was dissolved in dimethyl sulfoxide and then diluted.

[0069] (2) Experimental methods

[0070] 1) Sample preparation: Compound I was accurately weighed and dissolved in dimethyl sulfoxide solution to prepare a sample with an initial concentration of 1 mg / mL, and then diluted into samples of different concentrations by adding cell culture medium for later use.

[0071] 2) MTT (cytotoxicity) activity test: The cultured RAW264.7 cells were suspended in culture medium to prepare RAW264.7 suspension, and then evenly inoculated in a 96-well plate (5×10 4 cells / well) and continue incubation for 24 hours. Add 2 μL of the prepared compound I sample to each well (so that its final concentration is 3.0625-100 μM), continue to culture for 24 hours, then add 20 μL of the prepared MTT reagent to each well, and co-culture at 37°C in the dark for 4 hours. After absorbing the supernatant, add 150 μL of DMSO reagent to each well, shake and mix, and check its absorbance at 570 nm. Calculate the relative inhibition rate of compound 1 on the growth and proliferation of RAW264.7 cells. Each group of samples was tested in parallel three times (the results are shown in Table 2 and Appendix Figure 7 shown).

[0072] 3) Griess method to test the inhibitory activity of nitric oxide (NO) production: The cultured RAW264.7 cells were inoculated. The RAW264.7 cell supernatant was discarded under sterile conditions, and then PBS solution was added to it and washed twice repeatedly. At the same time, 700 μL of trypsin digestion solution was added to digest the cells to make them fall off, and finally 1 ml of culture medium was added to it. The digested cell suspension was transferred to a centrifuge tube, and then fresh culture medium was added to wash, followed by centrifugation at 800 rpm for three minutes in a centrifuge. After discarding the supernatant, 4 mL of culture medium was added to the centrifuge tube, and RAW264.7 cells (3.0×105 / ml) were inoculated in a 96-well plate after shaking and mixing, and placed in a 37°C, 5% CO2 incubator for culture. After 12 hours of cell attachment, the prepared test sample was added to the cells under sterile conditions to a final concentration of 3.0625-100 μM. After incubation for 1 hour, 10 μL of LPS was added to each well. According to the test method of the Griess detection kit, a NO standard curve was prepared. After the samples were incubated for 24 h, 50 μL of the cell suspension was transferred to a new 96-well plate, and Griess Reagent I / II was added at a ratio of 1:1. After the reaction was completed, the absorbance wavelength was recorded at 540 nm on a microplate reader, and the NO content and its inhibition rate were calculated (the results are shown in Table 2 and Appendix). Figure 7 shown).

[0073] Table 2. In vitro anti-inflammatory activity of compound I

[0074]

[0075] The results show that compound I of the present invention has certain in vitro anti-inflammatory activity and does not show obvious cytotoxic activity at an effective dose concentration. It can be used as an active ingredient of a natural anti-inflammatory agent and has wide application in biomedicine and food industry.

[0076] Although some embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A ketene fatty acid derivative, wherein the ketene fatty acid derivative is (E)-14-oxooctadec-12-enoic acid having a structural formula I, wherein the structural formula I is:

2. A salt that can be formed from a ketene fatty acid derivative, wherein the salt that can be formed from a ketene fatty acid derivative is formed from the ketene fatty acid derivative according to claim 1.

3. A method for preparing an ketene fatty acid derivative, wherein the ketene fatty acid derivative is the ketene fatty acid derivative according to claim 1, the preparation method comprising: 1) Preliminary extraction of actinomycetes: The actinomycete culture was extracted with an organic solvent and concentrated to dryness under reduced pressure at room temperature to obtain a crude extract; 2) Normal phase open chromatography column purification: the crude actinomycete extract obtained in step 1) is added to a chromatography column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and a petroleum ether-ethyl acetate solvent with a volume ratio of 0:100, and the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:10 is taken; 3) Purification by reverse phase medium and low pressure chromatography column: dissolve the fraction obtained in step 2) in an appropriate amount of the first solvent, add to a chromatography column filled with reverse phase silica gel, and sequentially elute with a methanol solution having a volume concentration of 40%, a methanol solution having a volume concentration of 50%, a methanol solution having a volume concentration of 60%, a methanol solution having a volume concentration of 70%, a methanol solution having a volume concentration of 80%, a methanol solution having a volume concentration of 90%, and a methanol solution having a volume concentration of 100%, and take the fraction eluted with the methanol solution having a volume concentration of 80%; 4) Normal phase HPLC purification: The fraction obtained in step 3) is separated and purified by normal phase HPLC to obtain enone fatty acid derivatives, the structure of which is shown in Formula 1.

4. The preparation method according to claim 3, characterized in that: The actinomycetes is Streptomyces undarbansensis.

5. The preparation method according to claim 3, characterized in that: In step 1), the organic solvent is ethyl acetate.

6. The preparation method according to claim 3, characterized in that: In step 4), the elution system used is a n-hexane-isopropanol system with a volume ratio of 95:

5.

7. A natural anti-inflammatory active substance, characterized in that: The natural anti-inflammatory drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the ketene fatty acid derivative as claimed in claim 1 or a salt thereof as claimed in claim 2.

8. The natural anti-inflammatory active substance according to claim 7, characterized in that: The pharmaceutically acceptable carrier is selected from any one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants, or any combination thereof.

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