Ketene fatty acid derivatives and their resulting salts, preparation methods and natural anti-inflammatory active substances

Abstract

The present invention provides an ene fatty acid derivative and its resulting salt, a preparation method, and a natural anti-inflammatory active substance, belonging to the fields of medicine, health, and the food industry. The ene fatty acid derivative is (E)-14-oxooctadec-12-enoic acid having a structural formula I, wherein the structural formula I is: The ene fatty acid derivative provided by the present invention has good in vitro anti-inflammatory activity, can be used as an active ingredient in anti-inflammatory drugs, and has good practical applications.

Description

Technical Field

[0001] The present 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, commonly referred to as "inflammation," is the body's defensive response to irritation, manifesting as redness, swelling, heat, pain, and functional impairment. Inflammation can be infectious, caused by infection, or non-infectious. During inflammation, a series of localized responses, centered around the vascular system, localize and eliminate damaging factors while also promoting the healing of damaged tissue. Fluid exudation dilutes toxins, phagocytizes and transports necrotic tissue to facilitate regeneration and repair, and confines pathogenic factors to the site of inflammation, preventing them from spreading throughout the body. Therefore, inflammation is a natural, localized, defensive response and, generally speaking, beneficial to the body. It's conceivable that without the inflammatory response, bacterial infection would be uncontrollable, the damage would never heal, and serious harm could result. However, in some cases, inflammation can be potentially harmful. Inflammation underlies the pathogenesis of some diseases. For example, severe hypersensitivity reactions, when excessive, can be life-threatening. 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 throat 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 medications used to treat inflammation that occurs after tissue damage. There are two main categories of anti-inflammatory drugs: steroidal anti-inflammatory drugs (SADAs) and non-steroidal anti-inflammatory drugs (NSADAs), which are referred to in medical practice as antipyretic, analgesic, and anti-inflammatory drugs such as aspirin. However, anti-inflammatory drugs currently used in clinical practice still have certain toxic side effects: 1. Side effects on the gastrointestinal tract, primarily manifested in 1) direct damage to the mucosal surface, 2) inhibition of systemic prostaglandin synthesis, and 3) antiplatelet aggregation effects; 2. Toxic side effects on the kidneys; 3. Effects on the central nervous system, with common central nervous system symptoms including 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 to provide a novel ketene fatty acid derivative and its salt, a preparation method and its natural anti-inflammatory active substance. According to in vitro activity results, the ketene fatty acid derivative has 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 structural formula I, wherein the structural formula I is:

[0007]

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

[0009] In a third aspect of the present invention, a method for preparing an alkenone fatty acid derivative, wherein the alkenone fatty acid derivative is the above-mentioned alkenone 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: the actinomycete extract obtained in step 1) was added to a chromatographic 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 was taken;

[0012] 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in an appropriate amount of the first solvent, added to a chromatography column filled with reverse-phase silica gel, and eluted with 40% methanol solution by volume, 50% methanol solution by volume, 60% methanol solution by volume, 70% methanol solution by volume, 80% methanol solution by volume, 90% methanol solution by volume, and 100% methanol solution by volume, and the fraction eluted with 80% methanol solution was taken;

[0013] 4) Normal-phase HPLC purification: The fraction obtained in step 3) was separated and purified by normal-phase HPLC to obtain an enone fatty acid derivative, 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 ketene 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 diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants, 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 the preferred embodiments taken in conjunction with the accompanying drawings, in which:

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

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

[0024] Figure 3 This is a two-dimensional nuclear magnetic resonance data diagram (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 spectrometry (HRESIMS) data diagram of the compound obtained in Example 1 of the present invention;

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

[0029] The technical solution of the present invention will be further described in detail below through 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 limiting the present invention.

[0030] 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 with reference to the examples.

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

[0032]

[0033] In another embodiment of the present invention, a salt of an ene fatty acid derivative is provided, wherein the salt of the ene fatty acid derivative is formed from the ene fatty acid derivative described in structural formula I.

[0034] In another embodiment of the present invention, a method for preparing an alkenone fatty acid derivative is provided, wherein the alkenone fatty acid derivative is the above-mentioned alkenone fatty acid derivative, 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: the actinomycete extract obtained in step 1) was added to a chromatographic 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 was taken;

[0037] 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in an appropriate amount of the first solvent, added to a chromatography column filled with reverse-phase silica gel, and eluted with 40% methanol solution by volume, 50% methanol solution by volume, 60% methanol solution by volume, 70% methanol solution by volume, 80% methanol solution by volume, 90% methanol solution by volume, and 100% methanol solution by volume, and the fraction eluted with 80% methanol solution was taken;

[0038] 4) Normal-phase HPLC purification: The fraction obtained in step 3) was separated and purified by normal-phase HPLC to obtain an enone fatty acid derivative, 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 ketene 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] Actinomycetes were cultured in a modified Gottfried's medium at 26°C and 120 rpm for 21 days. The medium contained 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 fractions 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 and added to a chromatographic column containing 120 g of reverse phase silica gel (120 angstroms, 30–50 mesh). Gradient elution was performed using a methanol-water system (40% to 100% methanol). The eluted fractions 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, ), using an isocratic n-hexane-isopropanol elution system (95:5, v / v) at a flow rate of 1.0 mL / min. Detection wavelength was 203 nm, and the injection volume was 20 μL. A secondary metabolite with the structural formula I was obtained, namely, a ketene fatty acid derivative.

[0057] 6) Structural Identification of Monomeric 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 hydrogen elements in compounds). 13 C NMR spectrum (carbon spectrum, as attached Figure 2As shown, it mainly provides relevant information of carbon element in the compound), homonuclear shift correlation spectrum (COSY, as shown in the attached Figure 3 As shown, it mainly provides hydrogen-hydrogen related information with coupling relationship in the compound) heteronuclear single quantum relationship spectrum (HSQC, as shown in the attached Figure 4 As shown, it mainly provides direct information about hydrogen and carbon in the compound) and heteronuclear multiple correlation spectrum (HMBC, as shown in the attached Figure 5 The spectral analysis is done using a Varian INOVA 500 MHz NMR instrument and deuterated methanol as the test reagent.

[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 a Finnigan LCQDECA mass spectrometer.

[0060] Compound I, (E)-14-oxooctadec-12-enoic acid, colorless needle-shaped crystals; H NMR and C NMR data are shown in 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 I.

[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. Cell culture medium was then added to dilute the sample to different concentrations 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 the suspension was evenly seeded in a 96-well plate (5×10 4 cells / well) and continue incubating for 24 hours. Add 2 μL of the prepared compound I sample to each well (to make its final concentration 3.0625-100 μM), continue to culture for 24 hours, add 20 μL of the prepared MTT reagent to each well, and co-culture at 37°C in the dark for 4 hours. After aspirating 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. Three parallel experiments were performed for each group of samples (the results are shown in Table 2 and the attached Figure 7 shown).

[0072] 3) Griess assay for nitric oxide (NO) production inhibition: Cultured RAW264.7 cells were plated. Aseptically aspirate the RAW264.7 cell supernatant and wash twice with PBS. Add 700 μL of trypsin solution to dislodge the cells, and then add 1 mL of culture medium. The digested cell suspension was transferred to a centrifuge tube and washed with fresh culture medium. The suspension was then centrifuged at 800 rpm for three minutes. The supernatant was aspirated and 4 mL of culture medium was added to the tube. After shaking to mix, RAW264.7 cells (3.0 × 105 cells / mL) were plated in a 96-well plate and incubated at 37°C with 5% CO2. After 12 hours of cell attachment, the prepared test sample was aseptically added to the cells at a final concentration of 3.0625–100 μM. After incubation for 1 hour, 10 μL of LPS was added to each well. A NO standard curve was prepared according to the test method of the Griess detection kit. 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 inhibition rate were calculated (the results are shown in Table 2 and the attached 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 applications 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 in the claims and their equivalents.

Claims

1. A ketene fatty acid derivative having the chemical structure shown in Formula I:

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

3. A method for preparing a ketene fatty acid derivative, wherein the ketene fatty acid derivative is The ketene fatty acid derivative according to claim 1, wherein the preparation method comprises: 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) was 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 was taken; 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in an appropriate amount of the first solvent, added to a chromatography column packed with reverse-phase silica gel, and eluted with 40% by volume methanol solution, 50% by volume methanol solution, 60% by volume methanol solution, 70% by volume methanol solution, 80% by volume methanol solution, 90% by volume methanol solution, and 100% by volume methanol solution, taking the fraction eluted with the 80% by volume methanol solution; 4) Normal-phase HPLC purification: The fraction obtained in step 3) is separated and purified by normal-phase HPLC to obtain an enone fatty acid derivative, the structure of which is shown in Formula I.

4. The preparation method according to claim 3, characterized in that The actinomycete 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 drug, 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 according to claim 1 or the salt thereof according to claim 2.

8. The natural anti-inflammatory drug 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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