Phenylpropionic acid derivative, preparation method thereof, antioxidant and application thereof

Abstract

The present invention provides a phenylpropionic acid derivative, a preparation method, an antioxidant, and applications thereof, belonging to the fields of medicine, health, and the food industry. The phenylpropionic acid derivative is ethane-1,2-diyl bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propanoate). The present invention provides a new use for a phenylpropionic acid derivative. In vitro tests have confirmed that the phenylpropionic acid derivative provided by the present invention has good in vitro antioxidant activity and can be used as an active ingredient in an antioxidant, thus having a wide range of applications.

Description

Technical Field

[0001] The present invention relates to the fields of medicine, health and food industry, and relates to a phenylpropionic acid derivative, a preparation method thereof, an antioxidant and application thereof. Background Art

[0002] The human body constantly produces free radicals due to constant contact with the outside world, including through respiration (oxidative reactions), pollution, and radiation exposure. Scientific research indicates that cancer, aging, and other diseases are often linked to excessive free radical production. Research into antioxidants can effectively overcome these harmful effects, making antioxidants a key research and development area for health supplement and cosmetic companies, and a key functional requirement in the market.

[0003] An antioxidant is any substance that, even at low concentrations, can effectively inhibit the oxidative reaction of free radicals. This mechanism of action can be either direct action on free radicals or indirect consumption of substances that are prone to free radical formation, preventing further reactions. While the human body inevitably produces free radicals, it also naturally produces antioxidants to counteract the oxidative attack of free radicals on human cells. Research has shown that the human antioxidant system is a comprehensive and complex system comparable to the immune system. The stronger the body's antioxidant capacity, the healthier and longer the lifespan.

[0004] With the development of economy and technology, commonly used synthetic antioxidants have toxic side effects, negatively impacting the heart, lungs, liver, and other organs. They are also susceptible to high temperature instability and poor thermal stability. These drawbacks include low antioxidant efficiency, limited laboratory labeling, a narrow application range (including export restrictions), and poor antibacterial effects. Consequently, their use has been restricted or banned in many countries.

[0005] Research has shown that natural food antioxidants are significantly less toxic than synthetic antioxidants, leading to a growing interest in finding antioxidants from natural sources. In recent years, scientists have intensified their research in this area, with a particular focus on discovering active ingredients found in nature. A large number of natural antioxidant products have emerged worldwide and are gaining widespread consumer favor. Representative natural antioxidants include vitamin E (VE), astaxanthin from shrimp shells, melanoidins from black rice, paprika extract from chili peppers, spice extracts from spices, and sugar alcohol antioxidants. These products, known for their natural and safe properties, are becoming a new favorite in the health food market. Consumers are increasingly choosing foods rich in natural antioxidants, believing them to offer not only health benefits but also a more nutritious nutritional value. Therefore, the trend toward natural antioxidants is expected to remain strong in the future, driving the food industry towards healthier and more environmentally friendly development. Summary of the Invention

[0006] The purpose of the present invention is to at least partially solve the above-mentioned technical problems and to provide a phenylpropionic acid derivative, a preparation method thereof, an antioxidant and its application. According to the in vitro activity results, this phenylpropionic acid derivative has good antioxidant activity in vitro.

[0007] In a first aspect of the present invention, a phenylpropionic acid derivative and a salt thereof are provided, wherein the phenylpropionic acid derivative is ethane-1,2-diyl bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propanoate), and the phenylpropionic acid derivative has a structure of formula I:

[0008]

[0009] In a second aspect of the present invention, a method for preparing the above-mentioned phenylpropionic acid derivative comprises the following steps:

[0010] 1) Primary extraction: The actinomycete culture is extracted with an organic solvent and then concentrated to dryness under reduced pressure at room temperature to obtain an extract;

[0011] 2) Normal phase open column purification: The extract obtained in step 1) was added to a chromatographic column packed with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixture with decreasing volume ratios of ethyl acetate solvent, and the fraction eluted with the petroleum ether-ethyl acetate solvent at a volume ratio of 100:10 was selected;

[0012] 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in methanol, added to a chromatography column packed with reverse-phase silica gel, and eluted with methanol-water solutions of increasing volume concentrations, and the fraction eluted with 70% methanol-water solution was selected;

[0013] 4) Reverse-phase high-performance liquid chromatography purification: The fraction obtained in step 3) was separated and purified by reverse-phase high-performance liquid chromatography to obtain a phenylpropionic acid derivative, the structure of which is shown in Formula 1.

[0014] In some embodiments, the organic solvent is ethyl acetate.

[0015] In some embodiments, the elution is carried out sequentially using a petroleum ether-ethyl acetate mixed solvent with a decreasing volume ratio of ethyl acetate solvent, specifically:

[0016] The elution was carried out in sequence using petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent with a volume ratio of 0:100.

[0017] In some embodiments, the elution is carried out sequentially using methanol aqueous solutions with increasing volume concentrations, specifically:

[0018] Elution was carried out using methanol solution with a volume concentration of 40%, methanol solution with a volume concentration of 50%, methanol solution with a volume concentration of 60%, methanol solution with a volume concentration of 70%, methanol solution with a volume concentration of 80%, methanol solution with a volume concentration of 90% and methanol with a volume concentration of 100%.

[0019] In some embodiments, the actinomycete is Streptomyces sundarbansensis;

[0020] In the third aspect of the present invention, there is provided a use of a phenylpropionic acid derivative or a salt thereof in the preparation of an antioxidant product. The phenylpropionic acid derivative is the above-mentioned phenylpropionic acid derivative or a phenylpropionic acid derivative prepared by the above-mentioned preparation method.

[0021] In the fourth aspect of the present invention, an antioxidant is provided, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned phenylpropionic acid derivative and its salt or the phenylpropionic acid derivative prepared by the above-mentioned preparation method.

[0022] In some embodiments, 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.

[0023] The phenylpropionic acid derivative and its salification, preparation method and application according to the embodiments of the present invention have at least one of the following advantages:

[0024] Compared with the prior art, the present invention provides a novel phenylpropionic acid derivative and a preparation method thereof. In vitro experiments have confirmed that the phenylpropionic acid derivative provided by the present invention has good in vitro antioxidant activity, can be used as an active ingredient of an antioxidant, and has a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] Figure 1 This is a hydrogen spectrum data diagram of the compound obtained in Example 1 of the present invention;

[0027] Figure 2 This is the carbon spectrum data of the compound obtained in Example 1 of the present invention;

[0028] Figure 3 This is a two-dimensional nuclear magnetic resonance data diagram (HSQC) of the compound obtained in Example 1 of the present invention;

[0029] Figure 4 This is a two-dimensional nuclear magnetic resonance data diagram (HMBC) of the compound obtained in Example 1 of the present invention;

[0030] Figure 5 This is a high resolution mass spectrum (HRESIMS) data diagram of the compound obtained in Example 1 of the present invention.

[0031] Add a description of the content and purpose of each figure in the detailed description. DETAILED DESCRIPTION

[0032] 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.

[0033] To further illustrate the present invention, the phenylpropionic acid derivatives and their salts, preparation methods, antioxidants and applications thereof provided in the embodiments of the present invention are described in detail below with reference to the examples.

[0034] In one embodiment of the present invention, a phenylpropionic acid derivative and a salt thereof are provided, wherein the phenylpropionic acid derivative is ethane-1,2-diyl bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propanoate), and the phenylpropionic acid derivative has a structure of Formula I:

[0035]

[0036] Accordingly, a method for preparing the above-mentioned phenylpropionic acid derivative is also provided, the method comprising the following steps:

[0037] 1) Primary extraction: The actinomycete culture is extracted with an organic solvent and then concentrated to dryness under reduced pressure at room temperature to obtain an extract;

[0038] 2) Normal phase open column purification: The extract obtained in step 1) was added to a chromatographic column packed with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixture with decreasing volume ratios of ethyl acetate solvent, and the fraction eluted with the petroleum ether-ethyl acetate solvent at a volume ratio of 100:10 was selected;

[0039] 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in methanol, added to a chromatography column packed with reverse-phase silica gel, and eluted with methanol-water solutions of increasing volume concentrations, and the fraction eluted with 70% methanol-water solution was selected;

[0040] 4) Reverse-phase high-performance liquid chromatography purification: The fraction obtained in step 3) was separated and purified by reverse-phase high-performance liquid chromatography to obtain a phenylpropionic acid derivative, the structure of which is shown in Formula 1.

[0041] Furthermore, in step 1), the organic solvent is ethyl acetate. Of course, those skilled in the art can also select other suitable types of organic solvents as needed, which will not be illustrated here.

[0042] In step 2), the elution is carried out using a petroleum ether-ethyl acetate mixed solvent with a continuously decreasing volume ratio of ethyl acetate solvent, specifically:

[0043] The elution was carried out in sequence using petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent with a volume ratio of 0:100.

[0044] In step 3), the elution is carried out sequentially using methanol aqueous solution with increasing volume concentration, specifically:

[0045] Elution was carried out using methanol solution with a volume concentration of 40%, methanol solution with a volume concentration of 50%, methanol solution with a volume concentration of 60%, methanol solution with a volume concentration of 70%, methanol solution with a volume concentration of 80%, methanol solution with a volume concentration of 90% and methanol with a volume concentration of 100%.

[0046] In a further example, the actinomycete is Streptomyces sundarbansensis. Of course, the actinomycete can also be any other type of actinomycete.

[0047] In another embodiment, a salt of a phenylpropionic acid derivative is provided. The phenylpropionic acid derivative is the above-mentioned phenylpropionic acid derivative or a phenylpropionic acid derivative prepared according to the above-mentioned preparation method.

[0048] In addition, the present invention also provides a use of a phenylpropionic acid derivative or a salt thereof in the preparation of an antioxidant product. The phenylpropionic acid derivative is a phenylpropionic acid derivative prepared according to the above or according to the above preparation method, and the salt thereof is a salt thereof according to the above phenylpropionic acid derivative.

[0049] In addition, an antioxidant is also provided, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the phenylpropionic acid derivative described above or prepared according to the above preparation method, or a salt that can be formed from the above phenylpropionic acid derivative.

[0050] 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.

[0051] Example 1. Preparation of the compound of formula I

[0052] In Example 1 of the present invention, laboratory culture, extraction under conventional conditions, purification and other steps are used to prepare the compound of the present invention, wherein the microorganism used is the actinomycete Streptomyces sundarbansensis.

[0053] The process for a specific compound is as follows:

[0054] 1) Cultivation

[0055] 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.

[0056] 2) Extraction

[0057] 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 an ethyl acetate extract weighing 10.3 g.

[0058] 3) Normal phase silica gel column chromatography separation

[0059] 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 with a petroleum ether-ethyl acetate solvent 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.

[0060] 4) Reverse phase silica gel column purification

[0061] 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 meshes). Gradient elution was performed using a methanol-water system (40% to 100% methanol). The eluted components were collected, of which 70% were the main collected fractions.

[0062] 5) Reverse-phase high performance liquid chromatography purification

[0063] The selected fractions were analyzed and purified by reverse phase high performance liquid chromatography. The analysis conditions were: chromatographic column Hedera C 18 A-5μm, 4.6mm ID x 250mm (Jiangsu Hanbang Technology) was used. The elution system was isocratic methanol-water (methanol-water ratio, 65:35, v / v) at a flow rate of 3.0 mL / min. Detection wavelength was 254 nm, and the injection volume was 20 μL. The secondary metabolite with the structural formula I, a phenylpropionic acid derivative, was obtained.

[0064] 6) Structural Identification of Monomeric Compound 1

[0065] 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 2 As shown, it mainly provides relevant information of carbon element in the compound), heteronuclear single quantum relationship spectrum (HSQC, as shown in the attached Figure 3 It mainly provides direct information about hydrogen and carbon in the compound) and heteronuclear multiple correlation spectrum (HMBC, as shown in the attached Figure 4 As shown, it mainly provides the hydrogen-carbon long-range information in the compound), the purpose of which is to Figure 1-4 ) were analyzed to determine the structure of the compound. The instrument used was a Varian INOVA 500MHz nuclear magnetic resonance instrument, and the test reagent was deuterated methanol.

[0066] The monomer compound I was subjected to high resolution mass spectrometry (HRESIMS, as shown in the attached Figure 5The molecular weight and molecular formula of the compound were determined by HPLC. The instrument used was a Finnigan LCQDECA mass spectrometer.

[0067] Compound I, ethane-1,2-diyl bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propanoate), is a pale yellow oily compound; H- and C-MR spectra are shown in Table 1; high-resolution ESI mass spectrometry data: (HRESIMS) m / z 639.4026 (m / z 581.3841 [MH] - ,605.3812[M+Na] + ).

[0068] Table 1 Proton spectrum (500 MHz) and carbon spectrum (125 MHz) data of compound 1 (deuterated methanol)

[0069]

[0070] The above results show that the structure of the obtained compound is as shown in Formula I.

[0071] Example 2: Antioxidant activity of compound 1 of the present invention

[0072] (1) Experimental materials

[0073] Instruments and reagents: DPPH reagent (1,1-diphenyl-2-picrylhydrazyl, 150 μM), phosphate buffer solution (0.2 M, pH 6.6), potassium ferricyanide reagent (1% concentration), trichloroacetic acid solution (10% concentration), ferric chloride solution (1% concentration), Bio-Rad 680 microplate reader, Shimadzu UV spectrophotometer (Shimadzu UV-2401PC).

[0074] Test sample: Actinomycete secondary metabolite I, the compound was dissolved in methanol and then diluted.

[0075] (2) Experimental methods

[0076] 1) Sample preparation: Compound I was accurately weighed and dissolved in methanol to prepare a sample with an initial concentration of 1 mg / mL. Methanol was then added to dilute the sample to various concentrations for later use.

[0077] 2) DPPH (free radical scavenging) activity test: Accurately pipette 0.1 ml of the prepared sample into a 96-well plate, add 0.1 ml of DPPH reagent to each well, place in a dark place, and incubate at room temperature for 30 minutes. After incubation, place the sample under a microplate reader and record the absorbance at 520 nm. Calculate its RSA value using the formula. 0.1 ml of methanol was used as a blank control, and the same concentration of vitamin E was used as a positive control. The results are shown in Table 2:

[0078] Calculation formula: RSA (%) = [1–(A1-A2) / A0] × 100, where A1 is the absorbance value of the sample to be tested, A2 is the absorbance value without adding DPPH reagent, and A0 is the value of the methanol blank control group.

[0079] Table 2: Free radical scavenging rate (RSA%) of compound I / II

[0080]

[0081] 3) Reducing Power Activity Test: Pipette 0.5 ml of the prepared sample solution, add 0.5 ml of phosphate buffered saline and 0.5 ml of potassium ferricyanide reagent, mix well, and incubate in a 50°C water bath. After 20 minutes, add 0.5 ml of trichloroacetic acid to the mixture to terminate the reaction. Centrifuge the sample at 3000 rpm for 10 minutes, aspirate 0.5 ml of the supernatant, mix with 0.5 ml of distilled water and 0.1 ml of ferric chloride solution, incubate for 5 minutes, and record the absorbance at 700 nm using a UV spectrophotometer. The larger the value, the higher the reducing power. The results are shown in Table 3:

[0082] Table 3: Reducing power test results of compound I (absorbance at 700 nm)

[0083]

[0084] The results show that the compound I of the present invention has good in vitro antioxidant activity, can be used as an active ingredient of a natural antioxidant, and has wide applications in biomedicine and food industry.

[0085] 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 method for preparing a phenylpropionic acid derivative, characterized in that: The chemical structural formula of the phenylpropionic acid derivative is shown in Formula I: The preparation method comprises the following steps: 1) Primary extraction: The actinomycete culture is extracted with an organic solvent and then concentrated to dryness under reduced pressure at room temperature to obtain an extract; 2) Normal phase open column purification: The extract obtained in step 1) was added to a chromatographic column packed with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixture with decreasing volume ratios of ethyl acetate solvent, and the fraction eluted with the petroleum ether-ethyl acetate solvent at a volume ratio of 100:10 was selected; 3) Purification by reverse-phase medium- and low-pressure chromatography: The fraction obtained in step 2) was dissolved in methanol, added to a chromatography column packed with reverse-phase silica gel, and eluted with methanol-water solutions of increasing volume concentrations, and the fraction eluted with 70% methanol-water solution was selected; 4) Reverse-phase high performance liquid chromatography purification: The fraction obtained in step 3) is separated and purified by reverse-phase high performance liquid chromatography to obtain the phenylpropionic acid derivative.

2. The preparation method according to claim 1, characterized in that The organic solvent is ethyl acetate.

3. The preparation method according to claim 1, characterized in that Said elution is carried out in sequence by using a petroleum ether-ethyl acetate mixed solvent with a continuously decreasing volume ratio of ethyl acetate solvent, specifically: The elution was carried out in sequence using petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, petroleum ether-ethyl acetate solvent with a volume ratio of 100:12, petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent with a volume ratio of 0:

100.

4. The preparation method according to claim 3, characterized in that Said elution is carried out in sequence using methanol aqueous solution with increasing volume concentration, specifically: Elution was carried out using methanol solution with a volume concentration of 40%, methanol solution with a volume concentration of 50%, methanol solution with a volume concentration of 60%, methanol solution with a volume concentration of 70%, methanol solution with a volume concentration of 80%, methanol solution with a volume concentration of 90% and methanol with a volume concentration of 100%.

5. The preparation method according to claim 1, characterized in that The actinomycete is Streptomyces undarbansensis.

6. The preparation method according to claim 1, characterized in that The phenylpropionic acid derivative is used as an active ingredient in the preparation of antioxidant products.

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