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

Abstract

The invention provides a phenylpropionic acid derivative, a preparation method thereof, an antioxidant and application, and belongs to the field of medicine and health and food industry. The phenylpropionic acid derivative is ethane-1, 2-diyl bis (3-(3, 5-di-tert-butyl-4-hydroxyphenyl)-propanoate, and the structural formula of the phenylpropionic acid derivative is shown in the specification, and the structural formula of the phenylpropionic acid derivative is shown in the specification, namely ethane-1, 2-diyl bis (3-(3, 5-di-tert-butyl-4-hydroxyphenyl)-propanoate. The invention provides a new application of the phenylpropionic acid derivative, and in-vitro tests prove that the phenylpropionic acid derivative provided by the invention has better in-vitro antioxidant activity, can be used as an active component of an antioxidant, and has wide application.

Description

Technical Field

[0001] The 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 is constantly exposed to the outside world, including breathing (oxidation reaction), external pollution, radiation exposure and other factors, which constantly produce free radicals in the human body. Scientific research shows that cancer, aging or other diseases are mostly related to the production of excessive free radicals. Research on antioxidants can effectively overcome the harm caused by them, so antioxidants are listed as one of the main research and development directions by health care products and cosmetics companies, and are also one of the most important functional demands of the market.

[0003] Antioxidants are any substances that can effectively inhibit the oxidation reaction of free radicals at low concentrations. Their mechanism of action can be to act directly on free radicals, or to indirectly consume substances that easily generate free radicals to prevent further reactions. While the human body inevitably produces free radicals, it also naturally produces antioxidant substances that resist free radicals to offset the oxidative attack of free radicals on human cells. Studies have shown that the human body's antioxidant system is a system with complete and complex functions comparable to the immune system. The stronger the body's antioxidant ability, the healthier it is and the longer its life will be.

[0004] With the development of economy and technology, commonly used synthetic antioxidants have toxic side effects, which have adverse effects on the human heart, lungs, liver, etc. They are not resistant to high temperatures and have poor thermal stability. The shortcomings of low antioxidant efficiency, experimental labeling, narrow application range (export restrictions), and poor antibacterial effect have emerged, so they have been restricted or banned in many countries.

[0005] Studies have shown that natural food antioxidants are significantly less toxic than synthetic antioxidants, which has led to more attention to finding antioxidants from natural sources in nature. In recent years, scientists have increased their research enthusiasm in this field, especially focusing on discovering effective ingredients in nature. Globally, many natural antioxidant products have emerged and are widely favored by consumers. Among them, representative natural antioxidants include vitamin E (VE), astaxanthin from shrimp shells, melanoidin extracted from black rice, red pepper extract from peppers, spice extracts extracted from spices, and sugar alcohol antioxidants. These products have gradually become the new darlings of the health food market with their natural and safe characteristics. When choosing food, consumers are increasingly inclined to choose products rich in natural antioxidants, believing that they are not only good for health, but also provide more pure nutritional value. Therefore, the development trend of natural antioxidants will continue to remain strong in the future, while also driving the food industry towards a healthier and more environmentally friendly direction. Summary of the invention

[0006] The purpose of the present invention is to at least partially solve the above-mentioned technical problems, and aims 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 the 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 1:

[0008]

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

[0010] 1) Preliminary extraction: extract the actinomycete culture with an organic solvent and then concentrate 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) is added to a chromatographic column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixed solvent with a decreasing volume ratio of ethyl acetate solvent, and the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:10 is taken;

[0012] 3) Purification by reverse phase medium and low pressure chromatography column: the fraction obtained in step 2) is dissolved in methanol, added to a chromatography column filled with reverse phase silica gel, and eluted with methanol aqueous solution with increasing volume concentration, and the fraction eluted with methanol aqueous solution with a volume concentration of 70% is taken;

[0013] 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 a phenylpropionic acid derivative, whose structure 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 in a volume ratio of 100:5, petroleum ether-ethyl acetate solvent in a volume ratio of 100:10, petroleum ether-ethyl acetate solvent in a volume ratio of 100:12, petroleum ether-ethyl acetate solvent in a volume ratio of 100:20, petroleum ether-ethyl acetate solvent in a volume ratio of 100:30, petroleum ether-ethyl acetate solvent in a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent in a volume ratio of 0:100.

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

[0018] The 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 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.

[0023] A phenylpropionic acid derivative and its salifiable salt, 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 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 a carbon spectrum data diagram 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 The 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 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.

[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 in conjunction with the embodiments.

[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 preparation method comprising the following steps:

[0037] 1) Preliminary extraction: extract the actinomycete culture with an organic solvent and then concentrate 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) is added to a chromatographic column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixed solvent with a decreasing volume ratio of ethyl acetate solvent, and the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:10 is taken;

[0039] 3) Purification by reverse phase medium and low pressure chromatography column: the fraction obtained in step 2) is dissolved in methanol, added to a chromatography column filled with reverse phase silica gel, and eluted with methanol aqueous solution with increasing volume concentration, and the fraction eluted with methanol aqueous solution with a volume concentration of 70% is taken;

[0040] 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 a phenylpropionic acid derivative, whose structure 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 described here.

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

[0043] The elution was carried out in sequence using petroleum ether-ethyl acetate solvent in a volume ratio of 100:5, petroleum ether-ethyl acetate solvent in a volume ratio of 100:10, petroleum ether-ethyl acetate solvent in a volume ratio of 100:12, petroleum ether-ethyl acetate solvent in a volume ratio of 100:20, petroleum ether-ethyl acetate solvent in a volume ratio of 100:30, petroleum ether-ethyl acetate solvent in a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent in 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] The 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, wherein 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 invention also provides a use of a phenylpropionic acid derivative or a salt that can be formed from a phenylpropionic acid derivative 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 that can be formed from the phenylpropionic acid derivative is a salt that can be formed from 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] Example 1 of the present invention adopts laboratory culture, extraction under conventional conditions, purification and other steps to prepare the compound of the present invention. The microorganism used is the actinomycete Streptomyces sundarbansensis.

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

[0054] 1) Cultivation

[0055] 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.

[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 equipped with 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, among which 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, 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 70% were the main collected fractions.

[0062] 5) Reverse-phase HPLC 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×250mm (Jiangsu Hanbang Technology), the elution system is methanol-water isocratic elution, the specific conditions are: methanol-water (65:35, V / V), the flow rate is 3.0mL / min. The detection wavelength is 254nm, and the injection volume is 20μL. The secondary metabolite with the structural formula I is obtained, that is, a phenylpropionic acid derivative.

[0064] 6) Structural Identification of Monomer 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 the hydrogen element in the compound). 13 C NMR spectrum (carbon spectrum, as attached Figure 2 HSQC, as shown in the attached Figure 3 It mainly provides direct information about hydrogen and carbon in compounds) and heteronuclear multi-correlation spectrum (HMBC, as shown in the attached Figure 4 As shown, it mainly provides information related to hydrogen-carbon long-range in compounds. Its purpose is to Figure 1-4 ) to determine the structure of the compound. The instrument used was a Varian INOVA 500MHz nuclear magnetic resonance instrument, and the test reagent used 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 using a Finnigan LCQDECA mass spectrometer.

[0067] Compound 1, ethane-1,2-diyl bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propanoate), light yellow oily compound; HMR and CMR data, see 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 Hydrogen 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 1.

[0071] Example 2: Antioxidant activity of compound I 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 ferrocyanide 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 metabolites 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 solution to prepare a sample with an initial concentration of 1 mg / mL, and methanol was added to dilute the sample into samples of different 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 the incubation, place the sample under an ELISA 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 solution, 0.5 ml of potassium ferricyanide reagent, mix well and place in a 50°C water bath for incubation. 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, take 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 use a UV spectrophotometer to record the absorbance at 700 nm. 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 700nm)

[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 a wide range of applications in biomedicine and food industries.

[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 by the claims and their equivalents.

Claims

1. A phenylpropionic acid derivative, 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:

2. A method for preparing a phenylpropionic acid derivative as claimed in claim 1, characterized in that: The preparation method comprises the following steps: 1) Preliminary extraction: extract the actinomycete culture with an organic solvent and then concentrate to dryness under reduced pressure at room temperature to obtain an extract; 2) Normal phase open column purification: the extract obtained in step 1) is added to a chromatographic column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate mixed solvent with a decreasing volume ratio of ethyl acetate solvent, 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: the fraction obtained in step 2) is dissolved in methanol, added to a chromatography column filled with reverse phase silica gel, and eluted with methanol aqueous solution with increasing volume concentration, and the fraction eluted with methanol aqueous solution with a volume concentration of 70% is taken; 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 a phenylpropionic acid derivative, whose structure is shown in Formula 1.

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

4. The preparation method according to claim 2, characterized in that: The elution is carried out sequentially using a petroleum ether-ethyl acetate mixed solvent with a decreasing volume ratio of ethyl acetate solvent, specifically: The elution was carried out in sequence using petroleum ether-ethyl acetate solvent in a volume ratio of 100:5, petroleum ether-ethyl acetate solvent in a volume ratio of 100:10, petroleum ether-ethyl acetate solvent in a volume ratio of 100:12, petroleum ether-ethyl acetate solvent in a volume ratio of 100:20, petroleum ether-ethyl acetate solvent in a volume ratio of 100:30, petroleum ether-ethyl acetate solvent in a volume ratio of 100:100, and petroleum ether-ethyl acetate solvent in a volume ratio of 0:

100.

5. The preparation method according to claim 4, characterized in that: The elution is carried out sequentially using methanol aqueous solution with increasing volume concentration, specifically: The 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%.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The actinomycete is Streptomyces sundarbansensis.

7. Use of the phenylpropionic acid derivative according to claim 1 or the phenylpropionic acid derivative prepared by the preparation method according to any one of claims 2 to 5 as an active ingredient in the preparation of an antioxidant product.

8. An antioxidant, characterized in that The antioxidant comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the phenylpropionic acid derivative according to claim 1 or a salt thereof, or the phenylpropionic acid derivative prepared by the preparation method according to any one of claims 2-5.

9. The antioxidant according to claim 8, 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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