A oryzanol derivative and its constituent salts, preparation method, natural antioxidant and application

Abstract

This invention provides a oryzanol derivative, its salts, a preparation method, a natural antioxidant, and its applications, belonging to the fields of medicine and food industry. The embodiments of this invention provide the oryzanol derivative and its salts, wherein the oryzanol derivative is a first compound cycloart-24-oxo-25-ene-3β-ol-3β-transferulate or a second compound cycloart-24-oxo-25-ene-3β-ol-3β-cis-ferulate. The oryzanol derivatives provided by this invention have good in vitro antioxidant activity and can be used as active ingredients in antioxidants, possessing good practical applications.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, health and food industry, and particularly relates to a oryzanol derivative, a salt formed by the oryzanol derivative, a preparation method, a natural antioxidant and application. BACKGROUND

[0002] Anti-oxidation is short for Anti-Oxidant. The human body produces free radicals due to continuous contact with the outside world, including breathing (oxidation reaction), external pollution, radiation exposure and other factors. Scientific research shows that cancer, aging or other diseases are mostly associated with the production of excess free radicals. Research on anti-oxidation can effectively overcome the harm caused by free radicals, so anti-oxidants are listed as one of the main research and development directions by health care and cosmetic companies, and are also one of the most important functional demands in the market. Anti-oxidation is any substance that can effectively inhibit the oxidation reaction of free radicals at a low concentration. Its mechanism of action can be directly acting on free radicals, or indirectly consuming substances that are prone to generate free radicals to prevent further reactions. While the human body inevitably produces free radicals, it also naturally produces anti-oxidants to counteract the oxidation attack of free radicals on human cells. Studies have shown that the human body's anti-oxidation system is a system with perfect and complex functions comparable to the immune system. The stronger the body's anti-oxidation ability, the healthier and longer the life.

[0003] With the development of economy and technology, the commonly used synthetic antioxidants have toxic side effects and have bad effects on the human heart, lungs, liver and other organs. They are not resistant to high temperature and have poor thermal stability. They also have low antioxidant efficiency, experimental labels, narrow application range (export restrictions), poor bacteriostatic effect and other shortcomings. Therefore, they have been limited or prohibited in many countries, such as BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), PG (propyl gallate) and TBHQ (tert-butyl hydroquinone). Studies have shown that natural food antioxidants have significantly lower toxicity than artificially synthesized antioxidants, which makes people pay more attention to finding natural antioxidants from nature. In recent years, scientists have increased their research in this field, especially focusing on exploring effective ingredients in nature. In the global market, a large number of natural antioxidant products have emerged, and are widely favored by consumers. Among them, representative natural antioxidants include vitamin E (VE), astaxanthin from shrimp shells, black rice extract, red pepper extract from chili peppers, spice extract from spices, and sugar alcohol antioxidants. These products have gradually become new favorites in the health food market due to their natural and safe characteristics. Consumers are increasingly inclined to choose products rich in natural antioxidants when choosing food, believing that they are not only healthy but also provide more pure nutritional value. Therefore, natural antioxidants will continue to maintain strong demand in the future. SUMMARY

[0004] The present application aims to at least partially solve the above technical problems, and aims to provide a phytosterol derivative and its possible salt, a preparation method, a natural antioxidant and an application. According to the in-vitro activity results, the phytosterol derivative has good antioxidant activity in-vitro.

[0005] In a first aspect of the present application, a phytosterol derivative and its possible salt are provided, the phytosterol derivative being a first compound cycloart-24-oxo-25-ene-3β-ol-3β-trans-ferulate or a second compound cycloart-24-oxo-25-ene-3β-ol-3β-cis-ferulate,

[0006] The first compound has a structural formula I, and the second compound has a structural formula II:

[0007]

[0008] In a second aspect of the present application, a preparation method of the phytosterol derivative is provided, and the preparation method comprises the following steps:

[0009] 1) Preliminary extraction: the plant is extracted by a first organic solvent, and then concentrated to dryness under reduced pressure at room temperature to obtain a plant extract;

[0010] 2) Solvent extraction: the plant extract is suspended in an aqueous solution, and then subjected to liquid-liquid extraction by using a second organic solvent, and then concentrated to dryness under reduced pressure at room temperature to obtain an organic solvent extract and a water extract;

[0011] 3) Normal phase open column purification: the organic solvent extract obtained in step 2) is added to a chromatographic column filled with normal phase silica gel, and then eluted with petroleum ether-ethyl acetate series solvents with increasing volume ratios of ethyl acetate, and then the flow fraction eluted by a predetermined volume ratio of petroleum ether-ethyl acetate is taken;

[0012] 4) Reverse phase medium-low pressure column purification: the flow fraction obtained in step 3) is dissolved in a predetermined amount of methanol, and then added to a chromatographic column filled with reverse phase silica gel, and then eluted with methanol series solutions with increasing volume concentrations, and then the flow fraction eluted by a predetermined volume concentration of methanol is taken;

[0013] 5) Reverse phase high performance liquid chromatography purification: the flow fraction obtained in step 4) is separated and purified by reverse phase high performance liquid chromatography to obtain the phytosterol derivative.

[0014] In some embodiments, the petroleum ether-ethyl acetate series solvents with increasing volume ratios of ethyl acetate are:

[0015] petroleum ether-ethyl acetate solvent in a volume ratio of 100:0, petroleum ether-ethyl acetate solvent in a volume ratio of 100:2, petroleum ether-ethyl acetate solvent in a volume ratio of 100:4, petroleum ether-ethyl acetate solvent in a volume ratio of 100:5, petroleum ether-ethyl acetate solvent in a volume ratio of 100:8, petroleum ether-ethyl acetate solvent in a volume ratio of 100:10, petroleum ether-ethyl acetate solvent in a volume ratio of 100:20, petroleum ether-ethyl acetate solvent in a volume ratio of 100:100, petroleum ether-ethyl acetate solvent in a volume ratio of 0:100.

[0016] In some embodiments, the predetermined volume ratio of petroleum ether-ethyl acetate solvent is petroleum ether-ethyl acetate solvent in a volume ratio of 100:5.

[0017] In some embodiments, the series of methanol solutions with increasing volume concentrations are a 40% volume concentration methanol solution, a 50% volume concentration methanol solution, a 60% volume concentration methanol solution, a 70% volume concentration methanol solution, an 80% volume concentration methanol solution, a 90% volume concentration methanol solution, and a 100% volume concentration methanol solution.

[0018] The predetermined volume concentration of the methanol solution is a 90% volume concentration methanol solution.

[0019] In some embodiments, the plant is a mature seed shell of rice.

[0020] In some embodiments, the first organic solvent is anhydrous ethanol.

[0021] The second organic solvent is ethyl acetate.

[0022] In a third aspect of the present application, there is provided a natural antioxidant comprising an active ingredient and a pharmaceutically acceptable carrier, the active ingredient being a oribec derivative as described above or a salt thereof or an oribec derivative prepared according to the preparation method described above.

[0023] In some embodiments, the pharmaceutically acceptable carrier is selected from any one or any combination of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorptive carriers, and lubricants.

[0024] In a fourth aspect of the present application, there is provided a use of an oribec derivative or a salt thereof in a natural antioxidant, the oribec derivative being an oribec derivative as described above or a salt thereof or an oribec derivative prepared according to the preparation method described above.

[0025] The oat fiber derivative, the salt thereof, the preparation method, the natural antioxidant and the application according to the embodiments of the present application have at least one of the following advantages:

[0026] The embodiments of the present application provide a preparation method of a new oat fiber derivative and the use thereof, and the oat fiber derivative provided by the present application has good in-vitro antioxidant activity, can be used as an active ingredient of an antioxidant, and has a wide range of applications. BRIEF DESCRIPTION OF DRAWINGS

[0027] These and / or other aspects and advantages of the present application will become apparent and readily understood from the following description, taken in connection with the accompanying drawings, in which:

[0028] Figure 1 HPLC liquid chromatogram of the oat fiber derivative prepared according to the embodiment 1 of the present application;

[0029] Figure 2 Hydrogen spectrum data graph of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0030] Figure 3 Carbon spectrum data graph of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0031] Figure 4 Two-dimensional nuclear magnetic resonance data graph (COSY) of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0032] Figure 5 Two-dimensional nuclear magnetic resonance data graph (HSQC) of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0033] Figure 6 Two-dimensional nuclear magnetic resonance data graph (HMBC) of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0034] Figure 7 Two-dimensional nuclear magnetic resonance data graph (NOESY) of the oat fiber derivative obtained according to the embodiment 1 of the present application;

[0035] Figure 8 High-resolution mass spectrum (HRESIMS) data graph of the oat fiber derivative obtained according to the embodiment 1 of the present application. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further specifically explained below by examples in combination with the drawings. In the description, same or similar reference numerals indicate same or similar components. The following description of the embodiments of the present application with reference to the drawings is intended to explain the general inventive concept of the present application, and should not be understood as a limitation of the present application.

[0037] In order to further illustrate the technical solutions of the present application, the phytosterol derivative and the preparation method thereof provided by the present application are described in detail below in combination with examples.

[0038] In one embodiment, a phytosterol derivative is provided, which is a first compound cycloart-24-oxo-25-ene-3β-ol-3β-trans-ferulate or a second compound cycloart-24-oxo-25-ene-3β-ol-3β-cis-ferulate,

[0039] The first compound has a structural formula I, and the second compound has a structural formula II:

[0040]

[0041] Correspondingly, a preparation method of the above-mentioned phytosterol derivative is also provided, which comprises the following steps:

[0042] 1) Preliminary extraction: after the plant is extracted by a first organic solvent, it is concentrated to dryness under reduced pressure at room temperature to obtain a plant extract;

[0043] 2) Solvent extraction: the plant extract is suspended in an aqueous solution, liquid-liquid extraction is performed by using a second organic solvent, and the mixture is concentrated to dryness under reduced pressure at room temperature to obtain an ethyl acetate extract and a water extract;

[0044] 3) Normal phase open column purification: the ethyl acetate extract obtained in step 2) is added to a chromatographic column filled with normal phase silica gel, and eluted with petroleum ether-ethyl acetate series solvents with increasing volume ratios of ethyl acetate, and the flow fraction eluted by a predetermined volume ratio of petroleum ether-ethyl acetate is taken;

[0045] 4) Reverse phase medium-low pressure column purification: the flow fraction obtained in step 3) is dissolved in a predetermined amount of methanol, and added to a chromatographic column filled with reverse phase silica gel, and eluted with methanol series solutions with increasing volume concentrations, and the flow fraction eluted by a predetermined volume concentration of methanol is taken;

[0046] 5) Reverse phase high performance liquid chromatography purification: the flow fraction obtained in step 4) is separated and purified by reverse phase high performance liquid chromatography to obtain the phytosterol derivative.

[0047] In step 1), the plant is a mature seed shell of rice; the first organic solvent is anhydrous ethanol.

[0048] In step 2), the second organic solvent is ethyl acetate.

[0049] In step 3), the ethyl acetate series solvents with increasing volume ratio are: petroleum ether-ethyl acetate solvent with a volume ratio of 100:0, petroleum ether-ethyl acetate solvent with a volume ratio of 100:2, petroleum ether-ethyl acetate solvent with a volume ratio of 100:4, petroleum ether-ethyl acetate solvent with a volume ratio of 100:5, petroleum ether-ethyl acetate solvent with a volume ratio of 100:8, petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, petroleum ether-ethyl acetate solvent with a volume ratio of 0:100.

[0050] The predetermined petroleum ether-ethyl acetate solvent is petroleum ether-ethyl acetate solvent with a volume ratio of 100:5.

[0051] In step 4), the methanol series solutions with increasing volume concentration are: 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 solution with a volume concentration of 100%.

[0052] The predetermined methanol solution is methanol solution with a volume concentration of 90%.

[0053] In another embodiment, a pharmaceutically acceptable salt of a derivative of oryzanol is also provided, wherein the derivative of oryzanol is the derivative of oryzanol described above or prepared according to the preparation method described above.

[0054] In addition, a natural antioxidant is also provided, which comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the derivative of oryzanol described above or prepared according to the preparation method described above or the pharmaceutically acceptable salt of the derivative of oryzanol described above.

[0055] The pharmaceutically acceptable carrier is selected from any one or any combination of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0056] Finally, there is provided the use of a derivative of oroselamine or a salt formable from a derivative of oroselamine in a natural antioxidant, the derivative of oroselamine being a derivative of oroselamine prepared according to the above or according to the above preparation method, the salt formable from a derivative of oroselamine being a salt formable from a derivative of oroselamine according to the above.

[0057] Preparation of derivatives of oroselamine of structural formula I and structural formula II

[0058] The present application uses conventional extraction, purification and other steps under laboratory culture conditions to prepare the compounds of the present application. The plant used is the mature seed coat of Oryza sativa L.

[0059] The process for a specific compound is as follows:

[0060] 1) Extraction

[0061] Fresh mature seed coat of Oryza sativa L. (10 kg) was collected, crushed and extracted with anhydrous ethanol (5 L*3) at room temperature. The extract was concentrated to dryness under reduced pressure to obtain about 187 g of residue.

[0062] 2) Extraction

[0063] The extract was suspended in 1.5 liters of water and liquid-liquid extraction was performed with an equal volume of ethyl acetate. The ethyl acetate layer was collected and concentrated to dryness under reduced pressure to obtain 83 g of ethyl acetate extract.

[0064] 3) Normal phase silica gel column chromatography separation

[0065] The ethyl acetate extract was loaded onto a silica gel column containing 200 g of normal phase silica gel (200-300 mesh) and eluted with a petroleum ether-ethyl acetate system gradient (100:0-0:100). The elution fractions were collected, with the petroleum ether-ethyl acetate fraction at a volume ratio of 100:5 being the main collection fraction.

[0066] 4) Reverse phase silica gel column chromatography purification

[0067] The selected fraction was dissolved in an appropriate amount of methanol and loaded onto a column containing 120 g of reverse phase silica gel (120 angstrom, 30-50 mesh) and eluted with a methanol-water system gradient (40%-100% methanol). The elution fractions were collected, with the 90% fraction being the main collection fraction.

[0068] 5) Reverse phase high performance liquid chromatography purification

[0069] The selected fractions were analyzed and purified by reverse phase high performance liquid chromatography. The analytical conditions were as follows: column Hedera C18A-5 μm, 4.6 mm I.D x 250 mm (Jiangsu Hanbon Science and Technology), elution system was methanol-water isocratic elution, specific conditions: methanol-water (90:10, V / V), flow rate was 2.0 mL / min. The detection wavelength was 220 nm, column temperature was 25 °C, injection volume was 20 μL. The secondary metabolites with structural formula of formula I, formula II, i.e. derivatives of GOS, were obtained.

[0070] Compound I, cycloart-24-oxo-25-ene-3β-ol-3β-trans-ferulate, white amorphous powder: compound optical rotation; [α] +27° (c 0.2 CHCl3); nuclear magnetic resonance hydrogen spectrum and carbon spectrum data, see Table 1; high resolution ESI mass spectrum data: (HRESIMS) m / z 639.4026 ([M+Na]+, calc. for C40H56O5639.4025).

[0071] Compound II, cycloart-24-oxo-25-ene-3β-ol-3β-cis-ferulate, white amorphous powder: compound optical rotation; [α] +27° (c 0.2 CHCl3); nuclear magnetic resonance hydrogen spectrum and carbon spectrum data, see Table 1; high resolution ESI mass spectrum data: (HRESIMS) m / z 639.4026 ([M+Na]+, calc. for C40H56O5639.4025).

[0072] Table 1 Hydrogen spectrum (500 MHz) and carbon spectrum (125 MHz) data of compounds I and II (deuterated chloroform)

[0073]

[0074] The above results show that the obtained compounds have structures as shown in formula I and II.

[0075] Example 2: Antioxidant activity of compounds I and II of the application

[0076] (1) Experimental materials

[0077] 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 trichloride solution (1% concentration), Bio-Rad 680 enzyme marker, Shimadzu UV-2401PC ultraviolet spectrophotometer.

[0078] Test samples: Rice mature seed coat metabolites I and II, the compounds were dissolved in methanol and then diluted.

[0079] (2) Experimental methods

[0080] 1) Sample preparation: Accurately weigh the mixture of compound I / II, dissolve it in methanol solution to prepare a sample with an initial concentration of 1 mg / mL, and dilute it with methanol to prepare samples of different concentrations for later use.

[0081] 2) DPPH (Free Radical Scavenger) Activity Assay: Accurately pipette 0.1 mL of the prepared sample into each well of a 96-well plate, add 0.1 mL of DPPH reagent to each well, and incubate at room temperature for 30 minutes in the dark. After incubation, place the sample under a microplate reader and record the absorbance at 520 nm. Calculate the RSA value using the formula. 0.1 mL of methanol was used as a blank control, and vitamin E of the same concentration was used as a positive control. The results are shown in Table 2.

[0082] 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 DPPH reagent, and A0 is the value of the methanol blank control group.

[0083] Table 2: Free radical scavenging rates (RSA%) of compounds I / II

[0084]

[0085] 3) Reducing Power Activity Test: Take 0.5 mL of the prepared sample solution, add 0.5 mL of phosphate buffer solution and 0.5 mL of potassium ferricyanide reagent, mix well, and incubate in a 50°C water bath for 20 minutes. Then, 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 it with 0.5 mL of distilled water and 0.1 mL of ferric chloride solution, and incubate for a total of 5 minutes. Record the absorbance at 700 nm using a UV spectrophotometer; a higher value indicates higher reducing power. The results are shown in Table 3.

[0086] Table 3: Reducing power test results of compounds I / II (absorbance at 700 nm)

[0087]

[0088]

[0089] The results show that compounds I and II of the present invention have certain in vitro antioxidant activity and can be used as active ingredients of natural antioxidants, with wide applications in biomedicine and food industry.

[0090] The oryzanol derivative, its preparation method, its salts, natural antioxidants, and its application in natural antioxidants according to embodiments of the present invention have at least one of the following advantages:

[0091] The embodiments of the present invention provide a new use for oryzanol derivatives. In vitro experiments have confirmed that the oryzanol derivatives provided by the present invention have good in vitro antioxidant activity and can be used as active ingredients of antioxidants, with a wide range of applications.

[0092] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand 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 derivative of oryzanol, the derivative of oryzanol being a first compound cycloart-24-oxo-25-ene-3β-ol-3β-trans-ferulate or a second compound cycloart-24-oxo-25-ene-3β-ol-3β-cis-ferulate, the first compound having a structural formula I, and the second compound having a structural formula II: the derivative of oryzanol and a salt thereof being prepared by a method comprising the following steps: 1) preliminary extraction: extracting a plant with a first organic solvent, and concentrating the plant extract to dryness under reduced pressure at room temperature to obtain a plant extract, the plant being a mature husk of Oryza sativa L.; 2) solvent extraction: suspending the plant extract in an aqueous solution, and performing liquid-liquid extraction with a second organic solvent, and concentrating the organic solvent extract to dryness under reduced pressure at room temperature to obtain an organic solvent extract and an aqueous extract; 3) normal phase open column chromatography purification: adding the organic solvent extract obtained in step 2) to a chromatographic column filled with normal phase silica gel, eluting the chromatographic column with ethyl acetate-petroleum ether series solvents with increasing volume ratios of ethyl acetate, and collecting fractions eluted with a predetermined volume ratio of petroleum ether-ethyl acetate; 4) reverse phase medium-low pressure column chromatography purification: dissolving the fractions obtained in step 3) in a predetermined amount of methanol, and adding the solution to a chromatographic column filled with reverse phase silica gel, eluting the chromatographic column with methanol series solutions with increasing volume concentrations of methanol, and collecting fractions eluted with a predetermined volume concentration of methanol; and 5) reverse phase high performance liquid chromatography purification: separating and purifying the fractions obtained in step 4) by reverse phase high performance liquid chromatography to obtain the derivative of oryzanol. 3.The method of claim 2, wherein the ethyl acetate-petroleum ether series solvents with increasing volume ratios of ethyl acetate are: petroleum ether-ethyl acetate solvents with volume ratios of 100:0, 100:2, 100:4, 100:5, 100:8, 100:10, 100:20, 100:100, and 0:

100.

2. A method for producing the oryzanol derivative according to claim 1, characterized by, 4.The method of claim 3, wherein the petroleum ether-ethyl acetate solvent with the predetermined volume ratio is a petroleum ether-ethyl acetate solvent with a volume ratio of 100:

5. 5.The method of claim 4, wherein the methanol series solutions with increasing volume concentrations of methanol are methanol solutions with volume concentrations of 40%, 50%, 60%, 70%, 80%, 90%, and 100%. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The predetermined volume concentration of the methanol solution is a 90% volume concentration of methanol solution. 6.The method of claim 5, wherein the first organic solvent is anhydrous ethanol. The first organic solvent is anhydrous ethanol. The second organic solvent is ethyl acetate.

7. A natural antioxidant characterized in that, The antioxidant comprises an active ingredient which is the orpivaline derivative according to claim 1 and a pharmaceutically acceptable carrier. 8.The natural antioxidant of claim 7, wherein the pharmaceutically acceptable carrier is selected from any one or any combination of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorptive carriers, and lubricants. 9.Use of the orpivaline derivative and the pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a natural antioxidant. ​

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