阅读说明：本技术 一种二聚体二氢杨梅素及其制备方法和作为自由基清除剂的用途 (Dimer dihydromyricetin, preparation method thereof and application of dimer dihydromyricetin as free radical scavenger ) 是由 张友胜 秦亚茹 张业辉 焦文娟 于 2020-03-09 设计创作，主要内容包括：本发明属于天然产物应用领域,公开了一种二聚体二氢杨梅素及其制备方法和作为自由基清除剂的用途。该二聚体二氢杨梅素具有如下式(Ⅰ)所示结构。制备方法包括步骤：二氢杨梅素溶于二甲基亚砜溶液中制成0.5-5.0mol/L的DMY储备液；用磷酸盐缓冲液将DMY储备液稀释至0.01-1.0mol/L；稀释液置于37℃恒温生化培养箱中温育72h-140h；浓缩,加入甲醇,去掉溶液中析出的磷酸盐；重复操作2-3次；加入乙酸乙酯,萃取去掉二甲基亚砜；浓缩、干燥得二聚体二氢杨梅素混合物；应用制备型HPLC对二聚体二氢杨梅素混合物进行制备,以二聚体二氢杨梅素为目标产物,得到纯度超过99％的二聚体二氢杨梅素。<Image he="645" wi="571" file="DDA0002404805530000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the field of natural product application, and discloses a dimer dihydromyricetin, a preparation method thereof and an application of the dimer dihydromyricetin as a free radical scavenger, wherein the dimer dihydromyricetin has a structure shown in the following formula (I), and the preparation method comprises the steps of dissolving the dihydromyricetin in a dimethyl sulfoxide solution to prepare a DMY stock solution with the concentration of 0.5-5.0 mol/L molThe method comprises the steps of diluting DMY stock solution to 0.01-1.0 mol/L with phosphate buffer solution, placing the diluted solution in a constant-temperature biochemical incubator at 37 ℃ for incubation for 72-140 h, concentrating, adding methanol to remove phosphate separated out from the solution, repeating the operation for 2-3 times, adding ethyl acetate to extract and remove dimethyl sulfoxide, concentrating and drying to obtain a dimer dihydromyricetin mixture, and preparing the dimer dihydromyricetin mixture by using preparative HP L C to obtain the dimer dihydromyricetin with the purity of more than 99% by taking the dimer dihydromyricetin as a target product.)

1. A dimer dihydromyricetin, which is characterized in that: the dimer dihydromyricetin has a structure shown in the following formula (I):

2. the method for preparing dimeric dihydromyricetin according to claim 1, comprising the following steps:

(1) weighing dihydromyricetin with purity of more than 99%, dissolving in dimethyl sulfoxide solution, and making into DMY stock solution with concentration of 0.5-5.0 mol/L;

(2) diluting DMY stock solution to 0.01-1.0 mol/L with phosphate buffer solution with pH value of 7.4;

(3) putting the diluent obtained in the step (2) into a constant-temperature biochemical incubator at 37 ℃ for incubation for 72-140 h;

(4) concentrating the solution incubated in the step (3) to 1/3-2/3 of the original volume, then adding methanol with 2-3 times of the volume of the concentrated solution, and removing phosphate precipitated in the solution; repeating the step operation for 2-3 times;

(5) adding ethyl acetate into the solution subjected to the phosphate removal in the step (4) according to the volume of 1/3-2/3, and extracting to remove dimethyl sulfoxide in the solution;

(6) concentrating and drying the solution obtained in the step (5) after removing phosphate and dimethyl sulfoxide to obtain a dimer dihydromyricetin mixture;

(7) the preparation type HP L C is used for preparing the dimer dihydromyricetin mixture, and the dimer dihydromyricetin is used as a target product to obtain the dimer dihydromyricetin with the purity of more than 99%.

3. The method of claim 2, wherein: the incubation time in step (3) was 132 h.

4. The preparation method according to claim 2, wherein the step (7) of preparing the dimer dihydromyricetin mixture with preparative HP L C comprises using a C18 preparative column with a sample loading of 600 μ L, a flow rate of 8ml/min, a detection wavelength of 292nm, a mobile phase of methanol-water solution, and performing gradient elution with a elution procedure of 0-10min, 90-80% water, 10-20min, 80-70% water, 20-40min, 70-50% water, 40-55min, 50-90% water, 55-60min, and 90% water, a column temperature of 30 ℃ during preparation, a detection wavelength of 292nm, collecting a solution at 12-17 min during preparation, and freeze-drying to obtain dimer dihydromyricetin with a purity of more than 99%.

5. Use of dimeric dihydromyricetin according to claim 1 as a free radical scavenger.

Technical Field

The invention belongs to the field of natural product application, and particularly relates to dimer dihydromyricetin, a preparation method thereof and application thereof as a free radical scavenger.

Background

Dihydromyricetin is the main active component of Ampelopsis grossedentata (Ampelopsis grossedentata) in Ampelopsis of Vitaceae, has multiple peculiar effects of scavenging free radicals, resisting oxidation, resisting thrombus, diminishing inflammation, resisting tumor and the like, and has important significance for preventing and treating functional diseases of human, such as respiratory system, digestive system, cardiovascular system, nervous system, immune system, endocrine system and the like.

The dimer dihydromyricetin is a stable compound formed by polymerizing 2 molecules of dihydromyricetin through chemical bonds, and no research report exists at present on the preparation method and the application of the dimer dihydromyricetin.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention mainly aims to provide a dimer dihydromyricetin.

The invention also aims to provide a preparation method of the dimer dihydromyricetin.

The invention also aims to provide the application of the dimer dihydromyricetin as a free radical scavenger.

The purpose of the invention is realized by the following technical scheme:

a dimeric dihydromyricetin having the structure shown in formula (I):

the preparation method of the dimer dihydromyricetin comprises the following operation steps:

(1) weighing Dihydromyricetin (DMY) with purity of more than 99%, dissolving in dimethyl sulfoxide (DMSO) solution, and making into DMY stock solution with concentration of 0.5-5.0 mol/L;

(2) diluting DMY stock solution to 0.01-1.0 mol/L with Phosphate Buffered Saline (PBS) with pH value of 7.4;

(3) putting the diluent obtained in the step (2) into a constant-temperature biochemical incubator at 37 ℃ for incubation for 72-140 h;

(4) concentrating the solution incubated in the step (3) to 1/3-2/3 of the original volume, then adding methanol with 2-3 times of the volume of the concentrated solution, and removing phosphate precipitated in the solution; repeating the step operation for 2-3 times; (removal of phosphate from the solution as completely as possible)

(5) Adding ethyl acetate according to the volume of 1/3-2/3 into the solution subjected to the phosphate removal in the step (4), and extracting to remove dimethyl sulfoxide (DMSO) in the solution;

(6) concentrating and drying the solution obtained in the step (5) after removing phosphate and dimethyl sulfoxide to obtain a dimer dihydromyricetin mixture;

(7) the preparation type HP L C is used for preparing the dimer dihydromyricetin mixture, and the dimer dihydromyricetin is used as a target product to obtain the dimer dihydromyricetin with the purity of more than 99%.

The incubation time in step (3) was 132 h.

The specific step of preparing the dimer dihydromyricetin mixture by using the preparative HP L C in the step (7) is to use C₁₈Preparing column with sample amount of 600 μ L, flow rate of 8ml/min, detection wavelength of 292nm, mobile phase of methanol-water solution, gradient eluting with water solution of 90-80% for 0-10min, 80-70% for 10-20min, 20-40min, 70-50% for 40-55min, 50-90% for 55-60min, 90% for 40-55min, column temperature of 30 deg.C, wavelength of 292nm, collecting solution of 12-17 min, freeze dryingDrying to obtain dimer dihydromyricetin with purity over 99%.

The dimer dihydromyricetin can be used as free radical scavenger in the fields of food, medicine and cosmetics.

The principle of the invention is as follows:

under neutral or weakly alkaline conditions (pH 7.4), DMY reacts with molecular oxygen to form DMY radicals (DMY. DMYRadical and superoxide radical (. O.)^2-). The superoxide radical can react with the next molecule of DMY to form DMY and hydrogen peroxide (H)₂O₂). DMY may also react with molecular oxygen to form DMY quinone (DMY quinone). The DMY quinone reacts with another molecule of DMY to form DMY dimer. Two molecules of DMY quinone may also polymerize to form a dimer (DMY dimer). Yet another possible mechanism of formation is the direct generation of superoxide radicals by molecular oxygen, which react with a molecule of DMY to form DMY and H₂O₂. DMY-reacts with another molecule of DMY to form DMY quinone and superoxide radical, which in turn forms a dimer (see figure 1). The dimer dihydromyricetin is a compound formed by combining 2 molecules of dihydromyricetin through covalent bonds, and like other flavonoid compounds, the molecular structure also has a 3',4' -ortho-position dihydroxy structure possessed by antioxidant substances. In addition, the dihydromyricetin is polymerized together, so that the dihydromyricetin is more stable than dihydromyricetin, and the dihydromyricetin has better stability when being used as an antioxidant in the development and utilization processes.

Compared with the prior art, the invention has the following advantages and effects:

the dimer dihydromyricetin is a polymer formed by combining 2 molecules of dihydromyricetin in a covalent bond mode, and is an important raw material required for researching the oxidative decomposition and metabolic pathway of the dihydromyricetin; in addition, the dimer dihydromyricetin can play an anti-oxidation role in the fields of food, medicine and cosmetics. At present, no report exists about the preparation method and application of the dimer.

Drawings

FIG. 1 is a schematic diagram of a possible DMY degradation to produce DMY dimer.

FIG. 2 is a high performance liquid chromatogram (37 ℃, pH7.4) of dihydromyricetin in PBS solution at different incubation times.

FIG. 3 is a high performance liquid chromatogram of dihydromyricetin and its degradation products in PBS (37 ℃, pH 7.4).

FIG. 4 shows the secondary mass spectrum and possible cleavage pattern of the main degradation product of dihydromyricetin.

FIG. 5 is a diagram showing the collection of dimer dihydromyricetin prepared from HP L C.

FIG. 6 is a graph showing the scavenging effect of dimer dihydromyricetin mixture on ABTS free radicals.

Detailed description of the invention

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.