阅读说明：本技术 一种具有抗氧化活性的新酚酸类化合物的制备方法及应用 (Preparation method and application of novel phenolic acid compound with antioxidant activity ) 是由 李志峰 冯育林 王�琦 杨世林 杜伟东 黄文平 游梦 于 2020-07-14 设计创作，主要内容包括：本发明涉及一种新的酚酸类化合物在制备抗氧化药物中的应用,发明还涉及从中药小花清风藤中提取、分离、纯化本化合物的制备方法。该制备方法简单易行,所得化合物纯度高。药理研究表明该新化合物具有较好的体外清除自由基的能力,可以降低H<Sub>2</Sub>O<Sub>2</Sub>诱导的巨噬细胞RAW264.7内活性氧(reactive oxygen species,ROS)的生成。此外,还可以升高细胞内超氧化物歧化酶(SOD)的活性。表明本发明所述化合物具有明显的抗氧化活性,可用于抗氧化药物的制备。具有良好的经济效益和应用前景。(The invention relates to application of a novel phenolic acid compound in preparing an antioxidant medicine, and also relates to a preparation method for extracting, separating and purifying the compound from a traditional Chinese medicine caulis sinomenii. The preparation method is simple and feasible, and the obtained compound has high purity. Pharmacological research shows that the new compound has better capacity of eliminating free radicals in vitro and can reduce H 2 O 2 Induced generation of Reactive Oxygen Species (ROS) within macrophage RAW 264.7. In addition, the activity of intracellular superoxide dismutase (SOD) can be increased. Show thatThe compound of the invention has obvious antioxidant activity and can be used for preparing antioxidant drugs. Has good economic benefit and application prospect.)

1. A phenolic acid compound with antioxidant activity is characterized in that: the name is: 5- (3-carboxy-4-hydroxy) -5-oxo-2-methyl-2-pentenoic acid having the formula:

。

2. the method for preparing phenolic acid compounds with antioxidant activity according to claim 1, wherein the phenolic acid compounds are selected from the group consisting of: the method comprises the following specific steps:

s1, extracting the dried stems and leaves of the caulis Sinomenii with 70% ethanol for three times, wherein the mass ratio of the stems and leaves of the caulis Sinomenii to the solvent is about 1: (10-16), concentrating until no alcohol smell exists to obtain an extract;

s2, loading the obtained extract on macroporous resin, and eluting with 30%, 50%, 70%, 95% and ethanol respectively;

s3 separating the part eluted by 30% ethanol in HP-20 resin, separating the obtained extract by macroporous resin, reversed phase column chromatography, semi-preparative high performance liquid chromatography and preparative high performance liquid chromatography in turn to obtain the compound.

3. The method for preparing phenolic acid compounds with antioxidant activity according to claim 2, wherein: the extraction method in the step S1 includes a cold soaking method, a percolation method, a microwave extraction method, an ultrasonic extraction method, a reflux extraction method and a continuous reflux extraction method.

4. The method for preparing phenolic acid compounds with antioxidant activity as claimed in claim 2, wherein the macroporous resin comprises the following types: HP-20, HP-21, SP850, SP700, SP207, AB-8, D101, XDA1, X-5, NKA-2, ADS-2.

5. The method according to claim 2, wherein the reverse phase column chromatography comprises low pressure preparative chromatography, medium pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography, wherein the mobile phase used in the reverse phase chromatography is an aqueous solution of an organic solvent, and the organic solvent is methanol or acetonitrile.

6. The method for preparing phenolic acid compounds with antioxidant activity according to claim 2, wherein the elution of the reversed phase column chromatography is gradient elution, and the gradient elution procedure is as follows: 20%, 30%, 40%, 50%, 60%, 70%, 80% aqueous solution of organic solvent, eluted 2 column volumes each.

7. The method for preparing phenolic acid compounds with antioxidant activity as claimed in claim 2, wherein the liquid phase conditions are: a chromatographic column: YMC-Pack ODS-A column, mobile phase: 10% -30% acetonitrile, wavelength: 540nm and 210nm, flow rate: 10 ml/min.

8. The use of the phenolic acid compound with antioxidant activity of claim 1 in the preparation of antioxidant drugs.

9. A pharmaceutical composition characterized by: comprises the compound of claim 1 and pharmaceutically acceptable carriers, and is prepared into tablets, capsules, injections, powder injections, granules, fat emulsions, dropping pills, ointments or transdermal controlled release patches.

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method for extracting and separating phenolic acid compounds from stems and leaves of caulis Sinomenii, traditional Chinese medicine extracts or mixtures containing the compounds, and application of the compounds in preparing antioxidant medicines.

Background

Oxidative stress has a very significant influence on human aging and health, and excessive free radicals caused by various factors can initiate and aggravate various diseases, such as alzheimer's disease, cardiovascular diseases, malignant tumors, and the like, so that the application of antioxidants to maintain human health has a wide demand. Because synthetic antioxidants have potential risks of carcinogenicity, toxicity and the like, the search for safe, effective and nontoxic natural antioxidants becomes a necessary trend for the development of antioxidants in the current food science. H₂O₂Is a key factor for inducing an oxidative damage cell model, and can stimulate macrophages in vivo to synthesize and release various endogenous active factors. By means of H₂O₂Macrophage processing is a common modeling tool for in vitro oxidative damage cell models.

Caulis Sinomenii is of SinomeniiSabiaceaeCaulis Seu folium Gaultheriae YunnanensisSabiaCaulis SinomeniiSabia parvifloraThe dried stems and leaves of wall ex roxb grow mostly in the southwest region of Guizhou province and Guangxi province in China, and the southwest to southeast of Yunnan province. Xiaohuang is known as Xiaohuang medicine in folk, and is usually used for treating hepatitis, jaundice, rheumatic arthralgia and traumatic injury。

The invention combines modern separation technology and spectral analysis method to separate one kind of anti-H from caulis Sinomenii₂O₂The phenolic acid compound causing RAW264.7 oxidative damage cell model is very expected to be developed into a new antioxidant drug candidate with intellectual property rights and applied to the treatment of relevant diseases of oxidative stress reaction.

Disclosure of Invention

The invention aims to provide a novel phenolic acid compound and application thereof in preparing antioxidant drugs. The compound prepared by the method has better capacity of eliminating free radicals in vitro, and can reduce H₂O₂Induced generation of Reactive Oxygen Species (ROS) within macrophage RAW 264.7. In addition, the activity of intracellular superoxide dismutase (SOD) can be increased.

The technical scheme for implementing the invention is as follows:

a phenolic acid compound with antioxidant activity is characterized in that: the name is: 5- (3-carboxy-4-hydroxy) -5-oxo-2-methyl-2-pentenoic acid having the formula:

the invention provides a phenolic acid compound with antioxidant activity, and a preparation method thereof, wherein the preparation method comprises the following steps:

the method comprises the following specific steps:

s1, extracting the dried stems and leaves of the caulis Sinomenii with 70% ethanol for three times, wherein the mass ratio of the stems and leaves of the caulis Sinomenii to the solvent is about 1: (10-16), concentrating until no alcohol smell exists to obtain an extract;

s2, loading the obtained extract on macroporous resin, and eluting with 30%, 50%, 70%, 95% and ethanol respectively;

s3 separating the part eluted by 30% ethanol in HP-20 resin, separating the obtained extract by macroporous resin, reversed phase column chromatography, semi-preparative high performance liquid chromatography and preparative high performance liquid chromatography in turn to obtain the compound.

Preferably, the extraction method comprises a cold leaching method, a percolation method, a microwave extraction method, an ultrasonic extraction method, a reflux extraction method and a continuous reflux extraction method.

Preferably, the macroporous resin mainly comprises the following types: HP-20, HP-21, SP850, SP700, SP207, AB-8, D101, XDA1, X-5, NKA-2, ADS-2.

Preferably, the reverse phase column chromatography mainly comprises low pressure preparative chromatography, medium pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography, wherein the mobile phase used by the reverse phase chromatography is an aqueous solution of an organic solvent, and the organic solvent is methanol or acetonitrile.

Preferably, the elution of the reversed phase column chromatography is a gradient elution, and the gradient elution procedure is as follows in volume fraction: 20%, 30%, 40%, 50%, 60%, 70%, 80% aqueous solution of organic solvent, eluted 2 column volumes each.

Preferably, the mobile phase of the preparative high performance liquid chromatography is methanol-water solution or acetonitrile-water solution.

The phenolic acid compound prepared by the method or the traditional Chinese medicine extract or the mixture containing the compound is applied to preparing antioxidant medicines.

The phenolic acid compound or extract or mixture containing the compound prepared by the method and pharmaceutically acceptable carriers can be prepared into tablets, capsules, injections, powder injections, granules, fat emulsions, microcapsules, dropping pills, ointments, transdermal controlled release patches and other dosage forms.

The new compound prepared by the method has better capacity of eliminating free radicals in vitro, and the evaluation result of a cell model method shows that the compound can also reduce H₂O₂Induced generation of Reactive Oxygen Species (ROS) within macrophage RAW 264.7. In addition, the activity of intracellular superoxide dismutase (SOD) can be increased. Shows good antioxidant activityCan be used for developing anti-oxidation related medicaments.

Drawings

FIG. 1 is a structural formula of a novel phenolic acid compound provided by the invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.