CN109534984B

CN109534984B - Method for preparing p-coumaric acid by using spartina alterniflora

Info

Publication number: CN109534984B
Application number: CN201811566755.2A
Authority: CN
Inventors: 钦佩; 张鹤云
Original assignee: Nanjing University
Current assignee: Jiangsu Shibeitai Biotechnology Co.,Ltd.
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2020-08-14
Anticipated expiration: 2038-12-19
Also published as: CN109534984A

Abstract

The invention discloses a method for preparing p-coumaric acid by spartina alterniflora, which comprises the steps of extracting a spartina alterniflora extracting solution by ethyl acetate, extracting a water phase by using n-butyl alcohol, and concentrating n-butyl alcohol phase extracting solution to obtain n-butyl alcohol phase crude paste. And (3) sequentially carrying out normal-phase silica gel column chromatography, ODS (ODS) reverse-phase chromatography and gel column chromatography on the n-butanol phase crude paste, and finally carrying out separation and purification by a high performance liquid chromatography technology to obtain the p-coumaric acid. The pharmacological research result of the invention shows that the p-coumaric acid has obvious effects of reducing uric acid and blood sugar of hyperuricemia mice.

Description

Method for preparing p-coumaric acid by using spartina alterniflora

Technical Field

The invention belongs to the technical field of plant source functional products. In particular to a method for preparing p-coumaric acid by utilizing spartina alterniflora and application of the p-coumaric acid prepared by the method in preparing functional products for reducing blood uric acid.

Background

P-coumaric acid (p-coumaric acid) has the chemical name of p-hydroxycinnamic acid and is the most important one of hydroxycinnamic acid compounds. Studies show that the p-coumaric acid has various physiological functions, such as activities of resisting oxidation, bacteria, cardiovascular diseases, mutation and the like. At present, methods for producing p-coumaric acid include chemical synthesis, natural extraction and biotransformation. The chemical synthesis method is to obtain p-coumaric acid by reacting p-hydroxybenzaldehyde with diacid. The natural extraction method is to obtain p-coumaric acid by alkaline hydrolysis of plant cell wall. The biotransformation method is to convert coumaric acid by microbial enzyme. P-coumaric acid can be extracted from crop wastes such as coix seed bran, bagasse, corncob and the like, but the crops are annual plants, the yield of which is influenced by the growth period and growth speed and the seeding scale, and the extraction source of the p-coumaric acid is very limited.

Spartina alterniflora (Spartina alterniflora) is a perennial herb of the family Poaceae, native to the Atlantic coast of North America, distributed in the intertidal zones beach and marshland in the middle of south to Florida in North New York, Finland, and along the coast of the gulf of Mexico. Introduced into China by Zhong Chong Xin professor of Nanjing university at the end of 1979, aiming at making up for the short and low yield of the rice grass (Spartina anglica) plants introduced previouslyAnd inconvenient harvesting and utilization, etc. The 20 th century 80-90 years successfully introduced species in Jiangsu, Zhejiang, Fujian and other places of China along the coast, and gradually developed into large-area salt marsh vegetation (the current development is 55468 hm)²) The method makes great contribution to the coastal region of China to protect the beach and promote silt and land reclamation. However, as the spartina alterniflora grows to spread and excessively expand, the negative effects gradually appear, and the biological diversity of a part of coastal areas, particularly the aquaculture of beaches, is influenced. In order to realize the resource utilization of the spartina alterniflora, create interest and remove defects, create economic benefits and generate good ecological benefits, researchers represented by the halophyte laboratory of Nanjing university have made a lot of research and development work, for example, internationally solve the problem of eating the spartina alterniflora extract and obtain a batch of new resource food of the Ministry of health (Weixin food Kai 94, No. 06).

Disclosure of Invention

In order to explore the biological effects of functional compounds in the spartina alterniflora extract such as uric acid reduction, the biological mineral liquid of the spartina alterniflora extract is separated, purified and identified to obtain 18 monomer compounds, and the uric acid reduction activity of the monomer compounds is researched, and the result shows that the spartina alterniflora can be used for preparing the p-coumaric acid, and the p-coumaric acid prepared by the method has the blood uric acid reduction activity.

The specific technical scheme of the invention is as follows:

a method for preparing p-coumaric acid by using spartina alterniflora comprises the following steps:

(1) collecting aerial parts of spartina alterniflora, extracting with water, concentrating to obtain spartina alterniflora concentrated solution, extracting with ethyl acetate, extracting the water phase with n-butanol, and concentrating the n-butanol phase extract to obtain n-butanol phase crude extract;

(2) separating the n-butanol phase crude paste obtained in the step (1) by adopting normal phase silica gel column chromatography, performing gradient elution by adopting a dichloromethane/methanol system at a ratio of 100: 0-100, and collecting eluent under the gradient condition that the volume ratio of dichloromethane to methanol is 100: 8;

(3) separating the eluent in the step (2) by adopting an ODS (oxide dispersion strengthened) reversed-phase chromatographic column, wherein the mobile phase is a mixed solution of methanol and water, and collecting the eluent;

(4) separating the eluent in the step (3) by adopting gel column chromatography, wherein the mobile phase is methanol, and collecting the eluent;

(5) and (4) separating the eluent in the step (4) by adopting a reverse phase high performance liquid phase, wherein the mobile phase is a mixed solution of methanol and water, and isocratic elution is carried out to obtain the p-coumaric acid.

In the method of the invention, in the step (2), preferably, the n-butanol phase crude paste and the silica gel filler are mixed, the sample is loaded by a dry method, the sample is eluted by a dichloromethane/methanol system, and the eluent under the gradient condition that the volume ratio of dichloromethane to methanol is 100:8 is collected. More preferably, the n-butanol phase crude extract is subjected to normal phase silica gel column chromatography twice under the same chromatography conditions.

In the method of the present invention, step (3) preferably has a methanol to water volume ratio of 4:6, and the eluent of the 5 th column volume is collected.

In the method, the step (4) preferably adopts Sephadex column chromatography, preferably Sephadex LH-20.

According to the method, C18 reversed-phase high-performance liquid phase preparation column is preferably adopted in the step (5), the mobile phase is 50% methanol water solution, the flow rate is 2ml/min, and the product with the retention time of 9.7min is collected to be p-coumaric acid.

The invention also aims to provide application of the p-coumaric acid prepared by the method in preparing a product with the function of reducing uric acid in blood. The functional product is food, health product or medicine.

The determination of the level of the uric acid in the blood of the hyperuricemia mouse shows that the effect of reducing the uric acid of the coumaric acid is obvious, and the effect of the positive control benzbromarone is not obvious. The determination of the blood sugar level of the hyperuricemia mouse shows that the effect of reducing the blood sugar by the coumaric acid is very obvious, and the effect of the positive control benzbromarone is not obvious. The determination of the serum creatinine level of the mice with hyperuricemia shows that the effect of reducing the creatinine is very obvious for the coumaric acid group and the spartina anglica powder group, and the effect of other test groups and the benzbromarone group are obvious. The determination of the serum total protein level of the mice with hyperuricemia shows that the recovery effect of the serum total protein level of each test group and the benzbromarone group is very obvious, particularly the serum total protein level of the coumaric acid group and the desmodium saccharifolium group is very close to the normal level of the normal saline group.

The invention has the advantages that:

(1) the spartina alterniflora is a perennial herb, a single plant can propagate dozens or even hundreds of plants in one year, compared with annual crops, the spartina alterniflora does not need frequent sowing, has low maintenance and planting cost and high yield, and has incomparable advantages when being used as a plant extraction source of coumaric acid;

(2) the spartina alterniflora p-coumaric acid prepared by the method disclosed by the invention has the HPLC liquid phase purity of 95%, and also contains other active ingredients.

Drawings

FIG. 1 is a flow chart of the separation of the active ingredients of Spartina alterniflora.

FIG. 2 is a LC-MS diagram of p-coumaric acid prepared by the method of the present invention.

FIG. 3 shows that p-coumaric acid is obtained by the method of the present invention¹H NMR (400MHz) chart (Acetone-d)₆)。

FIG. 4 shows that p-coumaric acid is obtained by the method of the present invention¹³C NMR spectrum (400MHz) (methane-d)₄)。

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 Studies of active ingredients of Spartina alterniflora

Collecting 100kg of overground part of spartina alterniflora, drying in the sun, cutting into 2-3 cm long pieces, adding 20 times of hot water for extraction, and concentrating the extract to obtain dark brown liquid (about 10 kg).

Also refer to the technical scheme disclosed in patent application No. 201510266409.2, to obtain Spartina alterniflora fine powder, dissolving in purified water, and preparing into 1:10 sample mother liquor (aqueous solution, wherein the total saponin content is > 6.0%). The fine rice grass powder used in the invention is purchased from Jiangsu Haili Producer science and technology limited.

2500g of spartina alterniflora concentrated solution is taken, extraction is carried out for 3 times by using ethyl acetate with the same volume, an organic phase is taken for decompression rotary evaporation and concentration, 113.8g of ethyl acetate phase crude paste is obtained, a water phase is combined, extraction is carried out for 3 times by using n-butanol with the same volume, decompression rotary evaporation and concentration are carried out, and 159.6g of n-butanol phase crude paste is obtained.

Subjecting the ethyl acetate phase crude extract and n-butanol phase crude extract to silica gel column chromatography, and separating with petroleum ether/ethyl acetate (v/v 100:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, each 2500ml) and CH₂Cl₂Performing gradient elution with/MeOH (v/v 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32 and 1:1, each 2500ml) as a mobile phase, combining similar components according to TLC thin-layer chromatography results, combining to obtain 9 components of an ethyl acetate phase and 10 components of an n-butanol phase, performing silica gel column chromatography, ODS reverse phase chromatography and gel column chromatography on each component, further separating, and finally performing separation and purification by a high performance liquid chromatography technology to finally obtain 18 monomeric compounds. The specific separation process is shown in figure 1, and compound 12 in figure 1 is p-coumaric acid, and its molecular weight is 164 and molecular formula is C estimated by high resolution mass spectrometry₉H₈O₃The calculation shows that the unsaturation degree is 6, and the structure is as follows:

the preparation process of p-coumaric acid is as follows:

53.2g of the n-butanol phase crude paste was mixed with an equal mass of silica gel material. And after the sample is fully contacted with the silica gel and the methanol is removed through volatilization, the sample is loaded by a dry method. The first coarse separation is carried out by normal phase silica gel separation method, silica gel 10 times the mass of the sample-mixed silica gel is weighed as a separation column, the inner diameter r of the selected silica gel column is 6.5cm, and the calculated volume of the separation column is 4378 ml. After completion of column packing, elution was carried out with a dichloromethane/methanol system, and 2500ml of each of dichloromethane/methanol (v/v) was sequentially used at a ratio of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32 and 1:1, and the eluates were collected under a gradient of 100:8, and vacuum-concentrated to obtain 3.9g of the extract of this fraction. The extract is subdivided and the same normal phase separation is used. This time, silica gel column having an inner diameter r of 1.8cm and a column volume of 264ml was used. Dichloromethane was collected with the same elution system: the eluent was taken at a ratio of 100:8 methanol and the amount of cream was 1.33 g. This paste was separated by means of an ODS reverse phase chromatography column (column inner diameter 1cm, column volume 110ml), and the volume of the column was measured with methanol: the system with 4:6 water (v/v) was used for elution, and the eluent was collected from the 5 th column volume, the paste weight was 0.64 g. Then, the compound was subjected to impurity removal (column inner diameter 1cm, column volume 141.3ml) before liquid phase preparation by Sephadex column chromatography (Sephadex LH-20), and eluted with methanol to obtain a final paste having a mass of 0.36 g. The paste was prepared by HPLC using a liquid preparative column of eighteen carbons (Eclipse XDB-C18, 5 μm, 9.4X 250mm) packed in methanol: isocratic elution is carried out by water (v/v) at a ratio of 1:1, the flow rate is 2ml/min, the elution peak with the retention time of 9.7min is collected, and p-coumaric acid (with the purity of more than 95%) is prepared, and 41.0mg is obtained in total.

Example 2 Spartina alterniflora extract and animal test for lowering uric acid in coumaric acid

First, experimental material

1. Animals: 20-25 g male Kunming species mice, 60

2. A molding agent: hypoxanthine

3. Positive drugs: benzbromarone

4. Quasi-positive medicine: refined rice straw powder and refined rice straw powder without sugar

5. Negative agents: physiological saline

6. Test agents: p-coumaric acid separated and identified from spartina anglica extract

7. Solvent: sodium carboxymethylcellulose-CMC-Na

8. An injector: 1ml, 5ml

9. Centrifuging the tube: 1.5ml, 2ml, 5ml

10. Capillary vessel for blood collection

11. A feeder: flat head 9# needle

Second, testing instrument

Full-automatic biochemical analyzer of beckmann LX20

Third, Experimental scheme

1. Preparing a reagent:

(1) preparation of 0.8% CMC-Na (sodium carboxymethylcellulose): weighing 2.4 g of CMC-Na, firstly using a small amount of purified water to prepare into a lake shape, then using the purified water to fix the volume to 300ml, pouring the solution into a beaker to be boiled to be completely dissolved, then pouring the solution into a volumetric flask, and using the purified water to complement the solution to 300 ml.

(2) Preparation of 50mg/ml hypoxanthine: 1g of hypoxanthine was weighed out and dissolved in 20ml of CMC-Na solution.

(3)0.5mg/ml benzbromarone preparation: 20mg of benzbromarone was weighed and dissolved in 40ml of CMC-Na solution to a concentration of 0.5 mg/ml.

(4) Preparing the quasi-positive medicinal rice grass fine powder:

accurately weighing 1g of herba Euphrasiae Regelii fine powder, and making into 4.5mg/ml dosage test solution with the CMC-Na solution (based on total saponin content of 5.7%).

(5) Preparing the pseudo-positive medicine deglycosemium rice grass fine powder:

weighing 2g of fine rice grass powder, precipitating with 20ml of 70% ethanol, filtering to remove polysaccharide from the fine rice grass powder, concentrating to obtain extract of the refined rice grass powder, and preparing the extract of the refined rice grass powder with the CMC-Na solution into a dosage test solution of 4.5mg/ml according to the total saponin content of 5.7%.

(6) Preparing a test medicament: 20mg of p-coumaric acid is dissolved in 40ml of CMC-Na solution, and the concentration is 0.5 mg/ml.

2. Designing a model animal test group:

(1) blank control group (physiological saline) 10 animals

(2) Model group (CMC-Na)10 animals

(3) Fine powder group of herba Euphrasiae Regelii (4.5mg/ml) of 10 animals

(4) Desugarized spartina anglica fine powder group (4.5mg/ml) of 10 animals

(5) P-coumaric acid group (0.5mg/ml)10 animals

(6) Benzbromarone group (0.5mg/ml)10 animals

Fourthly, an experimental method:

each group of test animals was dosed once a day (weighing before dosing daily) for 7 days, and each group of animals was tested: animals of

groups

1, 3, 4, 5 and 6 were administered in stomach (i.g.), and in stomach (ig) of model group, CMC-Na was administered; the specific situation is as follows:

after the last administration 30min on day 8, 50mg/ml hypoxanthine which has been prepared is injected into the abdominal cavity (i.p.) of each group except the normal saline group, the administration is carried out according to the dose of 1000mg/kg body weight, blood is collected immediately after 30-40min after the administration, the eyeballs are removed, the centrifugal tube is placed in a centrifugal tube, serum is taken out until the serum exudes at room temperature, the upper layer of the serum is centrifuged (15000rpm, 4min), the upper layer of the serum is sucked into a sample tube with a cover, the serum is completely prepared into the required volume by normal saline according to the volume of the obtained serum, and indexes such as blood uric acid, blood sugar, urea nitrogen, creatinine, total cholesterol, triglyceride, total protein, glutamic pyruvic transaminase and the like are measured on a Beckman LX20 full-automatic biochemical analyzer.

All data were processed through the Spss11.5 software package using one-way analysis of variance, expressed as mean. + -. standard deviation (x. + -.s).

Fifth, experimental results

5.1 results of blood uric acid assay in laboratory mice

Tests show that the test sample and the two spartina anglica refined powder groups (including the desaccharified spartina anglica refined powder group) have different degrees of influence on the reduction of the serum uric acid level of the hyperuricemia mice, and in the experimental design range, the test sample has obvious effect on the coumaric acid group, and the positive control benzbromarone group has no obvious effect (Table 1).

TABLE 1 Effect of test samples on blood uric acid (μ M) of Experimental mice

Note: p <0.05, indicating significant differences.

5.2 results of blood glucose measurement in laboratory mice

Tests show that the test sample and the two spartina anglica refined powder groups (including the deglycra anglica refined powder group) have different degrees of influence on the reduction of the blood sugar level of the hyperuricemia mice, and in the experimental design range, the effect on the coumaric acid group is very obvious, and the effect of the positive control benzbromarone group is not obvious (Table 2).

TABLE 2 Effect of test samples on blood glucose (mM) of test mice

5.3 results of urea nitrogen determination in laboratory mice

Tests show that the test sample and the two rice grass fine powder groups (including the deglycosylated rice grass fine powder group) have different degrees of influence on the reduction of the serum urea nitrogen level of the mice with hyperuricemia, and in the experimental design range, the effect on the coumaric acid group is obvious, the effect on the positive control benzbromarone group is very obvious, and the effect on the two rice grass fine powder groups is very obvious (Table 3).

TABLE 3 Effect of test samples on Urea Nitrogen (mM) in laboratory mice

Note: p <0.05, indicating significant difference, # P <0.01, indicating very significant difference, and # P <0.001, indicating very significant difference, as in the tables below.

5.4 measurement of creatinine in laboratory mice

Tests prove that the test samples and the two rice grass fine powder groups (including the deglycated rice grass fine powder group) have different degrees of influence on the reduction of the serum creatinine level of the mice with hyperuricemia, in the experimental design range, the effects on the coumaric acid group and the rice grass fine powder group are very obvious, the effect on the benzbromarone group is obvious, and the serum creatinine of each test group is close to the normal level of the normal saline group (table 4).

TABLE 4 Effect of test samples on creatinine (mM) in laboratory mice

5.5 Total Cholesterol measurement results in laboratory mice

Tests show that the test sample and the two spartina anglica refined powder groups (including the desaccharified spartina anglica refined powder group) have different degrees of influence on the reduction of the serum total cholesterol level of the mice with hyperuricemia, and in the experimental design range, the effect on the coumaric acid group is very obvious, and the effect on the benzbromarone group is obvious (Table 5).

TABLE 5 Effect of test samples on Total Cholesterol in laboratory mice (mM)

5.6 results of measurement of Experimental mouse triglyceride

The test samples from each group were tested to have varying degrees of effect on the reduction of serum triglyceride levels in hyperuricemic mice (Table 6).

TABLE 6 Effect of test samples on triglyceride in laboratory mice (mM)

5.7 Total protein assay results in laboratory mice

Tests prove that the total serum protein of the mice with hyperuricemia is remarkably reduced, the test samples and the two spartina anglica powder groups (including the desaccharified spartina anglica powder group) have different degrees of influence on the increase of the total serum protein of the mice with hyperuricemia, and in the design range of the experiment, the effects of each test group and the benzbromarone group are very remarkable, particularly the total serum protein of the coumaric acid group and the desaccharified spartina anglica powder group is very close to the normal level of the normal saline group (Table 7).

TABLE 7 Effect of test samples on Total protein in laboratory mice (g/L)

5.8 results of glutamic-pyruvic transaminase assay in laboratory mice

Tests show that the test sample and the two rice grass refined powder groups (including the deglycated rice grass refined powder group) have different degrees of influence on the reduction of the serum glutamic-pyruvic transaminase level of the mice with hyperuricemia, and in the experimental design range, the two rice grass refined powder groups have obvious effects, and the glutamic-pyruvic transaminase of the two rice grass refined powder groups is very close to the normal level of the normal saline group (Table 8).

TABLE 8 Effect of test samples on test mouse glutamate pyruvate transaminase (U/L)

5.9 results of body weight measurement of laboratory mice

The test shows that the animals in each group (including the normal saline group, the CMC group and the benzbromarone group) have increased weight after being fed for 7 days, and the level of the weight gain of the animals in each group has little difference within the design range of the experiment (Table 8).

TABLE 9 Effect of test agents on body weight of laboratory mice (g)

Note: the weight gain of each group is compared, and no significant difference exists.

Example 3 comparative test of the Effect of lowering blood uric acid of coumaric acid and commercial products prepared by the method of the present invention

The blood uric acid lowering effects of p-coumaric acid prepared by the method of the present invention and commercially available p-coumaric acid (obtained from Shanghai leaf Biotech Co., Ltd.) were examined by referring to the method of example 2, and the results are shown in Table 10.

The 0.5mg/ml dose group of p-coumaric acid prepared by the invention and the 0.75mg/ml dose group of p-coumaric acid sold in the market can reduce the serum uric acid level of the hyperuricemia mouse, while the 0.5mg/ml dose group of p-coumaric acid sold in the market can not reduce the serum uric acid level of the hyperuricemia mouse (Table 10). The activity of the p-coumaric acid for reducing the serum uric acid level of the hyperuricemia mouse is higher than that of the commercially available p-coumaric acid.

TABLE 10 Effect of test samples on blood Uric Acid (UA) of Experimental mice

Note: p <0.05 indicates significant difference; p <0.01 indicates significant difference; p <0.001 indicates a very significant difference

The 0.5mg/ml dose group of p-coumaric acid prepared by the invention and the 0.75mg/ml and 1mg/ml dose groups of the commercially available p-coumaric acid group can reduce the serum creatinine level of the mice with hyperuricemia, while the 0.5mg/ml dose group of the commercially available p-coumaric acid group can not reduce the serum creatinine level of the mice with hyperuricemia (Table 11). This shows that the activity of the invention on the serum creatinine level of the mouse with the p-coumaric acid for reducing hyperuricemia is higher than that of the commercially available p-coumaric acid.

TABLE 11 Effect of test samples on Creatinine (CREA) in laboratory mice

According to tests, the 0.5mg/ml dose group of p-coumaric acid prepared by the invention and the 0.75mg/ml and 1mg/ml dose groups of the commercially available p-coumaric acid group can reduce the total cholesterol level of the serum of the mice with hyperuricemia, while the 0.5mg/ml dose group of the commercially available p-coumaric acid group can not reduce the total cholesterol level of the serum of the mice with hyperuricemia (Table 12). The activity of the p-coumaric acid for reducing the level of the total cholesterol in the serum of the hyperuricemia mouse is higher than that of the commercially available p-coumaric acid.

TABLE 12 Effect of test samples on Total Cholesterol (CHOL) in laboratory mice

Note: p <0.05 indicates significant difference; p <0.01 indicates significant difference; p <0.001 indicates a very significant difference.

Claims

1. A method for preparing p-coumaric acid by using spartina alterniflora is characterized by comprising the following steps:

(1) collecting aerial parts of Spartina alterniflora, extracting with water, concentrating to obtain Spartina alterniflora concentrated solution, extracting with ethyl acetate, extracting water phase with n-butanol, and concentrating n-butanol phase extractive solution to obtain n-butanol phase crude extract;

(3) separating the eluent in the step (2) by using an ODS (ODS) reverse phase chromatographic column, collecting the eluent, wherein a mobile phase is a mixed solution of methanol and water with a volume ratio of 4: 6;

(5) and (4) separating the eluent in the step (4) by adopting a reverse phase high performance liquid phase, wherein a mobile phase is a mixed solution of methanol and water with the volume ratio of 1:1, and isocratic elution is carried out to obtain the p-coumaric acid.

2. The method according to claim 1, wherein in the step (2), the n-butanol phase crude paste is mixed with a silica gel filler, dry-loaded, eluted by a dichloromethane/methanol system, and the eluate is collected under a gradient of a dichloromethane-methanol volume ratio of 100: 8.

3. The method according to claim 2, wherein in the step (2), the n-butanol phase crude extract is subjected to normal phase silica gel column chromatography twice under the same chromatography conditions.

4. The method of claim 1, wherein step (3) collects the 5 th column volume of eluate.

5. The method of claim 1, wherein step (4) comprises performing Sephadex column chromatography.

6. The method of claim 1, wherein step (5) comprises using a C18 reverse phase high performance liquid preparation column with a flow rate of 2ml/min, and collecting the product with a retention time of 9.7min as p-coumaric acid.

7. The application of p-coumaric acid in preparing functional products for reducing uric acid in blood is characterized in that the p-coumaric acid is prepared by the following method:

(3) separating the eluent in the step (2) by using an ODS (ODS) reverse phase chromatography column, collecting eluent with the volume of the 5 th column, wherein the mobile phase is a mixed solution of methanol and water, and the volume ratio of the methanol to the water is 4: 6;

(4) separating the eluent in the step (3) by adopting Sephadex LH-20 column chromatography, wherein the mobile phase is methanol, and collecting the eluent;

(5) and (3) separating the eluent in the step (4) by adopting a reverse phase high performance liquid phase, preparing a column by using C18 reverse phase high performance liquid phase, wherein the mobile phase is 50% methanol aqueous solution, the flow rate is 2ml/min, and collecting a product with the retention time of 9.7min, namely p-coumaric acid.

8. The use according to claim 7, wherein the functional product is a food, a health product or a pharmaceutical.