Preparation method of Chinese herbaceous peony seed extract and application of Chinese herbaceous peony seed extract in preparation of medicine for treating hyperuricemia

Abstract

According to the preparation method of the Chinese herbaceous peony seed extract with the uric acid reducing effect, after the Chinese herbaceous peony seeds are extracted with the ethanol water solution, effective uric acid reducing components are obtained through simple extraction and separation steps, and the Chinese herbaceous peony seed extract has the development prospect of drugs for reducing uric acid and gout.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and specifically relates to an application of a peony seed extract in preparing a drug for treating hyperuricemia and a preparation method thereof. Background Art

[0002] The root of peony has been used as "white peony" or "red peony" for more than 2,000 years. The Chinese Pharmacopoeia records that it has the following effects: clearing heat and cooling blood, dispersing blood stasis and relieving pain. It is used for heat entering the blood, fever and toxicity, vomiting blood and bleeding, red and swollen eyes, liver depression and flank pain, amenorrhea and dysmenorrhea, abdominal pain due to lumps and masses, injuries from falls, carbuncles and ulcers. Recent studies have found that peony seeds are rich in unsaturated fatty acids, as well as various ingredients such as astragalus, monostearate glycosides, flavonoids and organic acids, which have antioxidant, anti-aging, liver protection, antibacterial, antiviral, antiallergic, and neuraminidase inhibition effects.

[0003] Hyperuricemia (HUA) refers to excessive production and / or insufficient excretion of uric acid in the body under normal dietary conditions. Hyperuricemia is defined as fasting blood uric acid levels higher than 420 μmol / L for men and higher than 360 μmol / L for women on two different days. Hyperuricemia is the basis for the onset of gout. There are no reports on the use of peony seed extract for hyperuricemia. Summary of the invention

[0004] The present invention provides a peony seed extract having a uric acid-lowering effect, and provides a preparation method thereof. The technical scheme of the present invention is as follows: The present invention provides a use of a peony seed extract in preparing a drug for treating hyperuricemia. The peony seed extract of the present invention is prepared by the following method: Step 1: After the peony seeds are crushed, they are extracted by heating and refluxing with an ethanol aqueous solution, and the extract is filtered and concentrated under reduced pressure to obtain an extract; Step 2: extracting the extract obtained in step 1 with ethyl acetate, and concentrating the ethyl acetate layer extract under reduced pressure to obtain an ethyl acetate extract; Step 3: Separate the ethyl acetate extract in step 2 with a silica gel column, use chloroform and methanol as eluents, collect the eluate of the 2nd to 4th column volumes, and concentrate to dryness under reduced pressure to obtain the product.

[0005] Preferably, the ethanol content in the ethanol aqueous solution described in step 1 of the method for preparing peony seed extract is 70%.

[0006] Further preferably, the volume ratio of chloroform to methanol in the chloroform-methanol eluent described in step three is 5:1.

[0007] The present invention also provides application of the peony seed extract in preparing a xanthine oxidase inhibitor.

[0008] The present invention also provides application of the peony seed extract in preparing a medicine for treating gout.

[0009] The present invention has the following advantages: the present invention uses peony seeds with a wide source, the preparation method is convenient, and the XOD inhibition activity is significant, and has a prospect for the development of uric acid-lowering drugs. DETAILED DESCRIPTION

[0010] Example 1 Preparation of Peony Seed Extract Peony seeds were purchased from Bozhou market. 800 g of seeds were crushed and extracted with 70% ethanol aqueous solution under heating and reflux, with a solid-liquid ratio of 1:8 (weight ratio). The extraction time was 30 minutes. The extraction was performed three times. The extracts were combined, filtered, and concentrated under reduced pressure to obtain an extract. Part of the extract was concentrated under reduced pressure to dryness and marked as extract E01. The remaining extract was extracted with three times the volume of ethyl acetate for three times. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain ethyl acetate extract EA02. The obtained ethyl acetate extract was separated using a silica gel column and eluted with chloroform methanol (5:1, V:V). The eluent of the 2nd to 4th column volume was collected and concentrated to dryness under reduced pressure to obtain EAFr24.

[0011] Example 2 In vitro inhibition experiment of peony seed extract on xanthine oxidase 1. Experimental methods 1.1 Solution preparation Preparation of phosphate buffered saline (PB) Prepare K2HPO4 (weigh 5.23g K2HPO4 and dissolve it in 400mL water) and KH2PO4 solution (weigh 1.02g KH2PO4 and dissolve it in 100mL water) in advance, then accurately measure 324mL K2HPO4 and 76mL KH2PO4, mix them evenly, and you will get 50mM, pH7.5 phosphate buffer PB as the test solution for the blank control group.

[0012] Preparation of allopurinol solution Weigh 5 mg of allopurinol and dissolve it in 1 mL of DMSO to make a stock solution. After it is completely dissolved, dilute it with PB to 1.5 μg / mL and 10 μg / mL, and store it in aliquots of 1 mL / tube in a -20°C refrigerator as the test solution for the positive control group.

[0013] Take 1 tube for each experiment without dilution and use directly after thawing.

[0014] Among them, the allopurinol solution with a concentration of 1.5 μg / mL was used as the allopurinol low-dose group, and the allopurinol solution with a concentration of 10 μg / mL was used as the allopurinol high-dose group.

[0015] Preparation of xanthine oxidase solution According to the enzyme amount and volume indicated on the xanthine oxidase product package, dilute it with PB to 100U / L. After preparation, divide it into 8mL / tube and store it in a -20℃ environment.

[0016] Take one tube each time and dilute it to 25 U / L with PB for later use.

[0017] Preparation of substrate xanthine solution Accurately weigh 4.56 mg of xanthine, place in a 50 mL volumetric flask, add 150 µL of 1 M sodium hydroxide, 30 mL of PB, and sonicate until the xanthine is completely dissolved and becomes clear and transparent, then dilute to the mark with PB to obtain a 600 µM xanthine solution.

[0018] Preparation of test solution 20 mg of E01, EA02 and EAFr24 prepared in Example 1 were weighed respectively and added into a 1.5 mL centrifuge tube, and then 1 mL of DMSO was added and ultrasonically dissolved into a mother solution. 50 μL of the mother solution and 950 μL of PB were drawn and mixed to obtain a solution of the test drug.

[0019] 1.2 Reaction and termination of enzyme and substrate In the experimental group, 400 μL of xanthine oxidase (XOD) solution and 200 μL of test solution were added to the 1.5 mL centrifuge tube in sequence; in the blank control group, 400 μL of xanthine oxidase (XOD) solution and 200 μL of test solution of the blank control group were added to the 1.5 mL centrifuge tube in sequence; in the positive control group, 400 μL of xanthine oxidase (XOD) solution and 200 μL of test solution of the positive control group, low-dose group and high-dose group were added to the 1.5 mL centrifuge tube in sequence; then, each group performed the following experimental operations: incubated in a 37°C water bath for 10 min, 400 μL of xanthine (XAN) solution was added to start the reaction, after reacting in a 37°C water bath for 10 min, 80 μL of 1 M hydrochloric acid was added to terminate the reaction, and then 70-75 μL of 1 M NaOH solution was added to adjust the pH to neutral.

[0020] 1.3 Centrifugation and machine testing After the treated test solution of each group was centrifuged at 12000 rpm for 10 min at room temperature, 600 μL of the supernatant was aspirated into a liquid phase bottle, and the uric acid value of each group of test solution was detected by HPLC.

[0021] 2. Experimental data detection and processing The xanthine oxidase inhibition rate was calculated according to the following formula: inhibition rate = [(uric acid value of the blank control group - uric acid value of the test solution) / uric acid value of the blank control group] * 100.

[0022] 4. Experimental results The specific experimental results of the inhibition rate of each extract on xanthine oxidase are shown in Table 1.

[0023] Table 1 Specific experimental results of the inhibition rate of peony seed extract on xanthine oxidase Group Inhibition rate (%) Positive control low dose group 40.1% Positive control high dose group 97.2% Experimental group E01 40.7% Experimental group EA02 56.8% Experimental group EAFr24 88.3% From the inhibition rate results in Table 1, it can be seen that peony seed extracts all have a certain xanthine oxidase inhibitory effect, and the inhibition rate of xanthine oxidase is higher than that of the positive control low-dose group, with significant xanthine oxidase inhibitory effect, especially EAFr24, which has a better xanthine inhibitory effect, and can be used as a potential uric acid-lowering drug.

Claims

1. A use of a peony seed extract in preparing a drug for treating hyperuricemia, characterized in that: The peony seed extract is prepared by the following method: Step 1: After the peony seeds are crushed, they are extracted by heating and refluxing with an ethanol aqueous solution, and the extract is filtered and concentrated under reduced pressure to obtain an extract; Step 2: extracting the extract obtained in step 1 with ethyl acetate, and concentrating the ethyl acetate layer extract under reduced pressure to obtain an ethyl acetate extract; Step 3: Separate the ethyl acetate extract in step 2 with a silica gel column, use chloroform and methanol as eluents, collect the eluate of the 2nd to 4th column volumes, and concentrate under reduced pressure to dryness to obtain the product.

2. The use according to claim 1, characterized in that In the preparation method of peony seed extract, the ethanol content in the ethanol aqueous solution in step 1 is 70%, and the volume ratio of chloroform to methanol in the chloroform-methanol eluent in step 3 is 5:

1.

3. Use of the peony seed extract according to claim 1 and claim 2 in the preparation of xanthine oxidase inhibitors.

4. Use of the peony seed extract according to claim 1 and claim 2 in preparing a drug for treating gout.