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

Abstract

According to the preparation method of the Chinese herbaceous peony water extract with the uric acid reducing effect, after pure water is used for extracting Chinese herbaceous peony, an effective uric acid reducing component is obtained through a simple separation step, and an in-vitro XOD inhibition experiment shows that the extract is remarkable in XOD inhibition effect and has a uric acid reducing medicine development prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and particularly relates to a peony extract with uric acid-lowering effect, a preparation method and use thereof. Background Art

[0002] Red peony root is the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, a plant of the Ranunculaceae family. It is dug in spring and autumn, and the rhizome, fibrous roots and mud are removed and then dried in the sun. It tastes bitter and slightly cold. It enters the liver meridian. The Chinese Pharmacopoeia records that it has the following effects: clearing away heat and cooling blood, dispersing blood stasis and relieving pain. It is used for heat entering the blood, fever and rashes, vomiting blood and epistaxis, 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.

[0003] Modern research shows that the main chemical components of the water-soluble part of peony are paeoniflorin and other monoglycoside compounds, which is the main medicinal part of red peony root. The existing biological activities reported are as follows: 1. Immune regulation: such as inhibiting inflammatory factors; inflammatory injury diseases (arthritis, atherosclerosis, etc.); anti-inflammatory, treatment of rheumatoid arthritis; immunosuppressant; 2. Blood system: treatment or prevention of chronic peripheral vascular occlusive disease, proliferative vascular lesions, blood tonic, and blood coagulation; 3. Regulating smooth muscle: treatment of esophageal achalasia; erectile dysfunction; promoting postoperative intestinal function recovery to prevent intestinal pinching; relieving local pain; 4. Liver protection; 5. Acting on the nervous system: anti-depression; improvement of menopausal syndrome; improvement of sleep, etc.

[0004] 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 extract for hyperuricemia. Summary of the invention

[0005] The present invention provides a plant extract having the effect of reducing uric acid by inhibiting XOD activity, and provides a preparation method. The technical scheme of the present invention is as follows: The present invention provides a use of a peony extract in preparing a drug for treating hyperuricemia. The peony extract of the present invention is prepared by the following method: Step 1: extracting red peony root by heating and refluxing pure water, filtering and concentrating the extract under reduced pressure to obtain an extract, separating the obtained extract using a macroporous resin, eluting with water, 30%, 60%, and 95% ethanol aqueous solutions in sequence, and concentrating the eluent under reduced pressure to obtain an extract; Step 2: Separate the extract of the 30% ethanol aqueous solution elution component in step 1 using an MCI column, and sequentially use water, 30%, 60%, and 95% ethanol aqueous solutions to elute in sequence, collect the 30% and 60% ethanol aqueous solution elution components respectively, and concentrate to dryness under reduced pressure to obtain; Step 3: Separate the extract of the 60% ethanol aqueous solution elution component in step 1 using an MCI column, and sequentially use water, 50%, 70%, and 95% ethanol aqueous solutions to elute in sequence, collect the 50% and 70% ethanol aqueous solution elution components respectively, and concentrate to dryness under reduced pressure to obtain the product.

[0006] Preferably, in the method for preparing peony extract, the macroporous resin filler in step 1 is D101, and the MCI column filler model in step 2 is CHP20 / P30.

[0007] In a further preferred method for preparing peony extract, in step 1, pure water is heated under reflux for extraction, the solid-liquid ratio is 1:8, and the extraction is performed three times, each time for 45 minutes.

[0008] The present invention also provides application of the peony extract in preparing a xanthine oxidase inhibitor.

[0009] The present invention also provides application of the peony extract in preparing a medicine for treating gout.

[0010] The present invention has the following advantages: the present invention uses red peony root medicinal materials with a long history of traditional medicine and excellent safety, the preparation method is convenient, and the XOD inhibition activity is significant, and has the prospect of uric acid-lowering drug development. Example

[0011] Preparation of Peony Extract Take 1 kg of commercially available red peony root, crush it, add pure water and heat it under reflux for extraction 3 times, with an extraction solid-liquid ratio of 1:8, 45 minutes each time, filter, combine the three extracts, and concentrate under reduced pressure to obtain an extract. Take part of the extract and concentrate it to dryness, marked as WA0, and separate the remaining extract with D101 macroporous adsorption resin, and use water, 30% ethanol, 60% ethanol, and 95% ethanol to elute in sequence. After the eluents are concentrated under reduced pressure, the 30% ethanol elution component is separated using an MCI column (column packing model CHP20 / P30), and water, 30%, 60%, and 95% ethanol aqueous solutions are used for elution in sequence. The 30% and 60% elution components are collected, concentrated under reduced pressure to dryness, and marked as WA3-E30 and WA3-E60. The 60% ethanol elution component was separated using an MCI column (column packing model CHP20 / P30), and eluted with water, 50%, 70%, and 95% ethanol aqueous solutions in sequence. The 50% and 70% elution components were collected, concentrated to dryness under reduced pressure, and marked as WA6-E50 and WA6-E70.

[0012] Example 2 In vitro inhibition experiment of red peony root extract on xanthine oxidase 1. Experimental methods 1.1 Solution preparation Preparation of phosphate buffered saline (PB) Pre-prepared K 2 HPO 4 (Weigh 5.23g K 2 HPO 4 dissolved in 400 mL of water) and KH 2 PO 4 Solution (weigh 1.02g KH 2 PO 4 Dissolve in 100 mL of water), then accurately measure 324 mL of K 2 HPO 4 and 76 mL KH 2 PO 4 After mixing evenly, a 50 mM, pH 7.5 phosphate buffer solution PB was obtained, which was used as the test solution for the blank control group.

[0013] 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.

[0014] Take 1 tube for each experiment without dilution and use directly after thawing.

[0015] 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.

[0016] 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.

[0017] Take one tube each time and dilute it to 25 U / L with PB for later use.

[0018] 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.

[0019] Preparation of test solution Weigh 20 mg of WA0, WA3-E30, WA3-E60, WA6-E50, and WA6-E70 prepared in Example 1 respectively and add them to a 1.5 mL centrifuge tube, then add 1 mL of DMSO and ultrasonically dissolve it into a mother solution, draw 50 μL of the mother solution and 950 μL of PB and mix them to obtain a solution of the test drug.

[0020] 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.

[0021] 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.

[0022] 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%.

[0023] 4. Experimental results The specific experimental results of the inhibition rate of each extract on xanthine oxidase are shown in Table 1.

[0024] Table 1 Specific experimental results of the inhibition rate of the extracts in each experimental group on xanthine oxidase Group Inhibition rate (%) Positive control low dose group 42.2% Positive control high dose group 95.1% Experimental group WA0 39.8% Experimental group WA3-E30 44.2% Experimental group WA3-E60 82.6% Experimental group WA6-E50 89.3% Experimental group WA6-E70 40.7% From the inhibition rate results in Table 1, it can be seen that red peony root extracts all have a certain xanthine oxidase inhibitory effect, and the inhibition rates of WA3-E60 and WA6-E50 on xanthine oxidase are higher than that of the positive control low-dose group, which have significant xanthine oxidase inhibitory effects and can be used as potential uric acid-lowering drugs.

Claims

1. A use of a peony extract in preparing a drug for treating hyperuricemia, characterized in that: The peony extract is prepared by the following method: Step 1: extracting red peony root by heating and refluxing pure water, filtering and concentrating the extract under reduced pressure to obtain an extract, separating the obtained extract using a macroporous resin, eluting with water, 30%, 60%, and 95% ethanol aqueous solutions in sequence, and concentrating the eluent under reduced pressure to obtain an extract; Step 2: Separate the extract of the 30% ethanol aqueous solution elution component in step 1 using an MCI column, and sequentially use water, 30%, 60%, and 95% ethanol aqueous solutions to elute in sequence, collect the 30% and 60% ethanol aqueous solution elution components respectively, and concentrate to dryness under reduced pressure to obtain; Step 3: Separate the extract of the 60% ethanol aqueous solution elution component in step 1 using an MCI column, and sequentially use water, 50%, 70%, and 95% ethanol aqueous solutions to elute in sequence, collect the 50% and 70% ethanol aqueous solution elution components respectively, and concentrate to dryness under reduced pressure to obtain the product.

2. The use according to claim 1, characterized in that The macroporous resin filler model described in step 1 of the preparation method of peony extract is D101, and the MCI column filler model described in step 2 is CHP20 / P30.

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

4. Use of the peony extracts according to claim 1 and claim 2 in preparing medicines for treating gout.