CN108456136B

CN108456136B - Extraction and separation method and application of pimaric dienoic acid

Info

Publication number: CN108456136B
Application number: CN201810249956.3A
Authority: CN
Inventors: 许洪波; 唐志书; 王薇; 宋逍; 孙琛; 宋忠兴
Original assignee: Shaanxi University of Chinese Medicine
Current assignee: Shaanxi University of Chinese Medicine
Priority date: 2018-03-26
Filing date: 2018-03-26
Publication date: 2021-03-23
Anticipated expiration: 2038-03-26
Also published as: CN108456136A

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and discloses an extraction and separation method of pimaric dienoic acid, which has a structure shown in a formula (I) and is extracted and separated from acanthopanax gracilistylus of araliaceae, wherein the extraction and separation steps comprise: 1) extracting Acanthopanax gracilistylus material with organic solvent or organic solvent-water mixed solvent; 2) concentrating the extract of step 1) under reduced pressure; 3) extracting the concentrate of step 2) with a lipophilic organic solvent; 4) concentrating the extract obtained in the step 3) under reduced pressure to obtain an extract; 5) separating the extract of step 4) by column chromatography; 6) concentrating the eluate of step 5) under reduced pressure; 7) recrystallizing the resultant dried product of step 6). The invention separates and purifies the pimaric dienoic acid from the dried root bark of the acanthopanax gracilistylus for the first time, and the extraction and separation method can realize the industrial separation and high-purity preparation of the pimaric dienoic acid. The invention firstly verifies that the pimaric dienoic acid inhibits the activity of xanthine oxidase.

Description

Extraction and separation method and application of pimaric dienoic acid

Technical Field

The invention belongs to the technical field of medicinal chemistry, relates to an extraction and separation technology of natural active ingredients, and particularly relates to a method for extracting and preparing high-purity pimaric dienoic acid from cortex acanthopanacis and the inhibition activity of pimaric dienoic acid on xanthine oxidase.

Background

Pimaradienoic acid (pimaradienoic acid; ent-pimaraa-8 (14),15-dien-19-oic acid) has a chemical structure shown in formula (I) and belongs to pimarane type (pimarane) diterpenoid compounds, and Japanese scholars firstly report that the pimaradienoic acid is separated from radix angelicae pubescentis in 1967. Later pharmacological research shows that the pimaric dienoic acid has wide pharmacological actions of resisting inflammation, easing pain, resisting tumor, resisting arterial spasm, resisting bacteria, killing insects and the like, wherein the anti-inflammatory activity is most obvious, and the pimaric dienoic acid has better development and application prospects after being proved by various in-vivo and in-vitro inflammation models.

As far as the present, pimaric dienoic acids have been reported to exist in 20 or more plants such as Aralia racemosa, Aralia cordia Thunb (devil's muguet), Aralia fargesii (longum pubescens), Aralia continentalis, Jungermania thermomar, Jungermania hattoria, Mikania pyramidata, Mikania alvimiae, Helianthus strictus, Notopterygium incisium Ting (Notopterygium incisum), Herbertus sakuraii (Oriental cherry leaf moss), Viguiera arenaria, Acanthopanaxtifolius (Acanthopanax trifoliatus), and Arahalium gauudianum, in addition to the radix Angelicae Pubescentis. Because of the huge market demand of the pimaric dienoic acid as a reference substance or a research object, the preparation process (including chemical synthesis) of the pimaric dienoic acid is basically not researched and reported at home and abroad.

Acanthopanax gracilistylus W.W.Smith of Araliaceae family, and its dried root bark is cortex Acanthopanacis, and has effects of dispelling pathogenic wind, removing dampness, and strengthening tendons and bones. The chemical composition research of cortex acanthopanacis indicates that more than 40 compounds are separated and identified from the cortex acanthopanacis up to now, wherein the compounds are mainly kaurane diterpenes. To date, no report has been made on the isolation of pimaric acid from acanthopanax bark.

Disclosure of Invention

The invention aims to provide a method for extracting and separating pimaric dienoic acid. The pimaric dienoic acid (pimaradienoic acid; ent-pimara-8(14),15-dien-19-oic acid) has a chemical structure shown in a formula (I). The extraction and separation method has the advantages of simple and convenient operation, high efficiency, stable process, lower cost and the like, and can realize the industrial separation and high-purity preparation of the pimaric dienoic acid.

The extraction and separation method of the pimaric dienoic acid takes the dried root bark of acanthopanax gracilistylus (namely the cortex acanthopanacis) of the araliaceae as a raw material, and the specific extraction and separation steps comprise:

1) extracting the Acanthopanax gracilistylus material with an organic solvent or an organic solvent-water mixed solvent;

2) concentrating the extract of step 1) under reduced pressure;

3) extracting the concentrate of step 2) with a lipophilic organic solvent;

4) concentrating the extract obtained in the step 3) under reduced pressure to obtain an extract;

5) separating the extract of step 4) by column chromatography;

6) concentrating the eluate of step 5) under reduced pressure;

7) recrystallizing the resultant dried product of step 6).

Preferably, the organic solvent of step 1) is acetone; in the acetone-water mixed solvent, the volume ratio of acetone to water is 100: 0-50: 50.

Preferably, the material in the step 1) is dried root bark of acanthopanax gracilistylus.

Preferably, the lipophilic organic solvent of step 3) is ethyl acetate.

Preferably, the column chromatography in step 5) is performed by silica gel column chromatography, and the eluent is petroleum ether or n-hexane.

Preferably, the solvent used in the recrystallization in the step 7) is one of hexane, dichloromethane, acetone, methanol or a combination thereof.

The extraction and separation method of the pimaric dienoic acid is preferably mixed solvent extraction, and in other implementation modes, water extraction, organic solvent extraction, supercritical carbon dioxide extraction or other conventional modes can be selected. Organic solvent extraction may be used as another preferred way to obtain said pimaric acid, operating substantially as follows: pulverizing dried root bark of Acanthopanax gracilistylus W.W.W.W.Chen, extracting the pulverized material with organic solvent, and recovering the organic solvent from the extract to obtain concentrated solution of the extract. The organic solvent of the invention is preferably one of methanol, ethanol, chloroform, acetone, ethyl acetate or a mixture thereof, or other organic solvents capable of obtaining the extract. In another embodiment, the organic solvent may be extracted by hot reflux, generally 3 times, and the extracts may be combined. The organic solvent can be alcohols (such as methanol, ethanol, etc.), ketones (such as acetone, etc.), and ethers (such as diethyl ether, etc.).

It is another object of the present invention to explore the biological activity of said pimaric dienoic acid. Through rigorous research of the patent application inventor team, the pimaric dienoic acid has strong inhibitory activity on xanthine oxidase and can effectively reduce the generation of uric acid.

The invention comprises a pharmaceutical composition, the active ingredient of which comprises at least said pimaric acid or pharmaceutically acceptable salt thereof.

"pharmaceutically acceptable salts" are salts comprising the compound of formula (I) with other organic or inorganic substances and represent salts that retain the biological effectiveness and properties of the compound of formula (I). It will be readily understood that the pimaric acid has a carboxyl group (-COOH) and may form salts with organic or inorganic bases, i.e. salts formed by replacement of the acidic proton in the structure of formula (i) by a metal ion, such as an alkali metal ion, alkaline earth metal ion or aluminium ion, or by complexation with an organic base, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

"pharmaceutical composition" refers to a mixture of one or more compounds described herein, or their pharmaceutically acceptable salts and prodrugs, with other chemical components, such as pharmaceutically acceptable carriers and excipients. Pharmaceutically acceptable excipients or carriers, or pharmaceutical adjuvants for pharmaceutical preparations, such as water, magnesium stearate, talc, starch, organic acids, dextran or lipidoids, suitable for oral, enteral, parenteral or topical administration. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism. In order to facilitate the absorption of the drug or the delivery of the drug to a target site, the pharmaceutical composition can be prepared into suitable dosage forms, such as granules, tablets, pills, dragees, suppositories, capsules sustained-release preparations, sustained-release tablets, suspensions or injections.

In the pharmaceutical composition, the pimaric dienoic acid or the pharmaceutically acceptable salt thereof is used for preparing the xanthine oxidase inhibitor. Based on the biological activity of the pimaric dienoic acid, the pimaric dienoic acid or the pharmaceutically acceptable salt thereof can be used for preventing and treating xanthine oxidase related diseases, such as hyperuricemia, heart failure, cardiovascular diseases, hypertension, diabetes, renal diseases, arthrosis and the like.

The present invention has at least the following advantageous effects or advantages.

(1) The invention separates the pimaric dienoic acid with the structure of the formula (I) from the dry root bark of the acanthopanax gracilistylus for the first time, expands the preparation source of the pimaric dienoic acid and realizes the comprehensive utilization of the acanthopanax gracilistylus which is a traditional Chinese medicine.

(2) The extraction and separation method of the pimaric dienoic acid has the advantages of simple and convenient operation, high efficiency, stable process, lower cost and the like, and can realize the industrial separation and high-purity preparation of the pimaric dienoic acid.

(3) The invention firstly ascertains that the pimaric dienoic acid has strong inhibition effect on xanthine oxidase, and the pimaric dienoic acid or the pharmaceutically acceptable salt thereof can be used for preparing medicines for preventing or treating hyperuricemia, gout, diabetes, kidney diseases, arthropathy and the like.

Drawings

FIG. 1 is a schematic representation of a pimaradiene acid of the present invention¹H-NMR spectrum.

FIG. 2 shows the production of pimaradiene acid according to the invention¹³C-NMR spectrum.

FIG. 3 is an HPLC chromatogram of pimaric acid according to the invention.

FIG. 4 shows Xanthine Oxidase (XO) inhibition IC of pimaric acid according to the present invention₅₀Graph is shown.

The present invention will be described in further detail with reference to examples.

Detailed Description

Example 1

This example is one of the preferred embodiments of the extraction and separation of pimaric dienoic acid, and the specific operation process is as follows.

Weighing 10kg of dried root bark of Acanthopanax gracilistylus W.W.W.W.W.Chen, pulverizing into fine powder with a pulverizer, and pulverizing into powder with particle size of 3-5 mm. Soak 2 times with 80L acetone-water (70:30, v: v) for 72 hours each at room temperature. And (3) combining the two soaking solutions, concentrating the solution to a small volume by using a rotary evaporator under reduced pressure, and controlling the concentration temperature to be not higher than 55 ℃. The resulting concentrate was extracted 3 times with an equal amount of ethyl acetate, and the extracts of 3 times were combined. The obtained extract was concentrated to dryness to obtain 700g of an ethyl acetate-extracted portion. The ethyl acetate extract was dissolved in a small amount of methylene chloride-methanol (2:1, v: v) and adsorbed on 1.5kg of silica gel, and subjected to vacuum silica gel column chromatography (200-mesh, 300-mesh, 5kg, 18X 100cm) using petroleum ether for elution. Concentrating the petroleum ether eluate under reduced pressure to dryness, and recrystallizing the dried product with dichloromethane-methanol (1:2, v: v) to obtain the pimaric dienoic acid monomer product 112 g.

By adopting the extraction and separation method of the embodiment, the yield of the pimaric dienoic acid is as follows: 112/10000 × 100% ═ 1.12%.

Example 2

The embodiment is one of the preferable embodiments of the extraction and separation of the pimaric dienoic acid, and the specific operation process is as follows.

Weighing 2kg of dried root bark of Acanthopanax gracilistylus W.W.W.W.Chen, pulverizing into fine powder with a pulverizer, and pulverizing into powder with particle size of 3-5 mm. Soak 2 times with 72 hours each time in 20L acetone-water (80:20, v: v) at room temperature. And (3) combining the two soaking solutions, concentrating the solution to a small volume by using a rotary evaporator under reduced pressure, and controlling the concentration temperature to be not higher than 55 ℃. The resulting concentrate was extracted 3 times with an equal amount of ethyl acetate, and the extracts of 3 times were combined. The obtained extract was concentrated to dryness to obtain 180g of an ethyl acetate-extracted portion. The ethyl acetate extract was dissolved in a small amount of methylene chloride-methanol (2:1, v: v) and adsorbed on 400g of silica gel, and subjected to vacuum silica gel column chromatography (200-mesh 300-mesh, 4kg, 18X 100cm) and eluted with petroleum ether. The petroleum ether eluent is decompressed and concentrated to be dry, and the dried substance is recrystallized by dichloromethane-methanol (1:3, v: v) to obtain 27g of pimaric dienoic acid monomer product.

By adopting the extraction and separation method of the embodiment, the yield of the pimaric dienoic acid is as follows: 27/2000 × 100% ═ 1.35%.

Example 3

FIG. 1 shows the pimaradiene acid¹H-NMR spectrum. FIG. 2 shows the pimaric acid¹³C-NMR spectrum. Figure 3 shows the HPLC profile of said pimaric acid. It was confirmed that the pimaric acid isolated by extraction in examples 1 and 2 has the structure of formula (I), and the relevant physicochemical parameters and structural data are as follows.

The molecular formula is as follows: c₂₀H₃₀O₂；

Molecular weight: 302.45, respectively;

the characteristics are as follows: white massive crystals;

solubility: is easily dissolved in dichloromethane, chloroform and acetone, and is hardly dissolved in water;

melting point: 162 ℃ and 163 ℃;

optically active [ alpha ]]25D＝–129°(c＝0.8,CHCl₃)；

¹H-NMR(600MHz,CDCl₃)δ_H:5.72(1H,dd,J＝17.1,10.4Hz,H-15),5.13(1H,br s,H-14),4.93(1H,dd,J＝10.4,2.0Hz,H-16a),4.90(1H,dd,J＝17.1,2.0Hz,H-16b),1.24(3H,s,H-17),0.98(3H,s,H-18),0.64(3H,H-20)。

¹³C-NMR(CDCl₃,150MHz)δ_C:39.3(t,C-1),19.3(t,C-2),38.0(t,C-3),44.1(s,C-4),56.2(d,C-5),24.2(t,C-6),35.9(t,C-7),138.0(s,C-8),50.6(d,C-9),39.3(s,C-10),19.6(t,C-11),36.5(t,C-12),38.6(s,C-13),128.0(d,C-14),147.3(d,C-15),113.0(t,C-16),29.3(q,C-17),29.4(q,C-18),184.5(s,C-19),13.9(q,C-20)。

Example 4

This example demonstrates that pimaric dienoic acid inhibits xanthine oxidase activity.

Xanthine Oxidase (XO) is a protease that catalyzes the production of uric acid from hypoxanthine and xanthine. XO exists in various organisms, and when its activity is too high, uric acid production in the organism is increased, which in turn leads to Hyperuricemia (HUA). The HUA is closely related to gout and various metabolic diseases (diabetes, metabolic syndrome, hyperlipidemia and the like), cardiovascular diseases, chronic kidney diseases, stroke and the like. At present, there are two major clinically used XOIs, allopurinol (allopurinol) and febuxostat (febuxostat). This example tests pimaric dienoic acid for anti-XO activity by constructing an in vitro XO inhibitory activity evaluation model.

1 Material

Absorbance was measured by a full-wavelength microplate reader (Multiskan GO, Thermo Scientific, USA), and xanthine oxidase, xanthine, and allopurinol were purchased from Dow chemical Co., Ltd, EDTA-2Na, KH, respectively₂PO₄、K₂HPO₄And methanol and the like are all domestic analytical pure reagents.

2 method

2.1 preparation of the solution

a. Preparation of buffer solution

KH is precisely weighed₂PO₄ 0.4780g，K₂HPO₄3H2O 3.4730g and EDTA-2Na 10.00mg are put into a 250ml volumetric flask, and the volume is determined by distilled water.

b. Substrate formulation

Accurately weighing 3.65mg xanthine, adding buffer solution to constant volume of 50ml, and dissolving with ultrasound to obtain the final product.

c. Preparation of enzyme solution

Diluting xanthine oxidase (XO, 5U) with 50ml buffer solution, packaging in multiple EP tubes, storing at-70 deg.C, and taking out when necessary.

2.2 Activity assay

Accurately weighing a proper amount of pimaric dienoic acid, dissolving the pimaric dienoic acid in DMSO for later use, wherein the total volume of a reaction system is 200 mu l, sequentially adding 20 mu l of buffer solution, 20 mu l of sample and 80 mu l of enzyme solution into a 96-well plate during reaction, incubating for three minutes at 37 ℃ in an enzyme-linked immunosorbent assay, then adding 80 mu l of substrate xanthine, starting the reaction, and immediately recording the absorbance value (once every 30 s) at 295nm for 6 min. The experiment was set up with a sample set (plus sample, substrate and enzyme), an enzyme set (plus substrate and enzyme but no sample), a blank set (plus substrate only and sample plus no enzyme) and a positive control set (allopurinol).

The inhibition (%) was calculated by the following formula:

inhibition (%) (. DELTA.Aase-. DELTA.A sample)/(. DELTA.Aase-. DELTA.A blank) × 100.

Where Δ a refers to the difference in absorbance over time.

The IC of the inhibition results of 6 series concentrations of pimaric acid is calculated by GraphPad Prism 5 software₅₀. Xanthine Oxidase (XO) inhibition IC of said pimaric dienoic acid₅₀The curve is shown in figure 4.

3 results

As can be seen from FIG. 4, pimaric dienoic acid has significant xanthine oxidase inhibitory activity, IC₅₀55.4. mu.g/ml (IC of positive allopurinol)₅₀9.1. mu.g/ml). This example first concluded that pimaric dienoic acid has anti-XO activity, and thus speculated that pimaric dienoic acid may have potential anti-hyperuricemic effects.

The present invention has been further described with reference to the examples, but the present invention is not limited to the above-described embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims

1. Application of pimaric dienoic acid or its pharmaceutically acceptable salt with structure of formula (I) in preparing xanthine oxidase inhibitor medicine is provided, the pimaric dienoic acid is extracted and separated from Acanthopanax gracilistylus of Araliaceae,

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