Diterpene dimer compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a diterpenoid dimer compound as well as a preparation method and application thereof. The diterpenoid dimer compound has a structure as shown in a formula I. The preparation method comprises the following steps: extracting pond fir seeds by using ethanol, concentrating in vacuum until no alcohol exists, and extracting by using ethyl acetate to obtain an ethyl acetate extract; performing gel column chromatography separation on the ethyl acetate extract to obtain an enriched part of the terpenoid polymer, and performing silica gel column chromatography and ODS column chromatography purification in sequence to obtain a target fraction; sequentially carrying out silica gel column chromatography separation, ODS (octadecylsilyl) column chromatography separation and reversed-phase high-performance liquid chromatography purification on the target fractions to obtain the diterpenoid dimer compounds. In-vitro alpha-glycosidase inhibition experiments prove that the compound represented by the formula I has significant hypoglycemic activity, can be used as an anti-diabetic active component, and has wide applications. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a diterpene dimer compound and a preparation method and application thereof. Background Art

[0002] Diterpene dimers are a relatively rare natural product composed of the same or different diterpene units. They are widely distributed in a variety of medicinal plants such as the Taxaceae, Leguminosae, and Lamiaceae. Common diterpene dimers are mostly formed by the polymerization of diterpenes and diterpenes through different linking methods such as DA cyclization, [2+2], [5+2], CC single bonds, ether bonds, and ester bonds to form complex and variable skeleton types and species, thereby showing diverse and significant biological activities. Modern pharmacological studies have shown that diterpene dimers exhibit a variety of biological activities such as anti-tumor, anti-inflammatory, anti-tumor cell migration, inhibition of non-alcoholic fatty liver, and hypoglycemic effects. The polymerization method connected by oxazole rings is a rare type of polymerization. At the same time, terpene polymers are characteristic components of Taxaceae plants. Studies have found that terpene polymers found in Taxaceae have significant therapeutic potential for antibacterial, anti-tumor, and anti-diabetic diseases.

[0003] In terms of chemical structure, diterpene dimers are polymers formed by connecting diterpenes through oxazole rings. They are a novel class of terpene polymers. Due to their novelty and complexity of structure and significant biological activity, they are increasingly attracting the interest of pharmacology and synthetic chemistry researchers. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a diterpene dimer compound containing an oxazole ring and a preparation method and application thereof.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] In the first aspect of the present invention, the present invention provides a diterpene dimer compound, wherein the diterpene dimer compound has a structure of formula I:

[0007]

[0008] In the second aspect of the present invention, the present invention provides a method for preparing the above-mentioned diterpene dimer compound, comprising the following steps:

[0009] S1. Collect mature seeds of pond cypress, extract with ethanol, and concentrate in vacuo until there is no alcohol to obtain an extract, and extract the extract with ethyl acetate to obtain an ethyl acetate extract;

[0010] S2. separating the ethyl acetate extract by gel column chromatography to obtain an enriched portion of the terpene polymer;

[0011] S3. The enriched portion was purified by silica gel column chromatography and ODS column chromatography to obtain a target fraction;

[0012] S4. Sequentially subjecting the target fraction to silica gel column chromatography, ODS column chromatography and reverse phase high performance liquid chromatography purification to obtain a compound having the structural formula I.

[0013] Preferably, in step S1, the ethanol is 95% ethanol.

[0014] Preferably, in step S2, the gel column chromatography separation is eluted with methanol.

[0015] Preferably, in step S3, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution.

[0016] Preferably, in step S3, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, specifically: using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 100:0, 100:5, 100:10, 100:20, 100:30, 100:50, 100:100 and 0:100 for gradient elution.

[0017] Preferably, in step S3, the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

[0018] Preferably, in step S3, the ODS column chromatography uses a methanol / water mixed solvent for gradient elution, specifically: using a methanol / water mixed solvent with a methanol to water volume ratio of 60:40, 70:30, 80:20, 85:15, 90:10, 95:5 and 100:0 for gradient elution.

[0019] Preferably, in step S3, the reversed-phase HPLC uses an XB-C18 chromatographic column with a specification of 5 μm, 21.2×250 mm, and the mobile phase of the reversed-phase HPLC is a methanol-water solution, in which the volume ratio of methanol to water is 93:7.

[0020] In the third aspect of the present invention, the present invention provides a use of the above-mentioned diterpene dimer compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-diabetic drug.

[0021] Compared with the prior art, the present invention selects mature pond cypress seeds, performs reflux extraction with ethanol, extracts with ethyl acetate for multiple times, and then obtains the above-mentioned diterpene dimer compounds through various separation technologies and means. The diterpene dimer compounds are formed by connecting a rosinane-type diterpene unit with a modified rosinane-type unit through an oxazole ring (the 6' position and the 8' position are connected after the B ring is cleaved, and the 7' position forms an aldehyde group, thereby forming the carbon atom position of the oxazole ring) to form a diterpene dimer, which has a novel structure and significant hypoglycemic activity, can be used as an active ingredient of an anti-diabetic drug, and has a wide range of uses. DETAILED DESCRIPTION

[0022] To further illustrate the present invention, the preparation method of the terpene polymer containing an oxazole ring provided by the present invention and the application of the same in the preparation of anti-diabetic drugs are described in detail below in conjunction with examples.

[0023] Example 1: Preparation of a compound having the structural formula I

[0024] The present invention uses mature seeds of Taxodium truncatum of the Taxodiaceae family to perform reflux extraction with 95% ethanol, and prepares the compound of the present invention through the steps of extraction, enrichment, separation, etc. The seeds of Taxodium truncatum are collected from Nantong, Jiangsu.

[0025] Taking the seeds of pond cypress as an example, the process for preparing the compound of formula I is as follows:

[0026] 1) Extraction, enrichment and initial separation

[0027] The mature and dried seeds of the pond cypress were crushed (10 kg), refluxed and extracted three times with 95% ethanol, and then vacuum concentrated to alcohol-free, mixed with hot water (60°C), and then extracted multiple times with ethyl acetate in equal proportions. The extract layer (0.5 kg) was subjected to gel column chromatography, using methanol as the elution solvent, and each 200 ml was used as a fraction to obtain fractions Fr.1-8. After LC-MS identification, Fr.1-3 were combined and separated by silica gel column chromatography. The elution solvents were, in sequence, petroleum ether / ethyl acetate in a volume ratio of 100:0, petroleum ether / ethyl acetate of 100:5, petroleum ether / ethyl acetate of 100:10, petroleum ether / ethyl acetate of 100:20, petroleum ether / ethyl acetate of 100:30, petroleum ether / ethyl acetate of 100:50, petroleum ether / ethyl acetate of 100:100, and petroleum ether / ethyl acetate of 0:100, to obtain fractions Fr.AH, and each fraction was detected by LC-MS to obtain the target fraction Fr.B.

[0028] 2) ODS-C18 column chromatography separation

[0029] The target fraction Fr.B was separated by ODS-C18 column chromatography. Methanol / water with a volume ratio of 60:40, methanol / water with a volume ratio of 70:30, methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:10, methanol / water with a volume ratio of 95:5, and methanol / water with a volume ratio of 100:0 were used in sequence. The fractions were collected and numbered Fr.B1-7 respectively. After HPLC and LC-MS analysis, the target fraction Fr.B4 was locked.

[0030] 3) Reverse-phase HPLC purification

[0031] The target fraction Fr.B4 was purified by reverse phase high performance liquid chromatography. The preparation conditions were preparative chromatographic column XB-C18 (5μm, 21.2×250mm), methanol-water (93:7, V / V), flow rate 10.0mL / min, detection wavelength 230nm, and compound formula I was prepared. The mass spectrum and spectroscopy data of formula I are shown below. Compound I: taxodascaloid C; optical rotation [α] 2 D 5 +155.1(c 0.09MeOH); UV absorption(MeOH)λ max (logε)200(3.97)nm, 239(3.87)nm, main absorption peaks of infrared spectrum (KBr)ν max 3484, 2923, 2864, 1600 and 1656cm -1 ; High resolution mass spectrum m / z is 612.4059 [M+H] + (ca l cd for C 40 H 54 NO 4 ), the H NMR and C NMR data are shown in Table 1.

[0032] Table 1. H NMR and C NMR data of compound I

[0033]

[0034]

[0035] The above results show that the structure of the compound formula I is correct.

[0036] Example 2: Hypoglycemic Activity of Compound I

[0037] 1) Experimental Materials

[0038] Instruments and reagents: MD 384p l us microplate reader, constant temperature incubator, micropipette, 96-well cell culture plate (COSTAR), pH meter, Jasco 810 circular dichroism spectrometer. α-D-glucosidase, 4-nitrobenzene-α-D-pyranoglucopyranoside (p-NPG), 1-deoxynojirimycin and genistein were purchased from Sigma; KH 2 PO 4 , K 2 HPO 4 etc. are products of Guangzhou Chemical Reagent Factory; the water used is ultrapure water; other analytical grade reagents are products of Jiangsu Hanbang Technology Co., Ltd.

[0039] 2) Experimental methods

[0040] 140 μL pH 6.8 phosphate buffer (0.05 mol / L), 10 μL α-glucosidase solution (0.5 U / mL) and 2 μL sample solutions of different concentrations were added to a 96-well ELISA plate and incubated at 37°C for ten minutes. Then 48 μL p-NPG solution (0.2 mM) was added, and the absorbance of the reaction mixture was measured at 405 nm after reacting for another half an hour. Each group of experiments was repeated 3 times, and IC 50 The values ​​were averaged. 1-Deoxynojirimycin and genistein were used as positive controls.

[0041] Inhibition rate (%) = 100 × (A control -A sample ) / A control

[0042] 3) Experimental results

[0043] According to the above method, the IC of the sample of formula I for inhibiting α-glucosidase was calculated. 50 The results are shown in Table 2.

[0044] Table 2. Screening results of test samples inhibiting α-glucosidase activity

[0045]

[0046]

[0047] All the control samples and test samples were repeated three times in parallel.

[0048] The results show that the compound of formula I of the present invention has good glucose-lowering activity and can be used as an active ingredient of anti-diabetic drugs.

[0049] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A diterpene dimer compound, characterized in that: The diterpene dimer compound has a structure of formula I:

2. A method for preparing a diterpene dimer compound as claimed in claim 1, characterized in that: The steps include: S1. Collect mature seeds of pond cypress, extract with ethanol, and concentrate in vacuo until there is no alcohol to obtain an extract, and extract the extract with ethyl acetate to obtain an ethyl acetate extract; S2. separating the ethyl acetate extract by gel column chromatography to obtain an enriched portion of the terpene polymer; S3. The enriched portion was purified by silica gel column chromatography and ODS column chromatography to obtain a target fraction; S4. Sequentially subjecting the target fraction to silica gel column chromatography, ODS column chromatography and reverse phase high performance liquid chromatography purification to obtain the diterpene dimer compound.

3. The preparation method according to claim 2, characterized in that: In step S1, the ethanol is 95% ethanol.

4. The preparation method according to claim 2, characterized in that: In step S2, the gel column chromatography separation is eluted with methanol.

5. The preparation method according to claim 2, characterized in that: In step S3, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution.

6. The preparation method according to claim 5, characterized in that: The silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, specifically: using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 100:0, 100:5, 100:10, 100:20, 100:30, 100:50, 100:100 and 0:100 for gradient elution.

7. The preparation method according to claim 2, characterized in that: In step S3, the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

8. The preparation method according to claim 7, characterized in that: The ODS column chromatography uses a methanol / water mixed solvent for gradient elution, specifically: using a methanol / water mixed solvent with a methanol:water volume ratio of 60:40, 70:30, 80:20, 85:15, 90:10, 95:5 and 100:0 for gradient elution.

9. The preparation method according to claim 2, characterized in that: In step S3, the reversed-phase HPLC uses an XB-C18 chromatographic column with a specification of 5 μm, 21.2×250 mm, and the mobile phase of the reversed-phase HPLC is a methanol-water solution, in which the volume ratio of methanol to water is 93:

7.

10. Use of the diterpene dimer compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-diabetic drug.

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