Diterpene dimer compounds and their preparation method and application

Abstract

The present invention belongs to the field of medical technology and discloses a diterpene dimer compound, its preparation method, and application. The diterpene dimer compound has a structure of Formula I. The preparation method comprises: extracting the seeds of Pool Cypress with ethanol, concentrating them in vacuo until they are alcohol-free, and extracting them with ethyl acetate to obtain an ethyl acetate extract; separating the ethyl acetate extract by gel column chromatography to obtain a terpene polymer enrichment site, and sequentially purifying the extract by silica gel column chromatography and ODS column chromatography to obtain a target fraction; sequentially subjecting the target fraction to silica gel column chromatography, ODS column chromatography, and reverse-phase high-performance liquid chromatography to obtain a diterpene dimer compound. In vitro inhibition of α-glucosidase experiments confirmed that compound Formula I has significant hypoglycemic activity and can be used as an anti-diabetic active ingredient with a wide range of uses. #imgabs0#

Description

Technical Field

[0001] The present 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 class of natural products composed of identical or different diterpene units. They are widely distributed, for example, in various medicinal plants from the Taxodiaceae, Leguminosae, and Lamiaceae families. Common diterpene dimers are typically formed by the polymerization of diterpenes through various linkages, such as DA cyclization, [2+2], [5+2], C-C single bonds, ether bonds, and ester bonds, resulting in complex and diverse backbone types and species, which in turn exhibit diverse and significant biological activities. Modern pharmacological studies have shown that diterpene dimers exhibit a variety of biological activities, including anti-tumor, anti-inflammatory, anti-tumor cell migration, inhibition of non-alcoholic fatty liver disease, and hypoglycemic effects. The polymerization method of oxazole ring linkage is a rare type of polymerization. Furthermore, terpene polymers are characteristic components of Taxodiaceae plants. Studies have found that terpene polymers found in the Taxodiaceae family have significant therapeutic potential for antibacterial, anti-tumor, and anti-diabetic effects.

[0003] In terms of chemical structure, diterpene dimers are polymers formed by diterpenes connected by oxazole rings. They are a novel class of terpene polymers. Due to the novelty and complexity of their structure and their 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 a 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 to obtain an extract, which is extracted with ethyl acetate to obtain an ethyl acetate extract;

[0010] S2. The ethyl acetate extract was separated by gel column chromatography to obtain a terpenoid polymer enriched portion;

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

[0012] S4. The target fraction is sequentially subjected to silica gel column chromatography, ODS column chromatography and reverse-phase high performance liquid chromatography 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 is performed using a petroleum ether / ethyl acetate mixed solvent for gradient elution, specifically: gradient elution is performed 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.

[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 high performance liquid chromatography uses an XB-C18 chromatographic column with a specification of 5 μm, 21.2×250 mm, and the mobile phase of the reversed-phase high performance liquid chromatography 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 multiple times, and then uses various separation techniques and methods to obtain the above-mentioned diterpene dimer compound. The diterpene dimer compound is composed of a bietane-type diterpene unit connected by an oxazole ring and a modified bietane-type unit (after the B ring is cleaved, the 6' position is connected to the 8' position, and the 7' position forms an aldehyde group, which further forms the carbon atom position of the oxazole ring). The diterpene dimer has a novel structure and significant hypoglycemic activity. It can be used as an active ingredient in anti-diabetic drugs 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 its application in the preparation of anti-diabetic drugs are described in detail below with reference to examples.

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

[0024] The present invention uses mature Taxodium truncatum seeds of Taxodia genus of Taxodiaceae to carry out reflux extraction with 95% ethanol, and prepares the compound of the present invention through the steps of extraction, enrichment and separation. The Taxodium truncatum seeds 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] Mature, dried pond cypress seeds (10 kg) were crushed and extracted three times with 95% ethanol under reflux. The mixture was then vacuum concentrated to a concentration free of alcohol, mixed with hot water (60°C), and extracted multiple times with equal proportions of ethyl acetate. The extract (0.5 kg) was subjected to gel column chromatography using methanol as the elution solvent, yielding 200 ml fractions 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. The mixtures were sequentially separated using methanol / water ratios of 60:40, 70:30, 80:20, 85:15, 90:10, 95:5, and 100:0, respectively. Fractions were collected and numbered Fr.B1-7. After HPLC and LC-MS analysis, the target fraction, Fr.B4, was identified.

[0030] 3) Reverse-phase high performance liquid chromatography purification

[0031] The target fraction Fr.B4 was purified by reverse-phase high-performance liquid chromatography. The preparation conditions were a preparative column XB-C18 (5 μm, 21.2 × 250 mm), methanol-water (93:7, V / V), a flow rate of 10.0 mL / min, and a detection wavelength of 230 nm to obtain compound I. The mass spectrum and spectroscopic 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 1656 cm -1 ; High-resolution mass spectrum m / z 612.4059 [M+H] + (ca l cd for C 40 H 54 NO4), 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 obtained 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-nitrophenyl-α-D-glucopyranoside (p-NPG), 1-deoxynojirimycin, and genistein were purchased from Sigma; potassium phosphate and potassium hydroxide were purchased from Guangzhou Chemical Reagent Factory. Ultrapure water was used; all other analytical grade reagents were purchased from Jiangsu Hanbang Technology Co., Ltd.

[0039] 2) Experimental methods

[0040] 140 μL of pH 6.8 phosphate buffer (0.05 mol / L), 10 μL of α-glucosidase solution (0.5 U / mL), and 2 μL of sample solutions of different concentrations were added to a 96-well microtiter plate and incubated at 37°C for ten minutes. Then, 48 μL of p-NPG solution (0.2 mM) was added. After another half-hour reaction, the absorbance of the reaction mixture was measured at 405 nm over time. Each experiment was repeated three times. 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] The IC of the sample of formula I for inhibiting α-glucosidase was calculated according to the above method. 50 The results are shown in Table 2.

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

[0045]

[0046]

[0047] aAll controls 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 hypoglycemic activity and can be used as an active ingredient of antidiabetic drugs.

[0049] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended 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 according to claim 1, characterized in that: The steps include: S1. Collect mature seeds of pond cypress, extract with ethanol, and concentrate in vacuo to obtain an extract, which is extracted with ethyl acetate to obtain an ethyl acetate extract; S2. The ethyl acetate extract was separated by gel column chromatography to obtain an enriched portion of the terpenoid polymer, and the gel column chromatography was eluted with methanol; S3. The enriched portion was purified by silica gel column chromatography and ODS column chromatography to obtain the target fraction, the silica gel column chromatography using a petroleum ether / ethyl acetate mixed solvent gradient elution, the ODS column chromatography using a methanol / water mixed solvent gradient elution; S4. Separate the target fraction by silica gel column chromatography, ODS column chromatography and reverse phase high performance liquid chromatography to obtain the diterpene dimer compound.

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

4. The preparation method according to claim 2, 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.

5. The preparation method according to claim 2, 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 to water volume ratio of 60:40, 70:30, 80:20, 85:15, 90:10, 95:5 and 100:0 for gradient elution.

6. The preparation method according to claim 2, characterized in that In step S4, 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.

7. Use of the diterpene dimer compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an antidiabetic drug.

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