A triterpenoid compound containing a spiro ring, a preparation method thereof and application thereof in preparing an anti-diabetic drug

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a spirocyclic triterpenoid compound, its preparation method, and its application in the preparation of antidiabetic drugs. This invention uses the whole herb of *Euphorbia humifusa* as the isolation target and employs an efficient and systematic separation and preparation method to enrich terpenoid compounds. A spirocyclic triterpenoid compound was obtained through silica gel, ODS column chromatography, and HPLC. In vitro inhibition of α-glucosidase experiments confirmed that compound I has significant hypoglycemic activity and can be used as an active ingredient in antidiabetic drugs, with broad applications.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a triterpenoid compound containing a spirocyclic ring, its preparation method, and its application in the preparation of antidiabetic drugs. Background Technology

[0002] Diabetes mellitus, a global chronic metabolic disease, poses a serious threat to human health due to its high incidence and complications. Developing safe and effective new hypoglycemic drugs remains a hot research topic in the pharmaceutical field.

[0003] Against this backdrop, traditional herbal medicines, due to their long history of application and potential bioactivity, have become an important resource for discovering novel lead compounds. *Euphorbia humifusa* Willd., the dried whole herb of *Euphorbia humifusa* or *Euphorbia variegata*, is harvested in summer and autumn, impurities are removed, and it is sun-dried. According to traditional Chinese medicine texts, *Euphorbia humifusa* has the effects of cooling the blood and stopping bleeding, promoting blood circulation and removing blood stasis, and stopping bleeding without leaving blood stasis. It is often used for hemoptysis, epistaxis, hematochezia, hematuria, hemorrhoidal bleeding, metrorrhagia, and traumatic bleeding caused by blood heat. It is also used in folk medicine to treat diarrhea and diabetes. This record suggests that *Euphorbia humifusa* contains potential hypoglycemic active ingredients and possesses significant research and development value.

[0004] Therefore, there is an urgent need for an efficient and systematic separation and preparation method that can obtain a series of structurally diverse monomeric compounds from Euphorbia humifusa on a large scale to meet the needs of subsequent in-depth pharmacological research and drug development. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a spirocyclic triterpenoid compound, a method for its preparation, and its application in the preparation of antidiabetic drugs. This novel spirocyclic triterpenoid compound has significant hypoglycemic activity and can be used as an active ingredient in antidiabetic drugs.

[0006] To solve the above problems, the technical solution provided by the present invention is as follows:

[0007] In a first aspect, the present invention provides a triterpenoid compound containing a spirocyclic ring, having the structural formula as shown in Formula I:

[0008]

[0009] The triterpenoid compound with the structural formula I is disclosed for the first time.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned triterpenoid compound containing a spirocyclic ring, comprising the following steps:

[0011] S1. The mature and dried whole plant of Euphorbia humifusa is crushed, extracted by reflux with ethanol, concentrated under vacuum until alcohol-free, suspended in water, and extracted with ethyl acetate to obtain ethyl acetate extract;

[0012] S2. Using petroleum ether / ethyl acetate as the elution solvent, the ethyl acetate extract was separated by silica gel column chromatography to obtain fractions Fr.1-10;

[0013] S3. Using methanol / water as the elution solvent, fraction Fr.4 was separated by ODS-C18 column chromatography to obtain subfractions Fr.4A-4H.

[0014] S4. The subfluid Fr.4A-4D was purified by reversed-phase high-performance liquid chromatography to obtain the triterpenoid compound containing a spirocyclic ring.

[0015] In some embodiments of the present invention, the reflux extraction with ethanol solution specifically refers to: reflux extraction three times with an aqueous ethanol solution of 95% by volume.

[0016] In some embodiments of the present invention, step S2 specifically involves eluting the ethyl acetate extract with petroleum ether / ethyl acetate in volume ratios of 100:10, 100:15, 100:20, 100:25, 100:30, 100:40, 100:50, 100:60, 100:70, and 100:80.

[0017] In some embodiments of the present invention, step S3 specifically involves eluting fraction Fr.4 sequentially using methanol / water systems with volume ratios of 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, and 100:0.

[0018] In some embodiments of the present invention, step S4 specifically involves: using an XB-C18 chromatographic column with a particle size of 5 μm and a size of 21.2 × 250 mm, using a methanol-water solution with a volume ratio of 93:7 as the mobile phase, setting the flow rate to 10.0 mL / min, and detecting at a wavelength of 230 nm, thereby preparing compound I.

[0019] In a third aspect, the present invention provides the use of the above-mentioned spirocyclic triterpenoid compound as an active ingredient in the preparation of an antidiabetic drug.

[0020] In a fourth aspect, the present invention provides a medicament comprising the above-described spirocyclic triterpenoid compound or a pharmaceutically acceptable salt thereof.

[0021] In some implementations, the aforementioned drug also includes pharmaceutically acceptable excipients and carriers.

[0022] In some implementations, the pharmaceutical preparations of the above-mentioned drugs are granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels, or injections.

[0023] Compared with existing technologies, this invention selects dried whole plant of *Euphorbia humifusa*, extracts it by reflux with ethanol, extracts it multiple times with ethyl acetate, and then uses various separation techniques and methods to obtain the above-mentioned oleanane-type triterpenoid compound containing a spirocyclic ring, Formula I. This oleanane-type triterpenoid compound containing a spirocyclic ring is a novel spirocyclic triterpenoid discovered for the first time, enriching the skeleton and types of triterpenoid compounds, and has significant hypoglycemic activity. It can be used as an active ingredient in antidiabetic drugs, providing a candidate compound for the development of new antidiabetic drugs. Detailed Implementation

[0024] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Where specific conditions are not specified in the following embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0025] Example 1: Preparation of a compound with the structural formula of formula I

[0026] This invention uses dried whole plant of *Euphorbia humifusa*, extracted by reflux with 95% (v / v) ethanol-water, and prepares the compound of this invention through extraction, enrichment, and separation. *Euphorbia humifusa* was collected from Nantong City, Jiangsu Province.

[0027] Taking the whole plant of *Euphorbia humifusa* as an example, the process for preparing a compound with structural formula I is as follows:

[0028] 1) Extraction, enrichment, and initial separation

[0029] The dried whole plant of *Euphorbia humifusa* was chopped (5.0 kg), extracted three times by reflux with 95% ethanol, then concentrated under vacuum until alcohol-free, mixed with hot water (60 °C), and then extracted multiple times with ethyl acetate in an equal proportion.

[0030] The extract (0.64 kg) was subjected to silica gel column chromatography with petroleum ether / ethyl acetate as the elution solvent to obtain fractions Fr.1-10. After HPLC and LC-MS analysis, it was found that fraction Fr.4 mainly contained triterpenoids, and Fr.4 was identified as the target fraction.

[0031] 2) ODS-C18 column chromatography separation

[0032] The target fraction Fr.4 was separated by ODS-C18 column chromatography. Methanol / water ratios were successively applied at 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, and 100:0 (v / v). Fractions Fr.4A-4H were obtained, and the target fraction Fr.4A-4D was identified by HPLC and LC-MS analysis.

[0033] 3) Reversed-phase high-performance liquid chromatography purification

[0034] Fractions Fr.4A-4D were purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The preparation conditions were as follows: preparative column XB-C18 (5 μm, 21.2 × 250 mm), methanol-water (93:7, V / V), flow rate 10.0 mL / min, detection wavelength 230 nm, yielding compound I. The mass spectrometry and wavelength spectrometry data for Formula I are shown below.

[0035] Compound I: spiroeuphone A, optical rotation [α]25 D+25.3 (c 0.09 MeOH); UV absorption (MeOH) λ max (log ε) 200 (0.167) nm, the main absorption peak in the infrared spectrum (KBr) ν max 3990 cm -1 The high-resolution mass spectrometry m / z was 481.3661 [M+Na]. + (calcd for C 30 H 50 The hydrogen and carbon NMR spectra data are shown in Table 1.

[0036] Table 1. 1H and 1C NMR spectra of compound I

[0037]

[0038] The above results indicate that the structure of the obtained compound, Formula I, is correct.

[0039] Example 2: Hypoglycemic Activity of Compound I

[0040] 1) Experimental materials

[0041] Instruments and reagents: MD 384 plus, microplate reader, incubator, 96-well cell culture plate (COSTAR), micropipette, Jasco 810 circular dichroism chromatograph. α-D-glucosidase, 4-nitrobenzene-α-D-glucopyranoside (p-NPG), 1-deoxynojirimycin, and genistein were purchased from Sigma-Aldrich; KH2PO4, K2HPO4, etc., were products of Guangzhou Chemical Reagent Factory; ultrapure water was used; other analytical grade reagents were products of Jiangsu Hanbang Technology Co., Ltd.

[0042] 2) Experimental methods

[0043] The study used a 96-well plate reaction system, with three replicates and one background control well for each sample group, and a final reaction volume of 200 μL. The specific steps were as follows: a. Plate pretreatment: Add 140 μL (three replicates) and 150 μL (background well) of PBS buffer to each of the four wells; b. Sample loading: Add 10 μL of enzyme solution to each of the three replicates, and add 2 μL of the sample to each of the four wells (or 2 μL of DMSO for the blank group); c. Initial incubation: React at 37 ℃ for 10 min; d. Substrate reaction: Add 48 μL of p-NPG solution to each well and incubate at 37 ℃ for another 20 min; e. Reaction termination: Finally, add 50 μL of Na2CO3 solution to each well to terminate the reaction. The absorbance of each well was measured at 405 nm using a microplate reader, with three parallel measurements. 1-Deoxynojirimycin and genistein were used as positive controls.

[0044] Inhibition rate (%) = 100 × (A) control - A sample ) / A control

[0045] 3) Experimental Results

[0046] The above method was used to conduct tests, and the IC50 of sample I inhibiting α-glucosidase was calculated. 50 The values ​​are shown in Table 2.

[0047] Table 2. Screening results of α-glucosidase activity inhibition in test samples

[0048] compound Inhibition rate ± SD (%) <![CDATA[IC 50 ± SD (μM)]]> I 89.12 ± 1.21 1.92 ± 0.16 Gentian isoflavones 45.85 ± 1.81 61.02 ± 1.52 1-Deoxynojirimycin 91,02 ± 0.20 1.35± 0.06

[0049] The results show that compound I of the present invention has good hypoglycemic activity and can be used as an active ingredient in antidiabetic drugs.

[0050] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A triterpenoid compound containing a spirocyclic ring, characterized in that, The triterpenoid compound has the structure of formula I:

2. A method for preparing a triterpenoid compound containing a spirocyclic ring as described in claim 1, characterized in that, The preparation method includes the following steps: S1. The mature and dried whole plant of Euphorbia humifusa is crushed, extracted by reflux with ethanol solution, concentrated under vacuum until alcohol-free, suspended in water, and extracted with ethyl acetate to obtain ethyl acetate extract; S2. Using petroleum ether / ethyl acetate as the elution solvent, the ethyl acetate extract was separated by silica gel column chromatography to obtain fractions Fr.1-10; S3. Using methanol / water as the elution solvent, fraction Fr.4 was separated by ODS-C18 column chromatography to obtain subfractions Fr.4A-4H. S4. The subfluid Fr.4A-4D was purified by reversed-phase high-performance liquid chromatography to obtain the triterpenoid compound containing a spirocyclic ring.

3. The preparation method according to claim 2, characterized in that, The reflux extraction with ethanol solution specifically involves reflux extraction three times using a 95% (v / v) ethanol aqueous solution.

4. The preparation method according to claim 2, characterized in that, Step S2 specifically involves eluting the ethyl acetate extract with petroleum ether / ethyl acetate systems of volume ratios of 100:10, 100:15, 100:20, 100:25, 100:30, 100:40, 100:50, 100:60, 100:70, and 100:

80.

5. The preparation method according to claim 2, characterized in that, Step S3 specifically involves eluting fraction Fr.4 sequentially using methanol / water systems with volume ratios of 60:40, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, and 100:

0.

6. The preparation method according to claim 2, characterized in that, Step S4 specifically involves using an XB-C18 chromatographic column with a particle size of 5 μm and a size of 21.2 × 250 mm, with a methanol-water solution of 93:7 as the mobile phase, a flow rate of 10.0 mL / min, and detection at a wavelength of 230 nm to prepare compound I.

7. The use of a spirocyclic triterpenoid compound as described in claim 1 as an active ingredient in the preparation of an antidiabetic drug.

8. A drug, characterized in that, The drug comprises the triterpenoid compound containing a spirocyclic ring as described in claim 1, or a pharmaceutically acceptable salt thereof.

9. The medicament according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients and carriers.

10. The medicament according to claim 8, characterized in that, The dosage form of the drug is selected from one of granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels, and injections.

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