Beta-dihydroagarofuran sesquiterpene, preparation method and application

Abstract

The invention relates to the technical field of medicines, in particular to beta-dihydroagarofuran type sesquiterpenes, a preparation method and application, and the structural formula of the beta-dihydroagarofuran type sesquiterpenes is shown as a formula I: # imgabs0 #. The chemical structure and physicochemical properties of the beta-dihydroagarofuran type sesquiterpenoids are confirmed through nuclear magnetic resonance, mass spectrum, infrared and other determination methods, and the pharmacological activity of the beta-dihydroagarofuran type sesquiterpenoids is verified through cell experiments. The novel beta-dihydroagarofuran type sesquiterpenoids have an obvious inhibition effect on SGLT-2, show excellent hypoglycemic activity and can be used as a lead compound for developing a novel hypoglycemic drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly relates to a β-dihydroagarofuran sesquiterpene, a preparation method and an application thereof. Background Art

[0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by insulin secretion deficiency or impaired biological action thereof, or both. Persistent hyperglycemia over a long term causes chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels and nerves. Although there are relatively many current first-line clinical drugs with diverse targets, these hypoglycemic drugs are mainly Western medicines and have defects such as large toxic and side effects and a serious economic burden on patients caused by long-term medication.

[0003] The contribution of the kidney to gluconeogenesis is approximately 15 - 55 g / day. That is, after fasting overnight, the kidney can release 20 - 25% glucose into the human circulation. The kidney mainly maintains the glucose homeostasis in the body through the reabsorption of glucose by the glomerulus, and its reabsorption mainly occurs in the proximal tubule (PXT). The hyperglycemia in diabetic patients partly originates from the reabsorption of metabolized glucose by the glomerulus. Sodium-glucose cotransporter 2 (SGLT-2) is located on the luminal side of the first segment of the PXT and has the characteristic of high-capacity affinity for glucose transport. In a healthy human body, SGLT-2 will reabsorb about 90% of the filtered glucose. Therefore, in diabetic patients, inhibiting the reabsorption of metabolized glucose by SGLT-2 in the glomerulus can effectively reduce the blood glucose of diabetic patients. Currently, SGLT-2 inhibitors have been developed into a class of hypoglycemic drugs widely welcomed by diabetic patients.

[0004] Plants of the genus Salacia contain rich active ingredients. At present, more than 10 species of plants of this genus have been found in China, mainly distributed in Hainan, Yunnan and Guizhou regions of China. In China, India, Thailand, Sri Lanka and other places, many plants of this genus can be used as medicine, and the more common medicinal parts are roots, branches, leaves and fruits (seed kernels). During the previous hypoglycemic activity screening process, we found that the chloroform extract of the 90% ethanol extract of the branches and leaves of Salacia obovatilimba has significant hypoglycemic activity in a type 2 diabetic rat model. Therefore, based on literature research and the research accumulation of the research group, further in-depth activity tracking research obtained a series of natural small molecules, and the active molecule β-dihydroagarofuran sesquiterpene obovatilin K was isolated therefrom. This study conducted relevant chemical and pharmacological activity research on its chemical composition and activity screening, which is of great significance for comprehensively understanding the properties, action pathways and drug development of β-dihydroagarofuran sesquiterpene compounds. Summary of the Invention

[0005] In order to overcome the deficiencies of the above technical defects, the present invention provides a β-dihydroagarofuran sesquiterpene obovatilin K and a preparation method thereof, and studies its hypoglycemic activity and pharmaceutical uses.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a β-dihydroagarofuran sesquiterpene having the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, stereoisomer thereof:

[0008]

[0009] Further, the molecular formula of the β-dihydroagarofuran sesquiterpene is C 31 H 37 NO 10 , and this compound is named obovatilin K.

[0010] In the second aspect, the present invention provides the use of a β-dihydroagarofuran sesquiterpene having the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, stereoisomer thereof in the preparation of a hypoglycemic drug.

[0011] Further, the following uses are also within the protection scope of the present invention:

[0012] A β-dihydroagarofuran sesquiterpene having the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, stereoisomer thereof is used to prepare an inhibitor of SGLT-2.

[0013] The β-dihydroagarofuran sesquiterpene having the structure shown in Formula I or a pharmaceutically acceptable salt, tautomer, stereoisomer thereof can be used alone in the preparation of the drug, or can be used in combination with other components having hypoglycemic activity.

[0014] In the third aspect, the present invention provides a hypoglycemic drug, comprising a β-dihydroagarofuran sesquiterpene having the structure shown in Formula I and a pharmaceutically acceptable salt, tautomer, stereoisomer thereof and a pharmaceutically acceptable excipient.

[0015] In particular, the pharmaceutically acceptable carrier is generally recognized for this purpose and is an inactive ingredient of the medicament.

[0016] The carrier includes excipients such as starch, water, etc.; lubricants such as magnesium stearate, etc.; disintegrants such as microcrystalline cellulose, etc.; fillers such as lactose, etc.; binders such as pregelatinized starch, dextrin, etc.; sweeteners; antioxidants; preservatives; flavoring agents; fragrances, etc.

[0017] Among them, the drug exists in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, patches.

[0018] Among them, the drug is administered through enteral and parenteral administration routes.

[0019] In particular, the parenteral administration route is selected from injection administration, respiratory administration, cutaneous administration, mucosal administration or cavity administration.

[0020] Among them, parenteral administration preparations are selected from injections, sprays, aerosols, patches, etc.

[0021] In particular, the enteral administration preparations are selected from tablets, capsules, powders, granules, pills, solutions, emulsions or syrups, etc.

[0022] Furthermore, the drug with hypoglycemic activity provided by the present invention can be used in combination with components having other hypoglycemic activities.

[0023] The pharmaceutical composition of the invention contains 0.1 - 90% by weight of the active ingredient.

[0024] The pharmaceutical composition can be prepared according to methods known in the art. For this purpose, if necessary, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable administration form or dosage form for human use.

[0025] In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0026] Fourthly, the present invention provides a preparation method of a β-dihydroagarofuran sesquiterpene shown in formula I, comprising the following steps:

[0027] S1. Take a plant of the genus Salacia, crush it, add a first extraction solvent, reflux and extract multiple times, collect the extract, filter, and concentrate the filtrate to a viscous state to obtain a viscous product;

[0028] S2. Dissolve the viscous product in 1 - 2 times the amount of pure water, extract it multiple times with a second extraction solvent, collect the extract, and concentrate it under reduced pressure to obtain an extract;

[0029] S3. Perform silica gel column chromatography on the extract, elute it with a gradient of a mixed organic solvent with a volume ratio of 1:0 to 0:1, collect the target gradient eluate, and concentrate it under reduced pressure;

[0030] S4. Perform MCI column chromatography on the concentrate obtained in S3, elute it with a gradient of a methanol aqueous solution with a volume content of 60 - 100%, collect the target gradient eluate, and concentrate it under reduced pressure;

[0031] S5. Purify the concentrated solution obtained in S4 by semi-preparative HPLC to obtain the β-dihydroagarofuran sesquiterpenoid shown in Formula I.

[0032] Furthermore, in S1, the first extraction solvent is selected from one of acetone, ethanol or methanol with a concentration of 80-100%; and / or,

[0033] Furthermore, in S2, the second extraction solvent is selected from one of chloroform and ether.

[0034] Furthermore, in S3, gradient elution is carried out successively with a mixed organic solvent of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, 0:1. The target gradient eluent is a mixed organic solvent with a volume ratio of 7:3. The mixed organic solvent is selected from one of petroleum ether-acetone, n-hexane-acetone or petroleum ether-ethyl acetate.

[0035] Furthermore, in S4, the target gradient eluent is an aqueous methanol solution with a volume content of 70-85%.

[0036] Furthermore, in S5, the mobile phase is an aqueous methanol solution with a volume concentration of 75%, the stationary phase is Agilent Zorbax C18, the detection wavelength is 254 nm, the flow rate is 3 mL / min, and the injection volume is 50-100 μL.

[0037] Beneficial effects: The β-dihydroagarofuran sesquiterpenoid is isolated from Salacia hekouensis of the genus Salacia. The chemical structure and physicochemical properties of the β-dihydroagarofuran sesquiterpenoid compounds are confirmed by measurement methods such as nuclear magnetic resonance, mass spectrometry, and infrared. The pharmacological activities of the β-dihydroagarofuran sesquiterpenoid are verified through cell experiments. The new β-dihydroagarofuran sesquiterpenoid compounds have obvious inhibitory activity against SGLT-2, showing excellent hypoglycemic activity, and can be used as lead compounds for the development of new hypoglycemic drugs. Description of the Drawings

[0038] Figure 1 It is the HRMS spectrum of the β-dihydroagarofuran sesquiterpenoid compound;

[0039] Figure 2 It is for the 1 1H NMR spectrum of the β-dihydroagarofuran sesquiterpenoid compound;

[0040] Figure 3 It is for the 13 13C NMR spectrum of the β-dihydroagarofuran sesquiterpenoid compound;

[0041] Figure 4IR spectrum of β-dihydroagarofuran sesquiterpenoids;

[0042] Figure 5 Effect of β-dihydroagarofuran sesquiterpenoids on the in vitro inhibitory activity of SGLT-2. Detailed implementation manners

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific implementation manners. For the experimental methods without specific conditions noted in the following examples, they are usually in accordance with conventional conditions or the conditions recommended by the manufacturer. The test materials used in the following examples are, unless otherwise specified, obtained from regular biochemical reagent stores. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0044] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0045] Based on the present invention, the present invention aims to provide a β-dihydroagarofuran sesquiterpenoid, which uses the branches, leaves and fruits (seeds) of the dried Salacia obovata as raw materials, and obtains a white amorphous powder named obovatilin K through extract extraction, organic solvent extraction, silica gel column chromatography, and high-pressure liquid chromatography separation. This compound has obvious inhibitory activity on SGLT-2, can inhibit the reabsorption of metabolized glucose, and thus effectively reduce the blood sugar of diabetic patients.

[0046] In some specific embodiments, the β-dihydroagarofuran sesquiterpenoid shown in formula I or its pharmaceutically acceptable salts, tautomers, stereoisomers:

[0047]

[0048] In some embodiments of this implementation manner, the β-dihydroagarofuran sesquiterpenoid shown in formula I or its pharmaceutically acceptable salts, tautomers, stereoisomers are used to prepare an inhibitor of SGLT-2.

[0049] In some embodiments of this embodiment, the β-dihydroagarofuran sesquiterpenoid shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof can be used alone in the preparation of the drug or can be compounded with other components having hypoglycemic activity.

[0050] In another specific embodiment, a hypoglycemic drug is provided, which includes the β-dihydroagarofuran sesquiterpenoid shown in Formula I and a pharmaceutically acceptable salt, tautomer, stereoisomer, and pharmaceutically acceptable excipients.

[0051] In some embodiments of this embodiment, it also includes other components having hypoglycemic activity.

[0052] In some embodiments of this embodiment, it is an oral dosage form or an injection dosage form, and a controlled release or sustained release dosage form well-known in the modern pharmaceutical industry can also be adopted.

[0053] In another specific embodiment, a preparation method of the β-dihydroagarofuran sesquiterpenoid shown in Formula I is provided, which is characterized by including the following steps:

[0054] S1. Take a plant of the genus Salacia, crush it, add a first extraction solvent, reflux and extract multiple times, collect the extract, filter it, and concentrate the filtrate to a viscous state to obtain a viscous product;

[0055] S2. Dissolve the viscous product in 1-2 times the amount of pure water, extract it multiple times with a second extraction solvent, collect the extract, and concentrate it under reduced pressure to obtain an extract;

[0056] S3. Perform silica gel column chromatography on the extract, elute it with a mixed organic solvent with a volume ratio of 1:0 to 0:1 in a gradient manner, collect the target gradient eluate, and concentrate it under reduced pressure;

[0057] S4. Perform MCI column chromatography on the concentrate obtained in S3, elute it with a methanol aqueous solution with a volume content of 60-100% in a gradient manner, collect the target gradient eluate, and concentrate it under reduced pressure;

[0058] S5. Separate and purify the concentrate obtained in S4 by semi-preparative HPLC to obtain the β-dihydroagarofuran sesquiterpenoid shown in Formula I.

[0059] In some embodiments of this embodiment, in S1, the branches, leaves, and fruits (seeds) of Salacia hancei are crushed to 20-40 meshes, reflux-extracted with a first extraction solvent at 85-90 °C for 2-3 times, 60-90 minutes each time, and the extracts are combined; the extract is filtered, and when the extract is concentrated under reduced pressure to 1 / 10-1 / 8 of its volume, it is left to stand, the precipitate is filtered off, and it is concentrated to a viscous state to obtain a viscous product;

[0060] In some embodiments of this embodiment, the first extraction solvent is selected from one of acetone, ethanol or methanol with a concentration of 80-100%;

[0061] In some embodiments of this embodiment, in S2, water with a weight ratio of 1-2 times the amount of the viscous product is added, and extraction is carried out 3-4 times with a second extraction solvent having the same volume as water. The organic solvent extraction phases are combined and concentrated under reduced pressure to obtain an extract;

[0062] In some embodiments of this embodiment, the second extraction solvent is selected from one of chloroform and ether;

[0063] In some embodiments of this embodiment, in S3, the extract is dissolved in chloroform with a weight ratio of 1.5-2 times the amount, and then mixed with silica gel with a mesh size of 80-100 and a weight 0.8-1.2 times that of the extract. Then, silica gel column chromatography is carried out. The silica gel for column packing is 200-300 mesh, and the amount used is 6-8 times the weight of the extract; Gradient elution is carried out successively with mixed organic solvents with volume ratios of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, and 0:1. After TLC detection, the same parts are combined, the target gradient eluate is collected, and concentrated under reduced pressure;

[0064] In some embodiments of this embodiment, in S3, the target gradient eluate is a mixed organic solvent with a volume ratio of 7:3, and the mixed organic solvent is selected from one of petroleum ether-acetone, n-hexane-acetone or petroleum ether-ethyl acetate;

[0065] In some embodiments of this embodiment, in S4, the eluate obtained by eluting with an organic solvent with a ratio of 7:3 is subjected to MCI column chromatography for decolorization. The MCI column chromatography material is packed with a reverse-phase decolorization material MCI GEL (CHP20P, 75-150μm); Gradient elution is carried out with a methanol aqueous solution with a volume content of 60-100%. After TLC detection, the same parts are combined, the target gradient eluate is collected, and concentrated under reduced pressure;

[0066] In some embodiments of this embodiment, in S4, the target gradient eluate is a methanol aqueous solution with a volume content of 70-85%;

[0067] In some embodiments of this embodiment, in S5, the eluate obtained by eluting with a methanol aqueous solution with a volume content of 70-85% is separated and purified by high-performance liquid chromatography. A 75% methanol aqueous solution is used as the mobile phase, the flow rate is 2 ml / min, a 10×250 mm (5μm) Agilent Zorbax C18 preparative chromatographic column is used as the stationary phase, the detection wavelength of the ultraviolet detector is 254 nm, 50-100 μL is injected each time, the chromatographic peaks at 21.5-22.0 min are collected, and after multiple accumulations, it is evaporated to dryness to obtain the β-dihydroagarofuran sesquiterpenoid compound obovatilin K.

[0068] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0069] Example 1 Extraction and Separation of β-Dihydroagarofuran-Type Sesquiterpenoids

[0070] Take 5.0 kg of the branches and leaves of Salacia hekouensis, a plant of the genus Salacia, dry them, coarsely crush them to 40 mesh, reflux and extract them 3 times with 6 times the amount (mass ratio) of 90% ethanol at 85 - 90 °C for 60 minutes each time, and combine the extraction solutions; filter the extraction solutions, and concentrate them under reduced pressure to 1 / 10 of the original volume; let it stand, filter off the precipitates, and concentrate it into about 500 g of extract a; add 500 g of water to extract a, extract it 3 times with chloroform with the same volume as water, combine the extraction phases, and concentrate them under reduced pressure to 110 g of extract b; pack a column with 660 g of 300-mesh silica gel, dissolve 165 g of chloroform in extract b, then add 88 g of 80-mesh silica gel for sample mixing, dry it at room temperature and then load it onto the column; elute it with a gradient of petroleum ether-acetone mixed organic solvents with volume ratios of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, 0:1 respectively, collect the gradient elution solutions, concentrate them, monitor them by TLC, combine the same parts, and obtain 8 parts. The elution solution c of the petroleum ether-acetone mixed organic solvent with a volume ratio of 7:3 is 13 g; pack a column with 1000 g of decolorizing material MCI (CHP20P, 75 - 150 μm), load elution solution c (13 g) onto the reverse-phase column, and perform gradient elution with a methanol aqueous solution with a volume content of 60 - 100%, collect each part of the elution solution and concentrate it, monitor it by TLC, and combine the same parts; take the elution solution eluted with a methanol aqueous solution with a volume content of 70 - 85%, then use a 75% methanol aqueous solution as the mobile phase, with a flow rate of 2 ml / min, a 10×250 mm (5 μm) Agilent Zorbax C18 preparative chromatographic column as the stationary phase, the detection wavelength of the ultraviolet detector is 254 nm, inject 50 μL each time, collect the chromatographic peak at 21.5 minutes, accumulate it multiple times and then evaporate it to dryness to obtain the β-dihydroagarofuran-type sesquiterpenoid obovatilin K.

[0071] Example 2 Extraction and Separation of β-Dihydroagarofuran-Type Sesquiterpenoids

[0072] Take 2.0 kg of dried Salacia obovata fruits (seed kernels), coarsely crush them to 20 mesh, and reflux extract twice with 6 times the amount (mass ratio) of 80% acetone at 85 - 90 °C for 60 min each time. Combine the extraction solutions; filter the extraction solution, and concentrate it under reduced pressure to 1 / 8 of its original volume; let it stand, filter off the precipitate, and concentrate it into about 310 g of extract a; add 310 g of water to extract a, extract 3 times with an equal volume of diethyl ether as water, combine the extraction phases, and concentrate it under reduced pressure into extract b (about 65 g); pack a column with 520 g of silica gel of 200 mesh, dissolve 130 g of chloroform in extract b, then add 80 g of silica gel of 100 mesh for sample mixing, dry it at room temperature and load it onto the column; elute with a gradient of n-hexane - acetone mixed organic solvents with volume ratios of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, 0:1 respectively, collect the gradient elution solution, concentrate it, monitor by TLC, and combine the same parts; the elution solution c with a volume ratio of 7:3 of n-hexane - acetone mixed organic solvents is 5.3 g; pack a column with 1000 g of decolorizing material MCI (CHP20P, 75 - 150 μm), load the elution solution c (5.3 g) onto the column, and perform gradient elution with a methanol aqueous solution with a volume content of 60 - 100%, collect each part of the elution solution and concentrate it, monitor by TLC, and combine the same parts; take the elution solution eluted with a methanol aqueous solution with a volume content of 70 - 85%, then use a 75% methanol aqueous solution as the mobile phase, a flow rate of 2 ml / min, a 10 × 250 mm (5 μm) Agilent Zorbax C18 preparative chromatography column as the stationary phase, a detection wavelength of 254 nm for the ultraviolet detector, inject 70 μL each time, collect the chromatographic peak at 22.0 min, accumulate it multiple times and then evaporate to dryness to obtain the β-dihydroagarofuran-type sesquiterpenoid obovatilin K.

[0073] Example 3 Extraction and Separation of β-Dihydroagarofuran-Type Sesquiterpenoids

[0074] Take 1.5 kg of dried Salacia obovata Hook. f. fruits (seed kernels), coarsely crush them to 30 mesh, and reflux extract them 3 times with 6 times the amount (mass ratio) of 90% methanol at 85 - 90 °C for 60 min each time. Combine the extraction solutions; filter the extraction solution and concentrate it under reduced pressure to 1 / 9 of its original volume; let it stand, filter off the precipitate, and concentrate it into about 300 g of extract a; add 300 g of water to extract a, and extract it 3 times with chloroform of the same volume as the water. Combine the extraction phases and concentrate them under reduced pressure to 55 g of extract b; pack a column with 450 g of silica gel of 200 mesh, dissolve 55 g of extract b in chloroform, then add 55 g of silica gel of 100 mesh for sample mixing, dry it at room temperature and load it onto the column; elute it with a gradient of petroleum ether - ethyl acetate mixed organic solvents with volume ratios of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, 0:1 respectively, collect the gradient elution solution, concentrate it, monitor it by TLC, and combine the same parts; the elution solution c of the petroleum ether - ethyl acetate mixed organic solvent with a volume ratio of 7:3 is 4.8 g. Pack a column with 500 g of decolorizing material MCI (CHP20P, 75 - 150 μm), load the elution solution c (4.8 g) onto the column and elute it with a gradient of methanol aqueous solution with a volume content of 60 - 100%, collect each part of the elution solution and concentrate it, monitor it by TLC, and combine the same parts; take the elution solution eluted with a methanol aqueous solution with a volume content of 70 - 85%, and then use a 75% methanol aqueous solution as the mobile phase, with a flow rate of 2 ml / min, an Agilent Zorbax C18 preparative chromatographic column of 10 × 250 mm (5 μm) as the stationary phase, a detection wavelength of 254 nm for the ultraviolet detector, inject 100 μL each time, collect the chromatographic peak at 22.0 min, accumulate it multiple times and then evaporate to dryness to obtain the β-dihydroagarofuran-type sesquiterpenoid obovatilin K.

[0075] Example 4 conducts structural characterization on the β-dihydroagarofuran-type sesquiterpenoid

[0076] The compound obovatilin K prepared in Examples 1 - 3 is a white amorphous powder, and its structure is identified by high-resolution mass spectrometry, infrared, nuclear magnetic resonance, and combined with its ECD determination. The mass spectrometry, infrared, and nuclear magnetic resonance characterization results of obovatilin K prepared in Example 1 (as Figures 1-4 and Table 1 show) can confirm that the molecular formula of the β-dihydroagarofuran-type sesquiterpenoid is C 31 H 37 NO 10 .

[0077] Table 1 1 H (600 MHz) and 13 C NMR (150 MHz) data (solvent is CD3OD), chemical shift δ (ppm), coupling constant J (Hz)

[0078]

[0079] The ion peak m / z 584.2502 [M+H] given by the HRESIMS mass spectrometry data + shows that the quasi-molecular ion peak of the compound of the present invention is m / z 584.2502 [M+H] + (calculated value C 31 H 38 NO 10 + , 584.2490); Combining with the infrared spectrum( Figure 4 ), it can be seen that IR(KBr) ν max 3449, 1726, 1662, 1160, 1048 cm -1 ; Combining with 1 the 1H NMR spectrum( Figure 2 ), 1 1H NMR(CD3OD, 600 MHz) δ AcO-1 [1.69(3H, s)], PydO-6 [8.62(1H, d, J = 2.4 Hz, H-6'), 8.11(1H, dd, J = 2.5, 9.5 Hz, H-4'), 6.55(1H, d, J = 9.4 Hz, H-3'), 3.63(3H, s, N-CH3)], BzO-9 [8.07(2H, brd, J = 8.4 Hz, H-2', 6'), 7.61(1H, t, J = 7.7 Hz, H-4'), 7.47(2H, t, J = 7.9 Hz, H-3', 5')]; Combining with 13 the 13C NMR spectrum( Figure 3 ), 13 13C NMR(CD3OD, 150 MHz) δ AcO-1 [172.1(C, COO–), 21.2(CH3)], PydO-6 [165.4(C, C-2'), 165.1(C, COO–), 146.9(CH, C-6'), 141.0(CH, C-4'), 119.8(CH, C-3'), 111.8(C, C-5'), 39.0(CH3, N-CH3)], BzO-9 [167.7(C, COO–), 134.4(CH, C-4'), 131.6(CH, C-1'), 131.4(CH, C-2', 6'), 129.5(CH, C-3', 5'); Therefore, the structure of obovatilin K is deduced as formula I, and the molecular formula is C 31 H 37 NO 10 .

[0080] After detection, the structure of the compound obovatilin K prepared in Examples 2-3 is the same as that of the compound in Example 1, and its molecular formula is C 31 H 37 NO 10 。

[0081] Example 5 investigated the hypoglycemic activity of β-dihydroagarofuran sesquiterpenoids

[0082] Experimental cell line: The full-length complementary deoxyribonucleic acid (cDNA) sequence of human SGLT-2 was cloned using standard techniques and stably transfected into Chinese hamster ovary cells (CHO). A radiolabeled glucose analog 14 C]-methyl-α-D-glucopyranoside ( 14 C]-AMG, Perkin Elmer) was used as the transporter substrate.

[0083] Buffer: An experimental buffer was prepared to simulate the low-protein conditions of glomerular filtration. The buffer included: 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 1.2 mM magnesium chloride (MgCl2), 120 mM sodium chloride (NaCl), 4.7 mM potassium chloride (KCl), and 2.2 mM calcium chloride (CaCl2), pH = 7.4. A 6 μM 14 C]-AMG buffer was prepared using this buffer.

[0084] Experimental method: CHO cells stably expressing the human SGLT-2 gene were seeded into a 96-well cell culture plate containing F12 (1X) medium (Invitrogen) and incubated overnight in a 37 °C, 5% CO2 incubator. Two groups were set up: Administration group: 49 μL of buffer, 1 μL of obovatilin K solution (diluent was DMSO), and 50 μL of 6 μM 14 C]-AMG buffer; Positive group: Empagliflozin. The cells were incubated at 37 °C for 1 h, the uptake reaction was stopped by removing the culture medium, and the incubation was stopped by rinsing the cells with ice-cold termination buffer. Then, 50 μL of 10% NaOH ice-cold lysis buffer was added to lyse the cells, and the cell lysate was transferred to a picoproas-vial, and 2 mL of Ultima Gold Cocktail was added. The intracellular radioactivity was quantified using Tri-carb, and the data obtained were analyzed using Origin 8.0 software. The response curve was adjusted to fit the empirical four-parameter model to determine the half-maximal response concentration of the test compound, i.e., IC 50 。Results are as Figure 5 shown.

[0085] It can be seen that the IC of the positive drug group in inhibiting the further absorption of glucose by SGLT-250 was 4.92 nM, while the IC of obovatilin K in CHO cells to further inhibit glucose absorption by SGLT-2 50 reached 11.9 nM, which is extremely rare among natural products. It is the first reported β-dihydroagarofuran sesquiterpenoid compound to achieve hypoglycemic activity by inhibiting SGLT-2.

[0086] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without violating the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. A β-dihydroagarofuran sesquiterpene having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

2. Use of the β-dihydroagarofuran sesquiterpene having a structure as shown in Formula I in Claim 1 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof in the preparation of an antidiabetic drug.

3. The application according to claim 2, characterized in that For the preparation of an inhibitor of SGLT-2.

4. The application according to claim 2, characterized in that The β-dihydroagarofuran sesquiterpene having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof can be used alone in the preparation of the drug or can be used in combination with other components having antidiabetic activity.

5. A hypoglycemic drug, characterized in that, Comprising the β-dihydroagarofuran sesquiterpene having a structure as shown in Formula I and a pharmaceutically acceptable salt, tautomer, stereoisomer, and pharmaceutically acceptable excipients.

6. The preparation method of the β-dihydroagarofuran sesquiterpenoid with the structure shown in Formula I in Claim 1, characterized in that, Comprising the following steps: S1. Take a plant of the genus Salacia, crush it, add a first extraction solvent, reflux extract multiple times, collect the extract, filter it, and concentrate the filtrate to a viscous state to obtain a viscous product; S2. Dissolve the viscous product in 1 - 2 times the amount of pure water, extract it multiple times with a second extraction solvent, collect the extract, and concentrate it under reduced pressure to obtain an extract; S3. Subject the extract to silica gel column chromatography, elute it with a gradient of a mixed organic solvent with a volume ratio of 1:0 to 0:1, collect the target gradient eluate, and concentrate it under reduced pressure; S4. Subject the concentrate obtained in S3 to MCI column chromatography, elute it with a gradient of an aqueous methanol solution with a volume content of 60 - 100%, collect the target gradient eluate, and concentrate it under reduced pressure; S5. Separate and purify the concentrate obtained in S4 by semi-preparative HPLC to obtain the β-dihydroagarofuran sesquiterpene having a structure as shown in Formula I.

7. The preparation method according to claim 6, characterized in that, In S1, the first extraction solvent is selected from one of 80 - 100% acetone, ethanol, or methanol; and / or, In S2, the second extraction solvent is selected from one of chloroform and ether.

8. The preparation method according to claim 6, characterized in that, In S3, elute it successively with a mixed organic solvent with a volume ratio of 1:0, 20:1, 10:1, 8:2, 3:2, 1:1, 1:2, 0:

1. The target gradient eluate is a mixed organic solvent with a volume ratio of 7:

3. The mixed organic solvent is selected from one of petroleum ether - acetone, n-hexane - acetone, or petroleum ether - ethyl acetate.

9. The preparation method according to claim 6, wherein In S4, the target gradient eluate is an aqueous methanol solution with a volume content of 70 - 85%.

10. The preparation method according to claim 6, characterized in that, In S5, the mobile phase is an aqueous methanol solution with a volume concentration of 75%, the stationary phase is Agilent Zorbax C18, the detection wavelength is 254 nm, the flow rate is 3 mL / min, and the injection volume is 50 - 100 μL.

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