CN114617874B

CN114617874B - Application of schisandra lactone B in preparation of alpha-glucosidase and/or beta-glucuronidase inhibitor

Info

Publication number: CN114617874B
Application number: CN202210070490.7A
Authority: CN
Inventors: 应优敏; 刘秀秀; 冉坤; 于航飞; 单伟光
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2023-05-23
Anticipated expiration: 2042-01-21
Also published as: CN114617874A

Abstract

The invention relates to an application of schisandra lactone B in preparing alpha-glucosidase and/or beta-glucuronidase inhibitors. The schisandra lactone B has obvious inhibition effect on alpha-glucosidase and escherichia coli beta-glucuronidase, and IC ₅₀ The values are 29.49+/-0.70 and 14.70+/-0.10 mu M respectively, and the preparation method has wide application prospect in the aspects of development of the medicines for treating the diabetes and the drug-induced diarrhea caused by the medicines for treating the diabetic complications.

Description

Application of schisandra lactone B in preparation of alpha-glucosidase and/or beta-glucuronidase inhibitor

Technical Field

The invention belongs to the technical field of biological medicines, and in particular relates to an application of triterpene compounds separated and prepared from schisandra chinensis in preparation of an alpha-glucosidase and beta-glucuronidase dual inhibitor.

Background

Diabetes is a metabolic disease characterized mainly by hyperglycemia, of which type 2 diabetes (type 2 diabetes mellitus,T2DM) accounts for about 90%. Long-term hyperglycemia can cause complications such as cardiovascular disease, diabetic retinopathy, diabetic nephropathy, diabetic peripheral neuropathy, etc., which severely threaten human health. Furthermore, studies have shown that sustained hyperglycemia and insulin resistance also increase the risk of developing cancer (e.g., colorectal cancer). A retrospective study showed that the prevalence of type 2 diabetes in colorectal patients was 8.04%, whereas meta analysis showed that the total risk of mortality in colorectal cancer-complicated type 2 diabetes patients was 17% higher than in colorectal cancer-only patients. For the complications of type 2 diabetes and other diseases, hypoglycemic agents and complications therapeutic agents need to be simultaneously administered. Many complications are associated with drug-induced diseases. For example, irinotecan (CPT-11), a commonly used agent for the treatment of colon cancer, is metabolized in the liver to inactive SN-38 glucuronide (SN-38G) when it enters the human body, whereupon SN-38G is excreted via the bile duct into the intestine, hydrolyzed by the intestinal bacteria beta-glucuronidase to SN-38, which causes severe delayed diarrhea and intestinal damage when SN-38 accumulates in the intestine, severely affecting the chemotherapy process. In addition, many carboxylic acid-containing non-steroidal anti-inflammatory drugs such as ketoprofen, diclofenac, and indomethacin also cause similar intestinal toxicity, thereby causing severe drug-induced diarrhea.

A great deal of research at home and abroad has proved that the use of antibiotics to remove intestinal bacteria capable of producing beta-glucuronidase or the use of high-efficiency beta-glucuronidase inhibitors can slow down gastrointestinal adverse reactions such as diarrhea and the like caused by chemical medicaments such as irinotecan and the like to a certain extent. However, excessive use of antibiotics can cause serious disturbance of intestinal flora of the gastrointestinal system of patients and cause fatal damage to organs such as liver and kidney. Therefore, aiming at the target point of the beta-glucuronidase of the intestinal bacteria, the dual-function medicine which is safe and effective, has the functions of reducing blood sugar and reducing adverse reactions of the medicine for treating the diabetic complications is developed, and has important clinical application prospect.

Schisandra lactone B (schisanlactone B) is triterpene compound, white powder, and has molecular formula of C ₃₀ H ₄₂ O ₄ The molecular weight of the compound is 466, the compound is easily dissolved in chloroform, methanol and ethanol, and can be extracted from natural plant schisandra chinensis (Schisandra sphaerandra), and the chemical structural formula is shown in formula (I):

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there is little research on the pharmacological activity of Guan Wuwei lactone B, which is currently reported to have weak to moderate cytotoxicity in vitro on human promyelocytic acute leukemia cells, cervical cancer cells, lung cancer, prostate cancer, squamous cell carcinoma, and nasopharyngeal carcinoma cells (1.chen, y.g.; wu, z.c.; lv, y.p.; guide, S.P., wen, J., liao, X.R., yuan, L.M., halaweish, F.Triterpenoids from Schisandra henryi with cytotoxic effect on leukemia and hela cells in vitro [ J ], arch.Pharm.Res.,2003,26,912-916.2.Wang, W., liu, J.Z., han, J., xu, Z.R., liu, R.X., liu, P., wang, W.X., ma, X.C., guan, S.H., guo, D.A., new triterpenoids from Kadsura heteroclita and their cytotoxic activity [ J ], planta Med, 2006,72, 450-457.3.Lu, Y., li, Y.Q., liu, Y.N., lee, K.H., chen, D.F.Cytoxec, and potential anticancer constituents from the stems of Schisandra pubescens, pr, X.35, U.S. 35, U.S. 6, G, D.A., new triterpenoids from Kadsura heteroclita and their cytotoxic activity [ J., U.S. 35, K., U.S. 35, K, K.S. 35, U.S. 35, U.G, U.S. X, K., U.S. 35, K, K.E., U.G., U.S. 35, K, K., U.S. 35, K and K.

At present, research on whether schisandra lactone B has alpha-glucosidase and beta-glucuronidase inhibition activity has not been reported yet; that is, whether schisandra lactone B can be developed into an alpha-glucosidase inhibitor, a beta-glucuronidase inhibitor, and a dual inhibitor of alpha-glucosidase and beta-glucuronidase is still a blank area of research in the art.

Disclosure of Invention

The invention aims to solve the defects of the prior art, provides application of schisandra lactone B in preparing alpha-glucosidase and/or beta-glucuronidase inhibitors, is beneficial to research and development of a dual-function medicament with the functions of reducing blood sugar and reducing diabetic complications, and is used for treating adverse effects of the medicament on intestinal tracts.

The technical scheme adopted by the invention is as follows:

the invention provides an application of schisandra lactone B (schisanlactone B) shown in a formula (I) in preparing alpha-glucosidase and/or beta-glucuronidase inhibitors:

the schisandra lactone B (schisanlactone B) can be used for preparing an alpha-glucosidase inhibitor, a beta-glucuronidase inhibitor, an alpha-glucosidase and a beta-glucuronidase dual inhibitor.

Further, the inhibitor can be a medicament for treating diabetes, and has the function of reducing adverse reactions of intestinal tracts caused by the medicament for treating diabetes complications, namely the inhibitor is a medicament for treating diabetes and reducing side effects of the medicament for treating diabetes complications.

The preparation method of the schisandra lactone B is not particularly limited, and can be prepared by a conventional preparation method known in the art, and preferably the preparation method of the schisandra lactone B is carried out according to the following steps:

(1) Collecting stem of Schisandra chinensis, naturally air drying, pulverizing, leaching with organic solvent at room temperature, concentrating the extractive solution under reduced pressure until no liquid flows out or no ethanol smell exists, to obtain crude extract;

(2) Suspending the crude extract with water, extracting with organic solvent, collecting organic phase, concentrating under reduced pressure to dry to obtain extract;

(3) And (3) subjecting the extract of the step (2) to silica gel column chromatography, MCI CHP20P column chromatography and silica gel column chromatography in sequence, collecting elution parts in sections, detecting the elution parts by using a thin layer chromatography, combining components with Rf value of 0.4-0.6, and concentrating under reduced pressure to dryness to obtain the compound shown in the formula (I).

Wherein the organic solvent in step (1) is 95% ethanol, methanol or acetone, preferably 95% ethanol; the volume amount of the organic solvent is 2-5mL/g, preferably 3mL/g, based on the weight of the schisandra chinensis stem powder; the leaching is carried out for at least 3 times, and the time for each extraction is 3-5 days.

The water volume in the step (2) is 2-5mL/g (preferably 2.3 mL/g) based on the weight of the crude extract; the organic solvent is ethyl acetate, the volume ratio of the organic solvent to water is 1:0.5-2, and preferably the volume ratio of the organic solvent to water for each extraction is 1:1; extracting for 3-5 times.

The specific operation method of the step (3) is as follows: 1) Dissolving the extract with petroleum ether-ethyl acetate mixed solvent with the volume ratio of 20:1, performing open silica gel column chromatography, and performing gradient elution with petroleum ether/ethyl acetate mixed solvent with the volume ratio of 20:1, 10:1, 5:1, 2:1 and 1:1 as eluent, wherein each gradient elution is 2-5 column volumes, and the flow rate is 10-20mL/min; collecting petroleum ether-ethyl acetate eluting part with volume ratio of 2:1, concentrating under reduced pressure to dry to obtain concentrate; 2) Adding methanol into the concentrate obtained in the step 1) for dissolving, performing MCI CHP20P column chromatography, and sequentially performing gradient elution by taking methanol/water mixed solvent with volume ratio of 70:30, 80:20 and 90:10 as eluent, wherein each gradient elution is carried out for 5-10 column volumes, and the flow rate is 10-20mL/min; collecting methanol-water eluting part with volume ratio of 80:20, concentrating under reduced pressure to dry to obtain concentrate; 3) Step 2) adding petroleum ether-acetone mixed solvent with the volume ratio of 5:1 into the concentrate, dissolving, and then performing silica gel column chromatography, wherein 6 column volumes are eluted at equal degree by the petroleum ether-acetone mixed solvent with the volume ratio of 5:1, and the flow rate is 10-20mL/min; and (3) carrying out thin-layer chromatography monitoring by adopting a silica gel GF254 thin-layer plate, taking a petroleum ether-ethyl acetate mixed solvent with the volume ratio of 2:1 as a developing agent, developing by using 10% sulfuric acid-ethanol after the development is finished, combining components with the Rf value of 0.5, and concentrating under reduced pressure until the components are dried to obtain the compound shown in (I).

Further, the invention relates to an IC with the effect of inhibiting alpha-glucosidase by schisandra lactone B ₅₀ IC with a value of 29.49+ -0.70 μm for beta-glucuronidase inhibition ₅₀ The value was 14.70.+ -. 0.10. Mu.M.

Further, the alpha-glucosidase of the invention is derived from Saccharomyces cerevisiae and is available from the biochemistry company.

Further, the beta-glucuronidase of the present invention is derived from intestinal bacteria, preferably E.coli, and can be obtained from the purchase of biochemical products, or extracted from E.coli, such as Escherichia coli BL (DE 3).

Compared with the prior art, the invention has the beneficial effects that:

the invention provides an application of schisandra lactone B (schisanlactone B) extracted from schisandra sphenanthera in preparing alpha-glucosidase and/or beta-glucuronidase inhibitors, wherein schisandra lactone B has remarkable inhibition effects on alpha-glucosidase and beta-glucuronidase, and the activity of the schisandra lactone B is superior to that of positive medicines acarbose (alpha-glucosidase inhibitors) and D-glucaric acid-1, 4-lactone (DSL) (beta-glucuronidase inhibitors), and can be used for developing a difunctional medicine with hypoglycemic effect and effect for reducing adverse reactions of diabetes complications in the intestinal tract of a therapeutic medicine.

Drawings

FIG. 1 is a hydrogen spectrum of schisandra lactone B.

FIG. 2 is a carbon spectrum of schisandra lactone B.

FIG. 3 is a concentration-dependent inhibition curve of schisandra lactone B on alpha-glucosidase.

FIG. 4 is a graph showing the concentration-dependent inhibition of acarbose on alpha-glucosidase.

FIG. 5 is a graph showing the concentration-dependent inhibition of beta-glucuronidase by schisandra lactone B.

FIG. 6 is a graph showing the concentration-dependent inhibition of beta-glucuronidase by D-glucarate-1, 4-lactone (DSL).

Detailed Description

The technical solution of the present invention will be further specifically described below by means of specific examples, which are only for explaining the present invention and are not intended to limit the scope of the present invention, with reference to the accompanying drawings. The materials, reagents and the like used, unless otherwise specified, are those commercially available. Fructus Schisandrae Sphenantherae (school name: schisandra sphaerandra Stapf) is wood vine of Schisandra of Magnoliaceae. The room temperature is 25-30 ℃.

Example 1 preparation of Schisandra lactone B

(1) Taking 10kg of dried shizandra berry stem, crushing, leaching with 95% ethanol 30L for 4 times (4 days each time) at room temperature, combining the extracting solutions, and concentrating under reduced pressure until no liquid flows out or no ethanol smell exists, thus obtaining 846g of crude extract.

(2) Suspending 846g of the crude extract obtained in the step (1) in 2L of water, extracting with ethyl acetate for 3 times, wherein each time is 2L; the ethyl acetate phases were combined and the solvent recovered under reduced pressure to give 108g of ethyl acetate extract.

(3) And (2) dissolving 108g of ethyl acetate extract with 200mL of petroleum ether-ethyl acetate mixed solvent with the volume ratio of 20:1, carrying out open silica gel column chromatography (with the diameter d=12 cm and the height h=70 cm), sequentially carrying out gradient elution with petroleum ether-ethyl acetate mixed solvents with the volume ratios of 20:1, 10:1, 5:1, 2:1 and 1:1, and collecting elution parts of petroleum ether/ethyl acetate (2:1) at the flow rate of 15mL/min for each gradient elution of 25L (3 column volumes), and concentrating under reduced pressure until the elution parts are dry to obtain 20.2g of concentrate.

(4) Dissolving 20.2g of the concentrate in the step (3) with 100mL of methanol, performing MCI CHP20P column chromatography (diameter d=5 cm, height h=30 cm), sequentially performing gradient elution with a methanol/water mixed solvent with volume ratio of 70:30, 80:20 and 90:10, eluting 2.5L (4 column volumes) each with flow rate of 15mL/min, collecting methanol-water eluting parts with volume ratio of 80:20, merging, concentrating under reduced pressure to dryness, and obtaining 1.89g of the concentrate.

(5) 1.89g of the concentrate in the step (4) is dissolved by using a petroleum ether-acetone mixed solvent with the volume ratio of 5:1, then silica gel column chromatography (diameter d=4 cm, height h=30 cm) is carried out, 6 column volumes (namely, the total elution volume is 2.2L) are eluted in an isocratic manner by using the petroleum ether-acetone mixed solvent with the volume ratio of 5:1, the flow rates are 10mL/min, the elution parts are collected and spotted, and silica gel GF is adopted for spotting ₂₅₄ Thin layer chromatography is carried out on the thin layer plate, a petroleum ether-ethyl acetate mixed solvent with the volume ratio of 2:1 is used as a developing agent, after development is finished, the developing agent is developed by 10% sulfuric acid-ethanol, the components with the Rf value of 0.5 are combined, and the components are concentrated to dryness under reduced pressure, so that 307mg of the compound shown in the formula (I) is obtained.

Example 2 structural identification of Schisandra lactone B

Schisandra lactone B: white powder with molecular formula C ₃₀ H ₄₂ O ₄ The method comprises the steps of carrying out a first treatment on the surface of the Which is a kind of ¹ H-NMR ¹³ The C-NMR (FIGS. 1 and 2) data are as follows: ¹ H-NMR(600MHz,CDCl ₃ )δ _H 0.89(3H,s,28-CH ₃ ),0.97(3H,d,J＝6.6Hz, 21-CH ₃ ),0.98(3H,s,18-CH ₃ ),1.03(1H,d,J＝5.1Hz,H-19β),1.22(1H,d,J＝5.1Hz, H-19α),1.35(3H,s,30-CH ₃ ),1.37(3H,s,29-CH ₃ ),1.91(3H,s,27-CH ₃ ),4.45(1H,ddd,J＝ 13.2,3.5,3.5Hz,H-22),5.94(1H,d,J＝12.7Hz,H-2),6.12(1H,d,J＝12.7Hz,H-1),6.60 (1H,d,J＝6.5Hz,H-24)； ¹³ C-NMR(150MHz,CDCl ₃ )δ _C 167.5 (C-3), 166.6 (C-26), 150.7 (C-1), 139.5 (C-24), 128.5 (C-25), 120.5 (C-2), 84.6 (C-4), 80.6 (C-22), 48.8 (C-13), 48.1 (C-17), 46.4 (C-5), 45.6 (C-14), 45.1 (C-8), 39.3 (C-20), 35.1 (C-16), 33.6 (C-9), 32.6 (C-12), 32.3 (C-11), 29.3 (C-29), 29.0 (C-7), 28.8 (C-10), 27.0 (C-15), 24.5 (C-6), 24.1 (C-19), 23.6 (C-23), 22.2 (C-30), 19.1 (C-28), 17.3 (C-27), 17.1 (C-18), and 13.3 (C-21). The above nuclear magnetic data (1.Liu,J.S.and Huang,M.F.Schisanlactone B,a new triterpenoid from Schisandra sp. [ J)]Tetrahedron letters, 1983,24,2355-2358;2. liu Haitao, li Xingbo, zhangjin, zhu Yingxia, ji Yaodong, peng Yong, zhangben, shougen, chinese schizandra fruit Petroleum ether fraction chemical composition, chinese J.Chinese, 2012,37 (11): 1597-1601) reported that schisandra lactone B (schisanlactone B) is consistent.

EXAMPLE 3 inhibition of alpha-glucosidase by Schisandra lactone B

10. Mu.L of 0.075U/mL of potassium dihydrogen phosphate buffer (pH 6.8) of alpha-glucosidase, 10. Mu.L of potassium dihydrogen phosphate buffer (pH 6.8) containing shizandra lactone B at various concentrations (10, 20, 30, 40, 50, 60, 100. Mu.M), and 70. Mu.L of potassium dihydrogen phosphate buffer (pH 6.8) were added to a 96-well plate, mixed uniformly, 10. Mu.L of potassium dihydrogen phosphate buffer (pH 6.8) containing 5.0mM of 4-nitrophenol-alpha-D-glucopyranoside (pNPG) was added, and after incubation at 37℃for 30min, 150. Mu.L of Na at a concentration of 0.2M was added ₂ CO ₃ The reaction was terminated with an aqueous solution, and the absorbance at 405nm was read with an enzyme-labeled instrument and the inhibition ratio was calculated.

Wherein A is _{Sample 0min} To add the absorbance of the system when the sample to be tested is added but no enzymatic reaction occurs, A _{Blank for 0min} The absorbance of the system when no sample to be detected is added and no enzymatic reaction occurs; the positive control group replaced schisandra lactone B with acarbose. The median Inhibition Concentration (IC) was calculated from the inhibition ratios at the different concentrations ₅₀ ) (FIGS. 3 and 4). In vitro alpha-grape of schisandra lactone B in the inventionThe glycosidase inhibitory activity is shown in Table 1.

TABLE 1 in vitro alpha-glucosidase inhibitory Activity of Schisandra lactone B

From the data in Table 1, it is clear that the schisandrin B has a good inhibition effect on alpha-glucosidase in vitro, and is superior to positive control acarbose.

EXAMPLE 4 inhibition of E.coli beta-glucuronidase by Schisandra lactone B

1. Preparation of E.coli beta-glucuronidase (EcGUS):

LB liquid medium: trypsin 10g/L, yeast extract 5g/L, sodium chloride 10g/L, water as solvent, pH 7.0.

Lysate: 20mM 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 300mM NaCl,5mM imidazole, 10% glycerol (glycidol) by volume, water as solvent, pH 7.4.

NTA-0 buffer: 20mM Tris-HCl,0.5M sodium chloride, 10% glycerol by volume, water as solvent, pH 7.9.

NTA-20 buffer: 20mM Tris-HCl,0.5M sodium chloride, 10% glycerol by volume, 20mM imidazole, water as solvent, pH 7.9.

NTA-250 buffer: 20mM Tris-HCl,0.5M sodium chloride, 10% glycerol by volume, 250mM imidazole, water as solvent, pH 7.9.

Coli Escherichia coli BL (DE 3) was inoculated into 200mL of LB liquid medium containing 30. Mu.g/mL kanamycin and cultured at 200rpm and 37℃to OD ₆₀₀ To 0.5, isopropyl-. Beta. -D-thiogalactoside (IPTG) was added at a final concentration of 100mM, and the resulting mixture was incubated at 200rpm and 30℃overnight to induce the expression of EcGUS (enzyme expression was detected by SDS-PAGE electrophoresis).After the expression is completed, the culture solution is centrifuged for 5min at 4 ℃ and 9000rpm, the thalli are collected, washed by PBS (pH 7.4) for 2-3 times, then 20mL of the lysate is added according to the volume of 1/10 of the original thalli to resuspend the thalli, the thalli are placed on ice, the thalli are broken by ultrasound for 20min at intervals of 10s under 300W and ultrasound for 10s, and then the thalli are centrifuged for 10min at 4 ℃ and 8000rpm, and the supernatant is taken. Then, respectively washing 15mL of Ni-NTA agarose resin column materials (purchased from GE medical company) for 2-3 times by using 15mL of purified water and 15mL of NTA-0 buffer solution, chelating the supernatant with the Ni-NTA agarose resin column materials for 3 hours at 4 ℃, then respectively carrying out gradient elution by using 15mL of NTA-0 buffer solution, NTA-20 buffer solution and 15mL of NTA-250 buffer solution, respectively collecting 9 tubes of eluent, respectively carrying out SDS-PAGE electrophoresis on each tube of eluent, showing that 4 tubes of eluent collected by eluting the NTA-250 buffer solution contain EcGUS, the molecular weight of the EcGUS is about 71kD, then merging the 4 tubes of eluent, finally, filtering by using a Millipore protein ultrafilter tube with the molecular weight of 10kD (the interception molecular weight is not more than 1/3 of the target protein), collecting the interception solution which is the enzyme solution, obtaining 7mL of EcGUS enzyme solution, and obtaining the enzyme solution with the concentration of 500 mu g/mL by using a kit (BCA protein concentration measuring kit, beijing Buddha protein concentration measuring company).

2. Screening of EcGUS inhibitor (final inhibitor concentration 100. Mu.M)

(1) Reagent(s)

Inhibitors: the schisandra lactone B is prepared into a solution of 10mM by using dimethyl sulfoxide (DMSO) for later use.

A substrate: 4-Nitrophenyl- β -D-glucopyranoside (PNPG, purchased from Sigma-Aldrich) was prepared in 2.5mM solution in PBS buffer for use.

Reaction enzyme solution: diluting the EcGUS enzyme solution prepared in the step 1 with PBS buffer solution for 500 times to obtain a reaction enzyme solution, and measuring 1 mug/mL of protein concentration by using a kit (BCA micro protein concentration measuring kit, beijing Soy Bao technology Co., ltd.) to obtain the reaction enzyme solution; three replicates were made in a 10. Mu.L system of the reaction enzyme solution 10. Mu.L+PBS 80. Mu.L+2.5 mM substrate 10. Mu.L system, OD values were measured at 405nm for 0min and 30min, respectively, using a microplate reader, and delta. Was found by calculation _OD Between 0.2 and 0.3.

Positive control (DSL): d-glucaric acid-1, 4-lactone (D-sacchoric acid 1,4-lactone, DSL, available from Sigma-Aldrich) was prepared as a 10mM solution in dimethyl sulfoxide (DMSO) as a positive control.

(2) The reaction: the reaction was performed in 96-well plates as follows, blank: 10. Mu.L of the reaction enzyme solution+79. Mu.L of PBS+1. Mu.L of a 10% volume concentration aqueous solution of DMSO+2.5 mM substrate; experimental group: 10. Mu.L of the reaction enzyme solution+PBS 79. Mu.L+10 mM of the inhibitor 1. Mu.L+2.5 mM of the substrate 10. Mu.L; positive control group: 10. Mu.L of the reaction enzyme solution+79. Mu.L of PBS+10 mM of positive control 1. Mu.L+2.5 mM of substrate; 3 parallel samples are prepared for each group, the enzyme-labeled instrument is used for measuring OD values (incubation at 37 ℃ in the period) of 0min and 30min respectively at 405nm wavelength according to the sequence of the reaction enzyme solution, PBS, inhibitor/positive control and substrate, and the inhibition rate of schisandrin B on EcGUS is 75.86 percent and is higher than that of positive control DSL (inhibition rate is 66.76 percent) under the condition of 100 mu M of final concentration.

The specific calculation process is as follows:

ΔOD＝OD _30min -OD _0min ；

ΔC _PNP =Δod/0.003262 (0.003262 is the correlation coefficient of absorbance and PNP solubility of the present invention);

relative activity (%) = experimental group Δc _PNP Blank ΔC _PNP ；

Inhibition (%) =1-relative activity (%);

3、IC ₅₀ measurement of values: IC for measuring schisandra lactone B ₅₀ Values, a series of inhibitor concentration points (e.g., 1, 5, 10, 20, 50, 70, 90, 100. Mu.M) were set up within a final concentration of 0.1-500. Mu.M, and the reaction was performed in 96-well plates as follows: blank group: 10. Mu.L of the reaction enzyme solution+70. Mu.L of PBS+10. Mu.L of 1% DMSO in volume fraction+2.5 mM substrate; experimental group: 10 mu L of reaction enzyme solution, 70 mu L of PBS and 10 mu L of different concentration inhibitor, 2.5mM substrate; positive control group: 10. Mu.L of the reaction enzyme solution+70. Mu.L of PBS+10. Mu.L of positive control+2.5 mM substrate; 3 groups of the enzyme-labeled reagent are arranged in parallel, the enzyme solution, PBS, inhibitor/positive control and substrate are sequentially added, OD values (incubated at 37 ℃ in the period) of 0min and 30min are respectively measured at 405nm of an enzyme-labeled instrument, and the enzyme-labeled reagent is obtained by calculationThe relative activity value of the inhibitor to EcGUS under different concentration conditions, finally, taking the derivative of the inhibitor concentration point mu M with the base of 10 to obtain an lg value, taking the lg value as an abscissa and the relative activity as an ordinate, and using Graphad Prism 6.0 software to draw an IC ₅₀ Graphs (FIGS. 5 and 6) and analysis by this software gave the IC of inhibitor/positive control versus EcGUS ₅₀ The in vitro inhibitory activity of schisandra lactone B against EcGUS is shown in table 2.

TABLE 2 in vitro E.coli beta-glucuronidase inhibitory Activity of Schisandra lactone B

From the data in Table 2, the schisandra lactone B has good inhibition effect on escherichia coli beta-glucuronidase in vitro, and is superior to a positive control drug DSL.

Comparative example 1 inhibition of alpha-glucosidase and E.coli beta-glucuronidase by wing pedunculata Schisandrin B

The schisandra lactone B in example 4 was replaced with wing stem schisandrin B, and the other operations were the same, and the results are shown in table 3. Table 3 shows that wing-pedunculata B, which is structurally similar to schisandra lactone B, has inhibitory activity against alpha-glucosidase but not beta-glucuronidase.

TABLE 3 in vitro enzyme inhibition Activity of Schisandrin B

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the invention in any way, but other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. An application of schisandra lactone B shown in formula (I) in preparing medicine for treating diabetes,

2. the use according to claim 1, wherein the schisandra lactone B is prepared by the steps of:

(3) And (3) subjecting the extract obtained in the step (2) to silica gel column chromatography, MCI CHP20P column chromatography and silica gel column chromatography in sequence, collecting the elution part in a segmented mode, detecting the elution part by using a thin layer chromatography, combining components with Rf value of 0.4-0.6, and concentrating under reduced pressure to obtain schisandra lactone B shown in the formula (I).

3. The use according to claim 2, wherein the organic solvent in step (1) is 95% ethanol, methanol or acetone; the volume consumption of the organic solvent is 2-5mL/g based on the weight of the schisandra chinensis stem powder; the leaching is carried out for at least 3 times, and the time for each extraction is 3-5 days.

4. The use according to claim 2, wherein the water volume in step (2) is 2-5mL/g based on the weight of the crude extract; the organic solvent is ethyl acetate, and the volume ratio of the organic solvent to water is 1:0.5-2; extracting for 3-5 times.

5. The use according to claim 2, characterized in that the method of step (3) is: 1) Extraction processDissolving the paste by using a petroleum ether-ethyl acetate mixed solvent with the volume ratio of 20:1, performing open silica gel column chromatography, and performing gradient elution by using petroleum ether/ethyl acetate mixed solvents with the volume ratios of 20:1, 10:1, 5:1, 2:1 and 1:1 as eluent, wherein each gradient elution is carried out for 2-5 column volumes, and the flow rate is 10-20mL/min; collecting petroleum ether-ethyl acetate eluting part with volume ratio of 2:1, concentrating under reduced pressure to dry to obtain concentrate; 2) Adding methanol into the concentrate obtained in the step 1) for dissolving, performing MCI CHP20P column chromatography, and sequentially performing gradient elution by taking methanol/water mixed solvent with volume ratio of 70:30, 80:20 and 90:10 as eluent, wherein each gradient elution is carried out for 2-5 column volumes, and the flow rate is 10-20mL/min; collecting methanol-water eluting part with volume ratio of 80:20, concentrating under reduced pressure to dry to obtain concentrate; 3) Step 2) adding the concentrate into a petroleum ether-acetone mixed solvent with the volume ratio of 5:1, dissolving, and then performing silica gel column chromatography, wherein the petroleum ether-acetone mixed solvent with the volume ratio of 5:1 is used for isocratically eluting for 5-6 column volumes, and the flow rate is 10-20mL/min; using silica gel GF ₂₅₄ Thin layer chromatography is carried out on the thin layer plate, a petroleum ether-ethyl acetate mixed solvent with the volume ratio of 2:1 is taken as a developing agent, the components with the Rf value of 0.5 are combined, and the mixture is concentrated to dryness under reduced pressure to obtain the compound shown in the formula (I).