CN113082038A

CN113082038A - Application of 3, 6' -dibapinyl sucrose in preparing medicine for treating type 2 diabetes

Info

Publication number: CN113082038A
Application number: CN202110390430.9A
Authority: CN
Inventors: 徐志宏; 代爽; 刘静雅; 刘晓雨; 李丽; 岳媛; 陈丽琼; 程凯凯
Original assignee: Xihua University
Current assignee: Xihua University
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2021-07-09

Abstract

The invention relates to the technical field of new application of compounds, in particular to application of 3, 6' -dibapinyl sucrose in preparing a medicament for treating type 2 diabetes. The 3, 6' -dibapinyl sucrose can compete with glucagon for binding to a glucagon receptor, and then can effectively inhibit or antagonize the action of glucagon, and can then treat type 2 diabetes.

Description

Application of 3, 6' -dibapinyl sucrose in preparing medicine for treating type 2 diabetes

Technical Field

The invention relates to the technical field of new application of compounds, in particular to application of 3, 6' -dibapinyl sucrose in preparing a medicament for treating type 2 diabetes.

Background

Diabetes Mellitus (DM) is a complex chronic endocrine metabolic disease, which may be caused by hypofunction of pancreatic islets, Insulin Resistance (IR) and other pathological changes due to various pathogenic factors such as free radical toxin, genetic factor, immune dysfunction, mental factor and the like acting on the body, thereby causing a series of metabolic disorders such as sugar, protein, fat, water and electrolyte. Diabetes is generally classified in the prior art into type i diabetes (insulin dependent diabetes mellitus), type ii diabetes (T2DM), other specific types of diabetes, and gestational diabetes. According to the latest research on the pathogenesis of T2DM, the abnormal increase of glucagon is the true cause of the pathogenesis of T2DM, and T2DM can be effectively treated by regulating glucagon.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of 3, 6' -dibapinyl sucrose in preparing a medicament for treating type 2 diabetes. The embodiment of the invention discovers that 3, 6' -dibapinyl sucrose can compete with glucagon to combine with a glucagon receptor, so that the action of glucagon can be effectively inhibited or antagonized, and the type 2 diabetes can be treated.

The invention is realized by the following steps:

in a first aspect, the present invention provides the use of 3, 6' -dibapinyl sucrose in the manufacture of a medicament for the treatment of type 2 diabetes. The structural formula of the 3, 6' -dibapinyl sucrose is shown as follows:

in an alternative embodiment, the preparation of the 3, 6' -dimonoylsucrose comprises: extracting polygala tenuifolia, and separating to obtain the 3, 6' -dibapinyl sucrose. The 3,6 '-dibapinyl sucrose can be extracted from cortex et radix Polygalae or other natural product containing 3, 6' -dibapinyl sucrose, or obtained by direct purchase or synthesis by chemical synthesis.

In an alternative embodiment, the preparation of the 3, 6' -dimonoylsucrose comprises: mixing the polygala tenuifolia with a solvent, extracting under an ultrasonic condition, and separating to obtain the 3, 6' -brassica juncea acyl sucrose. The 3, 6' -myrosinyl sucrose can be extracted from polygala tenuifolia by the ultrasonic extraction method or by other extraction methods. The separation and purification method can be column chromatography separation, recrystallization, high performance liquid separation and other conventional separation and purification methods, and the embodiment of the invention is not described in detail.

In an alternative embodiment, the extraction conditions are: the ultrasonic time is 40-50 minutes, for example, any value between 40-50 minutes such as 40 minutes, 45 minutes and 50 minutes; the ratio of the polygala tenuifolia to the solvent is 1:20-40, and can be any ratio of 1:20-40 such as 1:20, 1:25, 1:30, 1:35, 1:40 and the like; the solvent is an alcohol-water solvent.

In an alternative embodiment, the sonication time is 45 minutes, and the ratio of polygala tenuifolia to solvent is 1:30, the solvent is a monohydric alcohol water solvent;

in alternative embodiments, the solvent is a methanol aqueous solvent, and an ethanol aqueous solvent may be used instead of a methanol aqueous solvent.

In an alternative embodiment, the solvent is a methanol-water solvent with a methanol volume fraction of 50-90%, preferably a methanol-water solvent with a methanol volume fraction of 90%.

In an alternative embodiment, the drug is a glucagon antagonist drug.

In a second aspect, the invention provides the use of 3, 6' -dibapinyl sucrose in the preparation of an antagonist against glucagon.

In a third aspect, the invention provides an application of 3, 6' -dibapinyl sucrose in preparing a medicine for treating diseases caused by glucagon-like excess.

The invention has the following beneficial effects: the embodiment of the invention discovers that 3,6 '-dibapinyl sucrose can effectively compete with glucagon to combine with a glucagon receptor, can effectively inhibit or antagonize the action of glucagon, can treat type 2 diabetes mellitus, expands the selection of active ingredients for treating type 2 diabetes mellitus, and expands the application range of the 3, 6' -dibapinyl sucrose.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of the test of Experimental example 1 provided by the present invention;

FIG. 2 is a schematic diagram of a 5XEZ-97V protein-ligand complex of Experimental example 2 provided by the present invention;

FIG. 3 is a schematic diagram showing the binding condition between chain A and 97V and the binding condition between chain B and 97V in Experimental example 2;

FIG. 4 is a schematic diagram of a ligand complex of 5XEZ-97V A catenin in Experimental example 2 provided by the present invention;

fig. 5 is a schematic diagram of the contact ratio between the downloaded 97V and the newly calculated 97V in experimental example 2 provided by the present invention;

FIG. 6 is a graph showing the results of molecular docking of DY-130 of Experimental example 2 provided herein;

FIG. 7 is a graph showing the results of molecular docking of DY-62 in Experimental example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment of the invention provides a preparation method of 3, 6' -dibapinyl sucrose, which comprises the following steps:

1.0g of polygala tenuifolia (screened by a 40-mesh sieve) is precisely weighed, the polygala tenuifolia is placed in a conical flask with a plug, 25mL of 70% methanol (volume percentage) is precisely added, the weight is weighed, ultrasonic treatment is carried out for 1h (500W, 40kHz), the polygala tenuifolia is placed still and cooled, then the weight is weighed, and the weight loss is compensated by 70% methanol. Shaking, filtering, and filtering with microporous membrane to obtain 3, 6-di-sinapoyl sucrose crude product.

And (3) taking the prepared 3, 6-dibastine acyl sucrose crude product, carrying out rotary evaporation for about 40min in water bath at the temperature of 45-50 ℃, then carrying out separation and purification to obtain a final product, and adding methanol into the product to reach a constant volume of 10 ml.

Experimental example 1

Cell: HEK-293 cells;

experimental drugs: 3, 6' -Disinapoyl sucrose is noted: DY-130 (available from Dow Tex Biotechnology Ltd.); amentoflavone is designated DY-62 (available from Dorster Biotechnology Co., Ltd.);

plasmid construction: CDS region of gene was amplified by PCR using 2 × AccuProof HiFi HotStart SuperMix kit under the following system conditions:

the PCR product was subjected to agarose gel electrophoresis using an OMEGA gel Extraction Kit, and the band of interest was recovered by cutting the gel.

pCDNA3.1 vector enzyme digestion reaction, at 37 ℃ under the conditions of 3h, system conditions as follows:

the cleavage product was electrophoresed on agarose gel using the OMEGA gel Extraction Kit, and the band of interest was recovered by gel cutting.

And (3) recombination reaction: the vector and gene were recombined using the ExonArt Seamless Cloning and Assembly Kit. Keeping at 50 deg.C for 20min, and the system conditions are as follows:

and (3) transformation of a recombinant product: the transformation was carried out using XL2-Blue competence. Taking a competent part from the liquid nitrogen, and placing the competent part on ice for unfreezing; adding 15 mu L of the recombinant product obtained in the step 6 after about 3min, slightly blowing and beating for 2-3 min, and then placing on ice for incubation for 30 min; thermally shocking for 45sec by using a 45 ℃ metal bath, immediately standing on ice for about 3min, and shaking for 45-60 min at 37 ℃ and 200rpm in 800 mu L SOC culture medium; all or part of the plates (containing the carboxybenzyl group) were placed in an incubator at 37 ℃ overnight.

Selecting a single colony to perform colony PCR detection positive cloning: using 2 XTaq Advanced PCR Master Mix kit, the system conditions are as follows:

and (3) carrying out electrophoresis on the colony PCR product, selecting positive clone, sending a sample for sequencing, obtaining a plasmid with a correct sequence, carrying out escherichia coli plasmid amplification on the plasmid to obtain a required quantity, and subpackaging and storing the plasmid in a refrigerator at the temperature of-20 ℃ for later use (the plasmid is subpackaged to avoid the influence on plasmid effect caused by repeated freezing of the plasmid).

Cell transfection:

(1) cell liquid change: the pre-incubated 6-well plate cells were changed to serum-free media without double antibody, as transfection efficiency may be affected in the presence of serum.

(2) Preparing a plasmid transfection reagent compound: adding 4 mu g of plasmid into 250 mu L of serum-free and double-antibody-free culture medium in the ep tube 1, and gently mixing; 250 μ L of serum-free and double antibody-free culture medium was added to ep tube 2, and 10 μ L of transfection reagent was mixed well. After waiting for 10min, ep1 and ep2 were gently pipetted and incubated at room temperature for 30 min.

(3) Transfection: taking out the six-hole plate, uniformly and slowly dripping the plasmid transfection reagent compound into the six-hole plate, slightly shaking the six-hole plate to uniformly mix the six-hole plate, putting the six-hole plate at 37 ℃ and 5% CO₂Incubate in incubator, control in well plate without any treatment.

(4) Liquid changing culture: after 4h incubation, the medium containing the transfection complex was aspirated, washed once with PBS, aspirated PBS was added with 2mL of medium containing 10% serum and placed at 37 ℃ in 5% CO₂The incubator was incubated for 48 hours.

RNA extraction:

grinding and homogenization of the sample:

(1) sucking out the original culture medium in a six-hole plate, and adding PBS to wash the culture medium once;

(2) adding 1.5mL of RNAioso Plus, and gently shaking the six-well plate;

(3) transferring the lysate containing the cells into a centrifuge tube, and repeatedly blowing and sucking the lysate by using a pipette gun until no obvious precipitate exists in the lysate;

(4) after standing at room temperature (15-30 ℃) for 5min, RNA was isolated from the nucleoprotein.

Extraction of Total RNA:

(1) adding chloroform (1/5 volume of RNAioso Plus) into the homogenate lysate, covering the centrifugal tube, mixing until the solution is milky, and standing for 5 min;

(2) centrifuging at 1200r/min and 4 ℃ for 15 min. The centrifuge tube was carefully removed from the centrifuge, and the homogenate was divided into three layers at this time, i.e.: a colorless supernatant (containing RNA), an intermediate white protein layer (mostly DNA) and a colored lower organic phase;

(3) pipette the supernatant into another new centrifuge tube (do not aspirate the white middle layer);

(4) adding isopropanol with volume of 0.5-1 time of RNAioso Plus into the supernatant, turning the centrifuge tube upside down, mixing, and standing at room temperature for 10 min;

(5) after centrifugation at 1200r/min and 4 ℃ for 10min, RNA precipitation can occur at the bottom of the test tube.

Washing of RNA precipitate: the supernatant was carefully discarded, and the precipitate was discarded without touching, leaving a small amount of isopropanol. Adding 75% ethanol equivalent to RNAioso Plus, washing the tube wall of the centrifuge tube by slightly turning upside down, centrifuging at 7500r/min at 4 deg.C for 5min, carefully discarding the supernatant, and cutting without touching the precipitate.

And (3) RNA dissolution: the centrifuge tube lid was opened and the pellet was dried at room temperature for several minutes. After the precipitate is dried, an appropriate amount of RNase-free water is added to dissolve the precipitate.

And (3) fluorescent quantitative PCR detection: firstly, reverse transcribing RNA extracted by Trizol into cDNA by using a reverse transcription kit; the cDNA that has been reverse transcribed is then diluted five times with water to serve as the qPCR template. The reagents used in the reaction system for amplification are shown in the following table.

Preparing a fluorescent probe solution: the fluorescent probe is prepared by using anhydrous DMSO (dimethylsulfoxide) to be a stock solution with the final concentration of 2mM, and the stock solution is stored at minus 20 ℃ in the dark for later use. The stock solution was diluted to 4 μ M working solution with HBSS (phenol red free, calcium magnesium free) before use, and Pluronic F127 was added to the probe solution, Pluronic F127 preventing Fluo-4, AM from polymerizing in HBSS and helping it enter the cells.

Grouping: (1) negative control (N1) (glucagon only was added to transfected cells, treated with Fluo-4 AM) 3 groups: the dosage is 0.1. mu.M, 0.2. mu.M, 0.5. mu.M of 10. mu.L, respectively;

(2) negative control (N2) (untransfected cells with glucagon only, Fluo-4AM treatment) 3 groups: the dosage is 0.1. mu.M, 0.2. mu.M, 0.5. mu.M of 10. mu.L, respectively;

(3) blank control group 1 (B) (Fluo-4 AM treatment): transfected cells without glucagon;

(4) original control group 1 (P) (without Fluo-4AM treatment): untransfected cells without glucagon;

(5) positive control (O) (transfected cells, Fluo-4AM treated) group 1: simultaneously, 10. mu.L of known antagonist GRA (0.1. mu.M) and 10. mu.L of glucagon (0.1. mu.M) were added.

(6) DY-130 group: 0.5 μ M (10 μ L) DY-130 was used as the optimal experimental dose of glucagon.

(7) DY-62 group: 0.5 μ M (10 μ L) DY-62 was used as the optimal experimental dose of glucagon.

All groups were performed in 96-well plates, seeded at 3X 10 per well⁴And respectively administering the cells according to the set groups, and incubating in an incubator for 2-4h after administration.

Loading a fluorescent probe: after incubation was complete, the drug in the 96-well plate was aspirated.

(1) Adding Fluo-4AM working solution (4 mu mol/L) to cover the cells, and incubating for 30-40min at room temperature in a dark place;

(2) removing Fluo-4AM working solution, and washing cells by HBSS;

(3) adding HBSS containing 10% serum to the covered cells, and incubating at room temperature in dark for 20-30 min.

Wherein the excitation wavelength of Fluo-4AM is 494nm, the emission wavelength is 516nm, and the excitation wavelength is adjusted to 484nm according to actual tests.

And (3) detecting the fluorescence intensity: 1x10 into which fluorescent probe loading has been performed⁵The cells are put into a 1.5mL centrifuge tube and centrifuged for 5 minutes at 3000r/min, and the supernatant is discarded; add FACS buffer 100. mu.L suspension cells; the cell suspension was transferred to FACS tubes and prepared for instrumental detection and analysis, and each experiment was repeated 3 times to average.

The principle of fluorescence detection is adopted: glucagon binds specifically to the glucagon receptor, stimulating the opening of calcium channels and thereby promoting an increase in intracellular calcium ion concentration. When the antagonist competitively binds to the glucagon receptor, the intracellular calcium concentration is reduced and glucagon is antagonized. The relative concentration of intracellular calcium ions is characterized by fluorescence values to evaluate the degree of glucagon receptor antagonism of the compounds.

Referring to fig. 1, it can be seen from fig. 1 that when the fluorescence value of the sample group is lower than that of the N1 group (negative control group), it indicates that the sample can compete with glucagon to bind glucagon receptor, and the fluorescence value of the sample group DY-130 is lower than that of the negative control group N1, which has a certain antagonistic effect on glucagon; the fluorescence value of DY-62 is larger than that of a negative control group N1, and the antagonism effect on glucagon is avoided, so that the 3, 6' -dibapinyl sucrose can be used as an antagonist of glucagon and can be used for treating the 2-type diabetes mellitus.

Experimental example 2

Adopting Autodock to simulate molecular docking, specifically:

preparation of protein GCGR:

(1) pdb format download of GCGR: downloading the needed protein GCGR from the RCSB PDB webpage, wherein the PDB number of the protein GCGR is 5XEZ, inputting the 5XEZ search target protein in the RCSB PDB webpage, and downloading the PDB format of the protein GCGR for later use.

(2) GCGR protein modification: the pdb file of 5XEZ was imported into PyMol software, since the downloaded GCGR is a complex structure of protein and ligand small molecule (5XEZ-97V), as shown in fig. 2, and one 5XEZ receptor protein binds one 97V molecule in the a chain and B chain, respectively.

(3) A, B chain amino acid sequence and 97V binding surrounding amino acids: through the amino acids showing 97V binding and peripheral action in PyMol, it was found that the binding environment of 97V in A, B chain was almost the same, as shown in FIG. 3. To speed up the computation, the A chain was chosen for molecular docking, as shown in FIG. 4. Protein modification of 5XEZ at PyMol, deletion of unwanted chains, leaving intact a chains for calculation, and elimination of small molecules 97V, leaving binding pockets, saving 5XEZ a chains in pdb format and 97V small molecules in pdb format.

(4) GCGR binding pocket determination: opening 5XEZA in AutoDock Tools, removing water molecules, hydrogenating, completing the charge, storing in a pdbqt format, and setting the pdbqt format as a target protein; opening the 97V micromolecules, completing charge and hydrogen atoms by 97V, selecting the 97V micromolecules as ligand micromolecules, and storing the ligand micromolecules in a pdbqt format; the center of the pocket of the Grid Box was set to be centered on the small molecule ligand (45.117, 12.09, -16.915), and the Box size was (40, 40, 40).

(5) Verifying whether the box size is available: since spacing in AutoDock is 0.375 and that of Vina calculation software is 1, the box size in AutoDock needs to be multiplied by 0.375 and put into Vina for calculation, so that the box center is (45.117, 12.09, -16.915) and the box size is (15, 15, 15). A97V molecule is downloaded newly for calculation, the combination of the compound and the original compound is compared, the fit degree is not enough, the size and the center of the box are slightly changed, and the calculation result with higher fit degree with the original ligand is finally obtained (as shown in figure 5), the center of the box is (45.117, 12.483, -15.686), the size of the box is (17,17,17), and the center and the size of the box are used as final butt joint data.

Preparing small molecules DY-130 and DY-62: downloading the sdf format of the small molecules from the PubChem website for later use; introducing small molecule sdf format into Chem 3D and storing as mol2 format; introducing the small molecule mol2 format into AutoDock Tools, adding charges and hydrogen atoms, selecting the small molecule as a ligand, and outputting the small molecule ligand in a pdbqt format for later use.

Molecule docking:

the Windows + R opens a computer command interface, and determines to enter the command interface after the cmd is input; newly building a folder (docking) on the desktop for storing the calculation result and the required ligand; copying the position of the folder, pasting the position of the folder into a command window, and determining the position of the folder for positioning and calculating; copying the position of the Vina of the computing software, pasting the position to a command window, and determining to open the Vina of the computing software; a note file named conf is created in the docking folder, where the dockerin name and format, the ligand name and format, the protein center position and the docking box size are entered and saved. And starting molecular docking, wherein the output result after the molecular docking is finished is in txt format in the note book, and the output of the docking result structure is a pdbqt format file.

See tables below and fig. 6-7 for docking results.

From the above results, it is known that DY-130 forms 9 hydrogen bonds with GCGR, wherein O17 and O19 atoms of DY-130 form two hydrogen bonds with Arg346, and the bond lengths are respectively

O17, O19 atom forms two hydrogen bonds with Lys405, the bond lengths are respectively

The O18 atom forms a hydrogen bond with Asn404 having a bond length of

O2, O3, O4 and O12 atoms form four hydrogen bonds with Thr353, and the bond lengths are respectively

DY-62 and GCGR form 4 hydrogen bonds, wherein O6 and O7 atoms of DY-62 and Lys405 form two hydrogen bonds with the bond lengths of

The O6 atom forms a hydrogen bond with Arg346 having a bond length of

The O7 atom forms a hydrogen bond with Leu403 with a bond length of

The results show that DY-130 can be successfully butted and firmly combined, DY-62 can be butted but is not firmly combined, cannot compete with glucagon in cells and cannot antagonize glucagon, and further the results show that DY-130 can antagonize glucagon and further treat type 2 diabetes.

Experimental example 3

Research on conditions for extracting 3, 6-brassicaceae sucrose;

the variables involved and the parameters for the design of the dependent variables are shown in the following table.

The results are given in the following table:

note: k is the average value of the extraction rate of the same level; r is extreme difference

From the above results, it can be seen that: the extraction rate of nine groups of experiments for extracting 3, 6-dibastine acyl sucrose from polygala tenuifolia by an ultrasonic method is subjected to range analysis by taking the extraction rate as an index, and the volume fraction of the factors influencing the extraction rate, namely the material-liquid ratio, the ultrasonic time and the methanol is obtained, the optimal levels of the three factors are A2, B2 and C3, and the optimal combination of the three levels of the three factors is A2B2C 3.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of 3, 6' -dibapinyl sucrose in preparing medicine for treating type 2 diabetes is disclosed.

2. The use according to claim 1, wherein the preparation of 3, 6' -dibapinyl sucrose comprises: extracting polygala tenuifolia, and separating to obtain the 3, 6' -dibapinyl sucrose.

3. The use according to claim 2, wherein the preparation of 3, 6' di-sinapoyl sucrose comprises: mixing the polygala tenuifolia with a solvent, extracting under an ultrasonic condition, and separating to obtain the 3, 6' -brassica juncea acyl sucrose.

4. Use according to claim 3, wherein the solvent is an alcohol-water solvent, preferably the solvent is a monohydric alcohol-water solvent.

5. The use according to claim 4, wherein the solvent is a methanol aqueous solvent.

6. Use according to claim 5, wherein the solvent is an aqueous methanol solvent with a methanol volume fraction of 50-90%, preferably an aqueous methanol solvent with a methanol volume fraction of 90%.

7. Use according to claim 3, characterized in that the extraction conditions are: the ultrasonic treatment time is 40-50 minutes, and the material-liquid ratio of the polygala tenuifolia to the solvent is 1:20-1: 40;

preferably, the ultrasonic time is 45 minutes, and the ratio of the polygala tenuifolia to the solvent is 1: 30.

8. the use of claim 1, wherein said drug is a glucagon antagonist drug.

9. Application of 3, 6' -dibapinyl sucrose in preparing glucagon antagonist is provided.

10. Application of 3, 6' -dibapinyl sucrose in preparing medicine for treating diseases caused by glucagon is provided.