CN114213504A

CN114213504A - Compound with anti-diabetic activity and application thereof

Info

Publication number: CN114213504A
Application number: CN202111604034.8A
Authority: CN
Inventors: 曾静; 吴建明; 叶馨源
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-22
Anticipated expiration: 2041-12-24
Also published as: CN116283702A; CN114213504B

Abstract

The invention relates to a compound with anti-diabetic activity and application thereof, wherein the compound is an analogue of diprotinA, has stronger anti-diabetic activity, belongs to a DPP-IV inhibitor, can improve the blood insulin level, and opens up a new way for treating diabetes; the compound is applied to the preparation of foods, health-care products and medicines for preventing or/and treating diabetes, and has higher social value and important significance.

Description

Compound with anti-diabetic activity and application thereof

Technical Field

The invention relates to the technical field of compounds with anti-diabetic activity, and particularly relates to compounds with anti-diabetic activity and application thereof.

Background

Type II diabetes (T2DM) is a common disorder of the metabolism of blood sugar, cholesterol, protein, water, electrolyte levels, as well as cell dysfunction, impaired insulin secretion and insulin resistance. Patients with poor glycemic control are prone to a variety of diabetic complications, including renal failure, ketoacidosis, and diabetic nonketotic hyperosmolar syndrome. Blood glucose regulation is a highly complex process controlled by a variety of enzymes, hormones, and nerves. It has been found that, among many enzymes, blood glucose levels are closely related to the activity of dipeptidyl peptidase IV (DPP-IV).

DPP-IV is a well-known drug target for the treatment of type II diabetes, as it degrades glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) with high selectivity in vivo. Meanwhile, DPP-IV inhibitor can enhance the activity of external GLP-1 and GIP, thereby improving blood insulin level. Some synthetic anti-diabetic molecules, such as sitagliptin, linagliptin and gemfibrogliptin, have been approved for the treatment of type II diabetes by DPP-IV inhibition. At present, a plurality of II-type diabetes patients are generated in the world each year, the disease course is long, stronger control force is needed, and the pain trouble is brought to more and more patients, so that the search for more compounds with anti-diabetic activity has important significance for the treatment of diabetes.

Disclosure of Invention

The invention aims to: aiming at the problem that diabetics suffer from pain, compounds with anti-diabetic activity and application thereof are provided. The compound disclosed by the invention has stronger anti-diabetic activity, and opens up a new way for preventing and treating diabetes.

In order to achieve the purpose, the invention adopts the technical scheme that:

a class of compounds having anti-diabetic activity, which compounds are analogs of diprotin A.

The diprotin A analogue disclosed by the invention has stronger anti-diabetic activity, belongs to a DPP-IV inhibitor, can improve the blood insulin level, and provides a new choice for treating diabetes.

Further, the chemical structure of diprotin A is

Further, the compound is selected from at least one of the following structural formulas;

further, the compound is isolated from Gynura divaricata but not limited to Gynura divaricata. Preferably, the compound is isolated from gynura divaricata.

Another object of the present invention is to provide the use of the above compounds.

The application of the compound with the anti-diabetic activity in food or health care products is disclosed.

The application of the compound with the anti-diabetic activity in preparing the medicine is the medicine for preventing or/and treating diabetes.

Another object of the present invention is to provide a class of medicaments comprising the above compounds.

A medicament comprising a compound as described above having anti-diabetic activity.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the invention discovers an anti-diabetic active ingredient from the medicinal and edible gynura divaricata, characterizes the chemical structures of 25 diprotin A analogues, and verifies the anti-diabetic activity; the compound to be protected belongs to DPP-IV inhibitor, IC₅₀The value was 0.40 mg/mL; molecular docking studies have also demonstrated the interaction between diprotin A analogues and DPP-IV; in addition, cell experiments and animal experiments also prove that the compound disclosed by the invention can improve the blood insulin level, has a better hypoglycemic effect, and opens up a new way for treating diabetes.

2. The invention also discloses application of the compound with anti-diabetic activity, and the compound has higher social value.

Drawings

Figure 1 is a molecular network of a diprotin a analogue.

FIG. 2 shows the secondary mass spectra and possible cleavage pathways of diprotin A (a), formula (2) (b) and formula (3) (c).

FIG. 3 shows the DPP-IV inhibition ratios of different concentrations of diprotin A analogues.

FIG. 4 is a molecular pair score plot of 25 diprotin A analogs.

FIG. 5 is a schematic diagram of molecular docking interactions between DPP-IV and diprotin A, and the three compounds of formula (2) and formula (3).

FIG. 6 is a graph of the effect of analogs of diprotin A in example 2 on GLP-1 levels in NCI-H716 cells.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the following examples, LC-MS grade acetonitrile and LC-MS grade formic acid were obtained from Fisher Scientific, Mass. Ethanol was purchased from Titan Scientific (shanghai, china). Methanol was purchased from the Jinshan chemical test (Chinese Chengdu). Anhydrous disodium hydrogen phosphate was purchased from a scientific test (chinese achievements). Hydrochloric acid was purchased from chemical judgu, China. Milli-Q water purification systems (Billerica, MA, USA) are used to produce ultra pure water. Gynura divaricata is collected from Luxian (Sichuan China), and species identification is performed according to morphological characteristics of Gynura divaricata.

Example 1

Natural products play a vital role in modern life sciences and new drug development. The discovery of active compounds from natural products has attracted the attention of scientists, but remains a great challenge as natural products are extremely complex systems.

Rapid analysis of antidiabetic component in gynura divaricata

Step 1, taking a gynura divaricata sample, and preparing a test solution by using a methanol-water solution as a solvent.

1kg of white back of the threeSoaking in 20L methanol-water (50:50, v v) containing 0.1% hydrochloric acid^-1) For two weeks. Filtering, concentrating, and lyophilizing Gynura divaricata extract (GD-E) for further treatment.

And 2, dissolving the test sample prepared in the step 1 and analyzing by using ultra-high performance liquid chromatography-mass spectrometry to obtain mass spectrum information of the crude extract.

GD-E was analyzed by ultra performance liquid chromatography. The procedure used Inertsil C₁₈A column (100X 2.1mm, 3 μm) was subjected to gradient elution at a flow rate of 0.3mL/min with an aqueous solution (A) of 0.1% formic acid and acetonitrile (B) as mobile phases, with the column incubator temperature set at 40 ℃. The gradient elution procedure was as follows: 5% B in 0-2 min, 5-70% B in 2-18 min, 50-100% B in 18-20 min, and 100% B in 20-25 min.

The eluent from the hplc was introduced into a mass spectrometer and the compound primary and secondary mass spectral data were collected using an X500R Q-TOF mass spectrometer. Electrospray ionization (ESI) parameters were as follows: temperature: 500 ℃; ion source gases 1 and 2: 50 psi; air curtain air: 35 psi; CAD gas: 7 psi. The IDA is specifically set as: collision energy 40V, maximum candidate ion 10; an intensity threshold; 400 cps; full scan mass range: 100-1500 Da; ion spray voltage: 5500V.

And 3, introducing the mass spectrum data obtained in the step 2 into a GNPS platform, utilizing a GNPS database to represent chemical components in a sample solution through searching the database, and confirming the chemical components with compounds reported in the literature to determine potential active components.

The raw data files were collected and exported using SCIEX OS 1.4 software. The original data file is converted to mzXML format using MSconver software and a Molecular Network (MN) is built. The MN operating parameters are as follows: mass deviation of precursor ions is +/-0.02 Da; the mass deviation of the daughter ions is +/-0.02 Da; minimum paired cosine value: 0.7; minimum cluster size: 2; minimum matching fragment ions: 6.

GNPS forms a network graph visualized based on the similarity of the secondary mass spectral data of a compound, where one node represents a compound and the colors of different nodes represent different sources or attributes. Having similar structure (class)Analog) are clustered in a cluster. Cosine values exist between the two nodes; the higher the cosine (0-1), the more similar the structure. Using this method, diprotin a was successfully identified. Diprotin A is a known DPP-IV inhibitor, the IC of which₅₀1.6-5.8 mu g/mL, and is generally used as a positive control in a DPP-IV inhibition test; it can also prevent degradation of GLP-1, thereby exerting hypoglycemic effect.

And 4, enriching the potential active ingredient analogues from the gynura divaricata extract by adopting a strong cation exchange SPE method.

First, Gynura divaricata extract is dissolved in ethanol-water (20:80, v v) containing 0.1% hydrochloric acid^-1) In (1). Separation was performed using a strong cation exchange Solid Phase Extraction (SPE) column. The column was activated with methanol and then 250mmol/LNa₂HPO₄Methanol-water (20:80, v v)^-1) The column was continued to be activated. Methanol-water (20:80, v v) was used before loading^-1) Washing the column to remove residual Na₂HPO₄. After loading, methanol-water (50:50, v)^-1) The column was washed to remove neutral compounds. Using a solution containing 150mmol/L Na₂HPO₄Methanol-water (20:80, v v)^-1) And (4) eluting. With C₁₈(10μm，

Acchrom, china) as a packing and collecting the methanol eluate.

Step 5, taking methanol eluent, analyzing by using ultra-high performance liquid chromatography, and introducing the eluent into a mass spectrometer to obtain mass spectrum data of the test solution; and then, mass spectrum data is transmitted into a GNPS platform, a molecular network is constructed, and potential active ingredients are characterized.

As shown in FIG. 1, the filled circles represent compounds identified by a library search, identified as diprotin A, since the cosine values are set to>0.7, indicating that the other node is an analog of diprotin A. As shown in FIG. 2(a), diprotin A produced ions m/z 229.1566, 86.0968, 72.0812 and 70.0815 and performed fragment assignment, where m/z 72.0812 and 70.0815 are characteristic ions of N5-containing rings, which were used to aid in analysis of similar typesA compound (I) is provided. When the molecule is disintegrated, isoleucine and valine are removed, and M-113 Da and M-99Da ions are generated respectively. In addition, the diprotin A analog exhibited a neutral loss of 159Da due to the elimination of isoleucine and formate composition. As shown in FIG. 2(b), the parent ion generated by formula (1) is m/z 328.2238 (-2.2 ppm, C)₁₆H₂₉N₃O₄) Daughter ions similar to the diprotin a fragment were generated, including m/z 229.1561, 86.0968, 72.0812, and 70.0815. Fragment ions at M/z 229.1561 correspond to [ M + H-99 ]]⁺. Furthermore, the molecular weight of formula (1) is 13Da less than diprotin A. Thus, by comparing the MS and MS/MS data, formula (1) was identified as diprotin C. In the positive mode, as in FIG. 2(C), formula (2) is at m/z 243.1709(2.4ppm, C)₁₂H₂₂N₂O₃) To generate [ M + H]⁺Ion, by fragmentation to give M/z 144.1028([ M + H-99)]⁺) Ions and fragment ions (m/z 70, 72) identical to formula (1), thus, successfully characterizing the structure of formula (2). Based on the fragmentation pathways described above, 25 diprotin a analogues were preliminarily characterized, as shown in table 1.

Table 125 diprotin A analogue structure and molecular information

Step 6, carrying out DPP-IV inhibitory activity determination on the potential active ingredient analogue to obtain IC₅₀(ii) a And the analogues are subjected to molecular docking with DPP-IV to obtain a plurality of moleculesThe binding energy values were docked to verify the anti-diabetic activity of several potential active ingredient analogues.

Molecular docking technology, as a computer-based method, can be used to predict drug-enzyme interactions. The interaction between analogs of diprotin A and DPP-IV (PDB crystal structure: 1WCY) was studied using molecular docking. A higher score (binding energy value) indicates a more rational and stable interaction between the ligand proteins. All receptor proteins were prepared as follows: water molecules were removed, hydrogen atoms were added to the protein, and CHARMM force field was applied. All compounds used for docking were prepared using the "ready ligand" module in DS 3.1. The molecular docking score statistics are presented in figure 4. All diprotin A analogues interacted with DPP-IV to varying degrees, suggesting that they are potential inhibitors of DPP-IV. DPP-IV appears as a dimer and forms two openings providing access to the cavity, which is the exact binding site for the 25 analogs. The three analogues scored higher than diprotin a. As shown in FIG. 5, in Table 1, formula (6) (FIG. 5b) is stably docked within the lumen of DPP-IV and interacts with several amino acid residues with a docking value of 40.5567 kcal/mol. Formula (6) forms five conventional hydrogen bonds with amino acid residues Ser 209, Glu 205, Arg 125, Ser 630 and Tyr 547, three carbon-hydrogen bonds with amino acid residues Glu 206, Glu 205 and Tyr 547, and two hydrophobic interactions with amino acid residues His 126 and His 740. Similarly, formula (3) (FIG. 5a) and formula (23) (FIG. 5c) also form hydrogen bonds and hydrophobic interactions with amino acids in DPP-IV. Other compounds also interact with DPP-IV through hydrogen bonding and hydrophobic interactions.

The inhibitory activity of DPP-IV was determined using the DPP-IV inhibitor screening kit (Merck, Germany). The total volume of the reaction mixture was 100. mu.L. First, the samples were solubilized using the buffer from the DPP-IV kit. Then, 25. mu.L of the sample and 1. mu.L of DPP-IV enzyme with 49. mu.L DPP-IV detection buffer were added to a 96-well black plate. Mixing, and incubating at 37 deg.C for 10 min. Subsequently, 25. mu.L of the enzyme reaction mixture (2. mu.L of DPP-IV substrate in 23. mu.L of buffer) was added to each well and mixed. Finally, detection was carried out using a microplate reader (JIYUANBIO-TECH, China).

The inhibitory activity of DPP-IV is calculated as follows:

wherein:

FLU1 is the fluorescence intensity at T1; FLU2 is the fluorescence intensity at T2; SlopesM is the slope of the sample inhibition group; SlopeEC is the slope of the enzyme control group. The Delta FLU value of the irreversible DPP-IV inhibitor is 0, and the relative inhibition rate is 100%.

The inhibition of DPP-IV by diprotin A analogues was determined using a known DPP-IV enzyme inhibitor (sitagliptin) as a positive control, and the IC calculated₅₀The value is obtained. IC of diprotin A analogues as shown in FIG. 3₅₀0.40mg/mL, and is dose-dependent (n-3). Several compounds (3, 6, 10, 12 and 16) with higher scores were scored by directed synthesis of molecular docking and tested for IC inhibition of DPP-IV by each compound₅₀The results are shown in Table 2.

TABLE 26 IC of representative diprotin A analogs DPP-IV₅₀(μg/mL)

n＝5

Group of

Sitagliptin

3

6

10

12

16

23

IC₅₀

0.18±0.07

150±5.05

120±6.32

310±6.12

301±7.36

450±8.08

160±7.07

Example 2

The analogs of diprotin A obtained in

step

5 and 6 directionally synthesized monomeric compounds in example 1 were subjected to cell experiments.

NCI-H716 cells at 1X 10⁶Culturing the cells/mL in a 12-well plate at a density, sucking supernatant after 48h, adding 1mL of buffer solution and a drug to be tested, taking alogliptin (2 mu g/mL) as a positive drug, taking a mixture of 25 compounds with the concentrations of 100, 200 and 300 mu g/mL respectively, incubating at 37 ℃ for 2h, sucking supernatant, and detecting the GLP-1 content by adopting an ELISA kit, wherein the test result of the diprotin A analogue is shown in figure 6. The analogs of Diprotin A showed significant differences in the concentration-dependent increase of GLP-1 concentration, with a GLP-1 concentration of 7.33. + -. 0.44pmol/L at a concentration of 200. mu.g/mL and a GLP-1 concentration of 7.87. + -. 0.25pmol/L at a concentration of 300ug/mL (P)<0.05). Alogliptin increased GLP-1 concentration from 6.73 + -0.14 pmol/L to 7.45 + -0.12 pmol/L and was significant (P)<0.05). Monomeric compounds with concentrations of 50 mug/mL are concentrated on GLP-1The results of the degree test are shown in table 3.

TABLE 3 Effect of diprotin A analogues and 6 representative monomeric Compounds on GLP-1 concentration (pmol/L)

n＝5

TABLE 3

Group of	Control group	Positive group		3	6
						GLP–1	6.8±0.23	7.35±0.54	7.53±0.36**	7.51±0.11**
Group of	10	12	16	23
					GLP–1	7.02±0.33*	7.23±0.02*	6.89±0.36	7.56±0.12**

Note: p <0.05, P <0.01, compared to control group

Example 3

Animal experiments were performed on analogs of diprotin a isolated in example 1 in step 5, i.e. a mixture of 25 compounds numbered 2-26 in table 1 and 6 monomeric compounds synthesized in a targeted manner.

After male rats with the same weight and age are fed with high fat for 4 weeks, 30mg/kg STZ (0.5mL/100g) is injected into the abdominal cavity once after 6 hours of fasting without water prohibition, the male rats are fed with high fat diet for 4 weeks, blood sugar is measured, and a molding success group is selected for grouping administration and replaced with common feed. 72 diabetic rats with successful modeling (blood sugar value of 15-20 mmol/L) are selected and randomly divided into 9 groups: alogliptin (3mg/kg) group, model group, drug group. The drug components are a Diprotin A analogue group (20mg/kg) and 6 monomer compound groups (5 mg/kg). Another 8 normal male rats of the same batch were selected as the normal group. After 7 days of continuous intravenous administration in the first 7 groups, the normal group was given an equal volume of physiological saline; fasting for 5h to determine blood sugar, injecting 2g/kg glucose respectively, taking blood from tail tip of rat 30min, 60min, 90min and 120min after injection, and measuring (strong life and steady luxury type) blood sugar with glucometer. The results are shown in Table 4.

TABLE 4 sugar tolerance of Diprotin A analogues and 6 monomer compounds

Note: compared to the model, P <0.05, P <0.01

TABLE 5 Diprotin A analogues and 6 monomeric compounds on fasting plasma glucose (mmol/L)

n＝8

Group of	Normal group	Positive group	Model set	Diprotin A analogues	3
						Blood sugar	4.61±0.80	14.32±1.33	17.80±1.25	16.19±0.32*	14.45±1.51**
Group of	6	10	12	16	23
						Blood sugar	15.02±1.44**	16.88±0.21*	15.96±1.33**	15.39±0.16**	14.13±2.01**

Note: p <0.05, P <0.01, compared to model group

As shown in Table 4, the blood glucose values at all points in the glucose tolerance test of the model group rats were higher than those of the normal group rats (P < 0.05). As shown in table 5, Diprotin a analogs and 6 monomeric compounds significantly reduced blood glucose levels after administration (P < 0.05).

The invention adopts the method to discover the compound with anti-diabetic activity from the natural products of gynura divaricata, efficiently and selectively separates 25 analogs of diprotin A, and performs the characterization of the anti-diabetic activity; the compound to be protected belongs to DPP-IV inhibitor, IC₅₀The value was 0.40 mg/mL; molecular docking research also proves the interaction between the diprotin A analogue and DPP-IV, the diprotin A analogue promotes NCI-H716 cells to release GLP-1, and has obvious effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A class of compounds having anti-diabetic activity, wherein the compounds are analogs of diprotinA.

2. The compound having anti-diabetic activity according to claim 1, wherein the compound is selected from at least one of the following structural formulae;

3. the compound having anti-diabetic activity according to claim 2, wherein the compound is selected from at least one of the following structural formulae;

4. the compound having anti-diabetic activity according to claim 3, wherein the compound is selected from at least one of the following structural formulae;

5. the compound having anti-diabetic activity according to any one of claims 1 to 4, wherein the compound is isolated from Gynura divaricata but not limited to Gynura divaricata.

6. Use of the compound having an antidiabetic activity according to any one of claims 1 to 4 for the preparation of foods, health products and medicines for the prophylaxis or/and treatment of diabetes.

7. A medicament comprising an effective amount of a compound as claimed in claims 1 to 4 having anti-diabetic activity.