CN114796192A

CN114796192A - Flavonoid compound with cholinesterase resisting and carbohydrase resisting dual activities and application thereof

Info

Publication number: CN114796192A
Application number: CN202210498984.5A
Authority: CN
Inventors: 杨静; 李娜娜; 王春雨; 武力扬; 段安邦; 刘永平
Original assignee: Shanxi Jingxi Biotechnology Co ltd; North University of China
Current assignee: Shanxi Jingxi Biotechnology Co ltd; North University of China
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2022-07-29

Abstract

The invention provides a flavonoid compound with double activities of cholinesterase resistance and carbohydrase resistance and application thereof, belonging to the technical field of chemical drugs. The quercetin, kaempferol and myricetin have high anticholinesterase and carbohydrase activities and good blood brain barrier penetration capacity, which not only provides raw materials for preparing enzyme inhibitors, but also provides a new idea for medicaments for preventing and/or treating Alzheimer's Disease (AD) and non-insulin dependent diabetes mellitus (T2 DM).

Description

Flavonoid compound with cholinesterase resisting and carbohydrase resisting dual activities and application thereof

Technical Field

The invention belongs to the technical field of chemical drugs, and particularly relates to a flavonoid compound with cholinesterase resisting and carbohydrase resisting activities and application thereof.

Background

Alzheimer's Disease (AD) is an irreversible neurodegenerative disease characterized by central cognitive, memory and motor disorders. Many studies have found that AD is associated with decreased levels of the brain central neurotransmitters acetylcholine (ACh) and butyrylcholine (BCh), which are the major enzymes of AD, and its major physiological function is to rapidly hydrolyze the neurotransmitters acetylcholine (ACh) to form acetate (a) and choline (Ch) (see fig. 1). Therefore, inhibition of these two enzymes leads to increased communication between cholinergic pathways and nerve terminal activity, thereby improving cognitive symptoms, and has become one of the major means for treating AD. Currently, several cholinesterase inhibitors such as tacrine, donepezil, rivastigmine, and galantamine have been approved by the FDA in the united states for the treatment of mild to moderate AD. However, these drugs cause side effects such as gastrointestinal disorders, insomnia, anorexia, hepatotoxicity and other adverse reactions, and their bioavailability is low. Therefore, there is a need to find and develop cholinesterase inhibitors with fewer side effects.

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia, and diabetes mellitus and its complications have become a major public health problem in the fastest growing world in the 21 st century. In recent years, the incidence of diabetes has become higher and higher, and the disease has become a major concern. 90% -95% of the non-insulin-dependent diabetes mellitus type II (T2DM), T2DM is due to hydrolysis of carbohydrates after meals, resulting in increased blood sugar. Therefore, prevention of an increase in blood glucose level by inhibiting rapid hydrolysis of carbohydrates after meals, reducing glucose absorption rate, is considered to be an effective treatment for diabetes. Glycemic control can be achieved by inhibiting alpha-amylase and alpha-glucosidase, which both key enzymes play a key role in carbohydrate hydrolysis processes. Alpha-amylase, an endogenous hydrolase, cleaves the alpha-1, 4 glucosidic chains within starch into oligosaccharides, which are further hydrolyzed by alpha-glucosidase located on the epithelium of the small intestine into absorbable monosaccharides (see fig. 2). Thus, inhibiting the activity of both enzymes may delay carbohydrate digestion, reducing the rate of glucose entry into the blood. Drugs that inhibit hyperglycemia by alpha-glucosidase and alpha-amylase, including acarbose, miglitol, and voglibose, have been developed. However, these synthetic inhibitors may have various side effects such as diarrhea, flatulence, nausea, liver disorders and abdominal cramps. Thus, there is an ongoing interest in natural bioactive compounds that inhibit the activity of key enzymes in sugar metabolism.

Clinically, T2DM and AD are two independent non-infectious diseases. However, there is evidence that Insulin Resistance (IR) or insulin deficiency directly affects glucose metabolism in the Central Nervous System (CNS), ultimately leading to neurodegenerative diseases such as AD. Meanwhile, T2DM is at 1.5 times the risk of dementia as in non-diabetic patients. Diabetes and AD also share many common risk factors, including metabolic disease (obesity), cardiovascular disease, aging, and progressive insulin resistance, as well as common pathogenic processes such as chronic hyperglycemia, tissue amyloid beta (a β) deposition and toxicity, cellular oxidative stress, and chronic inflammation. Thus, even the term "type 3 diabetes" is proposed to describe AD associated with IR and similar growth factor signaling in the brain. Unfortunately, the irreversible negative effects of IR on peripheral tissues and the brain may occur earlier than in clinically defined diabetes. At present, pharmacological studies have found that some typical polyphenols and flavonoids may inhibit the pathogenic cascade of AD and T2DM caused by oxidative stress and inflammation through antioxidant activity, as shown in fig. 3. Previous studies also found that the anti-AChE activity of 11 flavonoids was highly correlated with their antioxidant activity (FRAP) (R0.9221). Quercetin may target mitogen-activated protein kinase (MAPK) signaling through molecular docking, and has certain therapeutic effects in T2DM and AD. Galangin, kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin and rutin, have reversible inhibition effect on BChE. Furthermore, studies have been conducted to analyze the inhibitory activity of approximately 500 synthetic and natural compounds on α -amylase and α -glucosidase and to find that flavonoids are promising antidiabetic molecules. Therefore, the development of dual inhibitors against AD and T2DM with fewer side effects has been the focus of attention by the public and pharmacologists.

Disclosure of Invention

In view of the above, the present invention aims to provide a flavonoid compound with dual activities of cholinesterase and carbohydrase resistance, and provides a new idea for preventing and/or treating AD and T2DM simultaneously.

The invention provides a flavonoid compound with double activities of cholinesterase resistance and carbohydrase resistance, which comprises the following two or three flavonoid compounds: quercetin, kaempferol and myricetin.

Preferably, the flavonoids include quercetin, kaempferol and myricetin.

Preferably, the mass ratio of the quercetin to the kaempferol to the myricetin is 1-10: 1-10.

The invention provides application of quercetin, kaempferol, myricetin or the flavonoid compound in preparation of a preparation with cholinesterase resistance and carbohydrase resistance.

Preferably, the cholinesterase comprises acetylcholinesterase and/or butyrylcholinesterase.

Preferably, the anti-carbohydrase enzyme comprises an alpha-glucosidase and/or an alpha-amylase.

The invention provides application of quercetin, kaempferol, waxberry or the flavonoid compound in preparing a medicament for preventing and/or treating AD and T2 DM.

Preferably, the drug has inhibitory activity against acetylcholinesterase, butyrylcholinesterase, α -amylase and α -glucosidase simultaneously.

Preferably, the drug has blood-brain barrier penetration ability.

The invention provides a medicament for preventing and/or treating AD and T2DM, which comprises an active ingredient and pharmaceutically acceptable auxiliary materials;

the active ingredient comprises quercetin, kaempferol, myricetin or the flavonoid compound.

Preferably, the dosage form of the medicament comprises an oral preparation.

The flavonoid compound with the double activities of resisting cholinesterase and carbohydrase provided by the invention comprises the following two or three flavonoid compounds: quercetin, kaempferol and myricetin. The invention evaluates the inhibitory activity of 16 flavonoids compounds on AChE, BChE, alpha-glucosidase and alpha-amylase under the in vitro condition, and the result shows that the quercetin (IC) in flavonol _50-AChE ＝34.46μg/mL，IC _50-BChE 99.25. mu.g/mL), myricetin (IC) _50-AChE ＝32.23μg/mL，IC _50-BChE 99.64 μ g/mL) and kaempferol (IC) _50-AChE ＝40.44μg/mL，IC _50-BChE 124.56 μ g/mL) has a higher potential for anticholinesterase activity than other flavonoids; quercetin, quercitrin, kaempferol, myricetin and other flavonoids have high inhibitory activity to alpha-glucosidase; myricetin, quercetin, kaempferol, luteolin and the like have high inhibition activity on alpha-amylase; however, the positive control galantamine and donepezil have specific inhibitory effects on cholinesterase activity, but no anti-carbohydrase activity, whereas acarbose is the opposite. Therefore, the results of the invention show that a plurality of flavones, particularly quercetin, kaempferol and myricetin show double functions and have higher cholinesterase resistance and carbohydrase resistance.

The invention provides an application basis of quercetin, kaempferol, myricetin or the flavonoid compound in preparing a medicament for preventing and/or treating AD and T2 DM. Since quercetin, kaempferol and myricetin, alone or in combination, have good anticholinesterase and carbohydrase activities, and simultaneously, based on Topological Polar Surface Area (TPSA) results, the quercetin, kaempferol and myricetin, alone or in combination, have good blood brain barrier penetration capability, which provides a basis for the quercetin, kaempferol and myricetin, alone or in combination, to serve as candidate drugs for the prevention and/or treatment of AD and T2 DM.

Drawings

FIG. 1 is a schematic diagram showing the action process of acetylcholinesterase;

FIG. 2 is a schematic view of a starch hydrolysis process;

FIG. 3 is a schematic diagram of the inhibition process of a phenolic compound on cholinesterase, alpha-amylase and alpha-glucosidase;

FIG. 4 shows the enzyme inhibitory IC of standards and flavonoids ₅₀ Values, binding energies, TPSA and FRAP values;

FIG. 5 is a schematic representation of molecular docking simulation of compounds with enzymes, AChE-galantamine and AChE-myricetin (a), BChE-galantamine and BChE-quercetin (b), α -glucosidase-acarbose and α -glucosidase-quercetin (c), α -amylase-acarbose and α -amylase-myricetin (d);

FIG. 6 is a graph showing the relationship between TPSA and the inhibitory activity of 4 enzymes;

FIG. 7 shows anticholinesterase and anticarbohydrase activities IC ₅₀ The correlation of (c);

FIG. 8 is a graph showing the correlation between anticholinesterase and minimum Binding Energy (BE).

Detailed Description

In the present invention, the flavonoids include quercetin, kaempferol and myricetin. The mass ratio of the quercetin to the kaempferol to the myricetin is preferably 1-10: 1-10, and more preferably 1:1: 1.

In the invention, the effect of 16 flavonoids compounds on inhibiting the enzyme activities of cholinesterase, alpha-glucosidase and alpha-amylase is measured under in vitro conditions, and the result shows that: 1) aiming at the aspect of cholinesterase inhibition, quercetin, kaempferol and myricetin have higher cholinesterase activity resistance potential than other flavonoids, wherein the inhibition of the quercetin and the myricetin is equivalent and better than that of the kaempferol; while the lutein, catechin and epicatechin of flavonols have no or low inhibitory activity (IC) on AChE and BChE ₅₀ >1000. mu.g/mL). 2) Aiming at the inhibition effect of alpha-glucosidase and alpha-amylase, quercetin, kaempferol and myricetin have higher inhibition activity on the alpha-glucosidase and the alpha-amylase, while the lutein, catechin and epicatechin have higher inhibition activity on the alpha-glucosidase and the alpha-amylaseThe inhibition of the enzyme is low.

Based on the results, the invention provides the application of quercetin, kaempferol, myricetin or the flavonoid compound in preparing a preparation for resisting the enzymatic activities of cholinesterase and carbohydrase simultaneously.

In the present invention, the cholinesterase preferably comprises acetylcholinesterase and/or butyrylcholinesterase. The anti-carbohydrase preferably comprises an alpha-glucosidase and/or an alpha-amylase. The present invention is not particularly limited in kind and preparation method of the preparation, and it is sufficient to use the kind and preparation method of enzyme inhibitor well known in the art.

The prior art generally considers that the inhibition of the activities of acetylcholinesterase and butyrylcholinesterase can lead to the increase of communication between cholinergic pathways and nerve endings, thereby improving cognitive symptoms, and the inhibition of the activities of the acetylcholinesterase and the butyrylcholinesterase becomes one of the main means for treating AD, and simultaneously, the blood sugar control can be realized by inhibiting the activities of alpha-glucosidase and alpha-amylase, and the combination of the results of the invention, the quercetin, kaempferol and myricetin can simultaneously inhibit the activities of the acetylcholinesterase and the butyrylcholinesterase and inhibit the activities of the alpha-glucosidase and the alpha-amylase, so the invention provides the application of the quercetin, the kaempferol and/or the myricetin in the preparation of the medicine for preventing and/or treating AD and T2 DM.

In the present invention, since quercetin, kaempferol and myricetin, alone or in combination, have good acetylcholinesterase, butyrylcholinesterase, α -glucosidase and α -amylase activities, the prepared drug preferably has inhibitory activities against acetylcholinesterase, butyrylcholinesterase, α -glucosidase and α -amylase simultaneously. The invention estimates the affinity of the compound and enzyme by simulating the molecular docking condition and obtaining the binding energy, and the docking simulation result shows that the lowest BE of galantamine to AChE (-8.65 kcal/mol) and BChE (-6.92 kcal/mol) is lower than that of myricetin-AChE (-5.64 kcal/mol) and quercetin-BChE (-6.74 kcal/mol), which is consistent with the trend of the in vitro inhibitory activity. The BE value of quercetin to alpha-glucosidase is-8.86 kcal/mol, which is lower than acarbose, and is consistent with the trend of in vitro inhibitory activity.

In the present invention, it is considered that the drug exerts its pharmacological effect not only in consideration of the magnitude of the pharmacological effect but also in consideration of whether or not the site of action is reached. The present invention assesses the permeation capacity of a drug by analyzing the Topologically Polar Surface Area (TPSA). Since the focus of alzheimer's disease is in the brain, therapeutic drugs need to have good blood-brain barrier permeability to exert their effects. Tests show that the quercetin, kaempferol and myricetin all have ideal blood brain barrier permeability, so that the medicine can be ensured to exert the efficacy through the blood brain barrier.

In the invention, correlation coefficients of the anticholinesterase activity and the carbohydrase activity of the flavonoid compound and the antioxidant activity of the flavonoid compound are also researched, and results show that the FRAP has higher correlation with the inhibitory activities of AChE, BChE and alpha-glucosidase. Meanwhile, the four enzymes have high correlation with flavonol, and the BE values of the combination of acetylcholinesterase, butyrylcholinesterase, alpha-glucosidase and alpha-amylase with ligands have certain correlation.

The invention provides a medicament for preventing and/or treating AD and T2DM, which comprises an active ingredient and pharmaceutically acceptable auxiliary materials; the active ingredient comprises quercetin, kaempferol, myricetin or flavonoid compounds.

In the present invention, the dosage form of the drug preferably includes an oral preparation. The invention selects different auxiliary materials to prepare the medicine according to different dosage forms of the medicine. The method for preparing the drug is not particularly limited, and the method for preparing the drug well known in the field can be adopted.

The flavonoid compounds with dual activities of anticholinesterase and anticarbohydrase and the application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Test materials and Instrument description

1. Experimental materials and instruments

1.1 materials and reagents

The main drug specifications and manufacturers are shown in table 1, and the structures of the compounds are shown in table 2.

TABLE 1 Experimental drugs

TABLE 2 structures of flavonoids and standards

2 instruments and apparatus

The main instrument in the experiment is Readmax 1900 light absorption full-wavelength microplate reader (Shanghai flash spectrum Biotechnology Co., Ltd.).

Example 1

In this example, 16 representative flavonoids (shown in table 2) were selected, and the following tests were performed on a composition prepared by mixing equal amounts of flavonol, flavone, flavanone, and flavanol, as well as quercetin, kaempferol, and myricetin (shown in table 3 and fig. 4).

1. Determination of acetylcholinesterase (AChE) inhibitory Activity

Determination of the inhibitory Activity of acetylcholinesterase (AChE) A slight modification was made according to the method of the prior art 1(Yao J, Chen J, Yang J, et al free, soluble-bound and insoluble-bound phenolics and the same biological activity in raspberric place [ J ]. LWT-Food Science and Technology,2021,135: 109995). Specifically, a sample to be tested (20. mu.L, 7.5mg/mL), PBS (sodium phosphate solution, 120. mu.L, 0.1mol/LpH ═ 8.0), and AChE (15. mu.L, 1U/mL) were mixed in a 96-well plate and incubated at room temperature for 20 min. Acetylthiocholine iodide (ATCI, 20. mu.L, 5.18mg/mL) and the color reagent DTNB (70. mu.L, 0.3mg/mL) were then added, incubated at 37 ℃ for 20min to form a yellow substance, and the absorbance was measured at 412nm using a microplate reader, with galantamine as a positive control. AChE inhibitory activity I% was calculated according to formula 1:

wherein: c ₁ Absorbance representing inhibition reaction (+ enzyme + sample), C ₂ Representative of the absorbance of the sample (-enzyme + sample), B ₁ Represents the absorbance of the enzyme reaction (+ enzyme-sample), B ₂ Absorbance of buffer (-enzyme-sample).

2. Assay for butyrylcholinesterase (BChE) inhibitory Activity

Determination of BChE inhibitory Activity reference is made to prior art 2(Li N, Jiang H, Yang J, et al. Characterisation of phenolic compounds and anti-acetylcholinesterase activity of coconutshells [ J]Food Bioscience,2021,42:101204.) assay method with BTCI as substrate. Specifically, 20 μ L of the sample solution, 120 μ L of PBS (sodium phosphate solution, 0.1M, pH 8.0), and 15 μ LBChE (1U/mL) were mixed and incubated at room temperature for 20 min. Then 70. mu.L DTNB and 20. mu.L LBTCI were added to the mixture and the absorbance was recorded at 412nm after incubation for 20min at 37 ℃. BChE inhibitory activity was calculated according to equation 1 using donepezil hydrochloride and galantamine as positive controls. IC (integrated circuit) ₅₀ Concentration of sample at half inhibition, IC ₅₀ Smaller values represent higher inhibitory activity.

3. Measurement of alpha-glucosidase inhibitory Activity

Alpha-glucosidase inhibitory Activity according to prior art 3(Wang L, Wang L, Wang T, et al, Complex of peptides and fat inhibition on tartartartartargary butyl starch peptide derivatives in vitro and the same derivatives in binding sites with the same derivatives enzyme [ J]Food Chemistry,2021,367: 130762). Specifically, method ,20 μ L of sample was mixed with 15 μ L α -glucosidase solution (1U/mL) and 85 μ L PBS (sodium phosphate solution, 0.1M, pH 8.0) and incubated at 37 ℃ for 15 min. Then 30. mu.L pNPG (2.5mM) was added and incubated at 37 ℃ for 20 min. Using 40. mu.L of 0.2M Na ₂ CO ₃ TerminateThe reaction was carried out, and the absorbance was measured at 405 nm. Acarbose was used as a positive control, and α -glucosidase inhibitory activity was calculated according to formula 1.

4. Determination of alpha-Amylase inhibitory Activity

Alpha-amylase inhibitory activity was determined according to the method reported in Prior Art 4(Ranilla L G, Kwon Y I, Apostolisis E, et al, phenolic compounds, antioxidant activity and in vitro inhibition activity and amino acid in amino acid derivatives in biological enzymes release for hyperglycemic and hypertension used medical plants, herbs and spores in sodium America [ J ]. Bioresource Technology,2010,101(12): 4676-4689). The reaction mixture consisted of 25 μ L of sample, 35 μ L of LPBS (sodium phosphate solution, 0.1M, pH 8.0) and 50 μ L of α -amylase solution (5mg/mL), and the mixture was reacted at 37 ℃ for 5 min. Then, 100. mu.L of 1% pregelatinized starch solution was added to the mixture and incubated at 37 ℃ for 10 min. The reaction was stopped by adding 25 μ L DNS developer and incubated for 15 minutes in a boiling water bath. After cooling to room temperature, the absorbance was measured at 540 nm. The solution without sample extract was used as control and the solution without substrate was used as blank. Acarbose was used as a positive control and the α -amylase inhibitory activity was calculated according to formula 1.

5 determination of antioxidant Activity of Flavonoids

Referring to prior art 5(Katalinic M, Rusak G, Domacinovic Barovic J, et al. structural aspects of flavonoids as inhibitors of human butyrylchlorineersterase [ J ]. Europan Journal of medicinal Chemistry,2010,45(1):186-92.) Total antioxidant capacity/iron ion reduction (FRAP) assay method, absorbance (Y) was regressed with molar concentration (X) and standard curve was plotted with ferrous sulfate as standard. Ferrous sulfate is good in linearity within 0.02-0.12 mu mol, and the regression equation is as follows: Y5.114X +0.1402 and R0.999. And (3) measuring each plant extract, calculating the reduction amount of iron ions by referring to a ferrous sulfate standard curve, and expressing the total antioxidant capacity by the reduction amount of the iron ions. Unit: mu mol.

6. Data analysis

All experiments were performed at least 3 times and the data are expressed as mean standard deviation (n-3). Analysis of variance (ANOVA) was performed using IBM SPSS Statistics 22.0 software to measure statistical differences. Significant differences were determined using the Duncan method (P < 0.05).

In vitro evaluation of the inhibitory Activity of flavonoids on AChE, BChE, alpha-glucosidase and alpha-Amylase, IC ₅₀ The values are shown in Table 3 and FIG. 4.

TABLE 3 inhibitory Activity of Quercetin, Kaempferol, Myricetin and combinations thereof on 4 enzymes

According to FIG. 4, quercetin (IC) in flavonol _50-AChE ＝34.46μg/mL，IC _50-BChE 99.25. mu.g/mL), myricetin (IC) _50-AChE ＝32.23μg/mL，IC _50-BChE 99.64 μ g/mL) and kaempferol (IC) _50-AChE ＝40.44μg/mL，IC _50-BChE 124.56 μ g/mL) has higher potential for anticholinesterase activity than other flavonoids, whereas the flavonoids, the catechins and epicatechins of flavanols, had no or low inhibitory activity (IC) on AChE and BChE ₅₀ >1000. mu.g/mL). The positive control galanthamine shows stronger AChE and BChE inhibitory activity, IC ₅₀ 3.80 and 32.25. mu.g/mL, respectively.

Quercetin, quercetin, kaempferol and myricetin have high alpha-glucosidase inhibiting activity, IC ₅₀ Lower than acarbose (IC) _50-α-G 85.53 μ g/mL). Myricetin, quercetin, kaempferol, luteolin, quercetin and Epigallocatechin Gallate (EGCC) have high alpha-amylase inhibiting activity and IC ₅₀ Lower than acarbose (IC) _50-α-A 156.70 μ g/mL). In addition, IC of Everxanthin, Catechin and epicatechin against alpha-glucosidase and alpha-amylase ₅₀ The value was also the highest of all tested compounds.

However, galantamine and donepezil have specific inhibitory effects on cholinesterase activity, but no anti-carbohydrase activity, as opposed to acarbose. In addition, it was found in this study that quercetin, kaempferol and myricetin exhibit dual functions, with higher anticholinesterase and carbohydrase activities.

Example 2

Effect of molecular docking on in vitro inhibitory Activity

The possible binding patterns of the enzymes and flavonoids were evaluated by docking software (AutoDock 1.5.6). The 3D constructs of AChE (PDB code: 1C2O), BChE (PDB code: 4AQD), alpha-amylase (PDB code: 5EMY) and alpha-glucosidase (PDB code: 5ZCB) were downloaded from the RCSB protein database (https:// www.rcsb.org /) and saved in PDB format. Proteins are prepared as acceptors by removing all water molecules and ligands, and then adding hydrogen atoms and gastiger charges. The 3D structure of the ligand was downloaded from the PubChem database (https:// PubChem. ncbi. nlm. nih. gov /), and the format was saved as SDF. The SDF format is converted into the PDB format through Open Babel GUI. The rotatable bond and center of the ligand were detected by Autodock tool and the catalytic site of the protein was used as docking site. Docking calculation was performed using the Lamark Genetic Algorithm (LGA), docking 50 times to find the best docking site. Finally, the lowest binding energy pattern of the receptor-ligand complex is shown by PyMOL.

Analysis result of molecular docking on in vitro inhibitory activity

In order to analyze the multi-enzyme inhibition mechanism of the flavonoids, binding sites and BEs of 16 flavonoids and 3 standards with the 4 enzymes were calculated by molecular docking as shown in FIG. 4. The lower the BE value, the higher the affinity of the compound for the enzyme, i.e., the higher the inhibitory activity. As can BE seen from FIG. 4 (in the vertical direction), the minimum BE values of AChE, BChE, α -glucosidase, and α -amylase are-10.12 to-5.83 kcal/mol, -9.13 to-5.09 kcal/mol, -11.01 to-6.92 kcal/mol, and-11.14 to-8.66 kcal/mol, respectively. The BE of the glycoside-containing flavonol was lower than that of the glycoside-free flavonol, which is not consistent with the previous inhibitory activity. However, it has been reported that rutin glycosides may form hydrogen bonds with enzymes, and thus rutin has a high pKa value and low binding energy, but has a lower inhibitory activity against digestive enzymes than quercetin.

The binding sites of myricetin and quercetin in the enzyme were further investigated by molecular docking compared to standards (galantamine and acarbose) (fig. 5). Docking simulation results showed that galantamine had a lower minimum BE for AChE (-8.65 kcal/mol) and BChE (-6.92 kcal/mol) than myricetin-AChE (-5.64 kcal/mol) and quercetin-BChE (-6.74 kcal/mol) (FIG. 4), consistent with their tendency to inhibit activity in vitro. As shown in a in fig. 5, the binding sites of galantamine and myricetin on AChE overlap, but their number of hydrogen bonds is different, galantamine forms two hydrogen bonds with Ser293 and Phe 295, and myricetin forms 3 hydrogen bonds with Ser293, Phe 295 and Arg 296. However, the binding sites of galantamine and quercetin are different from BChE, galantamine and BChE bind Asn 228 by hydrogen bonds, while quercetin forms 6 hydrogen bonds with amino acid residues Gln 71, Thr 483, Asn 479, Pro 480, Arg 470 and Trp 490 (b in fig. 5). Inhibitory Activity of galantamine on BChE (IC) ₅₀ ) And BE lower than quercetin, probably because the binding site for galantamine is close to the active site of the enzyme. In addition, the BE values for alpha-glucosidase were calculated to BE-6.92 kcal/mol and-8.86 kcal/mol, respectively, for acarbose and quercetin. As shown in fig. 5 c, acarbose forms 5 hydrogen bonds with Asp 375, Lys 373, Asn 371, Asn 438 and Asn 440 of α -glucosidase, while quercetin forms 7 hydrogen bonds with residues Phe 276, Asn 277, Lys 242, Lys 7, Val 269, Glu 271 and Trp 318 of the active site of α -glucosidase through the-OH group. Meanwhile, possible binding sites of flavonoids and acarbose with alpha-amylase are analyzed through docking studies. Two binding sites were found, with the binding site for myricetin being close to the enzyme active site. Acarbose and myricetin were found to have-8.66 and-7.40 kcal/mol, respectively, for alpha-amylase calculated according to AutoDock. Acarbose forms six hydrogen bonds with residues Asp 197, Asp 300, Arg 195, Glu 233, His 299 and Thr 163 of alpha-amylase. Myricetin forms only four hydrogen bonds with Asn 250, Leu 211, Pro 228 and Asp 212 of α -amylase (d in fig. 5).

Example 3

Relationship of Topologically Polar Surface Area (TPSA) to anticholinesterase and anticarbohydrase Activity

TPSA is the surface sum of polar atoms in a molecule, and can be obtained from PubChem database (https:// PubChem. ncbi. nlm. nih. gov. /). TPSA is thought to characterize drug absorption and has applications in human intestinal absorption, Caco-2 monolayer permeability and the Blood Brain Barrier (BBB). Previous studies have shown that TPSA>140

Indicating lower intestinal absorption and cell membrane permeability, and studies have shown that the lower the TPSA value, the higher the Blood Brain Barrier (BBB) distribution.

As shown in fig. 6, there is no direct relationship between the TPSA value and the enzyme inhibitory activity. The lutein, catechin, epicatechin, and EGCC have strong permeability, but low inhibitory activity. Thus, the inhibitory activity (IC) of flavonoids on enzymes is considered ₅₀ 100 μ g/mL or less) and BBB permeability (about 140 for TPSA

) Quercetin, kaempferol and myricetin have high anticholinesterase activity, carbohydrase activity and blood brain barrier penetration capacity, and can be used as candidate drugs for preventing and/or treating AD and T2 DM.

Example 4

Relationship between antioxidant activity and anticholinesterase and carbohydrase activities

The correlation coefficient of the anticholinesterase activity and the carbohydrase activity of flavonoids and the antioxidant activity thereof is shown in figure 7. It can be seen that flavonol plays an important role in anticholinesterase and carbohydrase, and FRAP has a high correlation with inhibitory activities of AChE (R ═ 0.905), BChE (R ═ 0.850), and α -glucosidase (R ═ 0.931). In addition, the four enzymes have higher correlation and are also related to flavonol. For example, the correlation between AChE and the other three enzymes is R _AChE/BChE ＝0.992，R _AChE/α-G 0.895 and R _AChE/α-A 0.726, BChE and α -glucosidase (R) _BChE/α-G 0.846) and alpha-starchAmylase (R) _BChE/α-A 0.778). Meanwhile, it was also found that there was a certain correlation between the BE values of the binding of cholinesterase, α -glucosidase and α -amylase to the ligand, the change rule was the same as that in FIG. 7, but the correlation between the antioxidant activity (FRAP) and BE was weak (FIG. 8).

Example 5

Structure-activity relationship

16 flavonoid compounds are screened, and the inhibition of enzyme activity and a molecular docking calculation method in vitro are researched, so that the key structural characteristics of various flavonoid compounds have certain influence on the binding capacity of the flavonoid compounds with AChE, BChE, alpha-glucosidase and alpha-amylase (figure 4).

(1) Hydroxy and methoxy

Hydroxyl and methoxyl in the flavonoid compounds play an important role in inhibiting enzymes. As shown in fig. 4, the presence of hydroxylated forms at C3 'and C5' positions of the core scaffold may enhance the inhibitory activity of the enzyme. Such as quercetin and myricetin, have stronger inhibitory activity on cholinesterase and alpha-amylase, and the inhibitory activity is higher than that of kaempferol. Through molecular docking simulation, myricetin and quercetin were found to form 3 hydrogen bonds with Phe 295, Ser293 and Arg 296 amino acid residues of AChE, while kaempferol only forms 2 hydrogen bonds with Phe 295 and Ser293 amino acid residues. Furthermore, the addition of an-OH group at C-ring C3 appears to increase the anticholinesterase and anticarbohydrase activity of flavonoids, such as kaempferol IC ₅₀ Value (IC) _50-AChE 40.44 μ g/mL and IC _50-BChE ＝124.56μg/mL，IC _50-α-G 23.56. mu.g/mL and IC _50-α-A 56.32 mug/mL) is much lower than apigenin (IC) _50-AChE ＝958.40μg/mL，IC _50-BChE ＝212.88μg/mL，IC _50-α-G 154.25 μ g/mL and IC _50-α-A 440.50 μ g/mL). This is because the hydroxyl group added by quercetin at C3 on the C ring forms a hydrogen bond with AChE residue Phe 295. Compared with apigenin, after-OH is added to the luteolin B ring C5', inhibition on AChE is increased by 5.37 times, and inhibition on BChE is reduced by 2.15 times. In addition, a phenomenon similar to BChE was also observed in α -glucosidase and α -amylase. The results show that the flavoneThe hydroxylation pattern (number and location of hydroxylations) on the core scaffold plays a key role in its anticholinesterase and anticarbohydrase biological activities.

Unlike the positive effects of hydroxyl groups, methoxy groups have a negative effect on the inhibitory activity of enzymes. Such as quercetin, has higher inhibitory activity on AChE, BChE, alpha-glucosidase and alpha-amylase than that on lutein (IC) _50-AChE ＝ND，IC _50-BChE ＝2179.5，IC _50-α-G 682.0 and IC _50-α-A 787.75 μ g/mL). Furthermore, docking results showed that the sum of BE binding of quercetin and lutein to the four enzymes was-30.35 kcal/mol and-22.92 kcal/mol, respectively, which is consistent with the results of in vitro inhibition experiments (FIG. 4). According to the molecular docking results, quercetin and lutein are located at the active site of AChE and cause enzyme inhibition. But they may compete for the same binding site, the hydroxyl group of quercetin interacts more easily with the amino acid residues of the enzyme than the methoxy group of the enzyme, forming a more stable compound, which may explain why the complex formed by quercetin and enzyme has a better inhibitory effect than the lutein-enzyme complex. Shows that the methoxylation of the flavonoid compounds is also an important factor influencing the enzyme inhibition activity.

(2) Glycosylation

Dietary flavonoids occur in nature mainly in the form of 3-and 7-glycosides. As can be seen from FIG. 4, quercetin and kaempferol have weak anticholinesterase and anticarbohydrase effects after glycosylation at C3 position of C ring (quercetin, Q-3-R, K-3-G and K-3-R), while quercetin and kaempferol show higher anticholinesterase and anticarbohydrase effects. Similarly, C7-glycoside forms of flavonoids Q-7-G and luteolin also exhibited weaker inhibitory activity than quercetin and luteolin. Theoretically, the lower the BE value, the higher the inhibitory activity thereof on the enzyme. But the BE value (-10.09 kcal/mol) of Q-3-R to AChE was observed to BE lower than that of quercetin (-6.94 kcal/mol) by molecular docking calculation, while the inhibitory activity of Q-3-R to AChE was lower than that of quercetin. Similarly, BChE, α -glucosidase, and α -amylase are also present. This is probably because rutin, in addition to the hydroxyl groups on the quercetin units forming hydrogen bonds with enzymes, the glycosides of rutin can also form hydrogen bonds with enzymes. In addition, differences in glycosidic position have different effects on enzyme inhibitory activity. Q-7-G (C7-glycoside) has little enzyme inhibitory activity as compared to quercetin (C3-glycoside). These data indicate that glycosylated flavonols bind less to the active site of the enzyme than non-glycosylated flavonols, thus resulting in lower anticholinesterase and anticarbohydrase activity of the former. This is probably due to the fact that the introduction of sugar groups in flavonols increases the steric hindrance of the molecule, limiting its ability to enter the active center of the enzyme.

(3) Hydrogenation of the double bond C2-C3

C2 ═ C3 bound to the 4-carbonyl group, playing a key role in maintaining the planar molecular structure of flavonoids. As can be seen in FIG. 4, apigenin shows higher anti-BChE and anti-carbohydrase activities (IC) _50-BChE ＝212.88，IC _50-α-G 154.25 and IC _50-α-A 440.5 ug/mL) naringenin (IC) _50-BChE ＝257.73，IC _50-α-G 157.09 and IC _50-α-A 494.75 μ g/mL). Catechins and epicatechins have a lower anticholinesterase (IC) than other compounds except for lutein ₅₀ >1000. mu.g/mL) and anti-carbohydrase Activity (IC) ₅₀ >500. mu.g/mL). In addition, the sum of BE values of catechin (-27.36 kcal/mol) and epicatechin (-26.91 kcal/mol) bound to the four enzymes was also ranked second and third after the esflavin. It is likely that the presence of flavonoid saturated C2-C3 bonds makes the planar molecular structure more flexible, resulting in distortion of the B ring and steric hindrance, thereby reducing interaction with the enzyme catalytic site. Briefly, flavanones and flavanols exhibit weaker anticholinesterase and anticarbohydrase activity than unglycosylated flavonols and flavones. This suggests that hydrogenation of the C2-C3 double bond of flavonoids may reduce the inhibitory activity of the enzyme.

From the results of the above examples, it is understood that quercetin and kaempferol show high anticholinesterase and carbohydrase activities, and these activities have the highest correlation with their antioxidant ability. The cholinesterase and carbohydrase activities of catechin, epicatechin, and abrin are low. Further structure-activity relationship analysis shows that hydroxyl is introduced into the core scaffold of flavone to be beneficial to inhibiting the activity of enzyme, and methoxyl is introduced to be not beneficial to inhibiting the activity of enzyme. Furthermore, glycosylation at C3 and C7 significantly reduced the inhibitory effect of flavonols, and similar inhibitory effects were observed for hydrogenation of the C2 ═ C3 double bond. Therefore, the structure of the flavonoid compound greatly influences the combination of the compound and the enzyme active site, so that the inhibitory activity has certain difference. In addition, molecular simulations indicate that the hydroxyl groups of C4', C5' and C3 in flavonoids favor the formation of hydrogen bonds and interact with amino acid residues of enzymes, preventing substrate binding to enzymes. The results show that: quercetin, kaempferol and myricetin have higher anticholinesterase activity, carbohydrase activity and BBB permeability, which provides theoretical basis for the application of the quercetin, kaempferol and myricetin not only serving as a natural inhibitor, but also serving as functional food ingredients in the dietary auxiliary prevention and/or treatment of AD and T2 DM.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The flavonoid compound with the double activities of resisting cholinesterase and carbohydrase is characterized by comprising the following two or three flavonoid compounds: quercetin, kaempferol and myricetin.

2. The flavonoid compound according to claim 1, wherein said flavonoid compound comprises quercetin, kaempferol and myricetin.

3. The flavonoid compound according to claim 2, wherein the mass ratio of quercetin to kaempferol to myricetin is 1-10: 1-10.

4. Use of quercetin, kaempferol, myricetin or a flavonoid compound according to any one of claims 1 to 3 for the preparation of a formulation with anticholinesterase and anticarbohydrase activity.

5. Use according to claim 4, wherein the cholinesterase comprises acetylcholinesterase and/or butyrylcholinesterase.

6. Use according to claim 4, wherein the carbohydrase comprises an alpha-glucosidase and/or an alpha-amylase.

7. Use of quercetin, kaempferol, myricetin or a flavonoid compound according to any one of claims 1 to 3 for the preparation of a medicament for the prevention and/or treatment of both alzheimer's disease and non-insulin dependent diabetes mellitus.

8. The use according to claim 7, wherein the medicament has simultaneous inhibitory activity on acetylcholinesterase, butyrylcholinesterase, α -glucosidase and α -amylase.

9. The use according to claim 7, wherein said medicament has blood-brain barrier penetration capability.

10. A medicament for preventing and/or treating Alzheimer's disease and non-insulin dependent diabetes mellitus at the same time, characterized by comprising active ingredients and pharmaceutically acceptable auxiliary materials;

the active ingredient comprises quercetin, kaempferol, myricetin or a flavonoid compound according to any one of claims 1 to 3.