CN110256512B - Alpha-glucosidase inhibitor extracted from Potentilla chinensis Franch - Google Patents
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Abstract
The invention relates to the technical field of traditional Chinese medicines, in particular to an alpha-glucosidase inhibitor extracted from Potentilla chinensis Kuntze, which is obtained by extraction from Potentilla chinensis Kuntze, column chromatography separation and glycosidase inhibition activity verification. The alpha-glucosidase inhibitor in the application is extracted from a natural plant Potentilla chinensis, which is widely distributed in Qinghai-Tibet plateau, is a monomeric compound, has high purity and high inhibition rate, is simple and safe in extraction process, has the alpha-glucosidase inhibition activity discovered for the first time, has excellent inhibition effect, can provide a basis for preparing a novel hypoglycemic active medicine, and can be used as a medicine for reducing the postprandial blood sugar of diabetes.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to an alpha-glucosidase inhibitor extracted from Potentilla anserine, an extraction method and application thereof.
Background
Diabetes is one of the most serious world health diseases in the 21 st century, and brings great harm to human beings. Alpha-glycosidase inhibitor medicines are commonly used hypoglycemic medicines at present, but the synthesized medicines have more side effects and cause damage to human bodies to different degrees. The hypoglycemic active ingredients extracted and separated from natural plants have low toxicity and better hypoglycemic effect. The extraction and separation of the hypoglycemic active ingredients from the natural plants are beneficial to developing new drugs and promoting the development and utilization of the natural plants.
Potentilla chinensis (B.C.)Potentilla bifurca var.Humilior R) is Rosaceae (R)Rosaceae) Potentilla (Potentilla genus)Potentilla) The plants are short and scattered, the flower stems are shorter than 7 cm, the small leaves are usually 3-5 pairs and 6 pairs, the whole edges are more, the tops of the buds are 2 cracks, the flowers are usually single, and the flowering phase is 5-10 months. Produced in Qinghai, Gansu, Ningxia, Xinjiang, Sichuan and Tibet, etc., and grown on mountain grassland, riverbank sandy land and arid grassland with elevation of 1800 and 4000 m. Recorded in the national Chinese herbal medicine compilation, it is sweet, slightly pungent and cool in nature, and mainly used for stopping bleeding and dysentery, and for functional uterine bleeding, postpartum excessive bleeding, dysentery, etc. Modern researches show that the chemical components of the plants relate to flavone, triterpene, tannin, organic acid and the like, and pharmacological researches show that the plants mainly have the effects of reducing blood sugar, resisting bacteria, tumors, oxidation, cancers, cerebral ischemia injury, allergy, viruses, liver protection, osteoporosis treatment, pain relief and the like. The monomer compound of the Potentilla bifida has higher alpha-glucosidase inhibitory activity for the first time. Alpha-glucosidase (AG) is a type of disaccharide hydrolase distributed on the surface of the microvillous membrane of the epithelial mucosa of the small intestine, and comprises maltase, isomaltase, sucrase, trehalase and the like. The enzyme can convert disaccharide taken into human body into monosaccharide which is easy to be absorbed by human body, and has important effect on sugar catabolism. The alpha-glucosidase inhibitor is a medicine for reducing postprandial blood sugar and treating diabetes, and achieves the purpose of reducing postprandial blood sugar by competitive inhibition with AG.
The prior art has the problems that: at present, acarbose, which is the most commonly used blood sugar-reducing drug of an alpha-glucosidase inhibitor, can effectively control diabetes and reduce blood sugar, but is limited in applicable population and not suitable for population taking high-protein and high-fat foods such as meat, eggs and the like as staple food, meanwhile, as the acarbose is slowly decomposed and absorbed in small intestines, the retention time is prolonged, gas production is increased through glycolysis of intestinal bacteria, abdominal distension, abdominal pain, diarrhea and the like can be caused, and the price of the acarbose is higher, so that the acarbose is not suitable for population with lower income. Other hypoglycemic drugs also have side effects of varying degrees and social problems.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an alpha-glucosidase inhibitor extracted from Potentilla chinensis Kunth.
In order to solve the technical problem, the invention adopts the following technical scheme:
an alpha-glucosidase inhibitor extracted from Potentilla chinensis, wherein the inhibitor is 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose). The alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity measuring method to measure enzyme inhibition activity, and the result shows that: when the sample concentration is 2.5mg/mL, the alpha-glucosidase inhibition rate of 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose) is 99.41%, and IC is50It was 0.0019 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla anserine, the inhibitor being: myricetin (myricetin), the alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity measuring method to measure enzyme inhibition activity, and the result shows that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of myricetin (myricetin) is 90.67 percent, and IC is500.0561 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla anserine, the inhibitor being: tiliroside (tiliroside), and the enzyme inhibitory activity of the α -glucosidase inhibitor was measured by an α -glucosidase in vitro activity measurement method, and the results showed that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of tiliroside is 87.38%, IC500.1807 mg/mL.
Shi Sheng IIAn alpha-glucosidase inhibitor extracted from potentilla crassa is provided, wherein the inhibitor is: catechin ((+) -catechin), the alpha-glucosidase inhibitor adopts an alpha-glucosidase in vitro activity measuring method to measure the enzyme inhibition activity, and the result shows that: at a sample concentration of 1.25mg/mL, the α -glucosidase inhibition rate of catechin ((+) -catechin) was 96.52%, IC50It was 0.0652 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla bifida, said inhibitor is: quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) -beta-D-glucoside), the alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity determination method to perform enzyme inhibition activity determination, and the result shows that: at a sample concentration of 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) -beta-D-glucoside) is 92.06%, IC50It was 0.0498 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla bifida, said inhibitor is: quercetin (quercetin), the alpha-glucosidase inhibitor is subjected to enzyme inhibitory activity measurement by an alpha-glucosidase in vitro activity measurement method, and the results show that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin (quercetin) is 88.16%, IC500.2095 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla bifida, said inhibitor is: determination of enzyme inhibitory activity of gomisin A methyl ester (methyl ester of rugosin A) as an α -glucosidase inhibitor by α -glucosidase in vitro activity assay, results showed: at a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of gofferin A methyl ester (methyl ester of rugosin A) was 97.77%, IC50It was 0.0004 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla anserine, the inhibitor being: (-) -epicatechin ((-) -epicatechin), the alphaThe enzyme inhibitory activity of the glucosidase inhibitor was measured by the α -glucosidase in vitro activity assay, and the results showed that: at a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of (-) -epicatechin ((-) -epicatechin) was 103.29%, IC500.2087 mg/mL.
An alpha-glucosidase inhibitor extracted from Potentilla bifida, said inhibitor is: 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone), and the enzyme inhibitory activity of the α -glucosidase inhibitor was measured by an α -glucosidase in vitro activity measurement method, and the results showed that: when the sample concentration is 0.625mg/mL, the alpha-glucosidase inhibition rate of 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone) is 91.98%, and IC is50It was 0.0019 mg/mL.
Drawings
FIG. 1 is a flow chart of the process for extracting alpha-glucosidase inhibitor;
FIG. 2 is a schematic view of the structure of 1-O-coumaroyl- β -D-glucose (1-O-coumaroyl- β -D-glucose);
FIG. 3 is a schematic view of myricetin (myricetin) structure;
FIG. 4 is a schematic structural diagram of tiliroside (tiliroside);
FIG. 5 is a schematic representation of the structure of catechin ((+) -catechin);
FIG. 6 is a schematic diagram showing the structure of quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) - β -D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) - β -D-glucoside);
FIG. 7 is a schematic diagram of quercetin (quercetin) structure;
FIG. 8 is a schematic diagram of the structure of methyl ester of rugosin A;
FIG. 9 is a schematic representation of the structure of (-) -epicatechin ((-) -epicatechin);
FIG. 10 is a schematic diagram of the structure of 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone).
Detailed description of the invention
The methods and devices used in the following examples of the present invention are conventional methods and devices unless otherwise specified; the equipment and the reagent are conventional equipment and reagents purchased by a reagent company. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention is provided in connection with the specific embodiments. Examples of these preferred embodiments are illustrated in the specific examples.
It should be noted that, in order to avoid obscuring the technical solution of the present invention with unnecessary details, only the technical solution and/or processing steps closely related to the technical solution of the present invention are shown in the embodiments, and other details with little relation are omitted.
Example 1
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is 1-O-coumaroyl- β -D-glucose (1-O-coumaroyl- β -D-glucose), and the chemical structure of which is shown in FIG. 2. The alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity measuring method to measure enzyme inhibition activity, and the result shows that: when the sample concentration is 2.5mg/mL, the alpha-glucosidase inhibition rate of 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose) is 99.41%, and IC is50It was 0.0019 mg/mL.
Example 2
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: myricetin (myricetin), the alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity measuring method to measure enzyme inhibition activity, and the result shows that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of myricetin (myricetin) is 90.67 percent, and IC is500.0561mg/mL, the chemical structure is shown in figure 3.
Example 3
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: tiliroside, which is alpha-glucosidase inhibitorEnzyme in vitro activity assay method, enzyme inhibition activity assay, results show: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of tiliroside is 87.38%, IC500.1807mg/mL, and the chemical structure is shown in figure 4.
Example 4
This example provides an α -glucosidase inhibitor extracted from Potentilla bifida, said inhibitor being: catechin ((+) -catechin), the alpha-glucosidase inhibitor adopts the alpha-glucosidase in vitro activity determination method, the enzyme inhibition activity is determined, the result shows: at a sample concentration of 1.25mg/mL, the α -glucosidase inhibition rate of catechin ((+) -catechin) was 96.52%, IC500.0652mg/mL, the chemical structure is shown in figure 5.
Example 5
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) -beta-D-glucoside), wherein the alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity determination method to perform enzyme inhibition activity determination, and the result shows that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) -beta-D-glucoside) is 92.06%, IC is IC500.0.0498mg/mL, and the chemical structure is shown in figure 6.
Example 6
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: quercetin (quercetin), the alpha-glucosidase inhibitor is subjected to enzyme inhibitory activity measurement by an alpha-glucosidase in vitro activity measurement method, and the results show that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin (quercetin) is 88.16%, IC500.2095mg/mL, and the chemical structure is shown in figure 7.
Example 7
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: gofferin A methyl ester (methyl ester of rugosin A), the alpha-glucosidase inhibitor was measured for enzyme inhibitory activity using the alpha-glucosidase in vitro activity assay, and the results showed: at a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of gofferin A methyl ester (methyl ester of rugosin A) was 97.77%, IC50Is 0.0004mg/mL, and the chemical structure is shown in figure 8.
Example 8
This example provides an α -glucosidase inhibitor extracted from Potentilla bifida, said inhibitor being: (-) -epicatechin ((-) -epicatechin), which is an α -glucosidase inhibitor, and the enzyme inhibitory activity of the α -glucosidase inhibitor is measured by an α -glucosidase in vitro activity measuring method, and the results show that: at a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of (-) -epicatechin ((-) -epicatechin) was 103.29%, IC500.2087mg/mL, and the chemical structure is shown in figure 9.
Example 9
This example provides an α -glucosidase inhibitor extracted from Potentilla anserine, which is: 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone), and the enzyme inhibitory activity of the α -glucosidase inhibitor was measured by an α -glucosidase in vitro activity measurement method, and the results showed that: when the sample concentration is 0.625mg/mL, the alpha-glucosidase inhibition rate of 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone) is 91.98%, and IC is50Is 0.0019mg/mL, and the chemical structure is shown in figure 10.
Example 10
The present embodiment provides a method for extracting α -glucosidase inhibitor from potentilla anserine, as shown in fig. 1, comprising the following steps:
(1) preparation of the experiment: raw materials and auxiliary materials: potentilla anserine, silica gel plate (for thin layer), silica gel (for column chromatography), Sephadex LH-20 (25-100 μm), MCI CHP-20P (75-100 μm), and developing agent (ethyl benzoate: formic acid) three-phase solution; the developer is 10% sulfuric acid ethanol solution and 1% ferric chloride ethanol solution (wherein the developer is prepared by analytical purification), distilled water, methanol, petroleum ether, ethyl acetate, etc. The main equipment is as follows: 600MHz nuclear magnetic resonance instrument, Auto Spec-3000 mass spectrometer, R1002 rotary evaporator, SHZ-D (III) circulating water type vacuum pump, ARA520 electronic analytical balance, ALC-210.3 electronic analytical balance, BS-100A automatic receiver, BCD-215KA Hell refrigerator, Newstyle HPLC, crusher, vacuum pump, rotary evaporator, condensed circulating water pump, D101 resin column, Sephadex LH-20, MCI-gel CHP-20P, RP-18, HPLC.
(2) Extraction and extraction: an alpha-glucosidase inhibitor extracted from the whole herb of Potentilla fruticosa, wherein the inhibitor is extracted from the whole herb of Potentilla fruticosa; the inhibitor is prepared by the following steps: pulverizing whole plant of Potentilla chinensis, extracting with 95% methanol under reflux at 65 deg.C, concentrating the extractive solution, and recovering methanol to obtain extract. Extracting the extract with petroleum ether and ethyl acetate respectively according to polarity to obtain petroleum ether part, ethyl acetate part and water layer extraction part;
(3) and (3) column chromatography separation: dissolving the ethyl acetate part with water, dispersing, and further extracting with diethyl ether to obtain diethyl ether layer and water layer; eluting the water layer with D101 column with 0% -30% -60% -80% -100% methanol water solution to obtain water eluate, 30% methanol eluate, 60% methanol eluate, 80% methanol eluate, and 100% methanol eluate; subjecting 80% D101 methanol eluate of ethyl acetate part to Sephadex LH-20 column chromatography, subjecting to gradient elution with water-methanol (1: 0-0: 1) every 10% concentration to obtain 7 fractions, subjecting 07 fraction to MCI column chromatography, subjecting to gradient elution with water-methanol (1: 0-0: 1) every 10% concentration to obtain 20 fraction (1.3 g), subjecting to Sephadex LH-20 column chromatography, subjecting to gradient elution with water-methanol (1: 0-0: 1) every 10% concentration to obtain 21 fraction (87 mg), subjecting to MCI column chromatography, subjecting to gradient elution with water-methanol (1: 0-0: 1) every 10% concentration to obtain 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose), namely the alpha-glucosidase inhibitor. The other 8 compound extraction methods are shown in figure 1.
(4) And (3) verification of glycosidase inhibition activity: the alpha-glucosidase inhibitor adopts an alpha-glucosidase in-vitro activity determination method to perform enzyme inhibition activity determination. The specific determination method is as follows:
principle of AG inhibitor screening model: and (3) carrying out AG catalytic hydrolysis on PNPG to generate p-nitrophenol (PNP), wherein the PNP has an absorption peak at the wavelength of 400 nm, measuring the output of PNG, and calculating the inhibiting activity of the Potentilla chinensis monomer compound on AG.
The enzyme inhibitory activity was measured using a fully automatic enzyme calibrator and the reaction was performed in non-detachable 96-well plates. The reaction system is 200 mu L: in 125. mu.L of phosphate buffer and 8.92X 10-3Adding PNPG 25 μ L with concentration of 1.953 × 10-2After 25 mu L of mg/mL sample and 25 mu L of AG 0.05U/mL, the sample is incubated at 37 ℃ for 20 min, absorbance is measured at 400 nm wavelength by using a full-automatic enzyme calibration instrument, and distilled water is used as a blank control instead of enzyme solution. Each sample was subjected to 3 replicates and the average was taken.
AG activity unit definition: the amount of PNG released by enzymatic hydrolysis of PNPG (OD value) per minute was found to be within the range of pH 6.8 at 37 ℃. Definition of inhibitor activity units: the amount of inhibitor required to reduce 1 enzyme activity unit under the same conditions.
To eliminate the effect of the sample and substrate PNPG on the assay results, it is necessary to determine the background absorbance of the sample and substrate. Calibration was performed by replacing the sample and substrate with 0.05 mol/mL phosphate buffer.
Inhibition (%) = [ (A1-A3) - (A2-A4) ]/(A1-A3)
A1: the original enzyme activity; a2: adding inhibitor to improve enzyme activity; a3: a PNPG background; a4: sample background
The results show that: when the sample concentration is 2.5mg/mL, the alpha-glucosidase inhibition rate of 1-O-coumaroyl-beta-D-glucose (1-O-coumaroyl-beta-D-glucose) is 99.41%, and IC is50It was 0.0019 mg/mL.
When the sample concentration is 1.25mg/mL,the alpha-glucosidase inhibition rate of myricetin (myricetin) is 90.67%, IC500.0561 mg/mL.
When the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of tiliroside is 87.38%, IC500.1807 mg/mL.
At a sample concentration of 1.25mg/mL, the α -glucosidase inhibition rate of catechin ((+) -catechin) was 96.52%, IC500.0652 mg/mL.
When the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin-3-O- (6'' -O-trans-p-hydroxycinnamoyl) -beta-D-glucoside (quercetin-3-O- (6'' -O-trans-p-coumaroyl) -beta-D-glucoside) is 92.06%, IC is IC50It was 0.0498 mg/mL.
When the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin (quercetin) is 88.16%, IC50It was 0.2095 mg/mL.
At a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of gofferin A methyl ester (methyl ester of rugosin A) was 97.77%, IC50It was 0.0004 mg/mL.
At a sample concentration of 2.5mg/mL, the alpha-glucosidase inhibition rate of (-) -epicatechin ((-) -epicatechin) was 103.29%, IC500.2087 mg/mL.
When the sample concentration is 0.625mg/mL, the alpha-glucosidase inhibition rate of 3,5,7-trihydroxy-4'-methoxyflavone (3, 5,7-trihydroxy-4' -methoxyflavone) is 91.98%, and IC is50It was 0.0019 mg/mL.
Has the advantages that:
the alpha-glucosidase inhibitor has high inhibition rate, is extracted from natural plants, has simple, convenient and safe extraction process, has excellent effect, provides basis for preparing novel hypoglycemic active medicaments, and can provide a potential medicament for reducing the postprandial blood sugar of diabetes. The key technology is extraction method and screening of alpha-glucosidase inhibitor drugs, the method is reasonable, and finally the monomer compound with obvious inhibition effect on alpha-glucosidase is screened out. In addition, the inhibitor screened by the invention can be used for the research and development of products aiming at inhibiting alpha-glucosidase, especially for the development of drugs for reducing the postprandial blood sugar of diabetes, and has industrial significance.
The alpha-glucosidase inhibitor in the application is extracted from a natural plant Potentilla chinensis, which is widely distributed in Qinghai-Tibet plateau, is a monomeric compound, has high purity and high inhibition rate, is simple and safe in extraction process, has the alpha-glucosidase inhibition activity discovered for the first time, has excellent inhibition effect, can provide a basis for preparing a novel hypoglycemic active medicine, and can be used as a medicine for reducing the postprandial blood sugar of diabetes.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (1)
1. Use of Potentilla anserine extract in the preparation of alpha-glucosidase inhibitors is characterized in that the extract is the compound gofferin A methyl ester (methyl ester of rugosin A).
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| CN114504612A (en) * | 2020-11-17 | 2022-05-17 | 中国医学科学院药物研究所 | Application of potentilla anserine extract in preparation of medicine for treating gastric ulcer |
| CN115177663B (en) * | 2022-08-12 | 2023-07-21 | 青海师范大学 | Preparation method and application of herba Potentilla chinensis extract |
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