CN109876021B - Preparation method of Lyophyllum decastes extract rich in polyacetylene compounds and its blood sugar lowering application - Google Patents

Preparation method of Lyophyllum decastes extract rich in polyacetylene compounds and its blood sugar lowering application Download PDF

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CN109876021B
CN109876021B CN201910223433.6A CN201910223433A CN109876021B CN 109876021 B CN109876021 B CN 109876021B CN 201910223433 A CN201910223433 A CN 201910223433A CN 109876021 B CN109876021 B CN 109876021B
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lyophyllum decastes
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lyophyllum
decastes
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CN109876021A (en
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刘宏伟
魏生龙
韩俊杰
马项英
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Hexi University
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Abstract

The invention discloses a preparation method of a Lyophyllum decastes extract rich in polyacetylene compounds and a hypoglycemic application thereof, and the Lyophyllum decastes is taken as a research object, and the extraction process and the biological activity of the Lyophyllum decastes extract are researched, so that the obtained Lyophyllum decastes extract rich in polyacetylene compounds can remarkably promote the glucose uptake activity of 3T3-L1 cells, enhance insulin and inhibit insulin resistance, and can be used as a hypoglycemic medicament for preventing and treating diseases related to glycometabolism abnormality.

Description

Preparation method of Lyophyllum decastes extract rich in polyacetylene compounds and its blood sugar lowering application
Technical Field
The invention belongs to the field of biology, relates to a fungus extract, and particularly relates to a preparation method of a Lyophyllum decastes extract rich in polyacetylene compounds and application of the Lyophyllum decastes extract as an active ingredient in preparation of a hypoglycemic drug.
Background
With the development of socioeconomic, the dietary structure and the life style of people are changed, the incidence rate of diabetes mellitus is increasing, and the diabetes mellitus becomes one of the most common and serious metabolic diseases in the world and affects nearly 5 hundred million people. Diabetes is divided into four major forms, i.e., type I diabetes, type II diabetes, gestational diabetes, and other types of diabetes, according to different pathogenesis. Among them, type II diabetes is a prevalent subject, whose prevalence accounts for about 90-95% of the total number of diabetic patients, and the main pathogenesis is insulin resistance, with or without impaired function of islets to various degrees, often accompanied by other metabolic disorders. More and more children and young people have type II diabetes in recent years. According to the World Health Organization (WHO), about 1.1 hundred million diabetics in China account for 1/10 of the total number of adults in China. The number of people is estimated to be increased to 1.5 hundred million by 2040 years, and the health and social economy of people are seriously influenced. Therefore, there is an urgent need for the prevention and control of diabetes and its complications throughout society.
The current research focus is to find safe, low-toxicity and high-efficiency sugar metabolism regulating substances from natural products. Lyophyllum decastes (Fr.: Fr.) Sing belonging to Agaricales, Lyophyllaceae, Lyophyllum, Neurospora, Lyophyllum, Phellinus Linteus, Tricholoma matsutake, Phellinus igniarius, Pleurotus ostreatus, Phellinus crispus, and Monochorus ovis is a precious fungus used as both wild food and drug. A large number of researches prove that sporocarp and mycelium polysaccharide of the fungus have the biological activities of reducing blood fat and blood sugar, resisting oxidation and resisting cancer. But less research on the active small molecular substances.
Disclosure of Invention
The invention provides a preparation method of a Lyophyllum decastes extract rich in polyacetylene compounds by taking Lyophyllum decastes as a research object, and the obtained active micromolecules are used as active ingredients for reducing blood sugar.
The technical scheme adopted by the invention is as follows:
firstly, the invention provides a preparation method of a Lyophyllum decastes extract rich in polyacetylene compounds, which comprises the following steps:
step 1), taking Lyophyllum decastes fruiting body as a raw material, crushing, and adding into an organic solvent A or B according to a certain proportion to soak for 2-10 hours;
step 2), then extracting at 10-100 ℃, filtering and collecting an extracting solution;
step 3), carrying out reduced pressure concentration on the extracting solution to obtain a lyophyllum decastes extract;
step 4) carrying out ultrasonic dissolution on the lyophyllum decastes extract by using an organic solvent C, dripping the obtained dissolved solution into a column bed filled with macroporous resin for static adsorption for at least 2 hours, and carrying out gradient elution separation on the macroporous resin column after statically adsorbing a sample by using water and organic solvents with different concentrations;
and 5) collecting the eluent, and concentrating to obtain the extract rich in the polyacetylene compounds.
In step 1) of the present invention, the organic solvent a is C1-4 lower alcohol (for example: methanol, ethanol, butanol, etc.) or an aqueous solution of a C1-4 lower alcohol, preferably an aqueous solution of 30-80(v/v) ethanol.
The organic solvent B is 100% v/v ethyl acetate, acetone, dichloromethane or a mixture of any 2-3, preferably 100% ethyl acetate.
Preferably, the mass-to-liquid ratio of the Lyophyllum decastes fruiting body to the solvent is 1: 2.5-8 (w/v).
In the step 2), any one of hot water reflux extraction, cold water extraction, ultrasonic treatment or conventional extraction is selected for extraction, and each extraction lasts for 2-3 hours.
And 3) concentrating the extracting solution at 35-55 ℃ during reduced pressure concentration in the step 3).
In the step 4), the organic solvent C is any one of 100% v/v ethyl acetate, 70% to 100% v/v methanol, 70% to 95% v/v ethanol, 100% v/v acetone or 100% v/v dichloromethane.
In the step 4), the macroporous resin can be polar, medium polar, weak polar or non-polar macroporous resin.
In the step 4), the macroporous resin column after statically adsorbing the sample is subjected to gradient elution separation by using water and methanol solutions with the concentrations of 40% v/v, 75% v/v and 100% v/v respectively, and each concentration is eluted by 3-5 retention volumes; or gradient elution separation is carried out on the macroporous resin column after the static adsorption of the sample by using water and ethanol solutions with the concentration of 40% v/v, 75% v/v and 95% v/v respectively, and each concentration is eluted by 3-5 retention volumes; or gradient elution separation is carried out on the macroporous resin column after statically adsorbing the sample by using water and acetone solutions with the concentration of 35% v/v, 70% v/v and 95% v/v respectively, and each concentration is eluted by 3-5 retention volumes.
The invention also provides an extract prepared by the method as an active ingredient for preparing the hypoglycemic drug composition.
The product contains the Lyophyllum decastes extract rich in polyacetylene compounds with effective dose for treating and preventing diabetes. The composition for treating and preventing inflammatory, allergic or neoplastic diseases may comprise 0.1 to 99%, preferably 0.1 to 50% by weight, based on the total weight of the composition.
The pharmaceutical composition can comprise one or more pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sustained-release agents and the like in the pharmaceutical field.
The pharmaceutical composition can be in the form of injection, tablet, powder, granule, pill, capsule, oral liquid, ointment, cream, spray, etc. The various dosage forms can be prepared according to conventional methods in the pharmaceutical field, and can be administered by oral administration, gastrointestinal administration, injection, spraying, physical or chemical mediation, etc., or by mixing or coating with other substances.
The invention has the beneficial effects that:
1. the invention takes Lyophyllum decastes as a research object, and researches on the extraction process and the biological activity of the Lyophyllum decastes extract, and discovers the Lyophyllum decastes extract which is rich in polyacetylene compounds and is an active small molecule, can remarkably promote the glucose uptake activity of 3T3-L1 cells, sensitizes insulin, inhibits insulin resistance and can be used for hypoglycemic drugs.
2. The Lyophyllum decastes extract rich in the polyacetylene compounds can also be used for preventing and treating diseases related to glycometabolism abnormality.
3. According to the invention, researches show that the lyophyllum decastes can be extracted by any conventional solvent, including ultrasonic extraction, the extraction method is simple and efficient, the extract contains a large amount of polyacetylene compounds, and the extract has remarkable antioxidation effect according to the experiments, so that the lyophyllum decastes extract rich in the polyacetylene compounds has wide medicinal application prospect.
Drawings
FIG. 1 is a diagram showing the results of HPLC analysis of Lyophyllum decastes extract rich in polyacetylene compounds prepared by different solvents according to the present invention;
in the figure: the ultraviolet detection wavelength is 220 nm; wherein the absorption peak (peak a in the figure) with retention time of 15.2 minutes is compound 4; the absorption peak (peak b in the figure) at retention time of 21.9 min was compound 1; the absorption peak at a retention time of 22.2 minutes (peak c in the figure) is compound 2; the absorption peak (peak d in the figure) at a retention time of 26.8 minutes is compound 3; the retention time errors of the same substance of different samples in the graph are within an acceptable range;
FIG. 2 is a chemical structural formula of compounds 1-4 of FIG. 1;
FIG. 3 is a graph of enhancement of glucose uptake in 3T3-L1 cells by Lyophyllum decastes extract enriched with polyacetylene compounds;
FIG. 4 is a graph showing the effect of the Lyophyllum decastes extract enriched with the polyacetylene compounds on the sensitization of insulin.
Detailed Description
The present invention will be described in further detail with reference to examples.
The reagent of the invention:
the Lyophyllum decastes fruiting body is collected from the natural protection region of Yan Zhishan in Gansu, and identified as Lyophyllum decastes.
Materials, reagents and the like used in the following examples are all common commercially available products unless otherwise specified.
Example 1 a method for preparing a Lyophyllum decastes extract rich in polyacetylene compounds:
the Lyophyllum decastes fruiting body is crushed and weighed to 500 g. The content of the active carbon is 3L (6 times) by volume percentage of 4: soaking in 1 ethanol/water for 3 hr, and extracting under reflux for 2 hr for 3 times. Mixing extractive solutions, concentrating under reduced pressure at 40 deg.C, and drying to obtain 13.6 g Lyophyllum decastes extract. Heating and ultrasonically dissolving the lyophyllum decastes extract by using 100% methanol, dropwise adding the sample solution on D101 macroporous resin at the speed of 5mL/min, standing and adsorbing for 2 hours, sequentially performing gradient elution and separation by using water, 40% ethanol, 75% ethanol and 95% ethanol, eluting 4 retention volumes in each proportion, collecting 75% ethanol and 95% ethanol eluates, concentrating, and combining to obtain the lyophyllum decastes extract LPe which is rich in the polyacetylene compounds and has 3.8g, wherein the calculated yield is 0.76%.
Example 2 fingerprint detection experiment of Lyophyllum decastes extract rich in polyacetylene compounds:
the analysis conditions of the high performance liquid chemical fingerprint are as follows: a Waters 2998 hplc chromatograph, quaternary gradient pump, DAD detector, Waters chromatography workstation were used. Chromatographic column YMC 8 analytical column (4.6 mm. times.150 mm, 5 μm), acetonitrile-trifluoroacetic acid aqueous solution with 0.01% volume percentage content as mobile phase, and gradient elution (elution procedure and volume percentage content of each component are shown below) at room temperature (20-30 deg.C) with flow rate of 1.00 mL/min. Dissolving crude extract LPe with acetonitrile to obtain 10mg/mL solution, with sample volume of 10 μ L and ultraviolet detection wavelength of 210 nm.
Table 1. gradient elution step:
Figure 100002_DEST_PATH_IMAGE001
the HPLC profile of the extract LPe obtained according to the above elution conditions is shown in FIG. 1.
Example 3 preparation of a Lyophyllum decastes extract enriched in polyacetylenes:
the Lyophyllum decastes fruiting body is crushed and weighed to 500 g. The content of the active carbon is 3L (6 times) by volume percentage of 4: 1 for 3 hours in methanol/water and extracted 3 times under reflux for 2 hours each time. Mixing extractive solutions, concentrating under reduced pressure at 40 deg.C, and drying to obtain 12.1 g Lyophyllum decastes extract. Dissolving the Lyophyllum decastes extract with 100% methanol by heating and ultrasonic, dripping the sample solution on D101 macroporous resin at the speed of 5mL/min, standing for adsorbing for 2 hours, performing gradient elution separation with 40%, 75% and 100% v/v methanol in sequence, eluting 4 retention volumes in each proportion, collecting 75% ethanol and 100% ethanol eluent, concentrating, and combining to obtain the Lyophyllum decastes extract LPe rich in the polyacetylene compounds, wherein the LPe is 3.2g, and the calculated yield is 0.64%.
Example 4 preparation of a Lyophyllum decastes extract enriched in polyacetylenes:
the Lyophyllum decastes fruiting body is crushed and weighed to 500 g. Soaking in 3L (6 times) 100% dichloromethane for 3 hr, and extracting under reflux for 2 hr for 3 times. Mixing extractive solutions, concentrating under reduced pressure at 40 deg.C, and drying to obtain 11.9 g Lyophyllum decastes extract. Dissolving the Lyophyllum decastes extract with 100% methanol by heating and ultrasonic, dripping the sample solution on D101 macroporous resin at the speed of 5mL/min, standing for adsorbing for 2 hours, performing gradient elution separation with 40%, 75% and 100% v/v methanol in sequence, eluting 4 retention volumes in each proportion, collecting 75% ethanol and 100% ethanol eluates, concentrating, and combining to obtain the Lyophyllum decastes extract LPe rich in the polyacetylene compounds, wherein the LPe is 2.6g, and the calculated yield is 0.52%.
Example 5 preparation of a Lyophyllum decastes extract enriched in polyacetylenes:
the Lyophyllum decastes fruiting body is crushed and weighed to 500 g. Soaking in 3L (6 times) 100% acetone for 3 hr, and extracting under reflux for 3 times (2 hr each time). Mixing extractive solutions, concentrating under reduced pressure at 40 deg.C, and drying to obtain 11.9 g Lyophyllum decastes extract. Heating and ultrasonically dissolving the lyophyllum decastes extract by using 100% methanol, dropwise adding the sample solution on D101 macroporous resin at the speed of 5mL/min, standing and adsorbing for 2 hours, sequentially carrying out gradient elution separation by using water, 35% acetone, 70% acetone and 95% acetone at a v/v ratio, eluting 4 retention volumes in each ratio, collecting 70% acetone and 95% acetone eluates, concentrating, and combining to obtain the lyophyllum decastes extract LPe which is rich in the polyacetylene compounds and has the weight of 2.9g, wherein the calculated yield is 0.58%.
Example 6 Activity of Lyophyllum decastes extract enriched with polyacetylenes to promote glucose uptake in 3T3-L1 cells:
materials: the tested sample solution is the lyophyllum decastes extract LPe rich in the polyacetylene compounds described in examples 1 and 3-5.
3T3-L1 cells were subcultured in DMEM medium containing 10% calf serum and 1% diabase (penicillin and streptomycin) in a CO 2 incubator, and no cytotoxicity was observed for compounds 1-4 by the CCK8 method.
Inoculating 3T3-L1 cells to a 96-well plate at 10000 cell density per well, carrying out adherent growth in an incubator at 37 ℃ and containing 5% CO 2 and saturated humidity, carrying out induced differentiation after 3T3-L1 cells are basically full, firstly, using 10% of calf serum DMEM high-sugar culture solution containing 10 mu M dexamethasone, 0.5mM IBMX and 1.0 mu g/mL insulin to induce and differentiate for 48 hours, then using 10% of fetal calf serum DMEM high-sugar culture solution containing 1.0 mu g/mL insulin as the culture solution to culture, inducing and differentiating, wherein a large amount of fat particles can be seen under a 3T3-L1 cell scope after 10 days of continuous differentiation, and the differentiation is completed when the fat particles account for more than 90%, and then the cells are used for glucose uptake.
The NBDG method measures the well-differentiated adipocytes of 3T 3-L1. Washing differentiated 3T3-L1 cells with KRPH buffer solution, starving the cells in KRPH buffer solution for 1h, treating with different concentrations of extract LPe for 24h, replacing culture solution, culturing with DMEM containing 100 μ M2-NBDG for 30min, discarding supernatant, washing with PBS for 2 times, and immediately detecting at 466nm and 540nm wavelength of a fluorescence microplate reader. The effect of the hon-shimeji extract LPe enriched with polyacetylenic compounds on glucose uptake activity in 3T3-L1 cells was evaluated by comparing the relative ratio of fluorescence intensity to the blank group. Insulin was used as a control.
Experiments prove that when the concentration of the Lyophyllum decastes extract LPe is 50 mu g/ml, the glucose uptake of the Lyophyllum decastes extract LPe rich in the polyacetylene compounds is 1.3 times that of a blank group, and as shown in figure 3, the composition has a certain promotion effect on the glucose uptake.
Example 7 experiments on sensitization of insulin by Lyophyllum decastes extract rich in polyacetylene compounds to inhibit insulin resistance:
differentiated 3T3-L1 adipocytes as described in example 6, were washed with KRPH buffer and the differentiated 3T3-L1 cells were starved for 1h in KRPH buffer, followed by treatment with LPe containing 100nM insulin and different concentrations of extract for 24h, the culture medium was changed, cultured for 30min using DMEM containing 100. mu.M 2-NBDG, the supernatant was discarded, PBS washed 2 times, and immediately examined at 466nM and 540nM wavelength in a fluorescence microplate reader. The improvement effect of the lyophyllum decastes extract LPe rich in the polyacetylene compounds on the insulin resistance of 3T3-L1 adipocytes was evaluated by comparing the relative ratio of the fluorescence intensity to the blank group.
Experiments prove that the Lyophyllum decastes extract LPe (50 mu g/ml) rich in the polyacetylene compounds can sensitize insulin and further increase the glucose uptake, which is 1.17 times of that of the pure insulin, as shown in figure 4, the Lyophyllum decastes extract LPe can improve the insulin resistance state of 3T3-L1 fat cells and increase the glucose uptake and utilization capacity of the fat cells, and can show that the Lyophyllum decastes extract LPe rich in the polyacetylene compounds has the effect of improving the insulin resistance on the in vitro cell level.
It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will occur to those skilled in the art without departing from the spirit and scope of this invention as defined by the appended claims.

Claims (4)

1. A preparation method of a Lyophyllum decastes extract rich in polyacetylene compounds is characterized by comprising the following steps: bag (bag)
The method comprises the following steps:
step 1), taking Lyophyllum decastes fruiting body as a raw material, crushing, and adding into an organic solvent A or B according to a certain proportion to soak for 2-10 hours; the organic solvent A is an aqueous solution of C1-4 lower alcohol or C1-4 lower alcohol; the mass-to-liquid ratio of the Lyophyllum decastes fruiting body to the solvent is 1: 2.5-8 (w/v); an organic solvent B which is 100% v/v ethyl acetate, acetone, dichloromethane or a mixture of any 2 to 3;
step 2), then extracting at 10-100 ℃, filtering and collecting an extracting solution;
step 3), carrying out reduced pressure concentration on the extracting solution to obtain a lyophyllum decastes extract;
step 4) carrying out ultrasonic dissolution on the lyophyllum decastes extract by using an organic solvent C, dripping the obtained dissolved solution into a column bed filled with macroporous resin for static adsorption for at least 2 hours, and carrying out gradient elution separation on the macroporous resin column after statically adsorbing a sample by using water and organic solvents with different concentrations; the organic solvent C is any one of 100% v/v ethyl acetate, 70-100% v/v methanol, 70-95% v/v ethanol, 100% v/v acetone or 100% v/v dichloromethane;
step 5), collecting the eluent, and concentrating to obtain an extract rich in the polyacetylene compounds; the polyacetylene compound is as follows:
Figure DEST_PATH_IMAGE001
2. the method for preparing a Lyophyllum decastes extract rich in polyacetylenes according to claim 1, wherein: in the step 1), the organic solvent A is 30-80(v/v) ethanol aqueous solution.
3. The method for preparing a Lyophyllum decastes extract rich in polyacetylenes according to claim 1 or 2, wherein: when the vacuum concentration is carried out in the step 3), the concentration temperature of the extracting solution is 35-55 ℃.
4. The method for preparing a Lyophyllum decastes extract rich in polyacetylenes according to claim 3, wherein: in the step 4), the macroporous resin column after statically adsorbing the sample is subjected to gradient elution separation by using water and methanol solutions with the concentrations of 40% v/v, 75% v/v and 100% v/v respectively, and each concentration is eluted by 3-5 retention volumes;
or gradient elution separation is carried out on the macroporous resin column after the static adsorption of the sample by using water and ethanol solutions with the concentration of 40% v/v, 75% v/v and 95% v/v respectively, and each concentration is eluted by 3-5 retention volumes;
or gradient elution separation is carried out on the macroporous resin column after statically adsorbing the sample by using water and acetone solutions with the concentration of 35% v/v, 70% v/v and 95% v/v respectively, and each concentration is eluted by 3-5 retention volumes.
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CN106674299A (en) * 2016-12-27 2017-05-17 西安交通大学 Polyacetylenic glycoside compounds as well as preparation method and application thereof
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CN108324744A (en) * 2018-03-17 2018-07-27 启东祥瑞建设有限公司 A kind of preparation method of radix bupleuri polyacetylene chemical composition

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