CN109876021A - The preparation method and its hypoglycemic purposes of Lyophyllum decastes extract rich in poly- acetylene compound - Google Patents
The preparation method and its hypoglycemic purposes of Lyophyllum decastes extract rich in poly- acetylene compound Download PDFInfo
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Abstract
The invention discloses the preparation methods and its hypoglycemic purposes of a kind of Lyophyllum decastes extract rich in poly- acetylene compound, by using Lyophyllum decastes as research object, the research of extraction process and bioactivity has been carried out to Lyophyllum decastes extract, it was found that the obtained Lyophyllum decastes extract rich in poly- acetylene compound, 3T3-L1 grape cell Sugar intake activity can be remarkably promoted, enhanced sensitivity insulin, inhibit insulin resistance, can be used as the prevention and treatment of hypoglycemic medicine, functional food or health care product for abnormal carbohydrate metabolism related disease.
Description
Technical field
The invention belongs to biological fields, are related to a kind of fungal extract, relate in particular to a kind of rich in carbene class chemical combination
The preparation method of the Lyophyllum decastes extract of object and the extract are used to prepare hypoglycemic medicine, functional food as active constituent
Or the application in health care product.
Background technique
With the development of social economy, people's dietary structure and living-pattern preservation, the disease incidence of diabetes are increasingly increased,
As most common in the world and most serious one of metabolic disease, nearly 500,000,000 people is affected.According to different onset mechanism, glycosuria
Disease is divided into four principal foum, i.e. type-1 diabetes mellitus, type-2 diabetes mellitus, gestational diabetes and other types diabetes.Wherein II
Patients with type Ⅰ DM is popular main body, and number of patients accounts for about the 90-95% of diabetic's sum, and main pathogenesis is insulin
It resists, companion or not impaired with different degrees of islet function, usually occur together other metabolic disorders.In recent years more and more children and
Population of adolescent suffers from type-2 diabetes mellitus.It is reported according to the World Health Organization (WHO), there are about 1.1 hundred million diabetics for China, about
Account for the 1/10 of Chinese adult sum.1.5 hundred million people will be increased to by expecting the year two thousand forty, be brought seriously to common people's health and social economy
It influences.Therefore, there is an urgent need to whole society's preventions and control diabetes and its complication.
It is current research hot spot that safety, low toxicity, efficient glycometabolism regulation and control substance are found from natural products.Decastes
Umbrella [Lyophyllum decastes(Fr.: Fr.) Sing.], it is subordinate to for Agaricales (Agaricales), from Zhe San section
(Lyophyllaceae), from pleat umbrella category (Lyophyllum), there is lotus leaf mushroom, pilose antler mushroom, roadbed mushroom, railway mushroom, grain nest, freeze bacterium
Cold fragrant bacterium, a brood of sheep etc. are a kind of very precious wild food medicine dual-purpose bacterium also known as.A large amount of research has shown that the son of fungus is real
Body, mycelium polysaccharides have blood lipid-reducing blood sugar-decreasing, anti-oxidant, anticancer bioactivity.And its active small molecular substance is studied less.
Summary of the invention
The present invention provides a kind of Lyophyllum decastes rich in poly- acetylene compound and mentions using Lyophyllum decastes as research object
Obtained active small molecular is applied to hypoglycemic purposes by the preparation method for taking object.
The technical solution adopted by the invention is as follows:
Firstly, the present invention provides a kind of preparation methods of Lyophyllum decastes extract rich in poly- acetylene compound, including with
Lower step:
Step 1) is added in organic solvent A or B by a certain percentage after crushing using decastes destroying angel as raw material and impregnates 2-
10 hours；
Then step 2) is extracted in 10-100 DEG C, extracting solution is collected by filtration；
Extracting solution is concentrated under reduced pressure in step 3), obtains Lyophyllum decastes extract；
Step 4) carries out ultrasonic dissolution to Lyophyllum decastes extract with organic solvent C, and gained lysate is added drop-wise to and is equipped with
It is carried out staticaccelerator adsorption at least 2 hours in the column bed of macroreticular resin, with the organic solvent of water and various concentration to staticaccelerator adsorption sample
Macroporous resin column afterwards carries out gradient elution separation；
Step 5) collects eluent, and concentration, which obtains, is rich in poly- acetylene compound extract.
In step 1) of the present invention, organic solvent A is C1-4 lower alcohol (such as: methanol, ethyl alcohol, butanol) or C1-4
The aqueous solution of the aqueous solution of lower alcohol, preferably 30-80 (v/v) ethyl alcohol.
Organic solvent B, be 100%v/v ethyl acetate, acetone, methylene chloride or any 2-3 kind mixture, preferably 100%
Ethyl acetate.
Preferably, the matter liquor ratio of the decastes destroying angel and the solvent is 1:2.5~8(w/v).
In step 2) of the present invention, extracts and select hot water return extraction, cold water extraction, ultrasonication or traditional extraction
In any one, every time extract 2-3 hours.
When being concentrated under reduced pressure in the step 3), extracting solution thickening temperature is 35~55 DEG C.
In the step 4), the methanol of ethyl acetate, 70%~100%v/v that the organic solvent C is 100%v/v, 70%
Any one in the methylene chloride of the ethyl alcohol of~95%v/v, the acetone of 100%v/v or 100%v/v.
In the step 4), macroreticular resin can be polarity, middle polarity, low pole or non-polar macroporous resin.
It is respectively the methanol solution of 40%v/v, 75%v/v, 100%v/v to static suction with water and concentration in the step 4)
Macroporous resin column after attached sample carries out gradient elution separation, and each concentration elutes 3-5 retention volume；Or divided with water and concentration
Not Wei the ethanol solution of 40%v/v, 75%v/v, 95%v/v gradient elution point is carried out to the macroporous resin column after staticaccelerator adsorption sample
From each concentration elutes 3-5 retention volume；It or is respectively the acetone soln of 35%v/v, 70%v/v, 95%v/v with water and concentration
Gradient elution separation is carried out to the macroporous resin column after staticaccelerator adsorption sample, each concentration elutes 3-5 retention volume.
The present invention also provides a kind of extracts prepared by the above method to be applied to prepare hypoglycemic as active constituent
In pharmaceutical composition, functional food or health care product.
Contain the Lyophyllum decastes extraction rich in poly- acetylene compound to anti-diabetic effective amount is treated and prevented in product
Object.Total weight based on composition, the composition for treating and preventing inflammatory, anaphylaxis or tumor disease may include by weight
Meter 0.1 to 99%, preferably 0.1 to 50%.
Described pharmaceutical composition may include one or more pharmaceutically acceptable auxiliary materials, and auxiliary material includes pharmaceutical field routine
Diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, sorbefacient, surfactant, absorption carrier, profit
Lubrication prescription, sustained release agent etc..
Described pharmaceutical composition can for injection, tablet, pulvis, granule, pill, capsule, oral solution, paste, creme,
The dosage forms such as spray.Various dosage forms can be prepared according to the conventional method of pharmaceutical field, can by oral, gastrointestinal administration, injection,
Injection, physically or chemically the methods of mediation administration, or be administered after other material mixings or package.
The prevention that functional food or health care product containing extract have daily health caring, adjust immunity of organisms related fields
And adjuvant treatment, concrete form can be oral solution, tea-drinking, lozenge, capsule, drink, effervescent tablet etc.." prevention " includes by applying
Inhibit or postpone any behavior of the generation of certain diseases or illness disclosed herein with the product；" treatment " includes by applying
Alleviate or beneficially modify any behavior of symptom related with certain diseases disclosed herein or illness with the product.
The beneficial effects of the present invention are:
1. the present invention has carried out extraction process and bioactivity to Lyophyllum decastes extract using Lyophyllum decastes as research object
Research, it was found that active small molecular-be rich in poly- acetylene compound Lyophyllum decastes extract, 3T3- can be remarkably promoted
L1 grape cell Sugar intake activity, enhanced sensitivity insulin inhibit insulin resistance, can be used for hypoglycemic medicine.
2. the Lyophyllum decastes extract rich in poly- acetylene compound of the invention can also be used in functional food or health care product
Carry out the prevention and treatment of abnormal carbohydrate metabolism related disease.
3. the present invention is by mentioning the study found that any conventional solvent, including ultrasonic wave extraction can be used in Lyophyllum decastes
Take method to be simple and efficient, in extract contain larger amount of poly- acetylene compound, and it is verified have significant antioxidation,
Therefore, the Lyophyllum decastes extract rich in poly- acetylene compound has wide medicinal application prospect.
Detailed description of the invention
Fig. 1 is the high-efficient liquid phase color of Lyophyllum decastes extract of the different solvents of the present invention preparation rich in poly- acetylene compound
Spectrum analysis result figure；
In figure: ultraviolet detection wavelength is 220 nanometers；Wherein, retention time 15.2 minutes absorption peak (in figure peak a) be chemical combination
Object 4；Retention time 21.9 minutes absorption peak (in figure peak b) be compound 1；Absorption peak of the retention time at 22.2 minutes
(peak c) is compound 2 in figure；Retention time 26.8 minutes absorption peak (in figure peak d) be compound 3；Different samples in figure
Same substance retention time error within an acceptable range；
Fig. 2 is the chemical structural formula of the compound 1-4 of Fig. 1；
Fig. 3 is that the Lyophyllum decastes extract rich in poly- acetylene compound promotes 3T3-L1 grape cell Sugar intake figure；
Fig. 4 is the Lyophyllum decastes extract enhanced sensitivity insulin action figure rich in poly- acetylene compound.
Specific embodiment
Below with reference to embodiment, invention is further described in detail.
Reagent of the invention:
Decastes destroying angel is acquired from the Gansu nature reserve area Yan Zhishan, is identified asLyophyllum decastes。
Material used in following embodiment, reagent etc. are unless otherwise specified ordinary commercial products.
A kind of preparation method of the Lyophyllum decastes extract rich in poly- acetylene compound of embodiment 1:
Decastes destroying angel is crushed, is weighed 500 grams.Measured with 3L(6 times) ethanol/water of volumn concentration 4:1 impregnates 3
Hour, refluxing extraction 3 times, every time 2 hours.Combined extract is concentrated under reduced pressure at 40 DEG C and is dried to obtain 13.6 grams of decastes
Umbrella extract.100% methanol of Lyophyllum decastes extract is heated into ultrasonic dissolution, sample solution is added dropwise with 5mL/min speed
On D101 macroreticular resin, staticaccelerator adsorption 2 hours, gradient elution point successively is carried out with water, 40% ethyl alcohol, 75% ethyl alcohol, 95% ethyl alcohol
From each ratio elutes 4 retention volumes, and after collecting 75% ethyl alcohol, the concentration of 95% ethanol eluate, merging is obtained rich in carbene class
The Lyophyllum decastes extract LPe of compound is 3.8g, calculated yield 0.76%.
The finger-print test experience of Lyophyllum decastes extract of the embodiment 2 rich in poly- acetylene compound:
Efficient liquid phase Chemical fingerprinting analysis condition is as follows: using 2998 high performance liquid chromatograph of Waters, quaternary gradient
Pump, DAD detector, Waters chromatographic work station.Chromatographic column YMC C8 analytical column (4.6mm × 150mm, 5 μm), mobile phase is
The aqueous solution of the trifluoroacetic acid of acetonitrile-volumn concentration 0.01% carries out gradient under conditions of temperature room temperature (20 ~ 30 DEG C) and washes
De- (volumn concentration of elution program and each ingredient is as follows), 1.00 mL/min of flow velocity.By crude extract LPe second
Nitrile dissolution, is made into the solution of 10mg/mL, 10 μ L of sample volume, and ultraviolet detection wavelength is 210nm.
1. gradient elution step of table:
The HPLC map for obtaining extract LPe according to above-mentioned elution requirement is as shown in Figure 1.
The preparation method of Lyophyllum decastes extract of the embodiment 3 rich in poly- acetylene compound:
Decastes destroying angel is crushed, is weighed 500 grams.Measured with 3L(6 times) methanol/water of volumn concentration 4:1 impregnates 3
Hour, refluxing extraction 3 times, every time 2 hours.Combined extract is concentrated under reduced pressure at 40 DEG C and is dried to obtain 12.1 grams of decastes
Umbrella extract.100% methanol of Lyophyllum decastes extract is heated into ultrasonic dissolution, sample solution is added dropwise with 5mL/min speed
On D101 macroreticular resin, staticaccelerator adsorption 2 hours, successively with 40%, 75%, the methanol of 100%v/v carry out gradient elution separation, often
A ratio elutes 4 retention volumes, and after collecting 75% ethyl alcohol, the concentration of 100% ethanol eluate, merging is obtained rich in carbene class chemical combination
The Lyophyllum decastes extract LPe of object is 3.2g, calculated yield 0.64%.
The preparation method of Lyophyllum decastes extract of the embodiment 4 rich in poly- acetylene compound:
Decastes destroying angel is crushed, is weighed 500 grams.Measured with 3L(6 times) 100% methylene chloride impregnate 3 hours, reflux
It extracts 3 times, every time 2 hours.Combined extract is concentrated under reduced pressure at 40 DEG C and is dried to obtain 11.9 grams of Lyophyllum decastes extracts.
100% methanol of Lyophyllum decastes extract is heated into ultrasonic dissolution, it is with 5mL/min speed that sample solution dropwise addition is big in D101
On the resin of hole, staticaccelerator adsorption 2 hours, successively with 40%, 75%, the methanol of 100%v/v carry out gradient elution separation, each ratio is washed
4 retention volumes are taken off, after collecting 75% ethyl alcohol, the concentration of 100% ethanol eluate, merges and obtains the lotus leaf rich in poly- acetylene compound
It is 2.6g, calculated yield 0.52% from pleat umbrella extract LPe.
The preparation method of Lyophyllum decastes extract of the embodiment 5 rich in poly- acetylene compound:
Decastes destroying angel is crushed, is weighed 500 grams.Measured with 3L(6 times) 100% acetone soak 3 hours, refluxing extraction 3
It is secondary, 2 hours every time.Combined extract is concentrated under reduced pressure at 40 DEG C and is dried to obtain 11.9 grams of Lyophyllum decastes extracts.By lotus leaf
Ultrasonic dissolution is heated from 100% methanol of pleat umbrella extract, sample solution is added dropwise in D101 macroreticular resin with 5mL/min speed
On, staticaccelerator adsorption 2 hours, gradient elution separation, each ratio elution 4 successively were carried out with water, 35%, the acetone of 70%v, 95%v/v
A retention volume after collecting the concentration of 70%, 95% acetone eluant, merges the Lyophyllum decastes for obtaining being rich in poly- acetylene compound and mentions
Taking object LPe is 2.9g, calculated yield 0.58%.
Lyophyllum decastes extract of the embodiment 6 rich in poly- acetylene compound promotes 3T3-L1 grape cell Sugar intake living
Property test:
Material: sample solution is the Lyophyllum decastes extract that poly- acetylene compound is rich in described in embodiment 1 and 3~5
LPe。
DMEM culture medium of the 3T3-L1 cell containing 10% calf serum and 1% dual anti-(penicillin and streptomysin), CO2Culture
Subculture in case determines that compound 1-4 does not have cytotoxicity through CCK8 method.
Be inoculated with 3T3-L1 cell to 96 orifice plates with 10000 cell densities in every hole, 37 DEG C, containing 5% CO2And saturated humidity
Adherent growth in incubator can carry out induction differentiation after 3T3-L1 cell covers with substantially, first with the DMEM of 10% calf serum
High sugar culture solution uses 10% tire ox instead containing after 10 μM of dexamethasone, 0.5mM IBMX and the insulin-induced differentiation 48h of 1.0 μ g/mL
The sugared culture solution of the DMEM high of serum is cultivated containing 1.0 μ g/mL insulin as culture solution, induction differentiation, after continuously breaking up 10d
Visible significant quantities of fat particle under 3T3-L1 cell mirror, it is that differentiation is completed that lipochondrion, which accounts for 90% or more, is used further to glucose uptake
It uses.
The fat cell that NBDG method measurement 3T3-L1 has broken up.The 3T3-L1 cell broken up with KRPH buffer solution for cleaning,
And starvation 1h replaces culture solution after the extract LPe processing for 24 hours for next using various concentration in KRPH buffer by cell,
Using 100 μM of 2-NBDG DMEM culture solution culture 30min are contained, supernatant is abandoned, PBS is cleaned 2 times, immediately in fluorescence microplate reader
It is detected at 466nm and 540nm wavelength.It evaluates by comparing comparing for fluorescence intensity and blank group rich in poly- acetylene compound
Influence of the Lyophyllum decastes extract LPe to 3T3-L1 cell grape glucose picked-up activity.Insulin is as control.
Tests prove that the Lyophyllum decastes extract LPe rich in poly- acetylene compound is to glucose when 50 μ g/ml
Intake is 1.3 times of blank group, as shown in figure 3, illustrating that the composition has certain facilitation to glucose uptake.
Embodiment 7 is rich in the Lyophyllum decastes extract enhanced sensitivity insulin of poly- acetylene compound, inhibits insulin resistance real
It tests:
The 3T3-L1 fat cell broken up as described in Example 6, the 3T3-L1 cell broken up with KRPH buffer solution for cleaning, and
By cell in KRPH buffer starvation 1h, next with the extract LPe of insulin containing 100nM and various concentration handle for 24 hours,
Culture solution is replaced, using 100 μM of 2-NBDG DMEM culture solution culture 30min are contained, abandons supernatant, PBS is cleaned 2 times, immediately in glimmering
It is detected at light microplate reader 466nm and 540nm wavelength.It evaluates by comparing comparing for fluorescence intensity and blank group rich in carbene
Improvement result of the Lyophyllum decastes extract LPe of class compound to 3T3-L1 fat cell insulin resistance.
Tests prove that the Lyophyllum decastes extract LPe(50 μ g/ml rich in poly- acetylene compound) it then can be with enhanced sensitivity pancreas
Island element, further increases the intake of glucose, is simple 1.17 times for using insulin, as shown in figure 4, can improve 3T3-L1
The insulin-resistant states of fat cell increase fat cell to glucose uptake and the ability utilized, from cell in vitro level
On can illustrate that the Lyophyllum decastes extract LPe rich in poly- acetylene compound has the function of improvement insulin resistance.
It will be appreciated that the following examples are given for illustrative purposes only, it is not used to the present invention
Range limited.Those skilled in the art without departing from the spirit and purpose of the present invention, to the present invention into
Row various modifications and replacement, fall within the scope of the claimed invention.
Claims (10)
1. a kind of preparation method of the Lyophyllum decastes extract rich in poly- acetylene compound, it is characterised in that: including following step
It is rapid:
Step 1) is added in organic solvent A or B by a certain percentage after crushing using decastes destroying angel as raw material and impregnates 2-
10 hours；
Then step 2) is extracted in 10-100 DEG C, extracting solution is collected by filtration；
Extracting solution is concentrated under reduced pressure in step 3), obtains Lyophyllum decastes extract；
Step 4) carries out ultrasonic dissolution to Lyophyllum decastes extract with organic solvent C, and gained lysate is added drop-wise to and is equipped with
It is carried out staticaccelerator adsorption at least 2 hours in the column bed of macroreticular resin, with the organic solvent of water and various concentration to staticaccelerator adsorption sample
Macroporous resin column afterwards carries out gradient elution separation；
Step 5) collects eluent, and concentration, which obtains, is rich in poly- acetylene compound extract.
2. the preparation method of the Lyophyllum decastes extract according to claim 1 rich in poly- acetylene compound, feature
Be: in step 1), organic solvent A is the aqueous solution of C1-4 lower alcohol or C1-4 lower alcohol.
3. the preparation method of the Lyophyllum decastes extract according to claim 1 or 2 rich in poly- acetylene compound, special
Sign is: in step 1), organic solvent B is the mixture of 100%v/v ethyl acetate, acetone, methylene chloride or any 2-3 kind.
4. the preparation method of the Lyophyllum decastes extract according to claim 3 rich in poly- acetylene compound, feature
Be: in step 1), organic solvent A is the aqueous solution of 30-80 (v/v) ethyl alcohol.
5. the preparation method of the Lyophyllum decastes extract according to claim 1,2 or 4 rich in poly- acetylene compound,
Be characterized in that: in step 1), the matter liquor ratio of the decastes destroying angel and the solvent is 1:2.5~8(w/v).
6. the preparation method of the Lyophyllum decastes extract according to claim 5 rich in poly- acetylene compound, feature
It is: in step 2), extracts any one selected in hot water return extraction, cold water extraction, ultrasonication or traditional extraction,
It extracts 2-3 hours every time.
7. the preparation method of the Lyophyllum decastes extract rich in poly- acetylene compound according to claim 1 or 6, special
Sign is: when being concentrated under reduced pressure in step 3), extracting solution thickening temperature is 35~55 DEG C.
8. the preparation method of the Lyophyllum decastes extract according to claim 7 rich in poly- acetylene compound, feature
Be: in step 4), the organic solvent C is the ethyl acetate of 100%v/v, the methanol of 70%~100%v/v, 70%~95%v/
Any one in the methylene chloride of the ethyl alcohol of v, the acetone of 100%v/v or 100%v/v.
9. the according to claim 1, system of the Lyophyllum decastes extract described in 2,4,6 or 8 rich in poly- acetylene compound
Preparation Method, it is characterised in that: be respectively the methanol solution pair of 40%v/v, 75%v/v, 100%v/v with water and concentration in step 4)
Macroporous resin column after staticaccelerator adsorption sample carries out gradient elution separation, and each concentration elutes 3-5 retention volume；
It or with water and concentration is respectively the ethanol solution of 40%v/v, 75%v/v, 95%v/v to the macropore tree after staticaccelerator adsorption sample
Rouge column carries out gradient elution separation, and each concentration elutes 3-5 retention volume；
It or with water and concentration is respectively the acetone soln of 35%v/v, 70%v/v, 95%v/v to the macropore tree after staticaccelerator adsorption sample
Rouge column carries out gradient elution separation, and each concentration elutes 3-5 retention volume.
10. a kind of Lyophyllum decastes extract rich in poly- acetylene compound of method according to claim 11 preparation,
Be characterized in that: the extract is used to prepare the application in hypoglycemic medicine, functional food or health care product as active constituent.
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| CN117643372A (en) * | 2023-12-08 | 2024-03-05 | 河西学院 | Application of Lyophyllum decastes fruiting body in preparation of Lyophyllum decastes food or lipid-lowering medicine |
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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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| 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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| CN117643372A (en) * | 2023-12-08 | 2024-03-05 | 河西学院 | Application of Lyophyllum decastes fruiting body in preparation of Lyophyllum decastes food or lipid-lowering medicine |
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