CN109985072A - Application of the elm mushroom extract in the drug, health care product, food of preparation treatment fatty liver - Google Patents
Application of the elm mushroom extract in the drug, health care product, food of preparation treatment fatty liver Download PDFInfo
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- CN109985072A CN109985072A CN201910060819.XA CN201910060819A CN109985072A CN 109985072 A CN109985072 A CN 109985072A CN 201910060819 A CN201910060819 A CN 201910060819A CN 109985072 A CN109985072 A CN 109985072A
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- elm mushroom
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention relates to the purposes that a kind of elm mushroom extract is used to prepare the drug for the treatment of fatty liver.Present invention simultaneously provides a kind of purposes of elm mushroom extract in the drug, health care product and food that preparation prevents and treats alcoholic liver injury.
Description
Technical field
The present invention relates to a kind of elm mushroom extracts to be used to prepare the use in the drug, health care product, food for the treatment of fatty liver
On the way.The present invention relates to the wider medical usage of elm mushroom extract, obtain it in preparation treatment alcoholic liver medicine
Using.
Background technique
In recent years, Chinese tradition Chinese medicine is in treatment ALD Drug development and research, has had shown that its superiority and opposite
Safety.Chinese herbal medicine has filtered out a variety of active ingredients from traditional Chinese medicinal and active component with Liver protection and resisting alcoholic hepatopathy
Group is simultaneously researched and developed, and most of is Changbai mountain, Jilin genuine traditional Chinese medicine material, e.g., Northeastern Radix Gentianae, Radix Salviae Miltiorrhizae, wilsonii etc..Wherein
It is especially prominent to the research of Changbai Mountain Northeastern Radix Gentianae, have mature independent extraction process, quality control method, has established accuracy
Higher HPLC content assaying method.
Alcoholic liver disease is liver cell textural anomaly and (or) functional disorder disease as caused by long-term excessive consumption of alcohol.
Initial stage is usually expressed as fatty liver, and then can develop into alcoholic hepatitis, alcoholic fibrosis and alcoholic cirrhosis.Seriously
Extensive necrosis of liver cells even hepatic failure can be induced when excessive drinking.This disease is common one of liver diseases, and it is strong to seriously endanger the mankind
Health.Initial symptoms of the alcoholic fatty liver as alcoholic liver disease, are mainly shown as in liver cell significant quantities of fat deposition occur,
And there is with slight inflammation but not hepatic fibrosis-renal tubular ectasia syndrome.More importantly alcoholic fatty liver be it is reversible, herein
Process, which carries out pharmaceutical intervention, can prevent the further development of disease, restore the health status of liver.At present for alcoholic liver
The treatment of disease lacks effective drug mostly based on ethanol withdrawal, but alcoholic patients occur to ethanol withdrawal compliance difference and easily
Abstinence reaction, therefore exploitation has the drug or health care product of confrontation alcoholic fatty liver from medical and edible dual purpose plant or mushroom
With important theory and realistic meaning.
Whether there is excessive drinking history according to Patients with Fatty Liver, is broadly divided into alcoholic fatty liver and non-alcoholic fatty
Liver.Nonalcoholic fatty liver is mainly related to insulin resistance, metabolic syndrome, and illness rate is higher in obese people.
Clinically nonalcoholic fatty liver can be divided into acute and chronic.Although acute nonalcoholic fatty liver disease is more rare, by
It can lead to the diseases such as some renal failures in acute nonalcoholic fatty liver, therefore cannot ignore.Chronic non-alcoholic fatty
Liver is more common compared to for acute, and the state of an illness slowly develops and hidden the characteristics of being, and will increase the wind for suffering from cardiovascular and cerebrovascular disease
Danger.Acute alcoholic fatty liver, which refers to, once to be drunk excessive alcohol or spills alcoholic liver disease caused by class beverage.Often it is accompanied by
Hepatic cell fattydegeneration, hepatocellular injury and inflammatory cell infiltration can be further development of liver fibrosis, liver if continuing to drink
Hardening even liver cancer, and can concurrent liver failure and upper gastrointestinal bleeding etc..It is bad that extensive liver cell can be induced when serious excessive drinking
Extremely or even liver failure.Although pathogenesis and protective agents of many researchers to alcoholic fatty liver for many years
It has made extensive and intensive studies, but since the organism metabolism process of alcohol is extremely complex, Different Individual is to Ethanol intake
Response difference is larger, and the definite occurrence and development process of alcoholic liver disease and pathomechanism are also indefinite so far, and also shortage makes us full
The intervention means of meaning include effectively protective agents, and therefore, the therapeutic agent for urgently finding natural alcoholic liver disease becomes ten
Divide important.
Elm mushroom is widely distributed in Northeast China, South Korea and Japan.Elm mushroom is rich in protein abundant, vitamin and mine
Material composition, delicious flavour is one kind by favorite integration of drinking and medicinal herbs mushroom.Studies have shown that elm mushroom extract has antioxygen
The multiple pharmacological activity such as change, anti-inflammatory, antitumor and immunological regulation.
Currently, usually used ethanol withdrawal method is to treat the main method of alcoholic liver disease, but there are nutritional supports
Little, the problems such as ethanol withdrawal patient compliance is poor is acted on, in view of prior art disadvantage, the purpose of the present invention is study elm mushroom
The effect of extract confrontation alcoholic fatty liver disease and mechanism are simultaneously developed as a kind of drug with resisting alcoholic fatty liver
Or health food.
It is found after the present inventor concentrates on studies, elm mushroom extract is used for Alcoholic rouge for the first time by the present inventor
The prevention and treatment of fat hepatopathy；The present inventor proves that elm mushroom extract has the function of good confrontation alcoholic fatty liver disease for the first time；
The perfect pharmacological action of elm mushroom of the present inventor, also has developed a kind of great potential for alcoholic liver disease.
Summary of the invention
In view of prior art disadvantage, the purpose of the present invention is to provide a kind of elm mushroom extracts to treat fatty liver in preparation
Drug application.
Application of the elm mushroom extract of the present invention in treatment alcoholic fatty liver.
Application of the elm mushroom extract of the present invention in treatment nonalcoholic fatty liver.
Application of the present invention, wherein elm mushroom extract is included in single formulation.
Application of the present invention, wherein elm mushroom extract is configured to pharmaceutical composition respectively and is applied in combination.
The second aspect of the present invention provides a kind of elm mushroom extract and prevents and treats Alcoholic and non-alcoholic in preparation
The application of the drug of hepatic injury.
The third aspect of the present invention provides a kind of elm mushroom extract and prevents and treats Alcoholic and non-alcoholic in preparation
The application of the health care product of hepatic injury.
The fourth aspect of the present invention provides a kind of elm mushroom extract and prevents and treats Alcoholic and non-alcoholic in preparation
The application of the drink of hepatic injury.
Through the invention, it was found that elm mushroom extract significantly mitigates glutamic-pyruvic transaminase in Ethanol intake induced mice blood
With the raising of glutamic-oxalacetic transaminease, the lipidosis in mouse liver cell and inflammatory reaction are alleviated.Elm mushroom of the invention mentions
It takes object that there is excellent druggability, can be used as the exploitation of resisting alcoholic fatty liver medicament.Meanwhile elm mushroom of the invention is extracted
Object, raw material have no toxic side effect as a kind of edible and medicinal fungi, and patient compliance can be improved, and raw material is easy to get, cheap etc. excellent
Point.
Detailed description of the invention
Fig. 1 shows acute and chronic alcoholic mouse model and elm mushroom extract-treated methods, wherein Figure 1A is acute
Alcohol mouse fatty liver modeling schematic diagram；Figure 1B is Chronic Alcohol mouse fatty liver modeling schematic diagram；
Influence Fig. 2 shows elm mushroom extract to acute alcohol model mice liver organization morphology and biochemical indicator；
Fig. 3 shows elm mushroom extract and expresses fat metabolism in acute alcohol model mice liver and inflammation related proteins
Influence；
Fig. 4 shows influence of the elm mushroom extract to Chronic Alcohol model mice liver organization morphology and biochemical indicator；
Fig. 5 is the immunoblot experiment that lipopexia and inflammation related proteins are carried out to Chronic Alcohol model mice liver
As a result；
Fig. 6 shows HepG2 cell Nile red coloration result；
Fig. 7, which is shown, carries out HE dyeing and oil red O stain result；
Fig. 8 shows influence of the elm mushroom extract to non-alcoholic mouse liver biochemical indicator.
Specific embodiment
In the following, a detailed description of the technical solution in the embodiment of the present invention is provided, it is clear that described embodiment is only
It is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
It should be noted that in the present invention, term " basic " or the meaning that " complete " " substantially " is not precluded.Such as one
A ingredient substantially free Y, is also possible to be entirely free of Y.In the case where limiting specific value, refer to the specific value
With the range to float up and down based on the specific value, floating range can be +/- the 5% of the specific value, +/-
4%, +/- 3%, +/- 2%, +/- 1%, +/- 0.5%, +/- 0.2%, +/- 0.1%, +/- 0.05%, +/- 0.01% etc..Such as
Fruit needs, " basic " or " substantially " can above floating range replace or deleted from of the invention define.
" containing " had both included the factor mentioned, and also allowed to include additional, uncertain factor.
" about ", " about ", " left and right " in the case where limiting specific value, refer to the specific value have with the specific number
The range to float up and down based on value, floating range can be +/- the 5% of the specific value, and +/- 4%, +/- 3%, it is +/-
2%, +/- 1%, +/- 0.5%, +/- 0.2%, +/- 0.1%, +/- 0.05%, +/- 0.01% etc..
"and/or" indicates respectively be used alone by multiple terms of its connection, mutually can also arbitrarily combine.
In the present invention, the numberical range used for simplicity not only includes its endpoint value, also includes its all son
Range and individual numerical value all within the scope of this.For example, numberical range 1-6 not only includes subrange, such as 1-3,1-4,1-
5,2-4,2-6,3-6 etc., also including individual numerical value within the scope of this, such as 1,2,3,4,5,6.
In the present invention, Fig. 1 shows acute with chronic alcoholic mouse model and elm mushroom extract-treated method,
In, Figure 1A is acute alcohol mouse fatty liver modeling schematic diagram；Figure 1B is Chronic Alcohol mouse fatty liver modeling schematic diagram；Fig. 2
Influence of the elm mushroom extract to acute alcohol model mice liver organization morphology and biochemical indicator is shown；Fig. 3 shows elm Huang
The influence that mushroom extract expresses fat metabolism in acute alcohol model mice liver and inflammation related proteins；Fig. 4 shows elm Huang
Influence of the mushroom extract to Chronic Alcohol model mice liver organization morphology and biochemical indicator；Fig. 5 is to Chronic Alcohol model
Mouse liver carries out the result of the immunoblot experiment of lipopexia and inflammation related proteins；Fig. 6 shows HepG2 cell Nile red
Coloration result.
The purpose of the present invention is to provide a kind of elm mushroom extracts to prepare the application for treating the drug of fatty liver.
Application of the elm mushroom extract of the present invention in treatment alcoholic fatty liver.
Application of the elm mushroom extract of the present invention in treatment nonalcoholic fatty liver.
Application of the present invention, wherein elm mushroom extract is included in single formulation.
Application of the present invention, wherein elm mushroom extract is configured to pharmaceutical composition respectively and is applied in combination.
The second aspect of the present invention provides the medicine that a kind of elm mushroom extract prevents and treats alcoholic liver injury in preparation
The application of object.
The third aspect of the present invention provides the guarantor that a kind of elm mushroom extract prevents and treats alcoholic liver injury in preparation
The application of strong product.
The fourth aspect of the present invention provides the drink that a kind of elm mushroom extract prevents and treats alcoholic liver injury in preparation
The application of product.
Medicament of the invention can be formulated into, such as pharmaceutical composition, such as tablet (including sugar coated tablet, film coating
Piece), powder, granule, capsule (including soft capsule), liquid agent, injection, suppository, sustained release preparation is (for example, be sustained micro- glue
Capsule) or quick releasing formulation, and safely oral administration or parenteral administration (for example, part, rectum, intravenous administration etc.).Institute
Stating injection can be used in administration in intravenous, intramuscular, subcutaneous or organ, or can be directly applied to lesion.
In the present invention, include with the example of salt formed by inorganic base and alkali metal (such as sodium, potassium etc.), alkaline-earth metal
Salt formed by (such as calcium, magnesium etc.) and aluminium, ammonium etc..Example with salt formed by organic base includes and trimethylamine, triethylamine, pyrrole
Pyridine, picoline, ethanol amine, diethanol amine, triethanolamine, dicyclohexylamine, N, salt formed by N- dibenzyl-ethylenediamin etc..With
The example of salt formed by basic amino acid includes and arginine, lysine.
The pharmacologically acceptable carrier that can be used in preparing medicament of the invention includes conventional use of various
Organic or inorganic carrier substance, such as the excipient for solid pharmaceutical preparation, lubricant, adhesive and disintegrating agent are used for liquid system
Solvent, solubilizer, suspending agent, isotonic agent, buffer and soothing agent of agent etc..In addition, if necessary, can also use appropriate
Universal additive, such as preservative, antioxidant, colorant, sweetener, adsorbent, wetting agent etc..
In the present invention, the example of the excipient includes: lactose, sucrose, PEARLITOL 25C, starch, cornstarch, crystallization
Cellulose, light anhydrous silicic acid etc..
In the present invention, the example of the lubricant includes magnesium stearate, calcium stearate, talcum, colloidal silicon dioxide etc..
In the present invention, the example of described adhesive includes avicel cellulose, sucrose, PEARLITOL 25C, dextrin, hydroxypropyl fibre
Tie up element, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, starch, gelatin, methylcellulose, sodium carboxymethylcellulose etc..
In the present invention, the example of the disintegrating agent includes starch, carboxymethyl cellulose, calcium carboxymethylcellulose, carboxylic first
Base sodium starch, L- hydroxypropyl cellulose etc..
In the present invention, the example of the solvent includes water for injection, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn
Oil, olive oil etc..
In the present invention, the example of the solubilizer includes polyethylene glycol, propylene glycol, PEARLITOL 25C, Ergol, second
Alcohol, Trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate etc..
In the present invention, the example of the suspending agent includes: surfactant, such as stearyl triethanolamine, lauryl sulphur
Sour sodium, lauryl amino propionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate etc.；Hydrophilic polymer, such as
Polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose,
Hydroxypropyl cellulose etc.；Etc..
In the present invention, the example of the isotonic agent includes glucose, D-glucitol, sodium chloride, glycerol, PEARLITOL 25C
Deng.
In the present invention, the example of the buffer include such as phosphate, acetate, carbonate, citrate it is slow
Electuary etc..
Experimental example
Extract in accordance with the following methods to elm mushroom: 250mL is added in elm mushroom 50g, and 80 DEG C of water carries out water-bath, respectively
Three times, the time is respectively 2h, 1h, 1h for water-bath.The filtrate for collecting water-bath three times after the water bath is over, is filtered with gauze, filtering
80 DEG C of water-baths of filtrate afterwards are concentrated into 100mL.It is 40%, 4 that 95% ethyl alcohol to solution ethanol content is added into ethyl alcohol concentrate
DEG C precipitates overnight collects precipitating, and it is 60% that supernatant, which continuously adds 95% ethyl alcohol to solution ethanol content, and 4 DEG C of precipitates overnights are received
Collection precipitating, it is 80% that 95% ethyl alcohol to solution ethanol content, which is added, in supernatant again, and 4 DEG C of precipitates overnights abandon supernatant, and it is heavy to collect
It forms sediment.Merge the precipitating collected three times, washs precipitating twice with dehydrated alcohol again after precipitating is repeatedly washed with 95% ethyl alcohol, it is dry heavy
Forming sediment is elm mushroom extract.
By the concentrating sample extracted in Ultimat 3000Ultra Performance Liquid
It is analyzed in Chromatography system (Thermo scientific, USA).In ACQUITY UPLC BEH C18 column (2.1
× 100mm I.D., 1.7 μm) on separated.Mobile phase is made of the water containing 0.1% formic acid (A) and acetonitrile (B).Gradient
Condition is in 1-25 minutes 5-90%B, and in 25.1-26 minutes 100%B, in 26-26.5 minutes 100-5%B, flow velocity was
0.3mL/min。
Hereinafter, zoopery but is not meant to any limit of the invention to further description of the invention
System.Clearly as popularity possessed by selected experimental animal pharmacology feasibility express present invention application should trace back and it is dynamic
Object realm.
In the present invention, using 10 week old, the male C57BL/6 mouse of 20~25g of weight, purchased from Changchun hundred million, this experiment is dynamic
Object Science and Technology Ltd. (Jilin, China), all mouse keep the periodicity of illumination round the clock of 12h/12h, living environment during test
It is maintained at temperature ± 23 ± 2 DEG C, humidity 55 ± 5%.Guarantee balanced standard diet and sufficient drinking public water supply.All realities
Program is tested to be ratified by the zooscopy committee, Yanbian University.All experimental animals carry out humanistic care, and going forward side by side, all are exerted
Power reduces experimental animal usage quantity and minimizes its pain.This experimental study meets ARRIVE guide (McGrat et
Al., 2015).
The present invention uses acute and chronic alcoholic fatty liver model.Specifically, mouse C57BL/6 mouse in acute model
Adaptation is randomly divided into three groups after a week: normal group, alcohol group, alcohol adds elm mushroom extract administration group (100mg/kg).Alcohol
Group gives 31.5% alcoholic solution with the every 12h of administration group and carries out stomach-filling to mouse, altogether three times, while normal group equal heat quantity
Smart solution carries out stomach-filling to maltose recklessly.Half an hour after administration group mouse alcohol stomach-filling, with the stomach-filling again of elm mushroom extract.Most
Afterwards after an alcohol stomach-filling 4h, all mouse carry out heart puncturing extracting blood, anatomical isolation liver.It is stayed after blood 3000rpm centrifugation
Taking serum, liver great Ye is immersed in room temperature in neutral formalin solution and retains, and liver leaflet is dispensed into 1.5ml centrifuge tube-
80 DEG C of keepings are in case he uses；C57BL/6 mouse adapts to be randomly divided into four groups after a week in Chronic Alcohol liver model: normal group, wine
Smart group, alcohol adds elm mushroom that low dose group (50mg/kg) is administered, and alcohol adds elm mushroom that high dose group (100mg/kg) is administered.Just
Normal group gives solid feed, and while administration group gives elm mushroom extract stomach-filling, normal group carries out physiological saline stomach-filling；Alcohol
Group gives liquid alcohol feed for 5 days after giving liquid control feed, and the concentration of alcohol feed is gradually increased to 5%, 1% by 1%
Alcohol feed to 4% each concentration is given two days, persistently gives 5% liquid alcohol feed later 28 days；Administration group mouse
Feed mode is given with alcohol group, carries out elm mushroom extract (50 or 100mg/kg) while giving 5% liquid alcohol feed
Stomach-filling, administration time have altogether 28 days.It is administered after four weeks and heart puncturing extracting blood, anatomical isolation liver is carried out to all mouse.Blood
Serum is left and taken after 3000rpm centrifugation, liver great Ye is immersed in room temperature in neutral formalin solution and retains, and liver leaflet is dispensed into
- 80 DEG C of keepings in 1.5mL centrifuge tube are in case he uses.
It is found by staining pathologic section, two kinds of models successfully cause the steatosis of mouse liver.Mouse elm is yellow
The intake of mushroom extract has reversed the accumulation of liver fat caused by alcohol and the infiltration of inflammatory cell, and shows certain journey
The dose dependent of degree.The overexpression of SIRT1 and AMPK α can promote the decomposition of fat.Alcohol group mouse IHC coloration result is aobvious
Show, the dyeing of SIRT1 and AMPK α is shallower compared with normal group, illustrates the table of two kinds of albumen of SIRT1 and AMPK α in alcohol group
Up to being suppressed；SIRT1 and AMPK α coloration result is obviously than alcohol group depth in the elm mushroom extract administration group, and has
Dose dependent.Srebp1 is Fatty synthesis GAP-associated protein GAP, and the intake of alcohol can cause Srebp1 expression to increase.To acute and chronic
Alcohol-induced liver injury model in mice Srebp1 ImmunohistochemistryResults Results can be seen that in alcohol group, and more normal group of the dyeing of Srebp1
It is obvious to deepen；The dyeing of elm mushroom extract administration group Srebp1 then obviously shoals compared with alcohol group, illustrates Srebp1 in elm
Decline is expressed in yellow mushroom extract administration group.P2X7R plays a significant role in inflammation adjusting, the immunohistochemical staining of P2X7R
Obviously relatively normal group is deep for the dyeing of P2X7R in alcohol group as the result is shown, illustrates the expression of the P2X7R in mouse liver in alcohol group
Increase；Elm mushroom extract administration group, compared with model group, dyeing is shallower for the expression of P2X7R, and it is more to illustrate that the expression of P2X7R has
Decline.Therefore elm mushroom extract inhibit alcoholic fatty liver lipopexia may with have activated SIRT1-AMPK signal path
It is related, and inhibition of inflammation may be by lowering the expression of P2X7R, so that the release for reducing proinflammatory factor is related.
In HE dyeing of the invention, hematoxylin eosin staining method (abbreviation HE decoration method), is that paraffin section is most common
One of colouring method.Hematoxylin dye liquor is alkalinity, mainly makes endonuclear chromatin and intracytoplasmic nucleic acid in bluish violet；She
Red is acid dyes, and the ingredient of cytoplasm and extracellular matrix is mainly made to dye red.Need before dyeing to paraffin section into
Row dewaxing, after paraffin section dries piece then conventional dewaxing carries out haematoxylin dyeing, slice is used after uniformly catching color to water
PBS solution carries out returning indigo plant, until blue is presented in liver section, carries out eosin stains after rinsing 1min with tap water, slice is soaked
Enter and rinses out extra Yihong dyestuff in the dyestuff of Yihong after 2-3min with tap water.Slice is taken off after dyeing
Water, concentration alcohol is successively dehydrated gradient concentration alcohol from low to high, and slice is put into 10min in dimethylbenzene after dehydration
It carries out transparent, finally carries out mounting with neutral gum.
It is established in acute and chronic alcoholic mouse model and elm mushroom extract-treated in of the invention, in order to illustrate elm
Yellow mushroom extract establishes acute and chronic mouse alcoholic fatty liver model (Fig. 1) to the curative effect of alcoholic liver disease respectively.Fig. 1
Indicate acute and chronic alcoholic mouse model and elm mushroom extract-treated method, wherein Figure 1A is acute alcohol mouse rouge
Fat liver modeling schematic diagram；Figure 1B is Chronic Alcohol mouse fatty liver modeling schematic diagram.
In the present invention, elm mushroom extract is as follows to the effect of alcohol-induced acute hepatic injury:
Acute alcohol-induced hepatic injury generally will appear the raising of transaminase, and Fig. 2A is in acute alcoholic liver disease mice serum
ALT and AST content detection result column diagram, the results show that alcohol group serum alt and the AST significant raising compared with normal group
(###P < 0.001)；ALT, which is compared with the content of AST compared with model group, in administration group mice serum is declined (#P < 0.05).
In addition, the measurement result (Fig. 2 B) to TG content in Models of Acute Alcoholic Liver Injury mice serum and liver shows alcohol group
TG content is even in mice serum and liver is significantly higher than normal group (###P < 0.001), TG in administration group mice serum and liver
Content be then significantly lower than alcohol group (* * * P < 0.001, * * * P < 0.01), illustrate that acute alcohol intakes cause mouse liver
Damage, leads to the raising of ALT and AST, and can be visual and clear in HE and oil red O stain result see, acute alcohol stomach-filling
Afterwards, there is apparent fat vacuole not of uniform size in alcohol group mouse liver, and oil red O stain is obviously deepened；Elm mushroom extract
Then have no fat vacuole in administration group liver, oil red O stain result is close to normal group (Fig. 2 C and Fig. 2 D), this more intuitive theory
Bright, elm mushroom extract can significantly inhibit the lipopexia phenomenon of acute alcoholic mouse liver injury process.Alcohol group is small
In mouse liver Masson coloration result, the dyeing of alcohol group blue collagenous fibres does not increase significantly compared with normal group, perhaps
Be since the acute alcohol model modeling time is shorter, though liver, which is had damage also, does not occur liver fibrosis (Fig. 2 E).
Fig. 2 indicates influence of the elm mushroom extract to acute alcohol model mice liver organization morphology and biochemical indicator.
Transaminase content detection result in Fig. 2A acute alcohol model mice serum；Fig. 2 B. acute alcohol model mice serum and liver
Middle content of triglyceride testing result；Fig. 2 C. acute alcohol model mice liver HE coloration result；Fig. 2 D. acute alcohol model
Mouse liver oil red O stain result；Fig. 2 E is acute alcohol model mice liver Masson coloration result.### indicate with it is normal
Group compare P < 0.001, * * * indicate with alcohol group compared with P < 0.001, * * indicate with alcohol group compared with P < 0.01, * expression and
Alcohol group compares P < 0.05.
In the present invention, influence of the elm mushroom extract to chmice acute alcoholic liver lipopexia is as follows: SIRT1 is in rouge
It plays an important role in the synthesis of fat, SREBP1 has regulating and controlling effect to lipolysis, carries out to acute alcoholic mouse model
The expression of immunohistochemical experiment observation SIRT1, SREBP1, AMPK.SIRT1, AMPK α coloration result more normally organize shallowly,
Illustrate in acute mouse liver injury model, the expression of both albumen is reduced；In elm mushroom extract administration group, two kinds of albumen
For immunohistochemical staining result close to normal group, i.e., the expression of two kinds albumen organizes no significant difference (Fig. 3 A and Fig. 3 B) with normal.
The immunohistochemistry coloring of SREBP1 is obviously deeper than normal group in acute alcoholic mouse model, illustrates SREBP1 in alcohol group mouse
It expresses and increases in liver；Elm mushroom extract administration group SREBP1 immunohistochemical staining is close to normal group (Fig. 3 C).P2x7R exists
It is played an important role in Inflammatory Pathway.In P2x7R immunostaining results, P2x7R coloration result in acute alcohol mouse liver
It is obviously deeper than normal group, illustrates that P2x7R is higher than normal group in the expression of alcohol group, i.e., in acute alcohol model, mouse occurs
The overexpression of P2x7R；Elm mushroom extract administration group liver dyes the overexpression (figure for not occurring P2x7R then close to normal group
3D)。
Fig. 3 indicates that elm mushroom extract expresses fat metabolism in acute alcohol model mice liver and inflammation related proteins
Influence, Fig. 3 A. acute alcohol model mice liver SIRT1 ImmunohistochemistryResults Results；Fig. 3 B. acute alcohol model mice liver
AMPK alpha immunization group result；Fig. 3 C. acute alcohol model mice liver SREBP1 ImmunohistochemistryResults Results；Fig. 3 D. acute alcohol mould
Type mouse liver P2x7R ImmunohistochemistryResults Results.
The elm mushroom extract is as follows to the effect of chronic alcoholic mouse liver injury, detects to Chronic Alcohol model mice
The significant product of hepatic injury, the level of ALT and AST, as a result as shown in Figure 4 A, ALT and AST in Chronic Alcohol group mice serum
Content compared with normal group, it is even apparent increase (###P < 0.001, P## < 0.01)；The high low dosage administration of elm mushroom extract
For group compared with alcohol group, serum alt level is decreased obviously (* * P < 0.01), and AST level is also declined (* P < in serum
0.05).And elm mushroom extract high dose group ALT and AST fall is greater than low dose group.
Fig. 4 B indicates the horizontal result of content detection of TG in mouse liver and serum.It can be seen that alcohol group serum and liver
The level of dirty middle TG is all significantly higher than normal group, illustrates in chronic alcoholic mouse liver injury model, mouse liver has occurred
Apparent lipopexia phenomenon.In elm mushroom extract administration group, TG content in elm mushroom extract low dose group mice serum
With model group no significant difference, liver TG has certain decline (* P < 0.05)；High dose serum and liver is administered in elm mushroom extract
The content of dirty middle TG then has more apparent decline (* * P < 0.01, * * * P < 0.001) compared with model group.
Mouse paraffin section and frozen section carry out HE dyeing and oil red O stain respectively, and the pathology for observing mouse liver becomes
Change.The results show that apparent inflammatory infiltration, liver rope disorder, oil red O occur for alcohol group mouse in chronic alcoholic mouse model
Dyeing is obviously deeper than normal group；Apparent inflammation immersion and fat vacuole are not found in the high low dosage administration group of elm mushroom extract
Appearance, liver cell queueing discipline, liver rope is high-visible, and the effect of elm mushroom extract have concentration dependent (Fig. 4 C
With Fig. 4 D).
Masson trichrome stain is the reliable method for observing collagen.Tri- color of Masson dye is carried out to Chronic Alcohol model mice
Color, from result (Fig. 4 E) as can be seen that the relatively normal group of alcohol group mouse blue cellulose dyeing is compared to fairly obvious；Elm mushroom is extracted
Stock-dye is considerably less than alcohol group and has dose dependent in object administration group.
Fig. 4 indicates influence of the elm mushroom extract to Chronic Alcohol model mice liver organization morphology and biochemical indicator,
Fig. 4 A. Chronic Alcohol model mice Biochemical Indices In Serum ALT, AST testing result；In Fig. 4 B. Chronic Alcohol model mice serum
The ELISA experimental result of IL-1 β；Fig. 4 C. Chronic Alcohol model mice HE coloration result figure；Fig. 4 D. Chronic Alcohol model mice
Oil red O stain result figure；Fig. 4 E. Chronic Alcohol model mice Masson coloration result figure.### indicates the P < compared with normal group
0.001, * * indicates that the P < 0.01 compared with alcohol group, * indicate the P < 0.05 compared with alcohol group.
Hereinafter, influence of the elm mushroom extract to Chronic Alcohol mouse model lipopexia is described in detail.
Fig. 5 is the immunoblot experiment that lipopexia and inflammation related proteins are carried out to Chronic Alcohol model mice liver
As a result.The overexpression of SIRT1 and AMPK α can promote the decomposition of fat.Alcohol group mouse immune histochemical staining the results show that
The dyeing of SIRT1 and AMPK α is shallower compared with normal group, illustrate the expression of two kinds of albumen of SIRT1 and AMPK α in alcohol group by
To inhibition；SIRT1 and AMPK α coloration result is obviously than alcohol group depth in elm mushroom extract administration group, and has dose-dependant
Property (Fig. 5 A and Fig. 5 B).SREBP1 is Fatty synthesis GAP-associated protein GAP, and the intake of alcohol can cause SREBP1 expression to increase.To slow
Property alcohol-induced liver injury model in mice SREBP1 ImmunohistochemistryResults Results can be seen that in alcohol group, and the dyeing of SREBP1 is more normal
Group is obvious to deepen；The dyeing of elm mushroom extract administration group SREBP1 then obviously shoals compared with alcohol group, illustrates that SREBP1 exists
Expression decline (Fig. 5 C) in elm mushroom extract administration group.Fig. 5 D is chronic alcohol liver injury model mice liver section
The immunohistochemical staining result of P2x7R.Obviously relatively normal group is deep for the dyeing of P2x7R in alcohol group, illustrates the mouse in alcohol group
The expression of P2x7R increases in liver；Elm mushroom extract administration group, the expression of P2x7R is compared with Chronic Alcohol model group, dyeing
It is shallower, illustrate that the expression of P2x7R is declined.
As shown in figure 5, elm mushroom extract is to fat metabolism and inflammation related proteins in Chronic Alcohol model mice liver
The influence of expression, A. Chronic Alcohol model mice liver SIRT1 ImmunohistochemistryResults Results；B. Chronic Alcohol model mice liver AMPK
Alpha immunization group result；C. Chronic Alcohol model mice liver SREBP1 ImmunohistochemistryResults Results；D. Chronic Alcohol model mice liver
P2x7R ImmunohistochemistryResults Results.
Influence of the elm mushroom extract to HepG2 liver cell lipopexia caused by alcohol, Nile red are a kind of common fluorescence
Dyestuff can dye fat.In HepG2 cell Nile red coloration result (Fig. 6), alcohol group cell Nile red fluorescence with just
Often group illustrates the accumulation that fat has occurred in alcohol group cell compared to being remarkably reinforced；It is thin that elm mushroom extract is administered alone group
Born of the same parents' fluorescence intensity is similar to normally organizing；Alcohol adds in elm mushroom extract administration group, and compared with alcohol group, three groups of cell fluorescences are equal
Weaken, and there is dose dependent.Melbine (metformin) is AMPK agonist, can promote the metabolism of fat.Diformazan is double
Guanidine and alcohol group ratio, Nile red fluorescent weakening, but with elm mushroom extract, fluorescence intensity is higher compared with high, middle dose group, explanation
Elm mushroom extract has the function of good inhibition lipopexia.
Fig. 6 indicates that HepG2 cell Nile red coloration result, alcohol are considered drawing by changing liver NADH/NAD+ ratio
Play fatty liver, that is, fatty acid oxidation and stimulation fat is inhibited to generate.It has recently been demonstrated that Ethanol intake may directly affect
The activity of AMP deopendent protein kinase (AMPK).AMPK is that energy and metabolic balance are adjusted in liver, heart, skeletal muscle
A kind of protein kinase is the key signal molecule that adiponectin plays biological effect.The activation of liver AMPK can increase fatty acid
Oxidation reduces the synthesis of fat, inhibits glycogen and cholesterol biosynthesis, adjusts energy homeostasis balance.AMPK, which is called, " adjusts cell
The switch of energetic supersession ", AMPK are the important target spots of liver regulating lipid metabolism balance.The mouse AMPK activity quilt of feeding ethyl alcohol
Inhibit, AMPK can reduce malonyl coenzyme A by inhibiting acetyl-CoA carboxylase (ACC) to control fatty acid metabolism.
AMPK also can be by inhibiting SREBP-1 to inhibit the synthesis of fatty acid, a kind of transcription factor that lipid can be promoted to generate related gene.
Research finds that the activation of AMPK needs upstream kinases to carry out phosphorylation to Thr172 on AMPK α subunit activation ring to complete.LKB1,
Being called STK11 (Serine Threonine Protein Linase 11) is the silk gas acid/threonine encoded by lkb1 gene
The member of protein kinase family is a kind of suppression cancer factor.When body is by that stress lead to energy consumption, LKB1 can be with phosphorylation
And activate AMPK, come alleviate stress, protect animal body.Identical conclusion can also be obtained in the present invention, damaged in acute alcohol liver
The western blot for hurting mouse model goes out the decline of alcohol group AMPK and LKB1 expression, the liter of ACC and SREBP-1 expression as the result is shown
It is high.Dihydroquercetin administration group is significantly raised as the increase AMPK and LKB1 of administration concentration are expressed, ACC and SREBP-1 expression
Apparent decline.
Hereinafter, illustrating the influence that elm mushroom extract is directed to nonalcoholic fatty liver.
1. experimental method
1.1 experimental animal
Zoopery uses the C57BL male mice of 8-10 week old, weight 18-25g or so, You Yisi experimental animal company
(Jilin Changchun, China) provides.All animals are all raised in animal housing, Yanbian University.Kept for 20-25 DEG C, relative humidity
40%-70%, free water, feeding manner are kept for the 12h/12h daily cycle using three-dimensional cage raising, experimentation.
1.2. experimental method
1.2.1 animal packet
C57BL male mice, adaptation are randomly divided into after a week: low fat feed group (LFD), high lipid food group (HFD) are high in fat
Feed/alcohol (HFD/ETOH), high lipid food/alcohol+elm mushroom (HFD/ETOH+YHM100,100mg/kg), high lipid food/
Alcohol+elm mushroom (HFD/ETOH+YHM50,50mg/kg), every group of 5 mouse.Keep 20-25 DEG C of temperature, relative humidity 40%-
70%, free water, sub-cage rearing.
1.2.2 animal model and experimental method
Low fat group gives low fat feed；High in fat group is given high lipid food；It is administered alone group and gives elm by 25mg/10ml weight
Yellow mushroom (50 or 100mg/kg)；Alcohol group and administration group give 31.25% alcohol；Low fat is given according to group respectively within first 8 weeks to raise
Material, high lipid food are raised, and free water weighs mouse weight, mouse food-intake daily.To from the 9th week, except grouping raising
It is outer daily according to group, each group stomach-filling records mouse weight, mouse food-intake to respective concentration medicine (elm mushroom) and daily.Mouse
Previous late fasting is put to death, it can free water.It puts to death morning on same day 7:00 last gastric infusion and gives alcohol stomach-filling, to mouse after 9h
Carry out heart puncturing extracting blood and anatomical isolation liver, fat.A part of liver, which is immersed in formalin solution, retains a part of liver
It is dirty to use OCT freezen protective, remaining -80 DEG C of keepings.
1.2.3 the measurement of biochemical indicator
Mouse whole blood 3000rpm is centrifuged 30min, isolates serum.Microplate reader detects NSC 334200 transferase (ALT), paddy
Oxamic acid transferase (AST), triglycerides (TG), total cholesterol (TC).
1.2.4H&E (hematoxylin-eosin staining) is dyed
After 5 μm of liver paraffin section de-waxing adds water, with haematoxylin and eosin stains.After dyeing, histotomy according to
Secondary to be dehydrated by low concentration to alcohol in high concentration, dimethylbenzene is transparent, finally uses neutral gum mounting.
1.2.5 oil red O (Oil red O) is dyed
Frozen section takes out from -80 DEG C, and room temperature rises again and washes away embedding with distilled water, and 60% isopropanol embathes, oil red work
Make liquid chamber temperature and be protected from light incubation 30min, the toning of 60% isopropanol, haematoxylin is redyed, and glycerin gelatine mounting is finally used.
1.2.6 statistical analysis
For statistical analysis, experimental data Mean ± SD table using GraphPad Prism Program software
Show, analyzed with single factor test variance analysis (One-way ANOVA) and Turkey ' s multiple comparative test, whether confirmation data
With statistical significance.
2. experimental result
As shown in fig. 7, carrying out HE dyeing with oil red O stain as a result, fat drips are heavy in liver after high lipid food raising 3 months
Product increases, and shows murine chronic nonalcoholic fatty liver modeling success；After high lipid food joint alcohol carousing in 3 months, in liver
Fat drips deposition increases again.With elm mushroom extract is given, fat deposition is obviously improved with fat drop in mouse liver, almost
It is restored to normal level.
The level of ALT, AST are raised with low fat in giving high lipid food joint alcohol nursing group (HFD+ETOH) mice serum
Material (LFD) group mouse, which is compared, to be obviously increased, after giving the elm mushroom extract of various concentration, the serum compared with HFD+ETOH group
ALT and AST level are substantially reduced (Fig. 8 A and 8B).Same high lipid food merges the TG water in alcohol nursing mouse liver and serum
Flat also obvious to rise, administration group is compared compared with model group (HFD+ETOH), and the content of TG is in and is decreased obviously in mouse liver and serum
Trend (Fig. 8 C, 8D).Fig. 8 E is the testing result of total cholesterol in mice serum (TC) content, it can be deduced that, with normal group phase
Than HFD+ETOH group TC is horizontal significantly raised, and compared with model group, TC content then significantly reduces administration group.
To sum up the result shows that, high lipid food joint alcohol carousing mouse liver injury model is successfully established nonalcoholic fatty liver
Nonalcoholic fatty liver is aggravated with alcohol, illustrates that elm mushroom carouses to nonalcoholic fatty liver and nonalcoholic fatty liver alcohol
With significantly improving effect.
In the research of the elm mushroom extract, we used chmice acute alcohol models and two kinds of Chronic Alcohol model
Model carry out elm mushroom extract to the ALD research acted on and demonstrate elm mushroom extract can reduce it is small caused by alcohol
Mouse lipopexia.A large amount of experiment show to be related to during alcohol mediates alcoholic fatty liver disease many transcriptional regulatories because
Son and regulatory factor etc..SIRT1 plays an important role in the metabolic function of mammal.The activation of SIRT1 can enhance
The oxidative function of mitochondria adjusts the balance of energetic supersession.AMPK and SIRT1 collective effect, to adjust organism in various conditions
Under energy homeostasis, SREBP1 adjust fat synthesis related gene expression, three fat synthesis and metabolism in play
Particularly important effect.
In the elm mushroom extract experiment, in acute and chronic alcohol model group mouse liver, the expression of AMPK and SIRT1 are equal
Relatively normal group decline；Then relatively normal group rises for the expression of SREBP1.There is a phenomenon where lipopexia kissings in Mice Body for this
It closes.After administration group, AMPK α, SIRT1 expression is compared with alcohol group compared to rising；The expression of SREBP1 then weakens than model group, illustrates elm
The effect of the inhibition alcoholic liver lipopexia of yellow mushroom extract may be related with SIRT1-AMPK signal path.
Through the invention, the elm mushroom extract stomach-filling is given to the mouse of alcohol induced synthesis fatty liver.With alcohol
Group mouse is compared, and the inflammation for giving hepatic steatosis caused by the mouse alcohol of elm mushroom extract and being accompanied by obtains pole
Big improvement, elm mushroom extract have preferable therapeutic effect to alcoholic fatty liver.
More than, it is to be understood that referring to the appended Detailed description of the invention present invention, still, invention of the invention is claimed
Range is not limited to embodiment above-mentioned and/or attached drawing.In addition, it is desirable to understand, it is claimed for being recorded in invention
Conspicuous improvement, change and modification also belong to invention of the invention and model are claimed for the technical staff of the invention of range
It encloses.
Claims (8)
1. a kind of elm mushroom extract is in the application of the drug of preparation treatment fatty liver.
2. elm mushroom extract according to claim 1 is in the application for the treatment of alcoholic fatty liver.
3. elm mushroom extract according to claim 1 is in the application for the treatment of nonalcoholic fatty liver.
4. application according to claim 1, wherein elm mushroom extract to be included in single formulation.
5. application according to claim 1, wherein elm mushroom extract to be configured to pharmaceutical composition and combine make respectively
With.
6. the application that a kind of elm mushroom extract prevents and treats the drug of Alcoholic and non-alcoholic hepatic injury in preparation.
7. the application that a kind of elm mushroom extract prevents and treats the health care product of Alcoholic and non-alcoholic hepatic injury in preparation.
8. the application that a kind of elm mushroom extract prevents and treats the drink of Alcoholic and non-alcoholic hepatic injury in preparation.
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Cited By (1)
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| CN113456679A (en) * | 2021-07-19 | 2021-10-01 | 北京市农林科学院 | Pleurotus citrinopileatus extract and application thereof in anti-inflammation |
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