CN109771411A - Use of dihydroquercetin in preparing medicine for treating fatty liver - Google Patents
Use of dihydroquercetin in preparing medicine for treating fatty liver Download PDFInfo
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Abstract
本发明涉及二氢槲皮素或其药学上可接受的盐在治疗脂肪肝的用途。详细而言，所述二氢槲皮素或其药学上可接受的盐在治疗酒精性脂肪肝的用途以及所述二氢槲皮素或其药学上可接受的盐在治疗非酒精性脂肪肝的用途。本发明涉及二氢槲皮素苷更广泛的医学用途，使其在制备治疗酒精性肝病药物中得到应用。通过二氢槲皮素可以在酒精性肝病的初期阶段的治疗作用；同时，该成分具有毒性小的特点，并且对酒精性肝病中脂质沉积和炎症反应有显著缓解作用。
The present invention relates to the use of dihydroquercetin or a pharmaceutically acceptable salt thereof in the treatment of fatty liver. In detail, the use of the dihydroquercetin or a pharmaceutically acceptable salt thereof in the treatment of alcoholic fatty liver disease and the use of the dihydroquercetin or a pharmaceutically acceptable salt thereof in the treatment of non-alcoholic fatty liver disease the use of. The present invention relates to the wider medical application of dihydroquercetin glycosides, so that it can be applied in the preparation of medicines for treating alcoholic liver disease. Dihydroquercetin can be used for the treatment of alcoholic liver disease in the early stage; at the same time, this component has the characteristics of low toxicity, and has a significant alleviation effect on lipid deposition and inflammatory reaction in alcoholic liver disease.
Description
Technical field
The present invention relates to the purposes that a kind of dihydroquercetin or its pharmaceutically acceptable salt are used to prepare treatment fatty liver.
The present invention relates to the wider medical usage of dihydroquercetin glycosides, answer it in preparation treatment alcoholic liver medicine
With.
Background technique
Flavone compound is a large amount of ingredients being widely present in most of Chinese traditional herbs, and dihydroquercetin is in ocean
The most abundant flavanone found in green onion, milk thistle and star-spangled banner pine bark (Pseudotsuga taxifolia).Dihydroquercetin
It is widely used in food and care industry as food additives, and studies have shown that dihydroquercetin has a variety of pharmacology
Activity, such as antiviral, anti-inflammatory, anti-oxidant and anti-fibrosis.So far, main research is around dihydroquercetin
Potent oxidation resistance, dihydroquercetin can be by inhibiting fatty acid synthetase to the rouge of prostate cancer and breast cancer cell
Fat generation shows effective rejection characteristic.In addition, dihydroquercetin is found in approved hepatic silymarin
A kind of unique flavonoids.
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.
Summary of the invention
In view of prior art disadvantage, the purpose of the present invention is to provide dihydroquercetins or its pharmaceutically acceptable salt to exist
The purposes for treating fatty liver.
Purposes of the present invention, the dihydroquercetin or its pharmaceutically acceptable salt are in treatment alcoholic fatty liver
Purposes.
Purposes of the present invention, the dihydroquercetin or its pharmaceutically acceptable salt are in treatment non-alcoholic fatty
The purposes of liver.
Purposes of the present invention, wherein dihydroquercetin or its pharmaceutically acceptable salt are included in single formulation
In.
Purposes of the present invention, wherein dihydroquercetin or its pharmaceutically acceptable salt are configured to medicine group respectively
It closes object and is applied in combination.
Purposes of the present invention, wherein the dihydroquercetin or its pharmaceutically acceptable salt concentration are
121.8mg/ daily/60kg or more.
Purposes of the present invention, wherein the dihydroquercetin or its pharmaceutically acceptable salt concentration are dihydro Mongolian oak
Skin element administration concentration is 25mg/kg~100mg/kg.
The second aspect of the present invention provides a kind of dihydroquercetin or its pharmaceutically acceptable salt in preparation prevention and controls
Treat the purposes in the drug of alcoholic liver injury.
A kind of dihydroquercetin of the third aspect of the present invention or its pharmaceutically acceptable salt are in preparation prevention and treatment wine
Purposes in the health care product of essence hepatic injury.
A kind of dihydroquercetin of the fourth aspect of the present invention or its pharmaceutically acceptable salt are in preparation prevention and treatment wine
Purposes in the drink of essence hepatic injury.
It is found after the present inventor concentrates on studies, dihydroquercetin can be changed by LKB1-AMPK signal path
Lipidosis caused by kind alcohol exposure；Dihydroquercetin can improve alcohol exposure by NLRP3 inflammation corpusculum signal path
Cause inflammatory reaction.There can be relaxation effect in the initial stage of alcoholic liver disease by dihydroquercetin；Meanwhile the ingredient
Have the characteristics that small toxicity, and has significant relaxation effect to lipidosis in alcoholic liver disease and inflammatory reaction.Specifically,
Dihydroquercetin to alcoholic fatty liver caused by alcohol have good inhibiting effect, while to alcoholic fatty liver with
Inflammation has good improvement result, and cheap.Dihydroquercetin can be by inhibiting fatty acid synthetase to prostate
The fat of cancer and breast cancer cell, which generates, shows effective rejection characteristic, in addition, dihydroquercetin is in approved liver protection
The unique a kind of flavonoids found in drug silymarin.
Detailed description of the invention
Fig. 1 shows dihydroquercetin to the testing result of chmice acute alcoholic fatty liver model biochemical indicator；
Influence Fig. 2 shows dihydroquercetin to carousing alcoholic fatty liver mouse liver tectology；
Fig. 3 shows influence of the dihydroquercetin to acute alcoholic fatty liver mouse liver tectology；
Fig. 4 shows the influence of inflammation corpusculum activation of the dihydroquercetin to chmice acute alcoholic fatty liver；
It is horizontal that Fig. 5 shows lipopexia and inflammatory factor expression in the HepG2 cell that dihydroquercetin stimulates alcohol
It influences；
Fig. 6 shows the influence of lipopexia in dihydroquercetin P2X7R dependence regulation alcohol stimulation HepG2 cell；
Fig. 7 shows influence of the dihydroquercetin to every biochemical indicator of mice serum and liver；
Fig. 8 shows influence (200 times) of the dihydroquercetin to murine liver tissue oil red fat stains.
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.
The dihydroquercetin is the compound that the double bond between 2 of the pyranoid ring of Quercetin and 3 is reduced, at this
In invention, the example of the pharmaceutically acceptable salt of the dihydroquercetin includes and salt formed by inorganic base and organic base institute
At salt, with salt etc. formed by basic amino acid.
Example with salt formed by inorganic base includes and alkali metal (such as sodium, potassium etc.), alkaline-earth metal (such as calcium, magnesium etc.)
And salt formed by aluminium, ammonium etc..Example with salt formed by organic base include with trimethylamine, triethylamine, pyridine, picoline,
Ethanol amine, diethanol amine, triethanolamine, dicyclohexylamine, N, salt formed by N- dibenzyl-ethylenediamin etc..With basic amino acid institute
At the example of salt include and arginine, lysine.
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.
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..
In the present invention, the dihydroquercetin significantly inhibits the liver cell lipopexia of AMPK mediation and P2X7R is mediated
Inflammatory response.The dihydroquercetin has no the side reaction for fat deposition and inflammation under 25mg/kg dosage.According to medicine
Object human trial safety risk management planning guide principle (U.S. FDA U.S. sanitary and the formulation of public service portion) for the first time, for
Adult healthy volunteer, it is every that the mouse dosage of 25mg/kg dihydroquercetin is equivalent to 121.8mg/ in clinical phase test
It/dosage of 60kg normal adult is the optimal dose that human body is normally taken in daily.
Embodiment
The present invention uses 10 week old, and the male C57BL/6 mouse of 20~25g of weight is purchased from this experimental animal section of Changchun hundred million
Skill Co., Ltd (Jilin, China), all mouse keep the periodicity of illumination round the clock of 12h/12h during test, and living environment is kept
In temperature ± 23 ± 2 DEG C, humidity 55 ± 5%.Guarantee balanced standard diet and sufficient drinking public water supply.In the present invention,
C57BL/6 mouse adapts to be randomly divided into six groups after a week: normal group, alcohol group is administered alone group, and alcohol adds low dose group, wine
Finishing middle dose group, alcohol increase dosage group, every group of 10 mouse.Method is as follows: normal group gives solid feed；It is administered alone
Group gives dihydroquercetin by 25mg/ml weight；Alcohol group gives 33% alcohol；Be administered low middle high dose group by 1mg/ml,
5mg/ml, 25mg/ml weight give 33% alcohol after giving dihydroquercetin respectively；Dihydroquercetin and alcohol are total every 12 hours
To 3 times, give for the 3rd time after 4 hours of dihydroquercetin and alcohol to heart puncturing extracting blood after all mouse etherizations, and
Anatomical isolation liver.Serum is left and taken after blood 3000rpm centrifugation, liver great Ye, which is immersed in neutral formalin solution, to be retained,
Liver leaflet is dispensed into -80 DEG C of keepings in 1.5ml centrifuge tube.
In C57BL/6 chmice acute alcoholic hepatic injury model, normal group mouse liver lobuli hepatis structure is complete；Alcohol group
Occur a large amount of fat bubbles not of uniform size around mouse liver portal area, liver cell balloon sample, which becomes apparent, waits pathological changes；It gives
Medicine group is significantly reduced with the increase steatosis of administration concentration, has been approached normal group.
Alcohol is considered causing fatty liver by changing liver NADH/NAD+ ratio, that is, inhibits fatty acid oxidation and stimulation
Fat generates.It has recently been demonstrated that Ethanol intake may directly affect the activity of AMP deopendent protein kinase (AMPK).
AMPK is a kind of protein kinase that energy and metabolic balance are adjusted in liver, heart, skeletal muscle, is that adiponectin plays biology
The key signal molecule of effect.The activation of liver AMPK can increase fatty acid oxidation, reduce the synthesis of fat, inhibit glycogen and gallbladder
Sterol synthesis adjusts energy homeostasis balance.AMPK is called " switch for adjusting cellular energy metabolism ", and AMPK is that liver adjusts rouge
The important target spot of matter metabolic balance.The mouse AMPK activity inhibited of feeding ethyl alcohol, AMPK can be by inhibiting acetyl-coa carboxylase
Enzyme (ACC) reduces malonyl coenzyme A to control fatty acid metabolism.AMPK also can be by inhibiting SREBP-1 to inhibit fatty acid
Synthesis, a kind of transcription factor that lipid can be promoted to generate related gene.Research finds that the activation of AMPK needs upstream kinases pair
Thr172 carries out phosphorylation to complete on AMPK α subunit activation ring.LKB1 is called STK11 (Serine Threonine
Protein Linase 11) it is the silk gas acid/Serineprotein kinase family member encoded by lkb1 gene, it is a kind of suppression
Cancer factor.When body is by that stress lead to energy consumption, LKB1 with phosphorylation and can activate AMPK, come alleviate stress, protection
Animal body.Identical conclusion can also be obtained in the present invention, it is aobvious in the western blot result of acute alcohol mouse liver injury models
The decline of alcohol group AMPK and LKB1 expression, the raising of ACC and SREBP-1 expression are shown.Dihydroquercetin administration group is with giving
The increase AMPK and LKB1 of concentration express significantly raised, ACC and the apparent decline of SREBP-1 expression.
In order to further confirm, it is respectively 100nM, 25nM, 12.5nM bis- that HepG2 cell, which gives concentration, in vitro experiment
Large dosage of alcohol inducing hepatocyte damage is given at hydrogen Quercetin, interval after 1 hour, albumen is scraped after 24 hours in -80 DEG C of keepings.
Also the identical conclusion of above-mentioned experiment in vivo is obtained in experiment in vitro, illustrates that dihydroquercetin can be influenced by adjusting the note living of enzyme
Lipid metaboli inhibits the synthesis of liver fat.
Alcoholic liver disease (ALD) is characterized in that liver lipid accumulation (steatosis) and inflammatory reaction, increases inflammatory cell
The expression of the factor.Two kinds in these cell factors, interleukin-1 ' beta ' (IL-1 β) and IL-18 are needed through NOD sample receptor
(NLR) member of family activates caspase-1 (caspase-1).When inflammatory reaction, local Extracellular ATP concentration is increased,
In conjunction with and activate P2X7R receptor formed duct, lead to K+Outflow and Ca2+Interior stream, cell membrane potential change, and make the dye being coupled with it
Material absorption duct pannexin-1 is open to form duct, allows bacterial product by entering cell with Extracellular ATP, while promoting core
Thuja acid combination oligomerization domain sample receptor family (Nucleotide-binding oligomerization domain like
Receptors, NLRs) inflammation corpusculum NLRP3 assembly, the pathogen-associated molecular patterns such as NLRP3 and virus, opsonigenous substance
(Pathogen Associated Molecular pattern, PAMP) changes in conjunction with rear conformation, its functional structure of exposure
Domain, then oligomerization, and Caspase-1 precursor is raised by The homotype interaction, lead to its conformational change, generates activity
Caspase-1, that is, IL-1 invertase, be responsible for for inactive IL-1 β, IL-18 precursor being cut into the functional inflammatory cell of tool because
Son.The expression of P2X7R and NLRP3 obviously increases around central vein in alcohol group；Administration group compared with alcohol group, the two
Expression is remarkably decreased；Compared with normal group, the expression of the two does not change significantly dihydroquercetin control group.Alcohol group with
Normal group compares, and the content of IL-1 β dramatically increases in serum；For administration group compared with alcohol group, the content of -1 β of serum IL is significant
Decline.The expression of Pro-IL-1 β and Caspase-1 in liver organization are observed, in alcohol group Pro-IL-1 β and
The expression of Caspase-1 significantly increases, and the expression of Pro-IL-1 β and Caspase-1 are obviously pressed down in liver organization after administration
System.HepG2 cell gives LPS after giving dihydroquercetin one hour, ATP is given after 24 hours, -80 DEG C of albumen are scraped after 30 minutes
It takes care of, is also obtained for above-mentioned identical conclusion in experiment in vitro.
Fig. 1 shows biochemical indicator testing result, in the present invention, the measurement of Triglycerides in Serum (Serum TG) according to
Following methods carry out: eyeball of mouse takes blood, stands, and 3000 turns/min is centrifuged 10min, isolates serum.
1) gradient dilution is carried out with the titer of 200mg/ml concentration, prepares 0mg/ml, 6.25mg/ml, 12.5mg/ altogether
The standard of ml, 25mg/ml, 50mg/ml, 100mg/ml, 200mg/ml various concentration is put into the Tube of 7 different 1.5ml
In.250 μ l working reagents are added in the Tube of each 1.5ml.It is and 250 μ l working reagents is only added in the Tube of blank.Into
Row is vortexed, after centrifugation, is put into 37 DEG C of water-baths 15 minutes.After 15 minutes, 500 μ l distilled water are added in each Tube, into
Row is vortexed, is centrifuged, and makes to be uniformly mixed.
2) in sample Tube, 48 different blood of 250 μ l working reagents and 5 μ l are put into the Tube of each 1.5ml
Clearly.After being vortexed, being centrifuged, it is put into 37 DEG C of water-baths 15 minutes.After 15 minutes, 500 μ l distilled water are added in each Tube,
It is being vortexed, is being centrifuged, making to be uniformly mixed.
3) after mixing, equipped with 7 standards, 1 blank, 48 samples the Tube of totally 56 1.5ml in every time
200 μ l to be inhaled to be added in 3 secondary orifices of 96 orifice plates, 96 orifice plates after adding are put into micropore board detector, wavelength 500nm is set, into
Row detection.
In the present invention, the measurement of triglycerides (Serum TG) carries out in accordance with the following methods in liver:
1) it after every 100mg liver adds 9 times of physiological saline, is homogenized.
2) in every 100 μ l homogenate suspension, the mixed liquor of 375 μ l chloroforms and 125 μ l methanol is added.It is vortexed 20 minutes.
3) 125 μ l chloroforms are added, are vortexed 20 minutes.
4) 125 μ l distilled water are added, are vortexed 15 minutes.
5) 1200 turns/min is centrifuged 5 minutes at room temperature.
6) it collects in 40 μ l to new 1.5mlTube of subnatant.
7) gradient dilution is carried out with the titer of 200mg/ml concentration, prepares 0mg/ml, 6.25mg/ml, 12.5mg/ altogether
The standard of ml, 25mg/ml, 50mg/ml, 100mg/ml, 200mg/ml various concentration is put into the Tube of 7 different 1.5ml
In.750 μ l working reagents are added in the Tube of each 1.5ml.It is and distilled water is only added in the Tube of blank.Be vortexed,
After centrifugation, it is protected from light and is put into 37 DEG C of water-baths 20 minutes.
8) in sample Tube, 48 different blood of 750 μ l working reagents and 7.5 μ l are put into the Tube of each 1.5ml
Clearly.After being vortexed, being centrifuged, it is put into 37 DEG C of water-baths 15 minutes.
9) after mixing, equipped with 7 standards, 1 blank, sample the Tube of totally 56 1.5ml in inhale 200 every time
μ l is added in 3 secondary orifices of 96 orifice plates, and 96 orifice plates after adding are put into micropore board detector, is set wavelength 500nm, is examined
It surveys.
The ALT (glutamic-pyruvic transaminase) and AST (glutamic-oxalacetic transaminease) be in human body sugar and the mutual phase transition of protein needed for
Enzyme.The degree of impairment of the ALT and AST reflection liver cell, ALT reflect liver cell acute injury.The AST reflection liver is thin
Cellular damage degree.TG (triglyceride) is also known as fat, is to be synthesized by food fat with liver, is long chain fatty acids and glycerol shape
At fat molecule, be most important one kind of blood lipid in blood.Serum is obtained after mouse blood centrifugation, is analyzed with full-automatic biochemical
Instrument has detected the level of serum alt, AST and TG respectively.As can be seen that alcohol nursing group more normally organizes phase from Figure 1A
Than the horizontal significant raising (###P < 0.01) of serum alt；Dihydroquercetin administration group serum alt and alcohol nursing group phase
Than being remarkably decreased (* * * P < 0.001).As can be seen that more normal group of AST level of alcohol nursing group than more significant raising in Figure 1B
(###P < 0.001)；Administration group serum AST is decreased obviously (* * * P < 0.01) compared with alcohol nursing group.Fig. 1 C is in mice serum
The testing result of TG content, it can be seen that alcohol group is compared with normal group, content apparent increase (the ##P < of TG in serum
0.01)；Administration group is compared with alcohol nursing group, and the content of TG is substantially reduced (* * * P < 0.01) in serum.Fig. 1 D is Mouse Liver
Dirty tissue T G content results.Alcohol nursing group liver TG content, which is more normally organized, rises (##P < 0.01) compared to obvious；Administration group compared with
Alcohol nursing group is compared and is decreased obviously (* * P < 0.01).
These results suggest that carousing mice alcoholic fatty liver model modeling success, dihydroquercetin is to alcohol fatty
Liver has excellent protective effect.
As shown in Fig. 2, HE coloration result can be seen that normal group lobuli hepatis structure complete, no inflammation cellular infiltration；Alcohol
Occurs obvious fat bubble not of uniform size around the portal area of nursing group, ballooning degeneration of liver cells is obvious；Administration group fat bubble is bright
Aobvious to become smaller and quantity is reduced, ballooning degeneration of liver cells phenomenon mitigates, and high dose administration group lobuli hepatis structure has been approached normal group；Two
Steatosis (Fig. 2A) is had no in hydrogen Quercetin control group.Oil red O stain is that the property of lipid is soluble in using dyestuff to observe
The content of tissue lipid.Find out from result, alcohol group cytolipin content is significantly raised；Give the mouse of dihydroquercetin with
The raising liver cell lipid content of administration concentration significantly decreases compared with alcohol group；Dihydroquercetin control group has no liver cell rouge
The increase (Fig. 2 B) of fat content.
Fig. 3 shows influence of the dihydroquercetin to acute alcoholic fatty liver mouse liver tectology, HE dyeing knot
It is complete that fruit can be seen that normal group mouse liver lobuli hepatis structure；There are a large amount of sizes around portal area in alcohol group mouse liver
Different fat bubble, liver cell balloon sample become apparent；Administration group is significantly reduced with the increase steatosis of administration concentration,
Close to normal group (Fig. 3 A).Oil red O stain be soluble in using dyestuff the property of lipid come in tissue visualization lipid content it is more
It is few.Fig. 3 B is oil red coloration result, as can be seen that alcohol group dyed color is obviously relatively normally organized deeply from experimental result；Administration
Oil red O stain obviously shoals afterwards, illustrates that the liver lipids content of mouse after being administered significantly declines.
As shown in Figure 3 C, alcohol can stimulate SREBP-1 to activate.From the point of view of the immune group coloration result of SREBP-1, give
The expression of SREBP-1 is significantly raised around mouse liver tissue central vein after alcohol forage feed 4 weeks；Administration group and alcohol group
It is obviously shallower compared to the coloration result of SREBP-1.
In addition, we, which are metabolized closely related protein expression with lipid synthesis to AMPK, LKB1 and ACC etc., has carried out albumen
Blot experiment, Fig. 3 E are protein blot experiment as a result, carry out gray analysis to it with quantity one software, can be with from Fig. 3 E
Find out, the expression of alcohol group AMPK, LKB1 significantly reduces (###P < 0.001), and the expression of ACC is more normally organized to be increased compared to significant
Add (###P < 0.001)；Administration group is compared with alcohol group, and the expression of AMPK, LKB1 significantly increase (* * * P < 0.001), ACC's
Expression significantly reduces (* * * P < 0.001).
Fig. 4 shows influence of the dihydroquercetin to the activation of the inflammation corpusculum of chmice acute alcoholic fatty liver, using immune
The expression of acute alcohol stomach-filling model mice P2X7R and NLRP3 are observed by histochemical staining method.From Fig. 4 A and Fig. 4 B
In as can be seen that in alcohol group the expression of P2X7R and NLRP3 obviously increased around central vein；Administration group and alcohol group phase
Than the expression of the two is remarkably decreased；Compared with normal group, the expression of the two does not become significantly dihydroquercetin control group
Change.It can inhibit the expression of inflammatory factor caused by alcohol to verify dihydroquercetin, we test detection blood by ELISA
The content of IL-1 β in clear.By alcohol group it can be seen from Fig. 4 C compared with normal group, the content of IL-1 β is dramatically increased in serum
(###P < 0.001)；Administration group is compared with alcohol group, and the content of IL-1 β is remarkably decreased (* * * P < 0.001) in serum.It utilizes
Western blotting observes the expression of Pro-IL-1 β and Caspase-1 in liver organization, as a result as shown in Figure 4 D: wine
The expression of Pro-IL-1 β and Caspase-1 significantly increase in smart group, Pro-IL-1 β and Caspase-1 in liver organization after administration
Expression be obviously suppressed.
It is horizontal that Fig. 5 shows lipopexia and inflammatory factor expression in the HepG2 cell that dihydroquercetin stimulates alcohol
It influences.The result of AMPK α, ACC and IL-1 β protein expression in the HepG2 cell stimulated using Western blotting detection alcohol.
Alcohol group is substantially reduced with the expression for normally organizing compared to AMPK α as can be seen from the results, ACC and IL-1 β expression is significant to rise
It is high.The expression of AMPK α significantly increases after administration, the expression of ACC and IL-1 β significantly reduces.
Fig. 6 shows the influence of lipopexia in dihydroquercetin P2X7R dependence regulation alcohol stimulation HepG2 cell.Rouge
Polysaccharide (LPS) is the main component of gram-negative bacteria cell wall, can act on inflammation and immunocyte, such as monokaryon/macrophage
Cell, endothelial cell etc., acute-phase response (acute phase reac-tion, APR) in inductor.P2X7R is extracellular
The ion-channel ligands of ATP activation, and ATP is the classical activator of NLRP3 inflammation body, research confirms, P2X7R is mediating ATP
Endogenous danger signal activating immune cell NLRP3 inflammation body intracellular promotes activity IL-18 and IL-1 β secretion, booster immunization thin
Born of the same parents' inflammation responsing reaction plays an important role.It can be seen that LPS+ATP group is compared with normally organizing from Fig. 6 A western blot result
The expression of P2X7R and pro-IL-1 β is significantly raised, administration group with LPS+ATP group compare with to want concentration increase P2X7R with
The expression of pro-IL-1 β gradually decreases.In addition, we are observed after silencing P2X7R with immunofluorescence dyeing in HepG2 cell
The expression of SREBP-1.It can be seen that the alcohol group of control group is with normally organizing the red fluorescence compared to SREBP-1 from Fig. 6 B result
It is remarkably reinforced, normal group of silencing P2X7R, the red fluorescence of model group and administration group SREBP-1 are obviously reduced.
Hereinafter, the dihydroquercetin or its pharmaceutically acceptable salt are treating non-alcohol during the present invention will be described in detail
The purposes of property fatty liver.
The present invention uses kunming mice, 18~20g of weight.At 25 DEG C of constant temperature, humidity 50%, 12h is dark, 12h illumination
Animal housing's raising, feeding manner is using three-dimensional cage raising.It is randomly divided into 10 groups: normal group (N, n=6), control feed group
(LFD, n=6), high lipid food group (HFD, n=6), high lipid food combine alcohol carousing group (HFD+ETOH, n=6), low dosage
Dihydroquercetin increases rouge Combined fodder alcohol carousing group (HFD+ETOH+TAX25, n=6), and middle dosage dihydroquercetin is increased
Rouge Combined fodder alcohol carousing group (HFD+ETOH+TAX50, n=6), high dose dihydroquercetin increase rouge Combined fodder alcohol
Carousing group (HFD+ETOH+TAX100, n=6) and high dose dihydroquercetin group (TAX100, n=6).Under standard environment into
After 4 days adaptive feedings of row, normal group and high dose dihydroquercetin group feed normal diet, and control feed group feeds low fat feed,
Remaining 5 groups are respectively fed 60% high lipid food, and the every group of drinking-water that all freers is raised 4 days again.
Above-mentioned mouse is randomly divided into 10 groups: normal group, control feed group, high lipid food group, high lipid food joint alcohol
Carousing group, low dosage dihydroquercetin increase rouge Combined fodder alcohol carousing group, and middle dosage dihydroquercetin adds high lipid food to join
Alcohol carousing group is closed, high dose dihydroquercetin increases rouge Combined fodder alcohol carousing group, high dose dihydroquercetin group.
In the present invention, the dihydroquercetin that low dosage dihydroquercetin increases rouge Combined fodder alcohol carousing group is administered dense
Degree is 25mg/kg, to increase rouge Combined fodder alcohol carousing group dihydroquercetin administration concentration be 50mg/ to middle dosage dihydroquercetin
Kg, high dose dihydroquercetin increase rouge Combined fodder alcohol carousing group and high dose dihydroquercetin group administration concentration is
100mg/kg。
Draw it was found by the inventors of the present invention that dihydroquercetin can improve alcohol exposure by LKB1-AMPK signal path
The lipidosis risen；Dihydroquercetin can improve alcohol exposure by NLRP3 inflammation corpusculum signal path and cause inflammatory reaction.
It can be in the therapeutic effect of the initial stage of alcoholic liver disease by dihydroquercetin；Meanwhile the ingredient has the spy of small toxicity
Point, and have significant relaxation effect to lipidosis in alcoholic liver disease and inflammatory reaction.Specifically, dihydroquercetin is to wine
Alcoholic fatty liver caused by essence has a good inhibiting effect, at the same to alcoholic fatty liver with inflammation have it is certain
Improvement result, and it is cheap.
Specify that dihydroquercetin passes through the activity influence Fatty synthesis and fat oxidation of AMPK according to the present invention, to press down
The accumulation of liver fat caused by preparing alcohol；Dihydroquercetin is to P2X7R-Caspase-1-NLRP3 inflammation corpusculum caused by alcohol
Activation have certain inhibitory effect；Dihydroquercetin makees alcoholic fatty liver caused by alcohol with good inhibition
With, at the same to alcoholic fatty liver with inflammation tool have some improvement.
Fig. 7 indicates influence and dihydroquercetin of the dihydroquercetin to triglycerides in mice serum and liver to small
The influence of transaminase in mouse serum, as shown in figs. 7 a-b, after mouse gives alcohol and maltodextrin stomach-filling respectively, normal group with
The active no significant difference of glutamic-oxalacetic transaminease (AST) activity and glutamic-pyruvic transaminase (ALT) of control feed group.High lipid food joint
Alcohol carousing group P < 0.05 compared with ALT activity with the AST of high lipid food group, there is significant difference.High, medium and low dosage dihydro
Although Quercetin, which is increased compared with rouge Combined fodder alcohol carousing group combines alcohol carousing group with high lipid food, not to be had statistically
Significant difference, but have the visible trend for increasing AST and the decline of ALT activity with dihydroquercetin concentration.Mouse point
After not giving alcohol and maltodextrin stomach-filling, as seen in figure 7 c, normal group, control feed group and high dose dihydroquercetin group it
Between serum TG content there is no apparent difference.It is each that the serum TG content of high lipid food joint alcohol carousing group is apparently higher than other
Group is also increased compared with normal group, illustrates modeling success.In addition, high, medium and low dosage dihydroquercetin adds high lipid food
Serum TG content is all declined compared with joint alcohol carousing group combines alcohol carousing group with high lipid food, and is shown according to two
The concentration of hydrogen Quercetin, which increases serum TG content, successively downward trend.Mouse gives alcohol and maltodextrin stomach-filling respectively
Afterwards, as illustrated in fig. 7d, liver TG content is without apparent poor between normal group, control feed group and high dose dihydroquercetin group
Not.The liver TG content of high lipid food joint alcohol carousing group is apparently higher than high lipid food group, is also risen compared with normal group
Height illustrates modeling success.It is shown in addition, high, medium and low dosage dihydroquercetin increases rouge Combined fodder alcohol carousing group each group
Increasing liver TG content according to the concentration of dihydroquercetin has successively downward trend.
In the present invention, monoglyceride (TG) content in high lipid food group hepatic tissue and serum is with normal group than more significant
It increases, shows that high fat diet can cause fatty liver.But compared with combining alcohol carousing group with high lipid food, its fatty liver journey of the latter
It spends more obvious.It follows that the damage to liver cell, which becomes apparent from, to be also easier to lead when high fat diet and alcohol intake synergy
Cause fatty liver.Various dose dihydroquercetin is increased TG content in rouge Combined fodder alcohol carousing group serum and is combined with high lipid food
Alcohol carousing group compares, and TG content has the tendency that being substantially reduced.This show joint alcohol high in fat carouse in the case where, two
Hydrogen Quercetin can effectively improve fat deposition in liver, enhance liver cell free radical resisting attacking ability, play certain protection liver
Effect.Increase content of triglyceride feeding more high in fat in rouge Combined fodder alcohol carousing group liver in various dose dihydroquercetin
Material joint alcohol carousing group is increased, but is increased according to dihydroquercetin dosage and reduced, and shows dihydro quercitrin
Element also has curative effect.
Through the invention, the liver that alcohol carousing group is combined in HE dyeing with oil red fat stains as a result, high lipid food is carried out
Interior fat drips deposition, inflammatory cell infiltration show mouse alcoholic fatty liver modeling success.And the difference fed with high lipid food
Fat deposition makes moderate progress with the increase of dihydroquercetin administration concentration in the liver of concentration dihydroquercetin stomach-filling mouse,
Although liver does not return to normal level, but it can illustrate that dihydroquercetin has certain treatment in alcoholic fatty liver
Effect.
The inventors discovered that although various concentration dihydroquercetin increases rouge, Combined fodder alcohol carousing group can reduce wine
The biochemical indicator of essence fatty liver and liver cell prevent to tend to normalization, these indexs and prevention fail to be completely recovered to normal water
It is flat, but dihydroquercetin, in terms for the treatment of alcoholic fatty liver and nonalcoholic fatty liver, right and wrong are often with potential day
Right antioxidant.
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 (10)
1. dihydroquercetin or its pharmaceutically acceptable salt are in the purposes for treating fatty liver.
2. purposes according to claim 1, the dihydroquercetin or its pharmaceutically acceptable salt are in treatment Alcoholic
The purposes of fatty liver.
3. purposes according to claim 1, the dihydroquercetin or its pharmaceutically acceptable salt are treating non-alcohol
The purposes of property fatty liver.
4. purposes according to claim 1, wherein be included in dihydroquercetin or its pharmaceutically acceptable salt single
In preparation.
5. purposes according to claim 1, wherein dihydroquercetin or its pharmaceutically acceptable salt to be configured to respectively
Pharmaceutical composition is simultaneously applied in combination.
6. purposes according to claim 2, wherein the dihydroquercetin or its pharmaceutically acceptable salt concentration are
121.8mg/ daily/60kg or more.
7. purposes according to claim 3, wherein the dihydroquercetin or its pharmaceutically acceptable salt concentration are two
Hydrogen Quercetin administration concentration is 25mg/kg~100mg/kg.
8. a kind of dihydroquercetin or its pharmaceutically acceptable salt are in the drug that preparation prevents and treats alcoholic liver injury
Purposes.
9. the health care product that a kind of dihydroquercetin or its pharmaceutically acceptable salt prevent and treat alcoholic liver injury in preparation
In purposes.
10. a kind of dihydroquercetin or its pharmaceutically acceptable salt are in the drink that preparation prevents and treats alcoholic liver injury
Purposes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113559113A (en) * | 2021-04-02 | 2021-10-29 | 广东昊邦医药健康有限责任公司 | Application of NAD + supplement in preparation of medicine for treating acute alcoholic liver injury |
| CN114007604A (en) * | 2019-06-25 | 2022-02-01 | 独立行政法人国立病院机构 | Liver fibrosis inhibitor and brown adipocyte activator containing taxifolin |
| CN118806871A (en) * | 2024-06-20 | 2024-10-22 | 瀚天生物(北京)科技有限公司 | Composition with alcohol sobering and hangover-resistant effects, preparation method and application |
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| CN102477453A (en) * | 2010-11-22 | 2012-05-30 | 天津药物研究院 | Method for preparing taxifolin monomer from engelhardtia leaves and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114007604A (en) * | 2019-06-25 | 2022-02-01 | 独立行政法人国立病院机构 | Liver fibrosis inhibitor and brown adipocyte activator containing taxifolin |
| EP3991728A4 (en) * | 2019-06-25 | 2023-08-09 | National Hospital Organization | HEPATIC FIBROSIS INHIBITOR AND TAXIFOLINE-CONTAINING BROWN FAT CELL ACTIVATING AGENT |
| CN113559113A (en) * | 2021-04-02 | 2021-10-29 | 广东昊邦医药健康有限责任公司 | Application of NAD + supplement in preparation of medicine for treating acute alcoholic liver injury |
| CN118806871A (en) * | 2024-06-20 | 2024-10-22 | 瀚天生物(北京)科技有限公司 | Composition with alcohol sobering and hangover-resistant effects, preparation method and application |
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Application publication date: 20190521 |