CN107441078A - A kind of pharmaceutical composition for treating diabetes and its production and use - Google Patents

A kind of pharmaceutical composition for treating diabetes and its production and use Download PDF

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CN107441078A
CN107441078A CN201710577168.2A CN201710577168A CN107441078A CN 107441078 A CN107441078 A CN 107441078A CN 201710577168 A CN201710577168 A CN 201710577168A CN 107441078 A CN107441078 A CN 107441078A
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phellinus
pharmaceutical composition
dihydroxy
extract
polyphenol extract
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CN107441078B (en
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杨新洲
黄密
郑思建
赵平
黄蕴
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South Central Minzu University
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South Central University for Nationalities
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract

Invention provides a kind of pharmaceutical composition for treating diabetes and its production and use, belongs to field of medicaments.The pharmaceutical composition of this treatment diabetes, including Inoscavin C, 7, the methoxy isoflavone of 3 ' dihydroxy 5 ', 7,8 dihydroxycoumarins and 3, at least one of 4 dihydroxy benzylideneacetones, and pharmaceutically acceptable carrier or auxiliary material.This pharmaceutical composition it is widely used, including：Purposes in the activator for promoting glucose uptake is prepared, prepare purposes in the activator of adenosine monophosphate activated protein kinase, treated or prevented in the purposes in preparing the activator of GLUT4 and preparing by the purposes in the medicine or health food of metabolic disorder associated diseases; to provide new treatment means and thinking by the treatment of metabolic disorder associated diseases, while also develop the new medical value of Phellinus.

Description

A kind of pharmaceutical composition for treating diabetes and its production and use
Technical field
The present invention relates to field of medicaments, in particular to a kind of pharmaceutical composition for treating diabetes and its preparation side Method and purposes.
Background technology
Phellinus also known as Sang Chen, mulberry ear, white heart-rot fungus, Phellinus mushroom, belong to Basidiomycota, Hymenochaetaceae (Phellinus Igniarus), it is a kind of main parasitic in large-scale medicinal true on the trunks such as mulberry tree, willow, syringa reticulata var mandshurica, pine tree, silver birch Bacterium.It is Phellinus nature and flavor slight bitter, cold, it is mainly used in treating prolapse of the anus in traditional medication and rushes down more than blood, married woman's strain, the puckery pain of navel abdomen, cold The symptoms such as carbuncle is festered, blood strangury, and there is the effect of changing drink, antidiarrheal.Found in being developed to its in-depth study, Phellinus has Good antitumor action, it is famous in the world, in addition, Phellinus also has a variety of work(such as protection liver, treatment gastric ulcer Effect.
On the extraction process of Phellinus, the extraction and separation of Phellinus polysaccharide are focused primarily upon at present.Nineteen sixty-eight Japanese Ikekawa is studied Phellinus, as a result finds that the wild fruiting body extract of Phellinus is high to small white mouse sarcoma S-180 inhibiting rate Up to 93%.Sasaki afterwards etc. confirms that anti-tumor active substance is Phellinus polysaccharide in Phellinus.The researchs such as Kimt find Phellinus Polysaccharide has the function that hypoglycemic.Zheng Yuan is isolated and purified to Phellinus polysaccharide and found during activity research, and Phellinus intracellular is more Sugar has good anti-aging effects, but has no research report to the anti-diabetic activity composition of medium and small polarity.
Because Phellinus has good antitumor activity, during the research for Phellinus both at home and abroad is just carried out in high gear. Beijing consonance institute of materia medica's stone is made contributions seminar isolated mulberry from the dichloromethane of Phellinus ethanol extract extraction part Flavine A-J, hispidin.Zhang Wanguo etc. has found that phellinus linteus extract has the function that to protect liver function.Lee I.K. etc. are from mulberry Isolated chlorophellins A-C in yellow cometabolism, and study and find that they have antagonism PPAR- γ effects, Especially chlorophellin C antagonistic effect is most strong, can be compared favourably with Rosiglitazone.Yeon Sil Lee (2008) etc. Multiple compounds are separated to from Phellinus, and studies and finds davallialactone, hypholomine B, ellagic acid There is antagonism aldose.
Chinese Traditional Medicine is the valuable cultural heritage in China, and tremendous contribution is made that for human health cause.China possesses Abundant Chinese herbal medicine resource and the thousands of years clinical experiences prevented and cured diseases using natural drug, except above-mentioned anti-oxidant, protection liver Outside the effect such as dirty, antitumor, the research for Phellinus medical value still has development space.
The content of the invention
The first object of the present invention is to provide a kind of pharmaceutical composition for treating diabetes, the work in the pharmaceutical composition Property ingredient origin is in Phellinus, available for treating or prevent diabetes.
The second object of the present invention is to provide a kind of preparation method of Phellinus polyphenol extract, can phase using this method To readily extraction obtains Phellinus polyphenol extract from Phellinus, to be further used for active component Inoscavin C, 7,3 '- Dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzylideneacetones isolate and purify.
The third object of the present invention is to provide a kind of application of aforementioned pharmaceutical compositions, is included in preparation and promotes glucose Purposes in the activator of intake, prepare purposes in the activator of adenosine monophosphate activated protein kinase, preparing glucose Purposes in the activator of transport protein 4 and preparing the medicine or the guarantor that treat or prevent by metabolic disorder associated diseases Purposes in health food, to provide new treatment means and thinking by the treatment of metabolic disorder associated diseases, while also develop The new medical value of Phellinus.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of pharmaceutical composition for treating diabetes, including Inoscavin C, 7, the different Huang of 3 '-dihydroxy -5 '-methoxyl group At least one of ketone, Daphnelin or 3,4- dihydroxy benzylideneacetone, and pharmaceutically acceptable carrier or auxiliary Material.
A kind of preparation method of Phellinus polyphenol extract, it includes：
Using alcohol-water mixed solution extraction Phellinus, resulting phellinus linteus extract is extracted with petroleum ether, dichloromethane successively Take, obtain Phellinus dichloromethane extract；And
Phellinus dichloromethane extract is crossed into polyamide column, with alcohol-water mixed solution gradient elution, it is 77 to collect eluant, eluent Cut during~83% ethanol solution, obtain Phellinus polyphenol extract.
A kind of purposes of pharmaceutical composition in the activator for promoting glucose uptake is prepared, pharmaceutical composition include Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins or 3,4- dihydroxy benzylideneacetones At least one.
A kind of purposes of pharmaceutical composition on GLUT4 transhipment is prepared in the activator of film, drug regimen Thing includes Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins or 3,4- dihydroxy benzyls Pitch at least one of acetone.
A kind of purposes of pharmaceutical composition in the activator for preparing adenosine monophosphate activated protein kinase, pharmaceutical composition Including Phellinus polyphenol extract, the Phellinus polyphenol extract includes Inoscavin C, 7, and 3 '-dihydroxy -5 '-methoxyl group is different At least one of flavones, Daphnelin or 3,4- dihydroxy benzylideneacetone.
A kind of pharmaceutical composition is preparing treatment or prevention by the medicine or health food of metabolic disorder associated diseases Purposes, pharmaceutical composition include Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, Daphnelin Or at least one of 3,4- dihydroxy benzylideneacetones.
Compared with prior art, beneficial effects of the present invention for example including：
The present invention isolates and purifies to obtain the active component for having diabetes therapeutic activity from traditional Chinese medicine Phellinus： Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, Daphnelin or 3,4- dihydroxy benzylideneacetone, No matter these four active components are single use or are used in combination, and are respectively provided with good hypoglycemic activity, clinical available for preparing Diabetic.
Research is found, Phellinus polyphenol extract and above-mentioned four kinds of active components, can reduce the disease caused by metabolic disorder The content of T-CHOL and triglycerides in disease, it can alleviate or treat hyperlipemia caused by disorders of lipid metabolism, sugar Urine disease.
Find simultaneously, Phellinus dichloromethane extract, polyphenol extract and above-mentioned four kinds of active components, can be Adenosine monophosphate activated protein kinase and membrane of glucose transporter 4 are activated to a certain extent, so as to promote taking the photograph for glucose Take, to treat or prevent the disease caused by metabolic disorder, such as diabetes, obesity, vascular hypertension and hyperlipemia.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the preparation flow figure of Phellinus polyphenol extract in embodiment 1~3；
Fig. 2 is the preparation flow figure of four kinds of active components in embodiment 4；
Fig. 3 is that (ordinate is that fluorescence is strong for influence of the Phellinus polyphenol extract to L6 grape cell Sugar intakes in experimental example one Degree),***P<0.001,**P<0.01,*P<0.05, compared with normal group；
Fig. 4 is that (ordinate is for the influence of Phellinus polyphenol extract and four kinds of active components to L6 grape cell Sugar intakes Fluorescence intensity),***P<0.001,**P<0.01,*P<0.05, compared with normal group；
Fig. 5 is the influence of Phellinus polyphenol extract and four kinds of active components to L6 muscle cell glucose uptakes：(A) mulberry Influence of the yellow polyphenol extract to film on L6 cells IRAP；(B) Phellinus polyphenol is to film on L6 cells IRAP before and after adding inhibitor Influence,***P<0.001,**P<0.01,*P<0.05, compared with starting point (ordinate is IRAP Fluorescence Increasings multiple)；(C) four Influence of the kind active component to film on L6 cells IRAP (ordinate is IRAP Fluorescence Increasings multiple)；
Fig. 6 be Phellinus polyphenol extract to mouse weight influence (ordinate is weight loss amount, and abscissa is the time, Unit is week),+++P<0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001,**P<0.01,*P<0.05, with Model group is compared；
Fig. 7 is that (ordinate is food ration, and abscissa is the time, single for influence of the Phellinus polyphenol extract to mouse food ration Position is week),+++P<0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001,**P<0.01,*P<0.05, with mould Type group is compared；
Fig. 8 is that (ordinate is blood sugar level, when abscissa is for influence of the Phellinus polyphenol extract to mouse fasting blood-glucose Between, unit is week),+++P<0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001, * * P<0.01,*P< 0.05, compared with model group；
(ordinate is blood sugar level, when abscissa is for influence of Fig. 9 Phellinus polyphenol extract to Mouse oral sugar tolerance Between, unit is minute),+++P<0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001,**P<0.01,*P< 0.05, compared with model group；
(ordinate is blood sugar level, when abscissa is for influence of Figure 10 Phellinus polyphenol extract to mouse islets element tolerance Between, unit is minute),+++P<0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001,**P<0.01,*P< 0.05, compared with model group；
Influence of Figure 11 Phellinus polyphenol extract to mice serum insulin,+++P<0.001,++P<0.01,+P<0.05, with Normal group is compared,***P<0.001,**P<0.01,*P<0.05, compared with model group；
Influence of Figure 12 Phellinus polyphenol extract to mice serum T-CHOL,+++P<0.001,++P<0.01,+P<0.05, Compared with normal group,***P<0.001,**P<0.01,*P<0.05, compared with model group；
Influence of Figure 13 Phellinus polyphenol extract to mice serum triglycerides,+++P<0.001,++P<0.01,+P<0.05, Compared with normal group,***P<0.001,**P<0.01,*P<0.05, compared with model group；
Influence of Figure 14 Phellinus polyphenol extract to mice skeletal p-AMPK albumen (A) and GLUT4 albumen (B),+++P< 0.001,++P<0.01,+P<0.05, compared with normal group,***P<0.001,**P<0.01,*P<0.05, compared with model group.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is The conventional products that can be obtained by commercially available purchase.
Present embodiment provides a kind of pharmaceutical composition for treating diabetes, and it includes：Inoscavin C, 7,3 '-dihydroxy At least one of base -5 '-methoxy isoflavone, Daphnelin or 3,4- dihydroxy benzylideneacetone, and pharmaceutically Acceptable carrier or auxiliary material.
Wherein, active component Inoscavin C structural formula is：
The structural formula of active component 7,3 '-dihydroxy -5 '-methoxy isoflavone is：
The structural formula of active component 7,8- dihydroxycoumarins is：
The structural formula of active component 3,4- dihydroxy benzylideneacetones is：
Further, the pharmaceutical composition includes Phellinus polyphenol extract, contains Inoscavin in Phellinus polyphenol extract C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins or 3,4- dihydroxy benzenes fork acetone.
Further, the pharmaceutical composition includes Phellinus dichloromethane extract, contains in Phellinus dichloromethane extract The Phellinus polyphenol extract.
Wherein, the preparation method of Phellinus dichloromethane extract includes：Using alcohol-water mixed solution extraction Phellinus, by institute Obtained phellinus linteus extract is extracted with petroleum ether, dichloromethane successively.Obtain Phellinus dichloromethane extract.
More specifically, the preparation method of Phellinus dichloromethane extract is：Flowed back with alcohol or alcohol-water mixture, dipping Or diacolation Phellinus powder, obtain extract solution after filtering；Solvent in extract solution is evaporated to obtain medicinal extract；Water is added to stir medicinal extract again Mix, obtain suspension；Finally, above-mentioned suspension is extracted with petroleum ether, obtains petroleum ether extract and water solubility Position, then water soluble part is extracted with dichloromethane, concentrate, obtain dichloromethane extract.
Further, the preparation method of Phellinus polyphenol extract is：It will be extracted as the Phellinus dichloromethane obtained by the above method Take thing to cross polyamide column, with alcohol-water mixed solution gradient elution, collect the elution during ethanol solution that eluant, eluent is 77~83% Liquid, obtain the Phellinus polyphenol extract.
More specifically, the preparation method of Phellinus polyphenol extract is：Phellinus dichloromethane extract is taken to cross polyamide column, And with 27~33%, 37~43%, 47~53%, 77~83%, 90~100% ethanol solutions progress gradient elution, or with 20%th, 40%, 60%, 80%, 95% ethanol solution carries out gradient elution.The component that different graded ethanol solutions are afforded Merge respectively, and carry out screening active ingredients respectively.Active ingredients result shows, the component that 77~83% ethanol solutions elute Activity is best, as Phellinus polyphenol extract (PI-PRE).
Present embodiment additionally provides the medical usage of aforementioned pharmaceutical compositions, i.e.,：
A kind of purposes of pharmaceutical composition in the activator for promoting glucose uptake is prepared.
Inventor, which studies, to be found, Phellinus polyphenol extract, Phellinus carbon dichloride extract and active component Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzal third Ketone, it can improve glucose uptake level, wherein mulberry polyphenol extract, Inoscavin C, 7,3 '-dihydroxy -5 '-methoxyl group Isoflavones, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzylideneacetone are to the grape under foundation level and under insulin stimulating Sugar intake has the facilitation of conspicuousness.
A kind of purposes of pharmaceutical composition in the activator of GLUT4 is prepared.
GLUT (GLUT4), it is a kind of transmembrane protein, it is mainly expressed in fat and muscle cell, It is to be responsible for the major protein that transhipment grape is warded off in animal body.In adipocyte and Skeletal Muscle Cell, GLUT4 is present in special Membrane structure in, referred to as GLUT4 vesicas.Insulin stimulating is the GLUT4 vesicas for promoting intracellular largely to the straight of cell membrane transporter Connect reason.Discharged on stimulation intracellular GLUT4 Vesicle transports to the film of insulin, cause the increase of GLUT4 contents on film.Have on film Glucose transport is entered metabolism, storage in the tissue such as muscle, fat by the GLUT4 of activity, to maintain the glycometabolism of body to balance. In the state of not stimulating, 95% GLUT4 is there are about in the vesica of storage intracellular.When motion or signal stimulus excite insulin Combined with acceptor, excite a series of signal reaction, cause the vesica rich in GLUT4 to be moved to plasma membrane, GLUT4 is indexed into plasma membrane And activity increase, the change of simultaneously occurred conformation is combined with glucose, now glucose is transported into the cell.
IRAP (aminopeptidase of insulin regulation) is another major protein on GLUT4 vesicas in addition to GLUT4, in pancreas Under the element incentive condition of island, it also can have high common location phenomenon, institute with GLUT4 Vesicle transports to cell membrane with GLUT4 It may also used to mark the GLUT4 vesicas of intracellular with it.The intracellular GLUT4's of IRAP dynamic transport process indirect reaction Transhipment.
Research finds, Phellinus polyphenol extract and active component Inoscavin C, 7,3 '-dihydroxy -5 '-methoxyl group Isoflavones, Daphnelin and 3,4- dihydroxy benzylideneacetone, this five kinds of compositions, GLUT4 vesicas can be promoted to turn It is transported on cell membrane, glucose combines and by glucose transport to intracellular, so as to increase intake of the cell to glucose.
A kind of purposes of pharmaceutical composition in the activator for preparing adenosine monophosphate activated protein kinase.
Adenosine monophosphate activated protein kinase (AMPK) is regulating cell and the important albumen of whole body energetic supersession.As thin The regulation and control person of born of the same parents' energy, AMPK play important in glycolipid metabolism and regulation and control metabolic disorder (such as diabetes, fat and cancer) Effect, therefore AMPK be treat metabolic disease important target.Activation AMPK paths can increase fatty acid oxidation, suppress fat Matter synthesizes, and can also improve the ability of insulin.Numerous studies show a large amount of different metabolic pathways of AMPK regulation and control, and it is that have safely The treatment type-II diabetes of effect and the target spot of Anomalous lipid metablism.GLUT4 is the speed limit that skeletal muscle and adipose tissue absorb glucose Step, and AMPK is one of GLUT4 major regulatory albumen.In glycolipid metabolism, environmental stimuli increase AMPK phosphorylations, AMPK signal paths are promoted to be activated, so as to promote GLUT4 by being transported in kytoplasm on cell membrane, the final cell that strengthens is to grape The intake of sugar.
Research finds that Phellinus polyphenol extract can increase AMPK phosphorylations, AMPK signal paths is activated, so as to promote Enter transferring film and the expression of GLUT4 albumen, finally play the effect for improving type II diabetes symptom.
A kind of pharmaceutical composition is preparing treatment or prevention by the medicine or health food of metabolic disorder associated diseases Purposes.
Because the pharmaceutical composition can activate AMPK, and AMPK is the important target for treating metabolic disease；And research hair It is existing, Phellinus polyphenol extract and active component InoscavinC, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxies Butylcoumariii and 3,4- dihydroxy benzylideneacetone, this five kinds of compositions can be reduced by total courage in metabolic disorder associated diseases The content of sterol and triglycerides, accordingly, it is capable to be enough in the disease alleviated or treated and treat or prevent caused by metabolic disorder, example Such as diabetes, obesity, vascular hypertension and hyperlipemia.Wherein, have significant therapeutic effect to type II diabetes.
Further, in order that medicine discharge active component rapidly, continuously and in a very long time, of the invention Pharmaceutical composition can manufacture according to those conventional methods in the art are disclosed in.The administration of the medicine of present embodiment Approach can be oral, nasal inhalation or parenteral.Preparation comprising said composition can be tablet, soft capsule, ebonite Capsule, oral liquid, pill, suppository, powder, particle, emulsion, syrup, aerosol, sterile injectable preparation and sterilized powder etc..
In the present invention, it is physiologically may be used that term " pharmaceutically acceptable ", which refers to the compound when compound is to human administration, Receive, and the allergic reactions such as gastrointestinal disturbance, dizziness or these similar anaphylactoid systemic anaphylaxis will not occur.
In the present invention, " pharmaceutically acceptable carrier or auxiliary material " includes but is not limited to：Adhesive (such as microcrystalline cellulose Element, alginates, gelatin and polyvinylpyrrolidone), filler (such as starch, sucrose, glucose and anhydrous lactic acid), disintegrant (such as cross-linked pvp, crosslinked carboxymethyl fecula sodium, Ac-Di-Sol and low-substituted hydroxypropyl cellulose), lubricant (magnesium stearate, aluminum stearate, talcum, polyethylene glycol, sodium benzoate), wetting agent (such as glycerine), surfactant (such as hexadecane Alcohol) and sorbefacient, flavouring, sweetener, diluent, coating agent etc..
The feature and performance of the present invention are described in further detail with reference to embodiments：
Embodiment 1
The present embodiment provides a kind of pharmaceutical composition, and the pharmaceutical composition includes Phellinus polyphenol extract and pharmaceutically may be used The carrier or auxiliary material of receiving.
Wherein, the preparation method of Phellinus polyphenol extract includes：
As shown in figure 1, taking Phellinus to dry bacterial strain 2kg, crushed with pulverizer, then by solid-liquid ratio 1:8 (1.0kg medicinal materials pair Answer 8.0L solvent) impregnated 3 days with the ethanol water that volumetric concentration is 80%, filtrate is filtered to take, filter residue uses equal volume instead 80% ethanol water impregnate 3 days, repeat five times, filtering merge five times extraction filtrates, be concentrated under reduced pressure to give yellow pulp Shape thing 340g.
By volume 1:10 are dissolved in water dilution, and sample, the decompression of dichloromethane extract are extracted with isometric dichloromethane Concentration, recycling design are evaporated, and obtain Phellinus dichloromethane extract 49g.
Phellinus dichloromethane extract 47g is taken, crosses polyamide column, and it is molten with 20%, 40%, 60%, 80%, 95% ethanol Liquid carries out gradient elution.The component that different graded ethanol solutions are afforded merges respectively, and carries out screening active ingredients respectively. Active ingredients result shows that component (Phellinus polyphenol extract, the PI-PRE) activity that 80% ethanol solution elutes is best, energy Enough it is obviously promoted film on GLUT4.Therefore, the component eluted with 80% ethanol solution, as Phellinus polyphenol extract.
Embodiment 2
The present embodiment provides a kind of pharmaceutical composition, and the pharmaceutical composition includes Phellinus polyphenol extract and pharmaceutically may be used The carrier or auxiliary material of receiving.
Wherein, the preparation method of Phellinus polyphenol extract includes：
As shown in figure 1, taking Phellinus to dry bacterial strain 2kg, crushed with pulverizer, then by solid-liquid ratio 1:7 (1.0kg medicinal materials pair Answer 7.0L solvent) impregnated 3 days with the ethanol water that volumetric concentration is 75%, filtrate is filtered to take, filter residue uses equal volume instead 75% ethanol water impregnate 3 days, repeat five times, filtering merge five times extraction filtrates, be concentrated under reduced pressure to give yellow pulp Shape thing 340g.
By volume 1:11 are dissolved in water dilution, and sample, the decompression of dichloromethane extract are extracted with isometric dichloromethane Concentration, recycling design are evaporated, and obtain Phellinus dichloromethane extract 50g.
Phellinus dichloromethane extract 47g is taken, crosses polyamide column, and it is molten with 17%, 37%, 47%, 77%, 90% ethanol Liquid carries out gradient elution.The component that different graded ethanol solutions are afforded merges respectively, and carries out screening active ingredients respectively. Active ingredients result shows that component (Phellinus polyphenol extract, the PI-PRE) activity that 77% ethanol solution elutes is best, energy Enough it is obviously promoted film on GLUT4.Therefore, the component eluted with 77% ethanol solution, as Phellinus polyphenol extract.
Embodiment 3
The present embodiment provides a kind of pharmaceutical composition, and the pharmaceutical composition includes Phellinus polyphenol extract and pharmaceutically may be used The carrier or auxiliary material of receiving.
Wherein, the preparation method of Phellinus polyphenol extract includes：
As shown in figure 1, taking Phellinus to dry bacterial strain 2kg, crushed with pulverizer, then by solid-liquid ratio 1:9 (1.0kg medicinal materials pair Answer 9.0L solvent) impregnated 3 days with the ethanol water that volumetric concentration is 85%, filtrate is filtered to take, filter residue uses equal volume instead 85% ethanol water impregnate 3 days, repeat five times, filtering merge five times extraction filtrates, be concentrated under reduced pressure to give yellow pulp Shape thing 341g.
By volume 1:9 are dissolved in water dilution, extract sample with isometric dichloromethane, the decompression of dichloromethane extract is dense Contracting, recycling design are evaporated, and obtain Phellinus dichloromethane extract 50g.
Phellinus dichloromethane extract 47g is taken, crosses polyamide column, and with 33%, 43%, 53%, 83%, 100% second Alcoholic solution carries out gradient elution.The component that different graded ethanol solutions are afforded merges respectively, and carries out activity respectively Screening.Active ingredients result shows that component (Phellinus polyphenol extract, the PI-PRE) activity that 83% ethanol solution elutes is most It is good, film on GLUT4 can be obviously promoted.Therefore, the more phenol extractions of the component eluted with 83% ethanol solution, as Phellinus Thing.
Embodiment 4
The present embodiment provides a kind of pharmaceutical composition, and it includes Inoscavin C, 7, and 3 '-dihydroxy -5 '-methoxyl group is different Flavones, Daphnelin and 3,4- dihydroxy benzylideneacetone, and pharmaceutically acceptable carrier or auxiliary material.
As shown in Fig. 2 taking 400mg Phellinus polyphenol extract (by being provided in embodiment 1~3), 2.0mL is dissolved in In DMSO, i.e., PI-PRE concentration is 200mg/mL.PI-PRE (200 μ L) is carried out half and prepares efficient liquid phase (HPLC) analysis, point Analysis condition is：The formic acid of+10% acetonitrile of 90% water+0.1%, gradient elution, the formic acid of 100% acetonitrile+0.1% after 25 minutes, 100% The formic acid of acetonitrile+0.1% persistently elutes 5min, i.e., analyzes 30min altogether, and combine mass spectral analysis.Analysis result obtains, PI-PRE There are four main peaks.HPLC preparations are then carried out according to liquid matter result, obtain 4 main peak components.First three main peak component passes through solidifying Glue post purify to obtain compound 1 (Inoscavin C, 55.9mg), compound 2 (7,3 '-dihydroxy -5 '-methoxy isoflavone, 27.1mg), compound 3 (7,8- dihydroxycoumarins, 34.8mg).Main peak component obtains chemical combination after purification by thin layer silica gel plate Thing 4 (3,4- dihydroxy benzylideneacetone, 36.2mg).
Experimental example
With reference to the test of pesticide effectiveness to provided in the embodiment of the present invention 4 Inoscavin C, 7,3 '-dihydroxy -5 '-first Epoxide isoflavones, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzylideneacetone, Phellinus polyphenol extract and Phellinus dichloro Methane extract carries out evaluating drug effect：
Experimental example one determines the shadow of Phellinus polyphenol extract and 4 kinds of active components to L6 muscle cell glucose uptakes Ring
The L6 cell recoveries that will be frozen, cultivated with the dual anti-α-MEM culture mediums containing 10%FBS and 1%, treat its life When length is to logarithmic phase, uses the dual anti-α-MEM culture mediums containing 2%FBS and 1% instead and broken up, treat that it is divided into myotube completely Afterwards, change with the α of serum-free containing 0.5%BSA-MEM cultivate 16 hours after, add each tester solution (Inoscavin C, 7,3 '-two Hydroxyl -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzylideneacetones are 10 μM of final concentration；PI- PRE is respectively 15 μ g/mL, 30 μ g/mL and 60 μ g/mL) handle 24 hours, blank control group adds isometric DMSO.24 is small Shi Hou, cell is cleaned three times with the 1*PBS of 37 DEG C of pre-temperatures, add be free of or insulin-containing (final concentration of 100nM) 0.5% FSA Krebs buffer solutions (NaCl 140mM, KCl 5mM, MgSO42.5mM, CaCl21mM, HEPES 20mM, pH7.4), 37 DEG C shake educate 40 minutes after, add 2- [1,2-3H (N)]-deoxy-D-glucose solution (final concentration of 0.5 μ Ci/ml), 37 DEG C work With 10 minutes, 3 times terminating reactions are washed with 1 × PBS of ice bath, add 0.15mL 0.1%Triton cell lysis afterwards, liquid dodges Count, the glucose uptake amount of calculating L6 cells after CPM values is corrected with protein content.
As a result Fig. 3 and Fig. 4 are seen, each tester can improve glucose uptake level, and wherein Phellinus polyphenol extract is with 15 μ G/mL, 30 μ g/mL and 60 μ g/mL, Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxy tonka-beans Element and 3,4- dihydroxy benzylideneacetone are with 10 μM of dosage, after L6 cytosiies 24 hours, its foundation level and insulin stimulating Under glucose uptake increase substantially.Data are represented (n=6) with mean+/-standard error (X ± SE).* P ＜ 0.05, * * P ＜ 0.01 is compared with blank control group under corresponding conditionses.
Thus illustrate, Phellinus polyphenol extract and four kinds of active components there is promotion to make the glucose uptake of L6 cells With wherein with 3,4- dihydroxy benzylideneacetone best results.
The influence and its Mechanism Study of the Phellinus polyphenol extract of experimental example two and four kinds of active components to film on GLUT4
Research shows that IRAP (aminopeptidase of insulin regulation) and GLUT4 (GLUT4) have the pass coexisted System, therefore the transhipment for observing IRAP can directly react GLUT4 motion.
Because the GLUT4 of the mankind and rat have the sequence homology of height, rat L6 can be used in testing in vitro Myotubes replace people's muscle cell.In the state of not stimulating, 95% GLUT4 is there are about in the vesica of storage intracellular.When Motion or signal stimulus excite insulin to be combined with acceptor, excite a series of signal reaction, cause be rich in GLUT4 vesica to Plasma membrane moves, and GLUT4 is indexed into plasma membrane and activity increase, the change of simultaneously occurred conformation is combined with glucose, now glucose quilt It is transported to intracellular.Based on this, inventor is created based on L6 cell GLUT4 fluorescence labelings and transfection and with laser co-focusing Membrane choosing system in the promotion GLUT4 transhipments of flying-spot microscope；It is thin that L6 is followed the trail of using laser confocal microscope by directly The IRAP of fluorescence labeling motion in born of the same parents, influence degree of the judgement sample to GLUT4 transferring films activity.
Stable expression IRAP L6 (L6IRAP-mOrange) cell is inoculated in 6cm culture dish, adds and contains 10% α-MEM- the culture mediums of hyclone, in 37 DEG C, 5%CO2 cell culture incubator cultures.
Before experiment starts, L6 cells are inoculated on slide, when cell density reaches 100%, abandon culture medium, added Serum free medium is hungry 2 hours, and PBS is washed three times.Add each tester solution (Inoscavin C, 7,3 '-dihydroxy -5 ' - Methoxy isoflavone, Daphnelin and 3,4- dihydroxy benzylideneacetone are with 10 μM of administrations, and insulin is as positive Control administration concentration is 100nM, and Phellinus polyphenol extract is administered with 30 μ g/ml), monitored with laser scanning co-focusing microscope The dynamic change that IRAP-mOrange after medicine is transported is added, observation intensity of cellular fluorescence change, is reacted with this on GLUT4 Film situation.
In addition, insulin signaling pathway and AMPK (adenosine monophosphate activated protein kinase) signal path are regulation glucose Transport protein 4 (GLUT4) indexing and two primary signal pathways of glucose uptake, Compound C are AMPK inhibition of phosphorylation Agent, wortmannin (Wortmannin) are PI3K inhibitor.
On above-mentioned experiment basis, from L6IRAP-mOrange cells, add Compound C before being administered and be incubated carefully Each testing drug is added after born of the same parents 30min, change in fluorescence on film is observed, reflects film situation on GLUT4 indirectly.At the same time, select With L6IRAP-mOrange cells, each testing drug is added after wortmannin incubated cell 30min is added before being administered, observes film Upper change in fluorescence, reflect film situation on GLUT4 indirectly.
As a result Fig. 5 is seen, each tester can effectively improve film water on cell GLUT4 and put down, and Phellinus polyphenol extract is with 30 μ g/ ML be administered, 7,3 '-dihydroxy -5 '-methoxy isoflavone, Daphnelin and 3,4- dihydroxy benzylideneacetone with 10 μM of administrations.
From Fig. 5 A, after adding Phellinus polyphenol extract, fluorescence substantially increases on film.In 20min, fluorescence on film Horizontal highest (Fig. 5 A, 5B), reflects that film now reaches maximum on GLUT4 with this.In figure 5b, before adding Compoud C, mulberry Yellow more phenol extractions can increase the fluorescence intensity on film, i.e. Phellinus polyphenol extract can promote GLUT4 upper film.Add After Compoud C, fluorescence intensity substantially weakens.Add Compoud C and be added without Compoud C fluorescence intensities and conspicuousness be present Difference * P<0.05, * * P<0.01.But fluorescence is without significant changes before and after adding Wortmannin.Thus illustrate, test compound is Film on GLUT4 is promoted by AMPK signal paths, so as to increase glucose uptake.
It can be observed from Fig. 5 C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, Daphnelin and 3,4- Dihydroxy benzylideneacetone can promote Fluorescence Increasing, that is, promote film on GLUT4.To fluorescence intensity carry out statistical disposition, data with Average value ± standard error (X ± SE) represents.*P<0.05, * * P<0.01, * * * P<0.001 compared with control group.
Experimental example three
Blood sugar reducing function of the Phellinus polyphenol extract to diabetic mice
1. material and instrument
1.1 experimental animal
KK-Ay mouse, SPF levels, 8 week old, male；C57BL/6J mouse, SPF levels, 8 week old, male, by Beijing Hua Fu Health biotech inc provides, licensing numbering:SCXK (capital) 2009-0004.Mouse material is through 60 DEG C of sterilizings, by north Capital Fukang biotech inc provides, and C57 mouse maintain feed, and KK-Ay mouse feed KK mouse material (high lipid food), Licensing is numbered:SCXK (capital) 2009-0008.
The selected KK-Ay mouse of this research are a kind of polygenes spontaneity genetic animal models, are the bases in KK mouse It is transferred to mutation coat color gene (Ay) on plinth to form, Ay genes not only influence the hair color of mouse but also can cause metabolic disorder, occur The pathological characters such as hyperglycaemia, hyperinsulinemia, insulin resistance, obesity, disorders of lipid metabolism, it is preferable spontaneous pancreas islet Element resistance diabetes animal model and spontaneous hereditary fat animal model.C57BL/6J has DNA homolog with KK-Ay mouse Property is as Normal group.
1.2 medicines and reagent
Phellinus polyphenol extract (Phellinus is purchased from Anhui Province Bozhou medicinal material market), insulin radioimmunoassay immune reagent kit (Tianjin Nine ancient cooking vessel bioengineering Co., Ltds), glucose (Chemical Reagent Co., Ltd., Sinopharm Group), metformin hydrochloride tablet (sun Shanghai Shi Guibao pharmaceutical Co. Ltds of Sino-U.S.), RIPA cell pyrolysis liquids (green skies Bioisystech Co., Ltd), PMSF (green clouds Its Bioisystech Co., Ltd), BCA protein concentrations kit (green skies Bioisystech Co., Ltd), GLUT4, β-actin, The antibody such as AMPK, p-AMPK, secondary antibody (Cell SignalingTechnology).
2. experimental method
The foundation of 2.1 experimental models
10 C57BL/6J mouse are as Normal group.70 KK-Ay mouse high lipid foods are fed 4 weeks, inducing mouse Hyperglycaemia is produced, afterbody takes the fasting plasma glucose concentration of hematometry mouse, and blood sugar concentration is qualified mould more than or equal to 11.1mmol/L Type mouse, available for testing in next step.The successful mouse of modeling is randomly divided into model control group, positive controls (hydrochloric acid two First biguanides piece 200mg/kg) and 5 groups, every group of the dosage group (40mg/kg, 80mg/kg, 160mg/kg) of Phellinus polyphenol extract three 10.Each group is not using (Normal group and model control group give isometric physiology salt to isoconcentration gastric infusion in equal volume Water), once, gavage volume is 0.1mL/10g to daily timed drug administrations, continuous gavage 28 days.
2.2 experimental datas gather
Respectively at administration 0d, 7d, 14d, 21d, 28d hematometry mouse blood sugar value and body are taken in tail vein Weight.It is administered 26d, after mouse fasting 12 hours, afterbody takes blood, blood glucose value of the measure mouse blood sugar concentration as 0min.Then, With 2.0g/kg glucose gavages, 30min, 60min, 90min, 120min mouse's blood sugar content are determined respectively, it is small to observe each group The change of mouse blood glucose value, compare and whether there is difference between each group.It is administered 28d, after mouse fasting 12 hours, afterbody takes blood, The blood glucose value that mouse blood sugar concentration is determined as 0min measures blood glucose, then, intraperitoneal injection 2IU/kg insulin, determines respectively 30min, 60min, 90min, 120min mouse's blood sugar content, the change of each group mouse blood sugar value is observed, compared between each group With the presence or absence of difference.
After experiment terminates, taken a blood sample by eyeball.With rotating speed 3000rpm centrifugation 15min separation serum.Using full-automatic Biochemical Analyzer determines the content of T-CHOL and triglycerides in mice serum respectively；With serum measured by radioimmunoassay pancreas islet Cellulose content.
After blood sampling terminates, mouse is put to death, then collects Muscle Tissue.
The detection of Muscle Tissue GAP-associated protein GAP：RIPA lysates extract total protein, and BCA protein reagent boxes determine albumen Concentration, each group sample equal quantities protein is taken, carry out SDS-PAGE glue protein isolates.And by albumen electrotransfer to pvdf membrane. Again with the TBST closings 1h containing 0.5% skimmed milk power；Add 4 DEG C of primary antibody GLUT4, AMPK, p-AMPK antibody of 1: 2000 dilution Overnight；Wash after film plus the goat anti-rabbit igg (1: 5 000 dilution) of horseradish peroxidase-labeled, room temperature jog 1h, fully washing ECL nitrite ions are added afterwards, and imaging analysis are carried out using gel imaging system (APLEGEN, INC, USA).
3. experimental result
Data represent that significant difference is examined with t inspections and correlation analysis between group with average value ± standard error (X ± SE).
The influence of 3.1 pairs of diabetic mice body weight
Experimental result is shown in Fig. 6, and after inducing mouse blood glucose rise, model group body weight significantly raises, and has compared with normal group There is significant difference (P ＜ 0.05).After being administered one week, each group mouse weight has declined, administration group and Metformin hydrochloride Group and model group bodyweight difference and unobvious.After two weeks, model group body weight is still higher compared to normal group body weight, exists aobvious Sex differernce is write, administration group and Metformin hydrochloride group body weight are without significant change.After being administered three weeks, model group body weight has still increased Height, administration group and Metformin hydrochloride group have significant difference compared with model group.After four weeks is administered, administration group and hydrochloric acid two First biguanides group, body weight have initially reduced compared with it, significant difference be present with model group.Thus illustrate, Phellinus polyphenol extract The body weight of diabetic mice can be reduced.
The influence of 3.2 pairs of diabetic mice food rations
Experimental result is shown in Fig. 7, after inducing mouse blood glucose rise, the food ration and model group of each administration group mouse during administration No significant difference, test result show that the polyphenol extract of Phellinus two is noiseless to the food ration of mouse.
The influence of 3.3 pairs of diabetic mice fasting blood-glucoses
After modeling success, model group blood sugar level compared with normal group is significantly raised, has significant difference (P ＜ 0.001), and the blood sugar level of administration group and model group there was no significant difference.After being administered one week, positive group and administration group blood glucose water Average to have declined, compared to model group, positive controls have significant difference.After two weeks, each administration group blood sugar level Begin to decline, compared with model group, significant difference occur.It can thus be seen that Phellinus polyphenol extract has II type that reduces The effect of blood glucose of diabetic mice.Experimental result is shown in Fig. 8.
The influence of 3.4 pairs of diabetic mice oral glucose tolerances
30min blood-sugar content of the normal group mouse after oral glucose reaches maximum, and 2h blood glucose recovers substantially It is horizontal before to administration.Model group blood glucose value after oral glucose 30min reaches maximum, is protected always although hereafter having declined Hold in higher level.Administration group and melbine group the 30min blood glucose values after oral glucose reach highest level, and diformazan is double Guanidine group has significant difference compared to model group, recovers level before oral glucose after 2h.Phellinus polyphenol extract it is basic, normal, high Administration group blood sugar level has the decline of conspicuousness after 30min, level before oral glucose is basically reached after 2h, with model group Compared to the difference (* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001) that conspicuousness be present.Thus illustrate, Phellinus polyphenol extract The oral glucose tolerance of type II diabetes mouse can be improved.Experimental result is shown in Fig. 9.
The influence of 3.5 pairs of diabetic mice insulin tolerances
After subcutaneous insulin injections, normal group mouse blood-sugar content after 30min reaches minimum value, and blood glucose starts extensive after 2h It is multiple.Though model group has a minimum blood glucose point after 60min, blood sugar level is constantly in a higher state, between each point There was no significant difference.Administration group is consistent with normal group with the situation of melbine group, i.e., in subcutaneous insulin injections 30min blood glucose Value reaches floor level, then gradually recovers blood sugar level before injection.Melbine group has significant difference, 2h compared to model group It is horizontal before recovery insulin injection afterwards.Each administration group blood sugar level has the decline of conspicuousness after 30min, is basically reached after 2h It is horizontal before insulin injection, the difference of conspicuousness, (* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ compared with model group be present 0.001).Thus illustrate, Phellinus polyphenol extract has the function that to improve diabetic mice insulin tolerance.Experimental result is shown in figure 10。
The influence of 3.6 pairs of diabetic mice Biochemical Indices In Serums
3.6.1 to the influence of diabetic mice serum insulin
Normal group mouse islets element is in normal level, and model group mouse islets element level is more obvious than normal group to be higher by very It is more, have significant difference (+++P ＜ 0.001), illustrate that diabetes B mouse has insulin resistance.Positive drug melbine Group and administration group insulin level are substantially much smaller than model group, significant difference (* P ＜ 0.05, * * P ＜ be present 0.01, * * * P ＜ 0.001).Thus illustrate, after insulin resistant mice is by administration, insulin resistance degree substantially reduces, pancreas Insulin resistance significantly improves.Experimental result is shown in Figure 11.
3.6.2 to diabetic mice serum total cholesterol (TC) influence
Compared with normal group, the horizontal conspicuousness rises of model group mouse TC, significant difference (P ＜ 0.001) be present, be administered Group has a certain degree of decline compared with model group TC levels, and has significant difference (* P ＜ 0.05, * * P ＜ 0.01, * * * P ＜ 0.001).Thus illustrate, medicine has the function that to reduce mouse TC levels.Experimental result is shown in Figure 12.
3.6.3 to diabetic mice serum triglyceride (TG) influence
Normal group mice serum TG levels are in normal level, and model group TG levels compared with normal group are significantly raised, sun Property medicine Metformin hydrochloride group slightly reduced than model group triglyceride levels, the TG levels of administration group reduce very than model group More, there is the difference of conspicuousness with high dose group in low dose group compared with model group.Thus illustrate, after administration, the blood of diabetic mice Clear TG levels can be reduced, and the medical instrument has the effect of improving diabetic mice TG levels.Experimental result is shown in Figure 13.
3.6.4 to the influence of Muscle Tissue GAP-associated protein GAP
GLUT4, AMPK, p-AMPK protein level in mouse muscle are detected by corresponding antibodies, as seen from Figure 14, In muscle, administration group mouse muscle GLUT4 expression increased compared with model group, and show dose-dependence.It is in addition, low The influence of dosage, middle dosage, high dose to p-AMPK protein expressions is different, but compared with model group, can improve p-AMPK It is horizontal.The outer Mechanism Study result of combination, it may be said that bright, Phellinus polyphenol extract is improved GLUT4 and turned by AMPK signal paths Film is transported, so as to promote intake of the mouse for glucose, blood glucose is reduced, improves diabetes mice symptom.Experimental result is shown in figure 14。
To sum up, present invention research shows, carries out gastric infusion with Phellinus polyphenol extract, has one to KK-Ay mouse weights Fixed inhibitory action, gastric infusion can reduce KK-Ay mouse blood sugars level for one week, and blood glucose value persistently reduces during administration, After successive administration 4 weeks, measure mouse blood sugar value is substantially close to normal water level values.In oral glucose tolerance experiment, it can be observed in reality During testing, Phellinus polyphenol extract can substantially reduce postprandial blood sugar.Insulin resistance is the main pathology of type II diabetes Feature, it plays key effect during the occurrence and development of diabetes, and research shows, hyperglycaemia can induce insulin and support It is anti-, promote the development of diabetes.The measurement result of serum insulin shows that administration group KK-Ay mouse islets element is horizontal obvious low In model group, there is significant difference, show that Phellinus polyphenol extract it is small can to improve KK-Ay by reducing mouse blood sugar level The effect of mouse insulin resistance.
Hyperglycaemia often easily causes hyperlipemia, and this is due to carbohydrate metabolism disturbance and causes disorders of lipid metabolism, so as to cause One or more lipid components contents raise extremely in blood.Sugar can be prevented or alleviate to a certain extent by improving lipid-metabolism Urinate the generation of disease.In this experiment, for Phellinus polyphenol extract gastric infusion after 4 weeks, serum TG, TC are horizontal equal compared to model group mouse Decrease, illustrate that Phellinus polyphenol extract can improve disorders of lipid metabolism caused by diabetes to a certain extent, play Prevention or the effect for alleviating diabetes.
AMPK is regulating cell and the important albumen of whole body energetic supersession.As the regulation and control person of cellular energy, AMPK is in sugar All played an important role in lipid metaboli and regulation and control metabolic disorder (such as diabetes, fat and cancer), therefore AMPK is treatment generation Thank to the important target of disease.Activation AMPK paths can increase fatty acid oxidation, suppress lipid synthesis, can also improve insulin Ability.Numerous studies show a large amount of different metabolic pathways of AMPK regulation and control, and it is safely and effectively to treat type-II diabetes and fat The target spot of metabolic disorder.GLUT4 is the rate-limiting step that skeletal muscle and adipose tissue absorb glucose, and AMPK is GLUT4 master Want one of modulin.
In cell experiment, inventor is by adding Compound C, Wortmannin have detected Phellinus polyphenol extract Rush GLUT4 on film activity, as a result show, after Compound C are added, film activity is inhibited on the rush GLUT4 of medicine, And after adding Wortmannin, film activity does not change on the rush GLUT4 of medicine.Thus illustrate, Phellinus polyphenol extract is logical AMPK paths are crossed to promote film in GLUT4 transhipments, so as to increase intake of the cell to glucose.
Western blot detection skeletal muscle GAP-associated protein GAPs also indicate that Phellinus polyphenol extract can increase p-AMPK albumen Expression, it is to reach increase grape cell Sugar intake by AMPK signalling channels and reduce small that this, which just more demonstrates said medicine, The effect of mouse blood sugar level.
Comprehensive cell experiment and the result of zoopery, Phellinus dichloromethane extract, Phellinus polyphenol extract and Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins and 3,4- dihydroxy benzylideneacetones In one or several kinds, there is to be obviously promoted L6 Skeletal Muscle Cell glucose uptakes, activate AMPK signal paths, Promote GLUT4 transferring films and expression, reduce blood sugar level, so as to have the function that prevention and treatment to metabolic disorder associated diseases.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

Claims (10)

  1. A kind of 1. pharmaceutical composition for treating diabetes, it is characterised in that described pharmaceutical composition includes Inoscavin C, 7, At least one of 3 '-dihydroxy -5 '-methoxy isoflavone, Daphnelin or 3,4- dihydroxy benzylideneacetone, and Pharmaceutically acceptable carrier or auxiliary material.
  2. 2. the pharmaceutical composition for the treatment of diabetes according to claim 1, it is characterised in that described pharmaceutical composition includes Phellinus polyphenol extract, Inoscavin C, described 7,3 '-dihydroxy -5 '-first are contained in the Phellinus polyphenol extract Epoxide isoflavones, the 7,8- dihydroxycoumarins or 3,4- dihydroxy benzenes fork acetone.
  3. 3. the pharmaceutical composition for the treatment of diabetes according to claim 2, it is characterised in that described pharmaceutical composition includes Phellinus dichloromethane extract, contain the Phellinus polyphenol extract in the Phellinus dichloromethane extract.
  4. 4. a kind of preparation method of Phellinus polyphenol extract, the Phellinus polyphenol extract includes Inoscavin C, 7,3 '-two Hydroxyl -5 '-methoxy isoflavone, Daphnelin and 3,4- dihydroxy benzylideneacetone, it is characterised in that it includes：
    Using alcohol-water mixed solution extraction Phellinus, resulting phellinus linteus extract is extracted with petroleum ether, dichloromethane successively, Obtain Phellinus dichloromethane extract；And
    The Phellinus dichloromethane extract is crossed into polyamide column, with alcohol-water mixed solution gradient elution, it is 77 to collect eluant, eluent Cut during~83% ethanol solution, obtain the Phellinus polyphenol extract.
  5. A kind of 5. purposes of pharmaceutical composition in the activator for promoting glucose uptake is prepared, it is characterised in that the medicine Composition includes Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins or 3,4- dihydroxies At least one of base benzylideneacetone.
  6. A kind of 6. purposes of pharmaceutical composition in the activator of GLUT4 is prepared, it is characterised in that the medicine Composition includes Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7,8- dihydroxycoumarins or 3,4- dihydroxies At least one of base benzylideneacetone.
  7. A kind of 7. purposes of pharmaceutical composition in the activator for preparing adenosine monophosphate activated protein kinase, it is characterised in that Described pharmaceutical composition includes Phellinus polyphenol extract, and Inoscavin C, described is contained in the Phellinus polyphenol extract 7,3 '-dihydroxy -5 '-methoxy isoflavone, the 7,8- dihydroxycoumarins, 3,4- dihydroxy benzenes fork acetone.
  8. 8. a kind of pharmaceutical composition is preparing treatment or prevention by the medicine or health food of metabolic disorder associated diseases Purposes, it is characterised in that described pharmaceutical composition include Inoscavin C, 7,3 '-dihydroxy -5 '-methoxy isoflavone, 7, At least one of 8- dihydroxycoumarins or 3,4- dihydroxy benzylideneacetone.
  9. 9. pharmaceutical composition according to claim 8 prepare treat or prevent by metabolic disorder associated diseases medicine or Purposes in person's health food, it is characterised in that the disease caused by metabolic disorder includes diabetes, obesity, high blood Press any one in disease and hyperlipemia.
  10. 10. pharmaceutical composition according to claim 9 is preparing medicine of the treatment or prevention by metabolic disorder associated diseases Or the purposes in health food, it is characterised in that the diabetes are type II diabetes.
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CN110882285A (en) * 2019-12-06 2020-03-17 浙江省农业科学院 Efficient preparation method of active substances in phellinus igniarius
CN111096983A (en) * 2020-01-17 2020-05-05 浙江工业大学 Phellinus igniarius phenolic extract with hypoglycemic activity and preparation and application thereof
CN111892568A (en) * 2020-09-03 2020-11-06 上海海洋大学 Daphnetin derivative serving as agonist as well as pharmaceutical composition and application of daphnetin derivative
CN111925349A (en) * 2020-09-03 2020-11-13 上海海洋大学 Daphnetin derivative as inhibitor and application and pharmaceutical composition thereof
CN111892568B (en) * 2020-09-03 2022-08-19 上海海洋大学 Daphnetin derivative serving as agonist as well as pharmaceutical composition and application of daphnetin derivative
CN111925349B (en) * 2020-09-03 2022-08-26 上海海洋大学 Daphnetin derivative as inhibitor and application and pharmaceutical composition thereof
CN112791108A (en) * 2021-02-07 2021-05-14 青岛农业大学 Preparation method of phellinus igniarius active ingredient and application of phellinus igniarius active ingredient in auxiliary hypoglycemic drugs or foods
CN116251129A (en) * 2021-12-09 2023-06-13 葡萄王生技股份有限公司 Use of Phellinus linteus GKPl mycelium for preparing composition for improving obesity and obesity related metabolic diseases

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