CN101926787A - Application of flavone compound in protecting liver - Google Patents
Application of flavone compound in protecting liver Download PDFInfo
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- CN101926787A CN101926787A CN2009100536041A CN200910053604A CN101926787A CN 101926787 A CN101926787 A CN 101926787A CN 2009100536041 A CN2009100536041 A CN 2009100536041A CN 200910053604 A CN200910053604 A CN 200910053604A CN 101926787 A CN101926787 A CN 101926787A
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Abstract
The invention relates to active traditional Chinese medicine monomer ingredients with the effect of protecting the liver, in particular to new application of a flavone active substance with a structural formula A in treating related diseases of liver inflammations. In the structural formula, R1, R2, R3 and R4 are respectively and independently selected from the following groups: -H, alkyl groups of -C1-(-C6), -OH, alkoxyl groups of -C1-(-C6), -SH, -NH2, fluorine, chlorine, bromine or iodine. Particularly, the invention relates to a flavone compound with the structural formula A, a pharmaceutically acceptable salt thereof or application of the combination of the compound and the salt thereof in preparing medical compositions for treating or preventing diseases (liver damages especially) related to serum glutamic pyruvic transaminase activity increase of targets. The flavone compound is preferably apigenin, luteolin and baicalein.
Description
Technical field
The present invention relates to biotechnology and field of medicaments, more specifically, relate to the new purposes of flavonoid active substance in disease prevention and treatment.
Background technology
Known multiple and serum glutamic pyruvic transminase are active in this area increases relevant disease, for example (but being not limited to): hepatic injury, intrahepatic cholestasis, obstruction of biliary tract, pancreatic diseases, hyperthyroidism, acute myocardial infarction, heart failure or polymyositis.Wherein, hepatic injury is the major disease of serious harm human health.
In these diseases, hepatitis is the major disease of serious harm human health.Hepatitis particularly viral hepatitis continues to raise at the sickness rate of China.With regard to hepatitis B, the whole world 2,000,000,000 people infect hepatitis B virus, and wherein 6.9 hundred million in China, and there are hepatitis B virus surface antigen (HBsAg) carrier 3.5 hundred million people in the whole world, and wherein 1/3rd in China; The whole world has 750,000 people to die from the disease that hepatitis B virus infection causes every year, and wherein 280,000 people are from China.So surprising data impel us to have to seek effective Drug therapy and prophylactic generation.
Hepatitis is seen from the cause of disease and is mainly contained viral hepatitis, alcoholic hepatitis, autoimmune hepatitis etc.But discover, no matter its initial cause of disease how, inflammation and immune-mediated hepatocyte and liver tissue injury be these diseases take place must be through process, and it is the main cause that influences disease severity and prognosis.
Therefore, explore the new method of reduction serum glutamic pyruvic transminase activity (especially hepatitis) and the research focus that medicine is field of medicaments always.
ConA (is a concanavalin A, Con A, a kind of phytohemagglutinin) inductive hepatitis model (ConA inducedhepatitis, CIH) be a kind of that extensively adopt in the world, experimental model (TiegsG, Hentschel J, Wendel A. in order to study immune-mediated hepatic injury
J Clin Invest1992,90:196-203).Induced the back 8 to 24 hours at model, glutamate pyruvate transaminase in the animal serum (ALT) activity sharply increases, pathological analysis (as HE dyeing and TUNEL) can be observed the liver normal configuration destroyed, a large amount of hepatic parenchymal cells apoptosis.Inducing in the process of CIH, cytokine tumor necrosis factor (TNF α), interferon gamma (IFN γ), il-1 2 and interleukin-4 have played pivotal role to the damage of liver.CIH is for the pathogeny of studying immune-mediated hepatic injury and seek the active drug treatment and the hepatic injury of epidemic prevention mediation provides favourable animal model.
Flavonoid (Flavonoids) chemical compound is distributed widely in plant kingdom, up to the present, has found kind more than 2000.Their majorities exist with the form of glycosides, and minority exists with free state, and its physiological action and biological activity are diversified.Conjugacy owing to the flavone compound structure all shows strong absorption to ultraviolet and visible light, and stablizes at visible and ultra-violet (UV) band inner height.Biological activity and pharmacological actions such as that the flavone compound of natural origin has is antibiotic, anti-photosensitive, antioxidation, free radical resisting, antiviral.
At present industrialization development many plant flavone class extracts, as silymarin, Semen Ginkgo extrac, tea polyphenols, Semen Vitis viniferae extract, Cortex Pini Massonianae extract, licoflavone, soybean isoflavone, Radix Puerariae isoflavone etc.
Yet, in treatment or object of prevention,, in the especially immune-mediated hepatic injury, still press for and develop effective medicine with the relevant disease of the active increase of serum glutamic pyruvic transminase.
Summary of the invention
One of purpose of the present invention is to develop the new purposes of some flavone compound in reducing gpt activity (especially prevent and treat immune-mediated hepar damnification relevant disease) just.
In a first aspect of the present invention, provide a kind of chemical compound with structural formula A, its pharmaceutically acceptable salt or they be combined in that preparation is used for the treatment of or object of prevention in the active medicine that increases relevant disease of serum glutamic pyruvic transminase in purposes:
In the formula:
R
1, R
2, R
3And R
4Independently of one another for be selected from down the group of organizing :-H ,-C
1-6Alkyl ,-OH ,-C
1-6Alkoxyl ,-SH ,-NH
2, fluorine, chlorine, bromine or iodine.
In an embodiment of the invention, described R
1, R
2, R
3And R
4Be independently of one another-H or-OH.
In yet another embodiment of the present invention, described chemical compound has the structural formula of the group of being selected from down:
In yet another embodiment of the present invention, the described disease relevant with the active increase of serum glutamic pyruvic transminase comprises: hepatic injury, intrahepatic cholestasis, obstruction of biliary tract, pancreatic diseases, hyperthyroidism, acute myocardial infarction, heart failure or polymyositis.In a preference, described hepatic injury is: the hepatic injury due to viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, drug induced hepatitis, the fatty liver.
In yet another embodiment of the present invention, described hepatic injury is immune-mediated hepatic injury.
In a preference, described immune-mediated hepatic injury is selected from: viral hepatitis, alcoholic hepatitis and autoimmune hepatitis.In another preference, described pharmaceutical composition also reduces the expression of serum cytokines in the object.In another preference, described serum cytokines is: tumor necrosis factor-alpha and/or gamma interferon.
In another embodiment, described to liking mammal.In a preference, described mammal is the people.
In yet another embodiment of the present invention, the chemical compound with structural formula A in the described pharmaceutical composition, its pharmaceutically acceptable salt or their combination account for the 0.001-99.9wt% of pharmaceutical composition gross weight.
In a preference, the chemical compound with structural formula A in the described pharmaceutical composition, its pharmaceutically acceptable salt or their combination account for the 1-95wt% of pharmaceutical composition gross weight, preferred 5-90wt%, more preferably 10-80wt%.
In yet another embodiment of the present invention, the dosage form of described pharmaceutical composition comprises: tablet, powder agent, injection, granule, syrup, capsule, solution, suppository or ointment.
In a preference, described pharmaceutical composition is unit dosage forms or multi-pharmaceutics, and the content that wherein has chemical compound, its pharmaceutically acceptable salt or their combination of structural formula A is the 1-4000mg/ agent, preferred 100-2000mg/ agent.
In another preference, with the 0.01-200mg/kg body weight, 0.1-150mg/kg body weight preferably, preferred 0.5-120mg/kg body weight, more preferably 1-70mg/kg body weight, more preferably 2-50mg/kg body weight, most preferably the 2.5-20mg/kg body weight gives described pharmaceutical composition.
In yet another embodiment of the present invention, described pharmaceutical composition also comprises the other medicines of alleviating or treating hepar damnification.
In a preference, the other medicines of described alleviation or treatment hepar damnification are selected from: Tanshinone I I A, glycyrrhizic acid preparation, green tea polyphenol, silymarin or tetrandrine.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it contains:
(1) chemical compound with structural formula A of effective dose, its pharmaceutically acceptable salt or their combination;
(2) be selected from down one or more materials of organizing: Tanshinone I I A, glycyrrhizic acid preparation, green tea polyphenol, silymarin, tetrandrine or their combination; With
(3) pharmaceutically acceptable carrier.
In a preference, described chemical compound, its pharmaceutically acceptable salt or their combination with structural formula A accounts for the 0.001-99.9wt% of composition total weight, and preferred 1-95wt% more preferably is 5-90wt%, more preferably 10-80wt%.
In another preference, described chemical compound is selected from: the chemical compound (being luteolin) of the chemical compound of structural formula B (being the celery flavin), structural formula C or chemical compound (being baicalin) or their combination of structural formula D.
In another preference, the weight ratio of component (1) and component (2) is 0.01: 100～100: 0.01, preferred 1: 95～95: 1.
In a third aspect of the present invention, treatment or prophylactic method are provided.
In a preference, the invention provides a kind of treatment or object of prevention and the active method that increases relevant disease of serum glutamic pyruvic transminase, described method comprises: the chemical compound with structural formula A of the object effective dose that needs, its pharmaceutically acceptable salt or their combination
In the formula:
R
1, R
2, R
3And R
4Independently of one another for be selected from down the group of organizing :-H ,-C
1-6Alkyl ,-OH ,-C
1-6Alkoxyl ,-SH ,-NH
2, fluorine, chlorine, bromine or iodine.
In another preference, described chemical compound is selected from: the chemical compound (being luteolin) of the chemical compound of the structural formula B as mentioned (being the celery flavin), the structural formula C shown in as mentioned or as mentioned shown in chemical compound (being baicalin) or their combination of structural formula D.
In one embodiment, the described disease relevant with the active increase of serum glutamic pyruvic transminase comprises: hepatic injury, intrahepatic cholestasis, obstruction of biliary tract, pancreatic diseases, hyperthyroidism, acute myocardial infarction, heart failure or polymyositis.
In a preference, described hepatic injury is: the hepatic injury due to viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, drug induced hepatitis, the fatty liver.
In another preference, described hepatic injury is immune-mediated hepatic injury, and is preferred: viral hepatitis, alcoholic hepatitis and autoimmune hepatitis.
In another preference, the invention provides a kind of method that serum cytokines is expressed that reduces, described method comprises: the chemical compound with structural formula A of the object effective dose that needs, its pharmaceutically acceptable salt or their combination.
In another preference, the invention provides the method for a kind of treatment or the prevention disease relevant with the serum cytokines over-expression, described method comprises: the chemical compound with structural formula A of the object effective dose that needs, its pharmaceutically acceptable salt or their combination.
In an embodiment of the invention, with the 0.01-200mg/kg body weight, 0.1-150mg/kg body weight preferably, preferred 0.5-120mg/kg body weight, more preferably 1-70mg/kg body weight, more preferably 2-50mg/kg body weight, most preferably the 2.5-20mg/kg body weight has chemical compound, its pharmaceutically acceptable salt or their combination of structural formula A.
In yet another embodiment of the present invention, the chemical compound, its pharmaceutically acceptable salt or their combination that have structural formula A with the form of tablet, powder agent, injection, granule, syrup, capsule, solution, suppository or ointment.
In yet another embodiment of the present invention, with the medicine of other alleviation or treatment hepar damnification or successively have chemical compound, its pharmaceutically acceptable salt or their combination of structural formula A.
In a preference of the present invention, the other medicines of described alleviation or treatment hepar damnification are selected from: tanshinone, glycyrrhizic acid preparation, green tea polyphenol, silymarin, tetrandrine or their combination.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1: the experiment flow sketch map of the CIH mice being implemented celery flavin (Api), luteolin (Lut) and baicalin (Bai) treatment.
Fig. 2: celery flavin (Api) is to the influence of ALT level in the CIH mice serum.Among the figure, " * * * " representative: compared with the control, P＜0.001.
Fig. 3: through the H﹠amp of the CIH mouse liver tissue of celery flavin (Api) treatment; E dyeing and TUNEL dyeing.Wherein, Fig. 3 A is control group mice liver tissue slices H﹠amp; The E coloration result; Fig. 3 B is Api processed group mice H﹠amp; The E coloration result; Fig. 3 C is a control group mice TUNEL coloration result; Fig. 3 D is an Api processed group mice TUNEL coloration result.Amplification * 100.
Fig. 4: celery flavin (Api) is to inflammatory cytokine (Fig. 4 A:TNF-α; Fig. 4 B:IFN-γ) influence.Among the figure, " * * " representative: compared with the control, and P＜0.01, " * * * " representative: compared with the control, P＜0.001.
Fig. 5: luteolin (Lut) is to the influence of ALT level in the CIH mice serum.Among the figure, " * * * " representative: compared with the control, P＜0.001.
Fig. 6: through the H﹠amp of the CIH mouse liver tissue of luteolin (Lut) treatment; E dyeing and TUNEL dyeing.Wherein, Fig. 6 A is control group mice liver tissue slices H﹠amp; The E coloration result; Fig. 6 B is Lut processed group mice H﹠amp; The E coloration result; Fig. 6 C is a control group mice TUNEL coloration result; Fig. 6 D is a Lut processed group mice TUNEL coloration result.Amplification * 100.
Fig. 7: luteolin (Lut) is to inflammatory cytokine (Fig. 7 A:TNF-α; Fig. 7 B:IFN-γ) influence.Among the figure, " * " representative: compared with the control, and P＜0.05, " * * " representative: compared with the control, P＜0.01.
Fig. 8: baicalin (Bai) is to the influence of ALT level in the CIH mice serum.Among the figure, " * * * " representative: compared with the control, P＜0.001.
Fig. 9: through the H﹠amp of the CIH mouse liver tissue of baicalin (Bai) treatment; E dyeing and TUNEL dyeing.Wherein, Fig. 9 A is control group mice liver tissue slices H﹠amp; The E coloration result; Fig. 9 B is Bai processed group mice H﹠amp; The E coloration result; Fig. 9 C is a control group mice TUNEL coloration result; Fig. 9 D is a Bai processed group mice TUNEL coloration result.Amplification * 100.
Figure 10: baicalin (Bai) is to inflammatory cytokine (Figure 10 A:TNF-α; Figure 10 B:IFN-γ) influence.Among the figure, " * " representative: compared with the control, and P＜0.05, " * * " representative: compared with the control, P＜0.01.
The specific embodiment
The inventor is by long-term and deep discovering: the flavone compound (as celery flavin, luteolin, baicalin) with structural formula A can reduce the serum glutamic pyruvic transminase activity, and immune-mediated hepatic injury is had protective effect.The inventor finds that further this type of flavone compound can reduce and the closely-related cytokine of inflammation---IFN-γ and TNF-α, thereby the clear and definite possible mechanism of its protective effect.On this basis, the inventor has finished the present invention.
Particularly, utilization of the present invention has the flavone compound (for example: celery flavin, luteolin, baicalin) of structural formula A, has confirmed their protective effects to immune-mediated hepatic injury on the CIH model.These flavone compounds all can suppress to induce behind the CIH activity of glutamate pyruvate transaminase (ALT) in the mice serum significantly, make the level of ALT be reduced to (CIH mice ALT meansigma methods is more than 8000U/L) about 1000U/L.The pathological analysis of liver shows, in the liver of the mice after these flavone compounds are handled respectively, almost can't see destruction, inflammatory infiltration and the hepatocellular apoptosis of tangible liver normal configuration.Studies show that further these flavone compounds pass through downward modulation and the closely-related cytokine of inflammation---IFN-γ and TNF-α, and suppress immune-mediated hepatic injury.
The invention provides flavone compound in prevention with treat new purposes in the immune-mediated hepatic injury, for reducing development cost and actively treating hepatic disease effectively provides a kind of new approach with structural formula A.
Flavonoid active substance with structural formula A
As used herein, term " the flavonoid active substance with structural formula A " and " flavone compound with structural formula A " are used interchangeably, be meant flavone structure-activity composition, its pharmaceutically acceptable salt or their mixture with structural formula A
In the formula:
R
1, R
2, R
3And R
4Independently of one another for be selected from down the group of organizing :-H ,-C
1-6Alkyl ,-OH ,-C
1-6Alkoxyl ,-SH ,-NH
2, fluorine, chlorine, bromine or iodine.Preferred described active substance has for as described in more detail below: celery flavin (structural formula B), luteolin (structural formula C) and/or baicalin (structural formula D).
In the present invention, have no particular limits, can extract from natural plants (for example Chinese crude drug), maybe can adopt the method for chemosynthesis or half chemosynthesis to prepare such as it for the method for preparing the flavonoid active substance.Employed flavonoid active substance can obtain by commercially available approach among the present invention, for example can be available from shanghai food drug inspection office, Sigma etc.
Representational flavonoid active substance following (but being not limited to) with structural formula A:
A. celery flavin
Celery flavin (apigenin) is a kind of flavone compound, claims apigenin or apiolin again, be distributed widely in the vegetable and fruit of warm band, and be height with content in the Herba Apii graveolentis especially.Very high content is also arranged in some medicinal plants such as Semen Plantaginis, Caulis Trachelospermi etc., in plant-derived beverage such as tea, wine and some flavoring agent distribution is arranged also.The chemical constitution of apigenin is 5,7,4 '-trihydroxyflavone (shown in the formula B as mentioned), relative molecular mass is 270.
Apigenin is water insoluble, is soluble in ethanol, dimethyl sulfoxide (DMSO).But the celery flavin is synthetic now, is a kind of abundance of originating, action range, natural plant composition with wide DEVELOPMENT PROSPECT.
As far back as the seventies in 20th century, external researcher is just studied celery flavin and related compound thereof.Yet, these researchs mainly concentrate on celery flavin antitumor and chemotherapy sensitizing effect, to aspects such as the influence of endocrine function, antibiotic, antivirus actions, and do not relate to its to the active effect that prevents and/or treats that increases relevant disease (especially immune-mediated hepatic injury) of serum glutamic pyruvic transminase.
Having proved among the present invention that celery flavin pair and serum glutamic pyruvic transminase are active increases relevant disease, especially immune-mediated hepatic injury has prevention and therapeutical effect, and the celery flavin of low dosage can suppress to induce behind the CIH activity of glutamate pyruvate transaminase (ALT) in the mice serum very significantly.And the pathological analysis of liver shows, in the liver of the mice after the celery flavin is handled, almost can't see destruction, inflammatory infiltration and the hepatocellular apoptosis of tangible liver normal configuration.Studies show that further the celery flavin passes through downward modulation and the closely-related cytokine of inflammation---IFN-γ and TNF-α, and suppresses immune-mediated hepatic injury.
The celery flavin that can be used among the present invention includes but not limited to: extract the celery flavin from natural plants (for example Herba Apii graveolentis, Semen Plantaginis, Caulis Trachelospermi etc.), the chemistry or the celery flavin of half chemosynthesis, or commercially available celery flavin (for example can available from shanghai food drug inspection office, Sigma etc.).
When making pharmaceutical composition, the celery flavin can be neutral form or salt form.These salt comprise acid-addition salts, with mineral acid, and for example hydrochloric acid, sulfonic acid or phosphoric acid, or and organic acid, for example salt of formation such as acetic acid, oxalic acid, tartaric acid, mandelic acid.Suitable base addition salts includes but not limited to: based on the cation of alkali metal or alkaline-earth metal, comprise lithium, sodium, potassium, calcium, magnesium and aluminum salt; Non-toxicity quaternary ammonium and amine cation comprise ammonium, tetramethylammonium, etamon, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine and ethamine.Other the representative organic amine that is used to form base addition salts comprises ethylenediamine, ethanolamine, diethanolamine, piperidines and piperazine etc.
B. luteolin
Luteolin (luteolin) is a kind of natural flavone compounds, is present in the various plants, shown in its structure structural formula C as mentioned.
This chemical compound has multiple pharmacologically active, as antiinflammatory, antiallergic, antitumor, antibiotic, antiviral etc., clinically is mainly used in cough-relieving, eliminates the phlegm, antiinflammatory, treatment cardiovascular disease, treatment " amyotrophic lateral sclerosis ", SARS etc.Yet this area does not relate to the effect that prevents and/or treats of the luteolin pair disease (especially immune-mediated hepatic injury) relevant with the active increase of serum glutamic pyruvic transminase.
Proved among the present invention that luteolin has protective effect to immune-mediated hepatic injury, the celery flavin of low dosage can suppress to induce behind the CIH activity of glutamate pyruvate transaminase (ALT) in the mice serum very significantly.And the pathological analysis of liver shows, in the liver of the mice after luteolin is handled, almost can't see destruction, inflammatory infiltration and the hepatocellular apoptosis of tangible liver normal configuration.Studies show that further luteolin passes through downward modulation and the closely-related cytokine of inflammation---IFN-γ and TNF-α, and suppresses immune-mediated hepatic injury.
The luteolin that can be used among the present invention includes but not limited to: extract the luteolin from natural plants (for example leaf of wooden slippers grass (ResedaodorataL.), stem and branch, Flos Lonicerae, Flos Chrysanthemi, Herba Schizonepetae, Herba Ajugae etc.), the chemistry or the luteolin of half chemosynthesis, or commercially available luteolin (for example can available from shanghai food drug inspection office, Sigma etc.).
When making pharmaceutical composition, luteolin can be neutral form or salt form.These salt comprise acid-addition salts, with mineral acid, and for example hydrochloric acid, sulfonic acid or phosphoric acid, or and organic acid, for example salt of formation such as acetic acid, oxalic acid, tartaric acid, mandelic acid.Suitable base addition salts includes but not limited to: based on the cation of alkali metal or alkaline-earth metal, comprise lithium, sodium, potassium, calcium, magnesium and aluminum salt; Non-toxicity quaternary ammonium and amine cation comprise ammonium, tetramethylammonium, etamon, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine and ethamine.Other the representative organic amine that is used to form base addition salts comprises ethylenediamine, ethanolamine, diethanolamine, piperidines and piperazine etc.
C. baicalin
Baicalin (Baicalein) is a kind of natural flavone compounds, is present in the various plants, shown in its structure structural formula D as mentioned.
Studies show that baicalin has the reduction cerebral vascular resistance, improve the effect of brain blood circulation, cerebral blood flow increasing amount and anti-platelet aggregation, clinically be used for the treatment of paralysing behind the cerebrovascular etc.Yet this area does not relate to the effect that prevents and/or treats of the baicalin pair disease (especially immune-mediated hepatic injury) relevant with the active increase of serum glutamic pyruvic transminase.
Proved among the present invention that baicalin has protective effect to immune-mediated hepatic injury, the baicalin of low dosage can suppress to induce behind the CIH activity of glutamate pyruvate transaminase (ALT) in the mice serum very significantly.And the pathological analysis of liver shows, in the liver of the mice after baicalin is handled, almost can't see destruction, inflammatory infiltration and the hepatocellular apoptosis of tangible liver normal configuration.Studies show that further baicalin passes through downward modulation and the closely-related cytokine of inflammation---IFN-γ and TNF-α, and suppresses immune-mediated hepatic injury.
The baicalin that can be used among the present invention includes but not limited to: extract the baicalin from natural plants (for example the leaf of the high Radix Scutellariae of labiate (Scutellaria altissimaL.), and the leaf of the seed of the Ye Hegen of head grass (S.galericulataL.), Bignoniaceae plant Semen Oroxyli (Oroxylum indicum (L.) Vent.) and peel of stem, Plantaginaceae plant Big Semen Plantaginis (Plantago majorL.) etc.), the chemistry or the baicalin of half chemosynthesis, or commercially available baicalin (for example can available from shanghai food drug inspection office, Sigma etc.).
When making pharmaceutical composition, baicalin can be neutral form or salt form.These salt comprise acid-addition salts, with mineral acid, and for example hydrochloric acid, sulfonic acid or phosphoric acid, or and organic acid, for example salt of formation such as acetic acid, oxalic acid, tartaric acid, mandelic acid.Suitable base addition salts includes but not limited to: based on the cation of alkali metal or alkaline-earth metal, comprise lithium, sodium, potassium, calcium, magnesium and aluminum salt; Non-toxicity quaternary ammonium and amine cation comprise ammonium, tetramethylammonium, etamon, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine and ethamine.Other the representative organic amine that is used to form base addition salts comprises ethylenediamine, ethanolamine, diethanolamine, piperidines and piperazine etc.
New purposes with flavonoid active substance of structural formula A
The flavonoid active substance has been used for diseases such as cardiovascular system, nervous system, hormonal system, tumor in former studies and clinical practice.
The cell-mediated hepatitis model " the inductive hepatitis model of ConA " of the T that the inventor utilizes this area to accept extensively (CIH); the flavone compound that has confirmed to have structural formula A has immune-mediated hepatic injury is had significant protective effect, and the flavone compound with structural formula A can make the active of CIH mice serum glutamate pyruvate transaminase (ALT) significantly reduce.The pathological analysis of liver also shows, compares with matched group all at the destruction and the male apoptotic cell of TUNEL of flavone compound processed group mouse liver normal configuration and obviously reduces.
The deep work of inventor further shows: the flavone compound with structural formula A is its inflammatory cytokine that causes mediating hepatic injury to the mechanism of the protective effect of immune-mediated hepatic injury; comprise the remarkable decline that gamma interferon and tumor necrosis factor are expressed, thereby the liver protecting is avoided damage.
As used herein, term " immune-mediated hepatic injury " is all general designations by immune-mediated hepar damnification, has all played important function in the pathogenic process of many hepatitis.Immune-mediated hepatic injury includes but not limited to: hepar damnification diseases such as viral hepatitis, alcoholic hepatitis, autoimmune hepatitis.Hepatic injury, hepatic fibrosis and hepatocarcinoma are the different pathological stages of hepar damnification, immune-mediated hepatic injury generally appears at the early stage of acute stage or chronic phase, if change many fibrosiss that can cause liver of chronic phase over to by acute stage, if and hepatocyte self-regeneration system disorders, a large amount of malignant proliferation of cell just changes hepatocarcinoma into.
The new purposes of the flavonoid active substance with structural formula A is provided among the present invention, its can be used for treating or object of prevention increase relevant disease with serum glutamic pyruvic transminase is active, comprising: the expression of the hepatic injury (especially immune-mediated hepatic injury) of treatment or object of prevention or reduction serum cytokines (for example tumor necrosis factor-alpha and/or gamma interferon).The described disease relevant with the active increase of serum glutamic pyruvic transminase includes, but is not limited to: hepatic injury, intrahepatic cholestasis, obstruction of biliary tract, pancreatic diseases, hyperthyroidism, acute myocardial infarction, heart failure or polymyositis.Wherein, hepatic injury includes, but is not limited to: the hepatic injury due to viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, drug induced hepatitis, the fatty liver.
In a preference, described hepatic injury is immune-mediated hepatic injury, is preferably viral hepatitis, alcoholic hepatitis and autoimmune hepatitis.
Correspondingly, also provide flavonoid active substance to be used for the treatment of or to prevent purposes in the pharmaceutical composition of above-mentioned disease among the present invention, and utilized and have the flavonoid active substance of structural formula A or the method that above-mentioned disease was treated or prevented to its pharmaceutical composition in preparation with structural formula A.
Should be understood that used term " alleviation " is meant among the present invention makes the symptom of disease or imbalance improve, even this disease or imbalance disappears or recovery from illness.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition at treatment or object of prevention and the active purposes that increases in the relevant disease (especially hepatic injury) of serum glutamic pyruvic transminase, described pharmaceutical composition contains the flavonoid active substance with structural formula A and the pharmaceutically acceptable carrier of effective dose, or optional other other active component that above-mentioned disease is had treatment or preventive effect known in the art that contains.
As used herein, term " contain " or " comprising " comprised " comprising ", " basically by ... constitute " and " by ... constitute ".
As used herein, the composition of term " pharmaceutically acceptable " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and allergy), the material of rational benefit/risk ratio is promptly arranged.
As used herein, term " effective dose " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art." Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences, Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991).
Acceptable carrier can contain liquid on combination of Chinese medicine is learned, as water, saline, glycerol and ethanol.In addition, also may there be complementary material in these carriers, as filler, disintegrating agent, lubricant, fluidizer, effervescent, wetting agent or emulsifying agent, correctives, pH buffer substance etc.Usually, these materials can be formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.
Can contain as effective ingredient in the pharmaceutical composition of the present invention: flavone compound, its pharmaceutically acceptable salt or their mixture with structural formula A.
The flavonoid active substance effective ingredient that has structural formula A in the compositions of the present invention accounts for the 0.001-99.9wt% of composition total weight; Being preferably the 1-95wt% of composition total weight, more preferably is 5-90wt%, more preferably 10-80wt%.Surplus is materials such as pharmaceutically acceptable carrier and other additive.
As used herein, term " unit dosage forms " is meant for taking convenience, becomes single to take required dosage form preparation of compositions of the present invention, includes but not limited to various solid formulation (as tablet), liquid agent, capsule, slow releasing agent.
In a preferred embodiment of the present invention, described compositions is unit dosage forms or multi-pharmaceutics, and the flavonoid content of active substance that wherein has a structural formula A is the 1-4000mg/ agent, preferred 100-2000mg/ agent.
In a preference of the present invention, contain the flavonoid active substance with structural formula A of low dosage in the described compositions, for example 1-10mg/ agent, more preferably 2-8mg/ agent further preferably contains the 3-6mg/ agent.
In another preference of the present invention, the flavonoid active substance with structural formula A of dosage in containing in the described compositions, for example its content is the 10-1000mg/ agent; More preferably 20-800mg/ agent, further preferred 50-600mg/ agent, more preferably 75-500mg/ agent.
In another preference of the present invention, contain the flavonoid active substance with structural formula A of high dose in the described compositions, for example its content is the 1000-3500mg/ agent; More preferably 1100-3200mg/ agent, further preferred 1200-3000mg/ agent, more preferably 1500-2000mg/ agent.
In another preference of the present invention, use 1-6 agent compositions of the present invention every day, preferably use the 1-3 agent; Take 1 dose preferred every day when high dose.
Compositions of the present invention can be solid-state (as granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (as oral liquid, injection) or other suitable form.
The effective dose that should be understood that used flavonoid active substance can change with the order of severity of object to be administered or treatment.Concrete condition decides according to the individual instances (for example object body weight, age, health, the required effect that reaches) of object, and this is in the scope that skilled practitioners or nutritionist can judge.
Method of application
Compositions of the present invention can be used by conventional route, comprising (but being not limited to): oral, intramuscular injection, subcutaneous injection etc.Preferred oral is used.Composition forms should be complementary with method of application.
Those skilled in the art can determine to have the dose of the flavonoid active substance of structural formula A as required.The amount of application of the common present composition, by active substance weight, be generally about 0.01-200mg active substance every day/kg body weight, preferably about 0.1-150mg active substance/kg body weight, preferred 0.5-120mg active substance/kg body weight, more preferably 1-70mg/kg body weight, more preferably 2-50mg active substance/kg body weight, most preferably 2.5-20mg active substance/kg body weight.
In one embodiment, the present composition contains the celery flavin as active substance, by celery flavin weight, be generally about 0.5-120mg celery flavin every day/kg body weight, more preferably 1-70 celery flavin mg/kg body weight, more preferably 2-50mg celery flavin/kg body weight, most preferably 2.5-20mg celery flavin/kg body weight drug administration.
In one embodiment, the present composition contains the luteolin as active substance, by luteolin weight, be generally about 1-200mg luteolin every day/kg body weight, more preferably 2-140 luteolin mg/kg body weight, more preferably 4-100mg luteolin/kg body weight, most preferably 5-40mg luteolin/kg body weight drug administration.
In one embodiment, the present composition contains the baicalin as active substance, by baicalin weight, be generally about 0.5-120mg baicalin every day/kg body weight, more preferably 1-70 baicalin mg/kg body weight, more preferably 2-50mg baicalin/kg body weight, most preferably 2.5-20mg baicalin/kg body weight drug administration.
Compositions of the present invention can directly be used, and also can unite use with other therapeutic agent or adjuvant.
In preferred implementation of the present invention, compositions of the present invention also can with effective dose (as 0.0005-0.1 gram/kg body weight/day; Preferably, be 0.001-0.05 gram/kg body weight/day) being selected under the group one or more materials co-administered: Tanshinone I I A, glycyrrhizic acid preparation, silymarin, green tea polyphenol or tetrandrine etc.
When two or more medication combined administration, generally have and be better than the individually dosed respectively effects of two kinds of medicines.Preferably, co-administered medicine or other preparation therapeutic activity of not disturbing flavonoid active substance effective ingredient of the present invention.
Advantage of the present invention
The present invention has disclosed the new purposes of flavonoid active substance in treatment or prevention hepar damnification with structural formula A, has the following advantages:
The present invention disclosed flavonoid active substance with structural formula A treatment or object of prevention with the active effect and the mechanism of action thereof that increases relevant disease (especially treatment or prevent hepar damnification) of serum glutamic pyruvic transminase, thereby provide new approach for the treatment and the prevention of relevant disease;
2. the flavonoid active substance with structural formula A that adopts among the present invention mostly is known drug, and this area has been known its pharmacokinetics, toxicology etc., has therefore reduced R﹠D costs; And known its process for producing in this area can prepare this medicine according to the prior art condition, has therefore reduced production cost.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experiment material and animal
Animal: C57BL/6 strain mice, 7-8 week, male, body weight 24-25g, available from Shanghai Slac Experimental Animal Co., Ltd., random packet;
ConA: available from Vector Laboratory Inc., be dissolved in PBS (pH 7.2-7.4), concentration is 2.25mg/ml.
Celery flavin (Api):,,, face with preceding with PBS (pH 7.2-7.4) dilution (concentration is 3.125mg/ml or 6.25mg/ml) in-20 ℃ of preservations earlier with DMSO dissolving (concentration is 0.17mg/ μ l) available from Shanghai City food and medicine calibrating institute.
Luteolin (Lut):,,, face with preceding with PBS (pH 7.2-7.4) dilution (concentration is 6.25mg/ml or 12.5mg/ml) in-20 ℃ of preservations earlier with DMSO dissolving (concentration is 0.17mg/ μ l) available from Shanghai City food and medicine calibrating institute.
Baicalin (Bai):,,, face with preceding with PBS (pH 7.2-7.4) dilution (concentration is 3.125mg/ml or 6.25mg/ml) in-20 ℃ of preservations earlier with DMSO dissolving (concentration is 0.17mg/ μ l) available from Shanghai City food and medicine calibrating institute.
Contrast solution: the PBS solution (DMSO concentration is 7.5%, and the pH of PBS is 7.2-7.4) that contains DMSO.
Test method:
The treatment experiment flow of flavone compound celery flavin (Api), luteolin (Lut) and baicalin (Bai) is as shown in Figure 1:
(1), gives contrast solution (200 μ l, matched group) or certain density flavone compound (administration group) through lumbar injection with the mice random packet;
(2) administration is after 2 hours, and each mice is accepted same administration and handles;
(3) subsequently, giving all mices immediately is the ConA of 15mg/kg body weight by tail vein injection dosage, to induce CIH;
(4a) after 2 hours, put to death mice, obtain serum, detect the level of TNF-α in the serum in injection ConA;
(4b) after 8 hours, put to death mice, obtain serum and liver, detect the level of serum alt and IFN-γ, and carry out the histopathological analysis of liver in injection ConA.
Embodiment 1 celery flavin (Api) is to the active reduction effect of CIH mice glutamate pyruvate transaminase (ALT)
(A) test
Carry out celery flavin (Api) treatment experiment according to record of above " test method " part, used Api dosage is respectively: 25mg/kg (low dosage Api group) and 50mg/kg (high dose Api group).Give ConA after 8 hours, put to death mice, get the level that serum detects ALT.
(B) glutamate pyruvate transaminase enzyme mensuration alive
Measuring process: the serum glutamic pyruvic transminase determination of activity test kit that adopts Shanghai Yi Hua medical science and technology company limited to produce, operation to specifications.
Concise and to the point step is as follows: serum is got 25 microlitres and is added to 96 orifice plates after suitably diluting, and adds 100 microlitre enzyme working solutions, hatched 15 minutes for 37 ℃, add 150 microlitre stop buffers, microplate reader 492nm reading, the standard serum and the extension rate that provide according to test kit calculate gpt activity numerical value.
(C) result of the test and analysis
Api treatment to the result of ALT activity influence respectively shown in Fig. 2 A and 2B.Data are represented with the form of average ± standard deviation, have represented the result (* * * P＜0.001) of 3 tests.
Low concentration Api group and high concentration Api group result all show: but the Api utmost point reduces the activity of glutamate pyruvate transaminase in the CIH mice serum significantly.Wherein, high dose Api more is better than low dosage Api to the active reduction effect of ALT.
Hence one can see that: Api can suppress to induce the level of ALT in the mice serum of CIH effectively.
The H﹠amp of the CIH mouse liver of embodiment 2 after celery flavin (Api) is handled; E dyeing and TUNEL dyeing are observed
(A) test:
Carry out celery flavin (Api) treatment experiment according to record of above " test method " part, used Api dosage is: 50mg/kg.Give ConA after 8 hours, put to death mice, get liver organization and cut into slices, carry out histopathological analysis.
(B) H﹠amp; E dyeing
H﹠amp is carried out in section to the mouse liver tissue; E dyeing.Dyeing course is as follows:
1. behind the mouse anesthesia, after removing remained blood behind the PBS heart perfusion, get liver, it is fixing to put into the pipe back that 4% paraformaldehyde is housed;
2. dehydration: the tissue after will fixing is cut into and passes through following steps after the suitable size successively:
2.1.70% ethanol, 30min,
2.2.95% ethanol, 1h 30min repeats once,
2.3.100% ethanol, 1h 30min repeats once;
3. transparent: the good tissue that will dewater is put into dimethylbenzene, and 15-20min repeats once;
4. waxdip: transparent good tissue is put into the paraffin (60 ℃) of thawing, and 1h repeats once;
5. embedding;
6. section: embedded piece of tissue is cut into the thick thin slice of 5 μ m;
7. exhibition sheet: the tissue of 5 μ m is layered on 40 ℃ the water surface, allows tissue fully flatten;
8. drag for sheet: the tissue that flattens is dragged on the slide that poly-D-lysine is handled well;
9. slide is placed on 37 ℃ the baking platform and dries;
10. dewaxing hydration: the slide that has a tissue that will dry is through the following steps hydration that dewaxes:
10.1. dimethylbenzene, 5min repeats once,
10.2.100% ethanol, 5min repeats once,
10.3.95% ethanol, 5min repeats once,
10.470% ethanol, 5min,
10.5 water, 5min;
The 7min 11. dye in the haematoxylin;
12. flowing water flushing;
13. inverse 5min in the water;
The 0.5min 14. dye in Yihong;
15. the dehydration transparence, mounting:
15.1.70% ethanol, 2min repeats once,
15.2.95% ethanol, 2min repeats once,
15.3.100% ethanol, 2min repeats once,
15.4. dimethylbenzene, 5min repeats once,
15.5. neutral gum mounting.
(C) TUNEL dyeing
TUNEL dyeing is carried out in the section of mouse liver tissue.The TUNEL dyeing course is as follows:
1. behind the mouse anesthesia, remove remained blood through the PBS heart perfusion, get liver, it is fixing to put into the pipe back that 4% paraformaldehyde is housed;
2. dehydration: the tissue after will fixing is cut into and passes through following steps after the suitable size successively:
2.1.70% ethanol, 30min,
2.2.95% ethanol, 1h30min repeats once,
2.3.100% ethanol, 1h30min repeats once;
3. transparent: the good tissue that will dewater is put into dimethylbenzene, and 15-20min repeats once;
4. waxdip: transparent good tissue is put into the paraffin (60 ℃) of thawing, and 1h repeats once;
5. paraffin embedding;
6. section: embedded piece of tissue is cut into the thick thin slice of 5 μ m;
7. exhibition sheet: tissue is layered on 40 ℃ the water surface, allows tissue fully flatten;
8. drag for sheet: the tissue that flattens is dragged on the slide that poly-D-lysine is handled well;
9. slide is placed on 37 ℃ the baking platform and dries;
10. dewaxing hydration: the slide that has a tissue that will dry is through the following steps hydration that dewaxes:
10.1. dimethylbenzene, 5min,
10.2.100% ethanol, 5min,
10.3.95% ethanol, 5min,
10.4.90% ethanol, 5min,
10.5.80% ethanol, 5min,
10.6.70% ethanol, 5min,
10.7. water, 5min,
10.8.PBS，5min；
11. unload the unnecessary liquid around the tissue on the slide with careful suction of filter paper, add E.C. 3.4.21.64 solution (20 μ g/ml), 37 ℃ of hydrolysis 15min in the wet box remove histone.Wash twice with PBS, each 5min;
12. add the methanol that contains 6% hydrogen peroxide in the color cylinder, in room temperature reaction 5min.Wash twice with PBS, each 5min;
13. unload the unnecessary liquid around the tissue on the slide with careful suction of filter paper, every specimen adds presses test kit (TUNEL apoptosis detection kit, available from Luo Shi) TdT enzyme reaction solution (the 5 μ lTdT enzymes+every specimen of 45 μ l marking fluids of description preparation, the negative control not enzyme-added liquid of liquid of only labelling), coverslip covers, 37 ℃ of reaction 1h wash twice with PBS in the wet box, each 5min;
14. unload the unnecessary liquid around the tissue on the slide with careful suction of filter paper, every specimen adds 50 μ l POD, 37 ℃ of reaction 30min in the wet box.Wash twice with PBS, each 5min;
15. unload the unnecessary liquid around the tissue on the slide with careful suction of filter paper, every specimen adds the DAB solution of the fresh configuration of 100 μ l, colour developing 3-10min;
16. the dehydration transparence, mounting:
16.1. water, 2min repeat once,
16.2.70% ethanol, 2min repeats once,
16.3.95% ethanol, 2min repeats once,
16.4.100% ethanol, 2min repeats once,
16.5. dimethylbenzene, 5min repeats once,
16.6. neutral gum mounting.
(D) experimental result and discussion
Liver tissue slices H﹠amp after the Api treatment; E dyeing or TUNEL coloration result are respectively shown in Fig. 3 A and 3B.
Can obviously be observed by figure: after the mice after handling through Api was induced CIH, the inflammatory infiltration and the hepatic necrosis at mouse liver position were starkly lower than control group mice.
Above result shows: Api can suppress to induce inflammatory infiltration and the hepatic necrosis in the mouse liver behind the CIH effectively.
Embodiment 3. celery flavin (Api) are to the influence of inflammatory cytokine
(A) concentration of tumor necrosis factor-alpha (TNF-α) detects in the serum
Carry out Api (dosage is 50mg/kg) treatment according to record of above " test method " part, give ConA after 2 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that provides with manufacturer detects the concentration of TNF-α in the serum.
(B) concentration of serum gamma interferon detects
Carry out Api (dosage is 50mg/kg) treatment according to record of above " test method " part, give ConA after 8 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that provides with manufacturer detects the concentration of gamma interferon (IFN-γ) in the serum.
(C) ELISA reagent and method
Use Duoset ELISA test kit (R﹠amp; D, MN, USA), according to the description operation that the manufacturer provides, concise and to the point step is as follows:
Getting 100 microlitres, one anti-wrapper sheet spends the night, 300 microlitres sealing buffer sealed 2 hours after washing plate, add 100 microlitre sample and standard substance after washing plate, hatched 2 hours, wash and add two anti-hatching 2 hours behind the plate, add enzyme mark streptavidin after washing plate, add the substrate colour developing, sulphuric acid is ended, microplate reader 450nm reading (reference wavelength is 540nm).Concentration according to each cytokine in the standard curve calculation sample.
(D) experimental result and discussion
Shown in Fig. 4 A and 4B, data have wherein been represented the result (* * P＜0.01, * * * P＜0.0001) of 3 tests respectively to the concentration test experience result of the concentration test experience of tumor necrosis factor-alpha and gamma interferon respectively in the mice serum.
The result shows: the water mean pole of two kinds of inflammatory cytokines significantly is lower than matched group in the Api processed group mice serum, can effectively suppress the expression of inflammatory cytokine.
This result shows: Api is its inflammatory cytokine that has caused the mediation hepatic injury to one of mechanism of the protective effect of immune-mediated hepatic injury, comprise the remarkable decline that gamma interferon and tumor necrosis factor are expressed, thereby the liver protecting is avoided damage.
Embodiment 4. luteolins (Lut) are to the active reduction effect of CIH mice glutamate pyruvate transaminase (ALT)
(A) test
Carry out luteolin (Lut) treatment experiment according to record of above " test method " part, used Lut dosage is respectively: 50mg/kg (low dosage Lut group) and 100mg/kg (high dose Lut group).Give ConA after 8 hours, put to death mice, get the level that serum detects ALT.
(B) glutamate pyruvate transaminase enzyme mensuration alive
Adopt method described in the embodiment 1 (B) and reagent to measure the ALT activity.
(C) result of the test and analysis
Lut treatment to the result of ALT activity influence respectively shown in Fig. 5 A and 5B.Data are represented with the form of average ± standard deviation, have represented the result (* * * P＜0.001) of 3 tests.
Low concentration Lut group and high concentration Lut group result all show: but the Lut utmost point reduces the activity of glutamate pyruvate transaminase in the CIH mice serum significantly.Wherein, high dose Lut more is better than low dosage Lut to the active reduction effect of ALT.
Hence one can see that: Lut can suppress to induce the level of ALT in the mice serum of CIH effectively.
Embodiment 5. is through the H﹠amp of the CIH mouse liver of luteolin (Lut) processing; E dyeing and TUNEL dyeing are observed
(A) test:
Carry out luteolin (Lut) treatment experiment according to record of above " test method " part, used Lut dosage is: 100mg/kg.Give ConA after 8 hours, put to death mice, get liver organization and cut into slices, carry out histopathological analysis.
(B) H﹠amp; E dyeing
Adopt method and the reagent described in the embodiment 2 (B) that H﹠amp is carried out in the section of mouse liver tissue; E dyeing.
(C) TUNEL dyeing
Adopt method described in the embodiment 2 (C) and reagent that TUNEL dyeing is carried out in the section of mouse liver tissue.
(D) experimental result and discussion
Liver tissue slices H﹠amp after the Lut treatment; E dyeing or TUNEL coloration result are respectively shown in Fig. 6 A and 6B.
Can obviously be observed by figure: after the mice after handling through Lut was induced CIH, the inflammatory infiltration and the hepatic necrosis at mouse liver position were starkly lower than control group mice.
Above result shows: Lut can suppress to induce inflammatory infiltration and the hepatic necrosis in the mouse liver behind the CIH effectively.
Embodiment 6 luteolins (Lut) are to the influence of inflammatory cytokine
(A) concentration of tumor necrosis factor-alpha (TNF-α) detects in the serum
Carry out Lut (dosage is 100mg/kg) treatment according to record of above " test method " part, give ConA after 2 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that adopts manufacturer to provide detects the concentration of TNF-α in the serum.
(B) concentration of gamma interferon detects in the serum
Carry out Lut (dosage is 100mg/kg) treatment according to record of above " test method " part, give ConA after 8 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that adopts manufacturer to provide detects the concentration of gamma interferon (IFN-γ) in the serum.
(C) ELISA reagent and method
Adopt the ELISA method described in the embodiment 3 (C) that TUNEL dyeing is carried out in the section of mouse liver tissue.
(D) experimental result and discussion
Shown in Fig. 7 A and 7B, data have wherein been represented the result (* P＜0.05, * * P＜0.01) of 3 tests respectively to the concentration test experience result of the concentration test experience of tumor necrosis factor-alpha and gamma interferon respectively in the mice serum.
The result shows: the level of two kinds of inflammatory cytokines significantly or extremely significantly is lower than matched group in the Lut processed group mice serum, can effectively suppress the expression of inflammatory cytokine.
This result shows: Lut is its inflammatory cytokine that has caused the mediation hepatic injury to one of mechanism of the protective effect of immune-mediated hepatic injury, comprise the remarkable decline that gamma interferon and tumor necrosis factor are expressed, thereby the liver protecting is avoided damage.
Embodiment 7. baicalins (Bai) are to the active reduction effect of CIH mice glutamate pyruvate transaminase (ALT)
(A) test
Carry out baicalin (Bai) treatment experiment according to record of above " test method " part, used Bai dosage is respectively: 25mg/kg (low dosage Bai group) and 50mg/kg (high dose Bai group).Give ConA after 8 hours, put to death mice, get the level that serum detects ALT.
(B) glutamate pyruvate transaminase enzyme mensuration alive
Adopt method described in the embodiment 1 (B) and reagent to measure the ALT activity.
(C) result of the test and analysis
Bai treatment to the result of ALT activity influence respectively shown in Fig. 8 A and 8B.Data are represented with the form of average ± standard deviation, have represented the result (* * * P＜0.001) of 3 tests.
Low concentration Bai group and high concentration Bai group result all show: but the Bai utmost point reduces the activity of glutamate pyruvate transaminase in the CIH mice serum significantly.
Hence one can see that: Bai can suppress to induce the level of ALT in the mice serum of CIH effectively.
Embodiment 8. is through the H﹠amp of the CIH mouse liver of baicalin (Bai) processing; E dyeing and TUNEL dyeing are observed
(A) test:
Carry out baicalin (Bai) treatment experiment according to record of above " test method " part, used Bai dosage is: 50mg/kg.Give ConA after 8 hours, put to death mice, get liver organization and cut into slices, carry out histopathological analysis.
(B) H﹠amp; E dyeing
Adopt method and the reagent described in the embodiment 2 (B) that H﹠amp is carried out in the section of mouse liver tissue; E dyeing.
(C) TUNEL dyeing
Adopt method described in the embodiment 2 (C) and reagent that TUNEL dyeing is carried out in the section of mouse liver tissue.
(D) experimental result and discussion
Liver tissue slices H﹠amp after the Bai treatment; E dyeing or TUNEL coloration result are respectively shown in Fig. 9 A and 9B.
Can obviously be observed by figure: after the mice after handling through Bai was induced CIH, the inflammatory infiltration at mouse liver position and hepatic necrosis will be starkly lower than matched group.
Above result shows: Bai can suppress to induce inflammatory infiltration and the hepatic necrosis in the mouse liver behind the CIH effectively.
Embodiment 9 baicalins (Bai) are to the influence of inflammatory cytokine
(A) concentration of tumor necrosis factor-alpha (TNF-α) detects in the serum
Carry out Bai (dosage is 50mg/kg) treatment according to record of above " test method " part, give ConA after 2 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that adopts manufacturer to provide detects the concentration of TNF-α in the serum.
(B) concentration of gamma interferon detects in the serum
Carry out Bai (dosage is 50mg/kg) treatment according to record of above " test method " part, give ConA after 8 hours, putting to death mice, getting serum, by the ELISA method described in (C), the ELISA test kit that adopts manufacturer to provide detects the concentration of gamma interferon (IFN-γ) in the serum
(C) ELISA reagent and method
Adopt the ELISA method described in the embodiment 3 (C) that TUNEL dyeing is carried out in the section of mouse liver tissue.
(D) experimental result and discussion
Shown in Figure 10 A and 10B, data have wherein been represented the result (* P＜0.05, * * P＜0.01) of 3 tests respectively to the concentration test experience result of the concentration test experience of tumor necrosis factor-alpha and gamma interferon respectively in the mice serum.
The result shows: the level of two kinds of inflammatory cytokines significantly or extremely significantly is lower than matched group in the Bai processed group mice serum, can effectively suppress the expression of inflammatory cytokine.
This result shows: Bai is its inflammatory cytokine that has caused the mediation hepatic injury to one of mechanism of the protective effect of immune-mediated hepatic injury, comprise the remarkable decline that gamma interferon and tumor necrosis factor are expressed, thereby the liver protecting is avoided damage.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
- Chemical compound with structural formula A, its pharmaceutically acceptable salt or they be combined in that preparation is used for the treatment of or object of prevention in the active medicine that increases relevant disease of serum glutamic pyruvic transminase in purposes:
In the formula:R 1, R 2, R 3And R 4Independently of one another for be selected from down the group of organizing :-H ,-C 1-6Alkyl ,-OH ,-C 1-6Alkoxyl ,-SH ,-NH 2, fluorine, chlorine, bromine or iodine. - 2. purposes as claimed in claim 1 is characterized in that, described R 1, R 2, R 3And R 4Be independently of one another-H or-OH.
- 3. purposes as claimed in claim 1 is characterized in that, described chemical compound has the structural formula of the group of being selected from down:
- 4. purposes as claimed in claim 1, it is characterized in that the described disease relevant with the active increase of serum glutamic pyruvic transminase comprises: hepatic injury, intrahepatic cholestasis, obstruction of biliary tract, pancreatic diseases, hyperthyroidism, acute myocardial infarction, heart failure or polymyositis.
- 5. purposes as claimed in claim 1 is characterized in that, described hepatic injury is immune-mediated hepatic injury.
- 6. purposes as claimed in claim 1 is characterized in that, and is described to liking mammal.
- 7. purposes as claimed in claim 1 is characterized in that, the chemical compound with structural formula A in the described pharmaceutical composition, its pharmaceutically acceptable salt or their combination account for the 0.001-99.9wt% of pharmaceutical composition gross weight.
- 8. purposes as claimed in claim 1 is characterized in that, the dosage form of described pharmaceutical composition comprises: tablet, powder agent, injection, granule, syrup, capsule, solution, suppository or ointment.
- 9. purposes as claimed in claim 1 is characterized in that, described pharmaceutical composition also comprises the other medicines of alleviating or treating hepar damnification.
- 10. pharmaceutical composition, it contains:(1) chemical compound with structural formula A of effective dose, its pharmaceutically acceptable salt or their combination;(2) be selected from down one or more materials of organizing: Tanshinone I I A, glycyrrhizic acid preparation, green tea polyphenol, silymarin, tetrandrine or their combination; With(3) pharmaceutically acceptable carrier.
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|---|---|---|---|
| CN2009100536041A CN101926787A (en) | 2009-06-23 | 2009-06-23 | Application of flavone compound in protecting liver |
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| CN2009100536041A CN101926787A (en) | 2009-06-23 | 2009-06-23 | Application of flavone compound in protecting liver |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103655929A (en) * | 2013-12-06 | 2014-03-26 | 江苏德和生物科技有限公司 | Pharmaceutical composition with liver-protecting effect and preparation method thereof |
| CN105919992A (en) * | 2016-05-03 | 2016-09-07 | 苏州大学 | Application of apigenin in preparing medicines or health care foods for preventing and/or treating alcoholic liver injury |
-
2009
- 2009-06-23 CN CN2009100536041A patent/CN101926787A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103655929A (en) * | 2013-12-06 | 2014-03-26 | 江苏德和生物科技有限公司 | Pharmaceutical composition with liver-protecting effect and preparation method thereof |
| CN103655929B (en) * | 2013-12-06 | 2017-01-18 | 江苏德和生物科技有限公司 | Pharmaceutical composition with liver-protecting effect and preparation method thereof |
| CN105919992A (en) * | 2016-05-03 | 2016-09-07 | 苏州大学 | Application of apigenin in preparing medicines or health care foods for preventing and/or treating alcoholic liver injury |
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