CN105078979A - Medicine for preventing and treating non-alcoholic steatohepatitis and preparation method of medicine - Google Patents
Medicine for preventing and treating non-alcoholic steatohepatitis and preparation method of medicine Download PDFInfo
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Abstract
The invention belongs to the field of Chinese herbal preparations, and specifically relates to a medicine for preventing and treating non-alcoholic steatohepatitis and a preparation method of the medicine. According to the medicine, puerarin and berberine are taken as active ingredients, and further, baicalin is selected as the active ingredient. The medicine is obvious in effect in improving hepatic tissue pathology, in particular for lipid droplet, also has large influences on transaminase, and basically achieves the effects of improving NASH dyslipidemia and actively regulating blood fat. The preparation method has good application prospect and great economic benefit for preparing the medicine for preventing and treating non-alcoholic steatohepatitis.
Description
Technical field
The invention belongs to field of traditional Chinese, be specifically related to a kind of medicine preventing and treating non-alcoholic stellato-hepatitis and preparation method thereof.
Background technology
In recent years, due to dietary structure and the lifestyle change of the masses, non-alcohol fatty liver (NAFLD) sickness rate is constantly climbed to a higher point, and replace hepatitis B virus gradually, become the Etiological of China's chronic hepatopathy, therefore, a medical problem is not only in the control of NAFLD, more becomes a social problem.Therefore, the preventing and controlling actively carrying out NAFLD are imperative.
Non-alcohol fatty liver (NAFLD) is a kind of insulin resistant (IR) and the closely-related metabolic stress hepatic injury of inheritance susceptible, its pathological change is similar to alcoholic liver disease (ACD), but patient is because of without excessive drinking history, this disease is now popular at crowd's camber at global rapid spread.Non-alcohol fatty liver comprises simple fatty liver, the non-alcoholic stellato-hepatitis (NASH) developed by it and non-alcoholic fatty liver and hardens.Wherein, non-alcoholic stellato-hepatitis plays important speed limit effect at simple fatty liver in non-alcoholic fatty liver sclerosis conversion process, it is one of major reason of cryptogenic cirrhosis, therefore, for the treatment of fatty liver disease, there is crucial effect to the Prevention and Curation of non-alcoholic stellato-hepatitis.
The pathogenesis of non-alcoholic stellato-hepatitis and endocrine and metabolic disorders and immune disorder closely related, therefore, QI and blood, the expectorant stasis of blood and suffocating of liver and gall damp and hot with the traditional Chinese medical science are lacked of proper care in close relations.At present for the treatment of NASH, enzyme, hepatoprotective and antioxidant fall in clinical widely using, and according to the cause of disease of NASH, pathogenesis and symptom thereof, impose distinct methods treatment.But the treatment at present to NASH, doctor trained in Western medicine aspect there is no effect method and determined curative effect, can for a long time for medicine safely and effectively that NASH treats.At present, utilize the effective treatment of Chinese medicine to NASH to progress into clinical stage, to the formation of prevention liver cirrhosis, there is important clinical meaning, and the research of Chinese medicine progressively becomes one of focus of fatty liver research field.
It is little that Chinese medicine prevention non-alcoholic stellato-hepatitis has side effect, the feature that clinical efficacy is definite, work out with the pai n nursing scheme of motion, the treatment of diet intervention Combined with Chinese Herbal according to different stadium, and give full play to the subjective initiative of patient self, carry out evaluation of clinical curative effect, to beginning to take shape the pai n nursing scheme that can for apply, improve clinical efficacy.In long-term clinical practice, tentatively summarize a large amount of clinical experience.It is reported, the medication for the treatment of non-alcoholic stellato-hepatitis is main mainly with clearing heat and expelling damp, adopts clearing heat and expelling damp one method or on the basis of clearing heat and expelling damp, drink the medicine adding some blood circulation promoting and blood stasis dispelling, but curative effect is not ideal, not yet make a breakthrough, there is no generally acknowledged radical cure medicine so far both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention there are provided a kind of medicine effectively can preventing and treating non-alcoholic stellato-hepatitis, and discloses the preparation method of described medicine.
The invention discloses a kind of medicine preventing and treating non-alcoholic stellato-hepatitis, described medicine with puerarin and berberine for active component.
Preferably, the weight part ratio of described berberine and described puerarin is 1:0.5-8.
Further, the active component of described medicine also comprises baicalin.
The weight part ratio of described berberine, puerarin and baicalin is 1:0.5-8:0.25-4.
Preferably, the weight part ratio of described berberine, puerarin and baicalin is 1:2:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:4:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:8:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:1:0.5; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:2:0.5; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:4:4; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:0.5:0.25; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:1:2; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:2:2.
The invention also discloses a kind of method preparing said medicine, comprise and measure each active component monomer according to selected prescription, add customary adjuvant, conveniently technique and make acceptable dosage form clinically.
Described acceptable dosage form is oral formulations, includes but not limited to the regular dosage forms such as capsule, tablet, granule.
Described adjuvant includes but not limited to the conventional adjuvant such as such as starch, dextrin etc. used in prior art.
The invention also discloses the purposes of above-mentioned medicine in the medicine preparing Prevention and Curation non-alcoholic stellato-hepatitis.
Technique scheme of the present invention has the following advantages compared to existing technology,
Dripped the change of content by cell experiment observation of cell fat, medicine of the present invention, can obviously lower fat content in cell, improve fat variation; Simultaneously to liver tissue injury area by reduction in various degree, intervene simultaneously to control NASH transaminase's rising aspect have positive role; And by the adjustment for serum total cholesterol, can be total to and significantly reduce blood fat, illustrate that this monomer compatibility can reach the effect of the original side of Chinese medicine in some aspects.Visible, medicine of the present invention with berberine and puerarin or berberine, puerarin and baicalin for active component, can effective Prevention and Curation non-alcoholic stellato-hepatitis.
Experimental example
Experimental example 1 puerarin, berberine drug combination intervene NASH cell model experimentation
1 instrument and material
1.1 cells and reagent
HepG2 cell granted by Chinese department of Chinese medicine institute basis;
DMED culture medium, hyclone, penicillin, streptomycin, 0.25%trypsin-EDTA are purchased from Gibco company;
Bovine serum albumin is purchased from Roche company;
DMSO, oleic acid, Palmic acid, oil red O available from Sigma; MTT test kit is purchased from Promega company;
Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) enzyme linked immunological kit are purchased from Wuhan Boster Biological Technology Co., Ltd.;
GLUT4 (GLUT4) enzyme linked immunological kit is purchased from upper sea blue base Bioisystech Co., Ltd.
1.2 key instrument
The full-automatic microplate reader of U.S. MULTISKANMK3 (ThermoScientific), vortex oscillation instrument (QL-902, its woods Bel instrument manufacturing company limited of Haimen City);
Centrifuge (Centrifuge5415D, Eppendorf), BT224S electronic balance (German Sartouris company);
-80 DEG C of refrigerators (Qingdao Haier);
HB-202 constant water bath box (Chinese and Western, Beijing is long-range);
Motor stirrer (JB300-D type, Shanghai Qi Wei Electronics Co., Ltd.);
MP200A electronic balance (balance equipment factory of Shanghai Precision Scientific Apparatus Co., Ltd);
YDS-50 type liquid nitrogen container (Beijing Xin Kang hundred million reaches development in science and technology company limited) etc.;
Other equipment and utensil comprise conventional magnetic agitator, filter, centrifuge tube, pipet, suction pipe, test tube, beaker, flask, Olympus camera system etc.
2. method
2.1. cell culture processes
Be inoculated in by HepG2 cell in 24 orifice plates containing the DMEM culture fluid of 10% hyclone, 100,000 U/L mycillins, cell culture puts into coverslip simultaneously, and cell attachment is grown on slide.Put 37 DEG C, the interior cultivation of 5%CO2 saturated humidity incubator.According to cell growth status, every 1-2 days with 0.25% trypsin-EDTA digest, carry out Secondary Culture.
2.2.NASH the foundation of cell model
HepG2 is in 24 orifice plates in inoculation, grows to after 60-70% until cell attachment, and giving free fatty 500 μm of ol/L (by Palmic acid: oleic acid is the mol ratio of 1:2) stimulates, and the time is 24h.If 3 parallel holes.After leaving and taking cell culture supernatant at the end of experiment, change with in oil red O stain Microscopic observation cytoplasm.
The alone impact on NASH cell model cell inhibitory rate of 2.3 puerarins, berberine
By HepG2 cell culture in 96 orifice plates, grow to after 60-70% until cell attachment, give puerarin/berberine (alone) Co stituation of free fatty 500 μm of ol/L (by Palmic acid: oleic acid is the mol ratio of 1:2) with variable concentrations, the time is 24h.Variable concentrations puerarin/berberine (alone) experimental group, blank group are established in experiment respectively, often organize and all establish 3 parallel holes.With MTT colorimetric method for determining cell proliferative conditions (representing with absorbance OD value), that understands medicine on cell proliferation affects situation, experiment repetition 3 times.
Suppression ratio IR (%)=(1-experimental group mean OD value/blank group mean OD value) * 100%.2.4. puerarin, berberine (the alone and drug combination) intervention to NASH cell model
According to MTT suppression ratio acquired results, choose suitable puerarin/berberine (alone and drug combination) and the intervention same period is carried out to NASH cell model.By HepG2 in 24 orifice plates, grow to after 60-70% until cell attachment, give free fatty 500 μm of ol/L (by Palmic acid: oleic acid is the mol ratio of 1:2)+puerarin/berberine (alone and drug combination) to stimulate, the time is 24h.If 6 parallel holes.After leaving and taking cell culture supernatant at the end of experiment, change with in oil red O stain Microscopic observation cytoplasm.
2.5. oil red O stain observation of cell lactone drips
Oil red O stain step is as follows: PBS liquid cleaning cell 3 times, and 4% paraformaldehyde fixes 10min, and PBS liquid cleaning cell 2 times, 60% isopropyl alcohol 1min, oil red O stain 10min under room temperature, distilled water stops dyeing.Neutral fat under microscope in visible cell can be dyed redness specifically.
2.6. the mensuration of IL-8, TNF-α and GLUT4 in cell culture supernatant
After each group of stimulation time arrives, collecting cell culture supernatant, and low-temperature centrifugation (3000rpm), carry out the detection of index of correlation by Elisa test kit description after centrifugal.
2.7. oil red O stain cell lactone drips assay
Oil red O stain step is the same, and after termination dyeing, repeatedly distilled water flushing is rinsed totally to extracellular oil red O, adds 100% isopropyl alcohol and carries out rupture of membranes, treats the complete stripping of oil red O in cell, after concussion evenly, carries out OD value and reads.Content is dripped with the content reacting cells lactone of oil red O.
2.8 date processing
Experimental result with
represent, adopt SPSS17.0 statistical software to carry out one factor analysis of variance (One-wayANOVA), P<0.05 is for there being statistical significance.
3. experimental result
3.1 puerarins, berberine are alone affects experimental result as shown in Figure 1 to NASH cell model cell inhibitory rate.
3.2 oil red O stains drip the observation of change to cell lactone
500 μm of ol/LFFA effect NASH model group, are dyed to red graininess fat and drip in visible cell endochylema, and in " signet ring " shape form.Variable concentrations puerarin, alone and the drug combination (p500 of berberine, p1000, p2000, b25, b50, b25p1000, b50p500, unit μm ol/L) intervention group, in visible cell, red fat drips and reduces to some extent, monomer drug combination group is better than the independent medication group of same concentration, and (namely b25p1000 is better than b25 and p1000, b50p500 is better than b50 and p500), substantially the object of low dose of drug combination effect no less than the independent medication effect of high dose is reached, specific experiment the results are shown in accompanying drawing 3 (in the present invention, it is individually dosed that namely b25 represents berberine, concentration is 25 μm of ol/L, namely b25p1000 represents berberine and puerarin administering drug combinations, and concentration is respectively 25 and 1000 μm of ol/L, lower same).
The change of IL-8, TNF-α and GLUT4 in 3.3 cell culture supernatants
500 μm of ol/LFFA stimulate the HepG2 cell after 24h, in its cell culture supernatant, inflammatory factor TNF-α and IL-8 content obviously increase, GLUT4 content obviously declines, and compared with blank group, difference has statistical significance (P<0.05).
Along with berberine, puerarin are used alone the increase of concentration, IL-8 level comparatively model group obviously reduces, with b50, b100, p2000 obvious difference (P<0.05); After composite reagent b25p1000, b50p500 all comparatively model group have statistical significance (P<0.05), b25p1000 is b25 successful (P<0.05) comparatively, and b50p500 is p500, p1000 successful (P<0.05) comparatively.
For TNF-a, along with berberine, puerarin are used alone the increase of concentration, its supernatant content significantly reduces, and all comparatively model group is with significant difference (P<0.05); After composite reagent, b50p500 is b50 successful (P<0.05) comparatively.
For GLUT4, along with berberine, puerarin are used alone the increase of concentration, its supernatant content significantly increases, with b50, b100, p500, p1000, p2000, comparatively model group is with significant difference (P<0.05), and puerarin is better than berberine for the effect of increase GLUT4 content; After composite reagent, the change of GLUT4 content is better than berberine and is used alone, b25p1000 after adding puerarin comparatively b25, b50 successful strengthen (P<0.05), b50p500 comparatively b50, b100 successful increase (P<0.05).Experimental result is in Table 1-1 and 1-2.
Alone and the drug combination of table 1-1 puerarin, berberine is to NASH cell model
The impact of IL-8/TNF-a/GLUT4 secretion
Note: Δ P<0.05vsN; AP<0.05vsM; BP<0.05vsb25; CP<0.05vsp1000; DP<0.05vsp2000; EP<0.05vsb25p1000
Alone and the drug combination of table 1-2 puerarin, berberine is to NASH cell model
The impact of IL-8/TNF-a/GLUT4 secretion
Note: Δ P<0.05vsN; AP<0.05vsM; BP<0.05vsp500; CP<0.05vsp1000; DP<0.05vsb50; EP<0.05vsb50p50
3.4 cytolipins drip changes of contents
Read OD value to analyze, puerarin, berberine drug combination group be model group and the equal significant difference of single medicine use group comparatively, has statistical significance (P<0.05).B25p1000, b50p500 lipid-lowering effect is better than model group and each monomer is used alone group.In table 2.
Alone and the drug combination of table 2 puerarin, berberine drips the impact of content to NASH cell model fat
Note: * P<0.05vsM; AP<0.05vsb25p1000; BP<0.05vsb50p500
3.5 result displays:
3.5.1. oil red O stain
Compared with 500 μm of ol/LFFA effect NASH model group, variable concentrations puerarin, berberine alone and drug combination (p500, p1000, p2000, b25, b50, b25p1000, b50p500, unit μm ol/L) intervention group, in visible cell, red fat drips and reduces to some extent, monomer drug combination group is better than the independent medication group of same concentration, substantially reaches the object of low dose of drug combination effect no less than the independent medication effect of high dose.
3.5.2. the change of TNF-α, IL-8 and GLUT4 in cell culture supernatant
Along with berberine, puerarin are used alone the increase of concentration, IL-8 level comparatively model group obviously reduces, with b50, b100, p2000 obvious difference; After composite reagent b25p1000, b50p500 all comparatively model group have statistical significance, b25p1000 is b25 successful comparatively, and b50p500 is p500, p1000 successful comparatively.Prompting is for IL-8, and puerarin, berberine exist synergism, and the more independent medication effect of its composite reagent effect strengthens.
For TNF-a, along with berberine, puerarin are used alone the increase of concentration, its supernatant content significantly reduces, all comparatively model group significant difference; After composite reagent, b50p500 is b50 successful comparatively.Prompting is for TNF-a, and puerarin, berberine are used alone and can obviously reduce its content, and when combinationally using, effect is more obvious, and synergism is remarkable, and the more independent medication effect of its composite reagent effect strengthens.
For GLUT4, along with berberine, puerarin are used alone the increase of concentration, its supernatant content significantly increases, and with b50, b100, p500, p1000, p2000 comparatively model group significant difference, and puerarin is better than berberine for the effect of increase GLUT4 content; After composite reagent, the change of GLUT4 content is better than berberine and is used alone, and b25p1000 is comparatively b25, b50 successful enhancing after adding puerarin, and b50p500 comparatively b50, b100 successful increases.Prompting is for GLUT4, and puerarin, berberine are used alone can obviously increase its content, and puerarin is better than berberine, and when combinationally using, effect is more obvious, and synergism is remarkable, and the more independent medication effect of its composite reagent effect strengthens.
3.5.3. cytolipin drips changes of contents
By measuring the optical density value of oil red O content in cell oil red O stain cell, reacting cells lactone drips the change of content.Result display puerarin, berberine drug combination group comparatively model group and the equal significant difference of single medicine use group, can obviously lower fat content in cell, improves fat and become.
This experiment is to comparatively easily to obtain in Radix Puerariae, Rhizoma Coptidis and the higher two kinds of monomer puerarins of content and berberine have carried out combination research.Result of study finds: variable concentrations puerarin, berberine alone and drug combination (p500, p1000, p2000, b25, b50, b25p1000, b50p500, unit μm ol/L) intervention group, in oil red O stain visible cell, red fat drips and reduces to some extent; Improving in TNF-α, IL-8, GLUT4, there is synergism in puerarin, berberine.In experiment in vitro, substantially can determine within the specific limits, berberine and puerarin portfolio ratio are good at 1:40-1:10.
Experimental example 2 puerarin, berberine, baicalin drug combination intervene the experimentation of NASH cell model
1 instrument and material
1.1 cells and reagent
HepG2 cell granted by Chinese department of Chinese medicine institute basis;
DMED culture medium, hyclone, penicillin, streptomycin, 0.25%trypsin-EDTA are purchased from Gibco company;
Bovine serum albumin is purchased from Roche company;
DMSO, oleic acid, Palmic acid, oil red O available from Sigma; MTT test kit is purchased from Promega company;
Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α) enzyme linked immunological kit are purchased from Wuhan Boster Biological Technology Co., Ltd.;
1.2 key instrument
The full-automatic microplate reader of U.S. MULTISKANMK3 (ThermoScientific), vortex oscillation instrument (QL-902, its woods Bel instrument manufacturing company limited of Haimen City);
Centrifuge (Centrifuge5415D, Eppendorf), BT224S electronic balance (German Sartouris company);
-80 DEG C of refrigerators (Qingdao Haier);
HB-202 constant water bath box (Chinese and Western, Beijing is long-range);
Motor stirrer (JB300-D type, Shanghai Qi Wei Electronics Co., Ltd.);
MP200A electronic balance (balance equipment factory of Shanghai Precision Scientific Apparatus Co., Ltd);
YDS-50 type liquid nitrogen container (Beijing Xin Kang hundred million reaches development in science and technology company limited) etc.;
Other equipment and utensil comprise magnetic stirring apparatus, filter, centrifuge tube, pipet, suction pipe, test tube, beaker, flask, Olympus camera system etc.
2 experimental techniques
The alone impact on NASH cell model cell inhibitory rate of 2.1 baicalin (Bai, bai)
By HepG2 cell culture in 96 orifice plates, grow to after 60-70% until cell attachment, give the baicalin Co stituation of free fatty 500 μm of ol/L (by Palmic acid: oleic acid is the mol ratio of 1:2) with variable concentrations, the time is 24h.Variable concentrations baicalin experimental group, blank group are established in experiment respectively, often organize and all establish 3 parallel holes.With MTT colorimetric method for determining cell proliferative conditions (representing with absorbance OD value), that understands medicine on cell proliferation affects situation, experiment repetition 3 times.
Suppression ratio IR (%)=(1-experimental group mean OD value/blank group mean OD value) * 100%.2.2 baicalin is alone and with puerarin (P, p), berberine (B, b) drug combination to the intervention of NASH cell model
According to MTT suppression ratio acquired results and experiment one gained puerarin and berberine best of breed, choose suitable baicalin, baicalin+puerarin+berberine carries out the intervention same period to NASH cell model.By HepG2 in 24 orifice plates, grow to after 60-70% until cell attachment, give free fatty 500 μm of ol/L (by Palmic acid: oleic acid is the mol ratio of 1:2)+baicalin/baicalin+puerarin+berberine and stimulate (unit μm ol/L), the time is 24h.If 6 parallel holes.After leaving and taking cell culture supernatant at the end of experiment, change with in oil red O stain Microscopic observation cytoplasm.
2.3 oil red O stain observation of cell lactone drip
Oil red O stain step is as follows: PBS liquid washed cell 3 times, and 4% paraformaldehyde fixes 10min, and again carry out PBS liquid cleaning cell 2 times, 60% isopropyl alcohol 1min, oil red O stain 10min under room temperature, distilled water stops dyeing.Under microscope, in visible cell, neutral fat is dyed redness specifically.
The mensuration of IL-8 and TNF-α in 2.4 cell culture supernatants
After each group of stimulation time arrives, collecting cell culture supernatant, and low-temperature centrifugation (3000rpm), carry out the detection of index of correlation by Elisa test kit description after centrifugal.
2.5 oil red O stain cell lactone drip assay
Oil red O stain step is the same, and after termination dyeing, repeatedly distilled water flushing is rinsed totally to extracellular oil red O, adds 100% isopropyl alcohol and carries out rupture of membranes, treats the complete stripping of oil red O in cell, after concussion evenly, carries out OD value and reads.Content is dripped with the content reacting cells lactone of oil red O.
2.6 date processing
Experimental result with
represent, adopt SPSS17.0 statistical software to carry out one factor analysis of variance (One-wayANOVA), P<0.05 is for there being statistical significance.
3. experimental result
The alone impact on NASH cell model cell inhibitory rate of 3.1 baicalins (bai), puerarin (p), berberine (b)
Experimental result as shown in Figure 2.
3.2 oil red O stains drip the observation of change to cell lactone
500 μm of ol/LFFA effect NASH model group, are dyed to red graininess fat and drip in visible cell endochylema, and in " signet ring " shape form.Alone and the puerarin of variable concentrations baicalin, berberine, baicalin drug combination (bai25, bai50, bai100, bai200, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100, unit μm ol/L) intervention group, in visible cell, red fat drips and reduces to some extent, (b25p1000bai100 is better than b25p1000 and bai100 to monomer drug combination group successful, b50p500bai100 is better than b50p500 and bai100), testing result is shown in Fig. 4.
The change of IL-8 and TNF-α in 3.3 cell culture supernatants
500 μm of ol/LFFA stimulate the HepG2 cell after 24h, and in its cell culture supernatant, inflammatory factor TNF-α and IL-8 content obviously increase, and compared with blank group, difference has statistical significance (P<0.05).
Along with baicalin is used alone the increase of concentration, IL-8 level comparatively model group obviously reduces, with bai200 obvious difference (P<0.05); After composite reagent b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 all comparatively model group have statistical significance (P<0.05), in each ratio combination, with b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 effect the most obviously (be all better than same concentration bai and be used alone effect).
For TNF-a, along with baicalin is used alone the increase of concentration, its supernatant content significantly reduces, and all comparatively model group is with significant difference (P<0.05); After composite reagent, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 all comparatively model group have statistical significance (P<0.05), and effect is better than baicalin when being used alone.In each ratio combination, the most obvious with b50p500bai50, b50p500bai100 effect.In table 3.
Alone and the puerarin of table 3 baicalin, berberine, baicalin drug combination are secreted NASH cell model IL-8/TNF-a
Impact
Note: Δ P<0.05vsN; AP<0.05vsM; BP<0.05vsb25p1000bai25; CP<0.05vsb25p1000bai50; DP<0.05vsb25p1000bai100; EP<0.05vsbai25; FP<0.05vsbai50; GP<0.05vsbai100; HP<0.05vsbai200; IP<0.05vsb50p500bai25
3.4 cytolipins drip changes of contents
Read OD value to analyze, puerarin, berberine, baicalin drug combination b25p1000bai100 group lipid-lowering effect are best, compare with model group, bai100, bai200, b25p1000, b50p500, all there is statistical significance (P<0.05), in table 4.
Table 4 baicalin alone and puerarin, berberine, baicalin drug combination pair
NASH cell model fat drips the impact of content
Note: * P<0.05vsA/B/D/E/H/I/K; Δ P<0.05vsF/G/J/L/M
3.5 result displays:
3.5.1. oil red O stain
Compared with 500 μm of ol/LFFA effect NASH model group, alone and the puerarin of variable concentrations baicalin, berberine, baicalin drug combination (bai25, bai50, bai100, bai200, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100, unit μm ol/L) intervention group, in visible cell, red fat drips and reduces to some extent, monomer drug combination successful.
3.5.2. the change of IL-8 and TNF-α in cell culture supernatant
500 μm of ol/LFFA stimulate the HepG2 cell after 24h, and in its cell culture supernatant, inflammatory factor TNF-α and IL-8 content obviously increase, and compared with blank group, difference has statistical significance (P<0.05).
Along with baicalin is used alone the increase of concentration, IL-8 level comparatively model group obviously reduces, with bai200 obvious difference (P<0.05); After composite reagent b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 all comparatively model group have statistical significance (P<0.05), in each ratio combination, the most obvious with b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 effect.Point out three kinds of combination of monomers medications to have synergism, enhance the effect of the independent medication of baicalin under Isodose.Meanwhile, on ratio combination, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 are better than b25p1000bai25, b25p1000bai50.
For TNF-a, along with baicalin is used alone the increase of concentration, its supernatant content significantly reduces, and all comparatively model group is with significant difference (P<0.05); After composite reagent, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100 all comparatively model group have statistical significance (P<0.05), and effect is better than baicalin when being used alone.In each ratio combination, the most obvious with b50p500bai50, b50p500bai100 effect.Pointing out three kinds of combination of monomers medication successfuls to be better than baicalin to be used alone, by combinationally using, intervention effect can be ensured under the prerequisite reducing Determination of baicalin.In portfolio ratio, b50p500bai50, b50p500bai100 combined effect is better than b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25.
3.5.3. cytolipin drips changes of contents
By measuring the optical density value of oil red O content in cell oil red O stain cell, reacting cells lactone drips the change of content.Result display puerarin, berberine, baicalin drug combination b25p1000bai100 group lipid-lowering effect are best, compare with model group, bai100, bai200, b25p1000, b50p500, all there is statistical significance (P<0.05).Can obviously lower fat content in cell, improve fat and become.
Variable concentrations baicalin alone and puerarin, berberine, baicalin drug combination intervention group, in oil red O stain visible cell, red fat drips and reduces to some extent, and monomer drug combination exists remarkable synergism; For TNF-α, IL-8, there is synergism in combination of monomers.In experiment in vitro, substantially can determine within the specific limits, berberine, puerarin, baicalin portfolio ratio are good at 1:10:1-1:10:2.
Experimental example 3 puerarin, berberine, baicalin combination to high lipid food feed induction Steatohepatitis in Rats pharmacodynamic observation
1 experimental drug and reagent
1.1 medicines and reagent
Puerarin (purity 99%), berberine (purity 98%), baicalin (purity 85%) are purchased from Nanjing Zelang Pharmaceutical Technology Inc..Dosage empirically animal and 60KG adult body surface area ratio equivalent adjusts ratio, calculates the dose,equivalent of animal;
Be mixed with desired concn with deionized water during experiment, put refrigerator cryopreservation;
Normal feedstuff and high lipid food to be pulled together feed corporation,Ltd's processing and fabricating by Beijing Australia of section, cleaning grade.Normal feedstuff room temperature is preserved;
High lipid food (88% normal feedstuff+10% Adeps Sus domestica+2% cholesterol), vacuum packaging, 10Kg/ wraps, 4 DEG C of cryopreservation.
Glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST), triglyceride (TG), T-CHOL (TC), HDL-C (HDL-C), low-density lipoprotein cholesterol (LDL-C) test kit are Zhongsheng Beikong Biological Science & Technology Co., Ltd. and produce;
Oil red O stain and HE dyeing related reagent purchased from American Sigma company.
1.2 key instrument
7160 full automatic biochemical apparatus (HIT);
The full-automatic microplate reader of U.S. MULTISKANMK3 (ThermoScientific);
U.S. Bio-Rad voltage stabilization and current stabilization electrophresis apparatus, the digital gel imaging system of U.S. Bio-radGelDoc2000, Image-ProPlusAnalysisSoftware computerized image analysis sgstem, vortex oscillation instrument (QL-902, its woods Bel instrument manufacturing company limited of Haimen City);
Centrifuge (Centrifuge5415D, Eppendorf);
Spectrophotometer (NANODROP2000, Thernoscientific);
Quantitative real time PCR Instrument (ABI7500, AppliedBiosystems);
UCT type ultramicrotome (German Lycra company);
BT224S electronic balance (German Sartouris company);
-80 DEG C of refrigerators (Qingdao Haier);
HB-202 constant water bath box (Chinese and Western, Beijing is long-range);
Motor stirrer (JB300-D type, Shanghai Qi Wei Electronics Co., Ltd.);
MP200A electronic balance (balance equipment factory of Shanghai Precision Scientific Apparatus Co., Ltd);
YDS-50 type liquid nitrogen container (Beijing Xin Kang hundred million reaches development in science and technology company limited) etc.;
Other equipment and utensil comprise conventional magnetic agitator, operating theater instruments, filter, centrifuge tube, pipet, suction pipe, test tube, beaker, flask, Olympus camera system etc.
2 methods
2.1 groupings and disposal
Experimental rat is divided into 11 groups at random: blank group, model group, drug combination group (see orthogonal table 5,6), positive control GEGEN QINLIAN TANG high dose group (HH).
Concrete steps are as follows: male SD rat (body weight is about 120g ± 20g) totally 66, and after adaptability feeds one week, randomly draw 6 for blank group, normal feedstuff is fed 8 weeks; Remain 60 and be divided into model group and each administration group at random, while giving high lipid food (88% normal feedstuff+10% Adeps Sus domestica+2% cholesterol), carry out pharmaceutical intervention, every day gavage 1 time, each group gavages and starts when modeling simultaneously, continues to for 8 weekends.Observe animal feed drinking-water, behavioral activity, the mental status, hair and two situation such as just every day, freely drink water, ingest, periodic dosages the weight of animals.
Table 5 orthogonal test calendar is (by orthogonal table L
9(3
4) grouping)
With reference to pertinent literature, to as follows after each experimental group assignment:
Table 6 orthogonal test calendar is (by orthogonal table L
9(3
4) grouping)
2.2 Indexs measure
2.2.2 serological index
Glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST), triglyceride (TG), T-CHOL (TC), high density lipoprotein (HDL), low density lipoprotein, LDL (LDL) adopt automatic clinical chemistry analyzer to measure.
2.2.3 liver histopathology detects
Fresh liver tissue frozen section, carries out conventional oil red O stain; Hepatic tissue paraffin section, row conventional H E dyes.Oil red O and HE cuts into slices and uses Olympus camera system to carry out observing and taking the photograph sheet.
2.4 data statistics
Application SPSS17.0 software carries out the statistical analysis of data, and enumeration data represents with x ± s, adopts one factor analysis of variance (One-wayANOVA), with P<0.05 for there being statistical significance.
3. experimental result
3.1 ordinary circumstance
The dimness of model group rats hair color is withered and yellow, and more blank group of rat reduces dietary amount, and large dry stool is rare uncomfortable, and activeness reduces.Blank group rat hair color is rich in gloss, physical agility, the diet water yield and Excreta without exception.Each administration group rat hair color, behavior, diet and Excreta are compared with blank group, and no significant difference, indivedual rat defecates rare soft as seen, and body weight increases between model group and blank group.Rats death is there is not in whole experimentation.
3.2 liver pathology changes
3.2.1 liver perusal situation
Blank group rat liver is bronzing, and smooth, matter is soft, is rich in elasticity, the sharp keen and smooth densification of liver edge.Model group rats liver is khaki, and volume increases, and surface-coating is nervous, and is dispersed in fatty spew as seen, and quality is more tough, and edge circle is blunt, and tangent plane loosens.Each administration group rat liver volume slightly increases, and more blank group of color and luster is slightly shallow, and more blank group of quality, elasticity is slightly poor, naked eyes no significant difference between each administration group.
3.2.2 hepatic tissue om observation situation
HE dyes display: blank group rats'liver leaflet structure, hepatocyte form are normal, and kytoplasm enriches, liver rope marshalling radially; There is obvious steatosis in model group rats hepatic tissue, lobules of liver boundary is unclear, and companion liver Cable Structure is disorderly, a large amount of neutrophil infiltration; Compared with model group, each intervention group pathological changes has and alleviates in various degree, and damaged area obviously reduces, and in born of the same parents, inflammatory cell infiltration reduces, and cavity and balloon sample become and reduce, and cell and leaflet structure recover in various degree, the results are shown in Figure 5.
Oil red O stain result shows: the fat of the non-show dye of blank group liver tissues of rats drips; The visible a large amount of red dye fat of model group rats hepatic tissue drips, and color is comparatively dark and fusion is in blocks.Also show red dye fat in GEGEN QINLIAN TANG high low dose group liver tissues of rats to drip, but quantity and degree are all few compared with model group, the results are shown in Figure 6.
The effect of 3.3 pairs of serum and hepatic tissue indices
3.3.1 Hepatic enzyme
Model group Serum ALT, AST level are all apparently higher than blank group, there were significant differences (P<0.01), each administration group Serum ALT, AST level all comparatively model group all significantly reduce (P<0.01), the results are shown in Table 7.
Table 7 respectively organizes Serum ALT, AST compares
Note: ▲ P<0.05, the blank group of ▲ ▲ P<0.01vs; * P<0.05, * * P<0.01vs model group; AP<0.05, aaP<0.01vsHH
3.3.2 blood fat
Compared with blank group, model group HDL level significantly reduces, and LDL, TG, TC content obviously increases, and has significant difference (P<0.01); A, B, D, E, I, HH group serum HDL levels compared with model group increases obviously, and significant difference (P<0.01), wherein HH group is obviously better than B, E group (P<0.01).Each administration group is compared with model group, and serum LDL levels declines obviously, and all have significant difference (P<0.01), wherein HH group is obviously better than E, F, H, I group (P<0.01).For serum triglycerides, between A, C, D, E, F, G, HH group and model group, equal significant difference (P<0.01), has no notable difference (P>0.05) therebetween.For serum total cholesterol, between A, B, C, D, E, F, G, HH group and model group, equal significant difference (P<0.01), has no notable difference (P>0.05) therebetween, the results are shown in Table 8.
Table 8 respectively organizes Serum HDL-C, LDL-C, TG, TC compare
Note: ▲ P<0.05, the blank group of ▲ ▲ P<0.01vs; * P<0.05, * * P<0.01vs model group; AP<0.05, aaP<0.01vsHH
3.4 result displays
3.4.1. liver histopathology
This result of study shows, and after each administration group intervenes 8 weeks, liver tissue injury area all has reduction in various degree, and in born of the same parents, inflammatory cell infiltration reduces, and cavity and balloon sample become and reduce, and liver interior accumulation fat drips content to be reduced, and cell and leaflet structure recover in various degree.Prompting GEGEN QINLIAN TANG high dose and each monomer administering drug combinations group all can significantly improve model hepatic tissue pathology.C, D group is the most obvious.Illustrate with puerarin to be the combination of monomers of principal agent, improve hepatic tissue pathology, particularly to drip improvement the most obvious for fat.
3.4.2. hepatocellular damage
During modeling 8 weeks, HFD rat model Serum ALT, AST level significantly raise, each administration intervention group, Serum ALT, AST level comparatively model group obviously reduce, difference has remarkable statistical significance, and prompting GEGEN QINLIAN TANG high dose group and monomer administering drug combinations group are intervened simultaneously has positive role to control NASH transaminase's rising aspect.In combination of monomers, be that main combination affects larger on transaminase with puerarin.
3.4.3. blood lipid metabolism
During modeling 8 weeks, HFD rat model serum LDL-C, TG, TC content obviously raise, and HDL-C level significantly reduces, and A, B, D, E, I, HH group serum hdl content compared with model group increases obviously, and significant difference, wherein HH group is obviously better than B, E group.Each administration group is compared with model group, and serum LDL levels declines obviously, and all have significant difference, wherein HH group is obviously better than E, F, H, I group.For serum triglycerides, between A, C, D, E, F, G, HH group and model group, equal significant difference, has no notable difference therebetween.For serum total cholesterol, between A, B, C, D, E, F, G, HH group and model group, equal significant difference, has no notable difference therebetween.Each combination of monomers of prompting GEGEN QINLIAN TANG and different ratio can reach the improvement result to NASH Anomalous lipid metablism substantially, demonstrate the positive regulating action of combination of monomers to blood fat, but along with the change of each monomer proportion, and have no special regularity, the usefulness of some combinations is basic and GEGEN QINLIAN TANG high dose group is suitable, illustrates that the clear and definite monomer compatibility of combination can reach the effect of the former side of Chinese medicine in some aspects.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein,
Fig. 1 is that puerarin in experimental example 1 of the present invention, berberine are to NASH cell model 24h cell inhibitory rate;
Fig. 2 is that baicalin in experimental example 2 of the present invention, puerarin, berberine are to NASH cell model 24h cell inhibitory rate;
Fig. 3 is in experimental example of the present invention, and puerarin, berberine intervene NASH cell model 24h oil red O color regime (× 400);
Fig. 4 is in experimental example of the present invention, and puerarin, berberine, baicalin intervene NASH cell model 24h oil red O color regime (× 400);
Fig. 5 is in experimental example 3 of the present invention, liver tissues of rats paraffin section HE coloration result (× 100) when 8 weeks, and wherein, A-I is each test results, and HH is positive controls, and M is model group, and N is blank group;
Fig. 6 is in experimental example 3 of the present invention, liver tissues of rats frozen section oil red O stain result (× 100) when 8 weeks, and wherein, A-I is each test results, and HH is positive controls, and M is model group, and N is blank group.
Detailed description of the invention
Embodiment 1-
Embodiment 1 capsule
Get berberine 100g, puerarin 100g is active component, add dextrin as adjuvant, conveniently technique is prepared into capsule.
Embodiment 2 tablet
Get berberine 100g, puerarin 50g is active component, add starch as adjuvant, conveniently technique tabletting is prepared into tablet.
Embodiment 3 granule
Get berberine 100g, puerarin 800g is active component, add starch as adjuvant, conveniently technique is prepared into granule.
Embodiment 4 powder
Get berberine 100g, puerarin 400g is active component, add customary adjuvant, conveniently technique is prepared into powder.
Embodiment 5 capsule
Get berberine 100g, puerarin 200g, baicalin 100g is active component, add starch as adjuvant, conveniently technique is prepared into capsule.
Embodiment 6 capsule
Get berberine 100g, puerarin 400g, baicalin 100g is active component, add customary adjuvant, conveniently technique is prepared into capsule.
Embodiment 7 granule
Get berberine 100g, puerarin 800g, baicalin 100g is active component, adding dextrin is adjuvant, and conveniently technique is prepared into granule.
Embodiment 8 tablet
Get berberine 100g, puerarin 100g, baicalin 50g is active component, add starch as adjuvant, conveniently technique is prepared into tablet.
Embodiment 9 capsule
Get berberine 100g, puerarin 200g, baicalin 50g is active component, add customary adjuvant, conveniently technique is prepared into capsule.
Embodiment 10 powder
Get berberine 100g, puerarin 400g, baicalin 400g is active component, add customary adjuvant, conveniently technique is prepared into powder.
Embodiment 11 capsule
Get berberine 100g, puerarin 50g, baicalin 25g is active component, add dextrin as adjuvant, conveniently technique is prepared into capsule.
Embodiment 12 capsule
Get berberine 100g, puerarin 100g, baicalin 200g is active component, add dextrin as adjuvant, conveniently technique is prepared into capsule.
Embodiment 13 granule
Get berberine 100g, puerarin 200g, baicalin 200g is active component, add customary adjuvant, conveniently technique is prepared into granule.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (8)
1. prevent and treat a medicine for non-alcoholic stellato-hepatitis, it is characterized in that, described medicine with puerarin and berberine for active component.
2. medicine according to claim 1, is characterized in that, the weight part ratio of described berberine and described puerarin is 1:0.5-8.
3. medicine according to claim 1 and 2, is characterized in that, the active component of described medicine also comprises baicalin.
4. medicine according to claim 3, is characterized in that, the weight part ratio of described berberine, puerarin and baicalin is 1:0.5-8:0.25-4.
5. medicine according to claim 4, is characterized in that:
The weight part ratio of described berberine, puerarin and baicalin is 1:2:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:4:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:8:1; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:1:0.5; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:2:0.5; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:4:4; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:0.5:0.25; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:1:2; Or
The weight part ratio of described berberine, puerarin and baicalin is 1:2:2.
6. prepare a method for the arbitrary described medicine of claim 1-5, it is characterized in that, measure each active component monomer according to selected prescription, add customary adjuvant, conveniently technique and make acceptable dosage form clinically.
7. preparation method according to claim 6, is characterized in that, described dosage form is oral formulations.
8. the arbitrary described purposes of medicine in the medicine preparing Prevention and Curation non-alcoholic stellato-hepatitis of claim 1-5.
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| CN113368107A (en) * | 2020-02-25 | 2021-09-10 | 中国医学科学院药物研究所 | Pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing non-alcoholic fatty liver disease |
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| CN1850153A (en) * | 2006-03-09 | 2006-10-25 | 中国药科大学 | Method for preparing Radix puerariae and cenlian decoction |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113368107A (en) * | 2020-02-25 | 2021-09-10 | 中国医学科学院药物研究所 | Pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing non-alcoholic fatty liver disease |
| CN113368107B (en) * | 2020-02-25 | 2024-02-06 | 中国医学科学院药物研究所 | Pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing nonalcoholic fatty liver disease |
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