CN118236421A - Application of traditional Chinese medicine composition in preparation of medicines for treating or preventing alcoholic fatty liver - Google Patents
Application of traditional Chinese medicine composition in preparation of medicines for treating or preventing alcoholic fatty liver Download PDFInfo
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- CN118236421A CN118236421A CN202211658339.1A CN202211658339A CN118236421A CN 118236421 A CN118236421 A CN 118236421A CN 202211658339 A CN202211658339 A CN 202211658339A CN 118236421 A CN118236421 A CN 118236421A
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Abstract
The invention relates to application of a traditional Chinese medicine composition in preparing a medicine for treating or preventing alcoholic fatty liver, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-30 parts of astragalus membranaceus, 1-20 parts of kudzuvine root, 1-20 parts of radix bupleuri, 1-10 parts of lucid ganoderma, 1-20 parts of red paeony root, 1-20 parts of turtle shell, 1-20 parts of radix salviae miltiorrhizae, 1-10 parts of pseudo-ginseng, 1-20 parts of abrus herb and 1-30 parts of phyllanthus urinaria. The traditional Chinese medicine composition used by the invention has obvious regulation effects on the reduction of cell viability caused by ethanol-induced BRL rat liver cell oxidative damage, the increase of cell MDA content, CAT activity reduction, GSH content reduction and ROS increase, and has good application prospect in medicines for treating or preventing alcoholic fatty liver.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to application of a traditional Chinese medicine composition in preparation of a medicine for treating or preventing alcoholic fatty liver.
Background
Alcoholic Liver Disease (ALD) is a liver injury disease caused by prolonged high volume drinking. Alcoholic Fatty Liver (AFLD) is an early stage of the pathological development of ALD and refers to a clinically common disease in which alcohol-induced fat accumulates in hepatocytes, leading to the development of hepatic steatosis. About 90% of ALD. World health organization issues global alcohol and health report of 2018 that the number of deaths from alcohol each year worldwide accounts for 5.3% of all deaths, with the incidence of alcoholic liver disease being as high as 20%. In recent years, due to the improvement of the living standard and the change of the living style of people, the prevalence of AFLD tends to rise year by year, and if the treatment is not intervened in time in the reversible AFLD stage, the disease which is not easy to reverse is extremely likely to be further worsened, and finally the alcoholic liver cancer is progressed. Thus, timely treatment of AFLD is of paramount importance.
The pathogenesis of alcoholic fatty liver is complex, is not completely clear at present, and may be related to factors such as toxic effects of alcohol and metabolites thereof on liver, malnutrition caused by excessive alcohol, unbalanced intestinal microecological environment, oxidative stress reaction, inflammatory reaction and the like.
At present, western medicine has no accepted special medicine for treating AFLD, mainly adopts symptomatic treatment, and long-term administration can cause serious damage to liver and kidney functions of human body and damage functions of human body.
Therefore, there is a need in the art to further develop a solution that can effectively treat or prevent alcoholic fatty liver disease while ensuring high safety without significant toxic or side effects.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides the application of the traditional Chinese medicine composition in preparing the medicine for treating or preventing the alcoholic fatty liver, can have an effective treatment or prevention effect on the alcoholic fatty liver, and has high safety and no obvious toxic or side effect.
In order to solve the problems, the invention adopts the following technical scheme:
The invention provides application of a traditional Chinese medicine composition in preparing a medicine for treating or preventing alcoholic fatty liver, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-30 parts of astragalus membranaceus, 1-20 parts of kudzuvine root, 1-20 parts of radix bupleuri, 1-10 parts of lucid ganoderma, 1-20 parts of red paeony root, 1-20 parts of turtle shell, 1-20 parts of radix salviae miltiorrhizae, 1-10 parts of pseudo-ginseng, 1-20 parts of abrus herb and 1-30 parts of phyllanthus urinaria.
Patent application CN101214288A discloses a traditional Chinese medicine composition which is prepared from ten traditional Chinese medicines of astragalus, turtle shell, kudzuvine root, bupleurum, lucid ganoderma, red paeony root, red sage root, pseudo-ginseng, abrus herb and phyllanthus urinaria, and further researches show that the traditional Chinese medicine composition has the following effects: (1) Improving lipid metabolism disorder, reducing excessive accumulation of liver lipid, and protecting liver; (2) Reducing liver lipid level of alcoholic fatty liver rat model, improving steatosis, reducing liver inflammatory reaction, reducing tissue fat and inflammation, improving lipid peroxidation, and reducing blood plasma and intrahepatic TNF-alpha level, thereby preventing and treating alcoholic fatty liver; (3) Has obvious protective effect on liver injury and liver fibrosis of rats; (4) Can effectively improve the insulin resistance and liver lipid metabolism of rats, reduce the malondialdehyde, liver index and leptin level of fatty liver rat model, reduce lipid deposition in liver, and protect liver.
The traditional Chinese medicine composition selected by the invention considers from the aspect of dialectical treatment of traditional Chinese medicine, combines clinical curative effect and disease treatment root thinking in compatibility, improves the clinical curative effect of treating alcoholic fatty liver, is different from western medicine which only treats symptomatic treatment, and has no toxic or side effect.
Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of astragalus membranaceus, 1-10 parts of kudzuvine root, 1-10 parts of radix bupleuri, 1-6 parts of lucid ganoderma, 1-10 parts of red paeony root, 1-10 parts of turtle shell, 1-10 parts of radix salviae miltiorrhizae, 1-6 parts of pseudo-ginseng, 1-10 parts of abrus herb and 5-15 parts of phyllanthus urinaria.
Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5 parts of astragalus, 3 parts of kudzuvine root, 3 parts of bupleurum, 1.5 parts of lucid ganoderma, 3 parts of red paeony root, 3 parts of turtle shell, 3 parts of red sage root, 1.5 parts of pseudo-ginseng, 3 parts of abrus herb and 5 parts of phyllanthus urinaria.
Preferably, the traditional Chinese medicine composition is a common tablet, a dispersible tablet, an effervescent tablet, an orally disintegrating tablet, a lozenge, a chewable tablet, a capsule, a soft capsule, a microcapsule, a granule, a pill, a powder, a drop pill, a sustained release preparation, a controlled release preparation, an oral liquid preparation or an injection.
According to the different dosage forms of the traditional Chinese medicine composition, different auxiliary materials, including diluents, disintegrants, excipients, binders, lubricants, surfactants, fillers, preservatives, stabilizers, flavoring agents, thickeners or glidants and the like are required to be added in the preparation process.
Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:
Extracting carapax Trionycis and Ganoderma with water; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae, and Notoginseng radix with ethanol, and recovering ethanol under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and herba Phyllanthi Urinariae with water; mixing the filtrates, concentrating, drying, pulverizing into fine powder, adding adjuvants, and making into any pharmaceutically acceptable dosage forms.
Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:
extracting carapax Trionycis and Ganoderma with 6-10 times of water for 2-3 times each for 3-4 hr, filtering, and mixing filtrates; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae and Notoginseng radix with 6-10 times of 70% ethanol for 2-3 times each for 1-2 hr, filtering, mixing filtrates, and recovering ethanol at 50-70deg.C under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and phyllanthus urinaria with 6-10 times of water for 2-3 times (each for 1-2 hr), filtering, mixing the filtrate with the above two extractive solutions, concentrating to obtain soft extract with relative density of 1.30-1.35 at 50deg.C, drying, pulverizing into fine powder, adding adjuvants, and making into any pharmaceutically acceptable dosage.
Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:
Extracting carapax Trionycis and Ganoderma with 8 times of water for 3 times each for 3 hr, filtering, and mixing filtrates; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae and Notoginseng radix with 8 times of 70% ethanol for 3 times each for 1 hr, filtering, mixing filtrates, and recovering ethanol at 60deg.C under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and phyllanthus urinaria with 8 times of water for 3 times each for 1 hr, filtering, mixing the filtrate with the above two extractive solutions, concentrating to soft extract with relative density of 1.30-1.35 at 50deg.C, drying, pulverizing into fine powder, adding adjuvants, and making into any one of pharmaceutical preparations.
Preferably, the Chinese medicinal composition is a granule, and the preparation method comprises the following steps of:
Extracting carapax Trionycis and Ganoderma with 8 times of water for 3 times each for 3 hr, filtering, and mixing filtrates; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae and Notoginseng radix with 8 times of 70% ethanol for 3 times each for 1 hr, filtering, mixing filtrates, and recovering ethanol at 60deg.C under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and phyllanthus urinaria with 8 times of water for 3 times each for 1 hr, filtering, mixing the filtrate with the above two extractive solutions, concentrating to obtain soft extract with relative density of 1.30-1.35 (50deg.C), drying under reduced pressure (60deg.C, -0.08 Mpa), pulverizing into fine powder, adding appropriate amount of dextrin, mixing, adding 5% PVPK30 ethanol solution as wetting agent, granulating, and drying to obtain granule.
Compared with the prior art, the invention has the following beneficial effects:
(1) The traditional Chinese medicine composition used by the invention has obvious regulation effects on the reduction of cell viability caused by ethanol-induced BRL rat liver cell oxidative damage, the increase of cell MDA content, CAT activity reduction, GSH content reduction and ROS increase, and has good application prospect in medicines for treating or preventing alcoholic fatty liver.
(2) The aqueous or organic solvent extract of each traditional Chinese medicine is a pure natural component, is safer and has no obvious side effect. Overcomes the defect of larger side effect of western medicines for treating alcoholic fatty liver, and when water and alcohol are adopted to prepare the extract, the preparation process is economical, simple and convenient, is suitable for industrialized mass production, and is convenient for patients to take.
Drawings
FIG. 1 is a graph comparing the effect of ethanol on BRL cell viability (mean+ -SD).
FIG. 2 is the effect of a Chinese medicinal composition on normal BRL cell proliferation (mean+ -SD).
FIG. 3 is the effect of a Chinese medicinal composition on ethanol-induced BRL-injured cell growth (mean+ -SD).
FIG. 4 is the effect of a Chinese medicinal composition on CAT in ethanol-damaged BRL cells (mean.+ -. SD).
FIG. 5 is the effect of a Chinese medicinal composition on ethanol-induced BRL-injured cell MDA (mean+ -SD).
FIG. 6 is the effect of a Chinese medicinal composition on GSH of ethanol-damaged BRL cells (mean+ -SD).
FIG. 7 is the effect of a Chinese medicinal composition on ROS in ethanol-damaged BRL cells (mean+ -SD).
FIG. 8 is a representative graph showing the effect of a Chinese medicinal composition on liver histopathology in an alcohol + high fat diet rat (HE staining)
Detailed Description
The present invention will be described in further detail by the following examples, which are only for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.
The traditional Chinese medicine composition (also called Huangjia liver softening particles) in the following examples is prepared by the following steps: 5 parts of astragalus, 3 parts of turtle shell, 3 parts of kudzuvine root, 3 parts of bupleurum, 1.5 parts of lucid ganoderma, 3 parts of red paeony root, 3 parts of red sage root, 1.5 parts of pseudo-ginseng, 3 parts of abrus herb and 5 parts of phyllanthus urinaria. The preparation method comprises the following steps:
Extracting carapax Trionycis and Ganoderma with 8 times of water for 3 times each for 3 hr, filtering, and mixing filtrates; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae and Notoginseng radix with 8 times of 70% ethanol for 3 times each for 1 hr, filtering, mixing filtrates, and recovering ethanol at 60deg.C under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and phyllanthus urinaria with 8 times of water for 3 times each for 1 hr, filtering, mixing the filtrate with the above two extractive solutions, concentrating to obtain soft extract with relative density of 1.30-1.35 (50deg.C), drying under reduced pressure (60deg.C, -0.08 Mpa), pulverizing into fine powder, adding appropriate amount of dextrin, mixing, adding 5% PVPK30 ethanol solution as wetting agent, granulating, and drying to obtain granule.
Example 1: medicine efficacy experiment 1
1. Experimental method
1.1 Cultivation of BRL cells
BRL cells were cultured in DMEM high sugar medium containing 10% fetal bovine serum and 1% diabody at 37 ℃ with 5% co 2. When the cell density reaches 75-80%, the cells are passaged, and 4-10 generations of cells are taken for experiments.
1.2 Ethanol-induced BRL cell model establishment
1.2.1 Ethanol mother liquor preparation: filtering 2mL of absolute ethanol with an organic filter membrane to an EP tube, and collecting 1162.8 mu L
2837. Mu.L of absolute ethanol and serum-free DMEM medium were added to obtain a 5M ethanol stock solution.
1.2.2 Cell handling and grouping
The BRL cells in the logarithmic phase are inoculated into a 96-well plate according to the density of 5 multiplied by 10 3 per well, the culture medium is sucked after the culture medium is used for 24 hours, the culture medium is sucked after the culture medium is replaced by the complete culture medium for further incubation for 24 hours, the cells are divided into a normal group and an ethanol group (250-450 mM), 200 mu L of serum-free DMEM culture medium is added to a normal control group, 200 mu L of corresponding ethanol-containing DMEM culture medium is added to each concentration group of ethanol, the ethanol-containing DMEM culture medium is added to a cell culture plate, 6 compound wells are arranged in each group, after the culture medium is incubated for 24 hours at 37 ℃ and 5% CO 2, the activity of the cells is detected by a CCK-8 method, and the activity=the OD value of the test well/the average OD value of the normal group.
1.3 Effect of Chinese medicinal composition on proliferation of Normal BRL cells
The logarithmic phase BRL cells were inoculated into 96-well plates at a density of 5X 10 3/well, and after addition of complete medium 37℃and 5% CO 2, the medium was aspirated. The cells are divided into normal groups and Chinese medicinal composition groups (0.01, 0.1, 1, 10, 50, 100, 500, 1000 and 10000 mug/mL), the working solution of the Chinese medicinal composition groups is prepared by using a complete culture medium, 100 mug of the complete culture medium is added into the normal groups to serve as a contrast, the corresponding medicinal culture medium is respectively added into the Chinese medicinal composition groups, each group is cultured for 24 hours by 6 compound holes, and the temperature is 37 ℃ and the concentration of CO 2 percent. Cell viability was measured by CCK-8 method and the effect of the traditional Chinese medicine composition on BRL cell proliferation was observed, cell viability = test well OD/normal group average OD.
1.4 Effect of Chinese medicinal composition on growth of ethanol-damaged BRL cells
1.4.1 Ethanol mother liquor preparation: the same procedure as in item "1.2.1".
1.4.2 Ethanol working solution preparation: the 5M ethanol stock was diluted to 275mM ethanol working solution in serum-free DMEM medium.
1.4.3 Preparation of sample working solution: about 1g of the Chinese medicinal composition is precisely weighed by an analytical balance before the experiment
A mother liquor of 1g/mL was prepared with distilled water, and the supernatant was collected by centrifugation at 0.22 μm and filtered into an EP tube. The mother liquor of the test sample was diluted with DMEM complete medium according to the ratio.
1.4.4 Glutathione working solution preparation: a100 mM stock solution was prepared with physiological saline for injection. 100mM stock solution was diluted to 625. Mu.M glutathione working solution in DMEM complete medium.
1.4.5 Cell treatment
The logarithmic BRL cells were inoculated into 96-well plates at a density of 5X 103/well, and the medium was aspirated after addition of complete medium 37℃and 5% CO 2 for 24 hours. Cells were divided into a normal group, a model group and a treatment group (1, 10, 25, 50, 100, 250, 500. Mu.g/mL) with 7 concentrations of the Chinese medicinal composition and a positive glutathione (625. Mu.M) group, 6 duplicate wells were added to the normal group and the model group, DMEM complete medium was used as a control, 100. Mu.L of the corresponding drug-containing complete medium was added to each treatment group, 37 ℃ and 5% CO 2 were incubated for 24 hours, then the medium was aspirated, 200. Mu.L of DMEM medium with ethanol concentration of 275mM was added to the model group and each treatment group was molded, DMEM medium control was added to the normal group, and after 24 hours incubation with 5% CO 2, the medium was aspirated, PBS was washed twice, and cell viability was detected by the CCK-8 method, cell viability=well OD value/average OD value of the normal group.
1.5 Effect of Chinese medicinal composition on ethanol damage to BRL cells CAT, MDA, GSH
The logarithmic BRL cells were seeded at a density of 3.0X10 5/well in 6-well plates, and after addition of complete medium 37℃and 5% CO 2, the medium was aspirated. Cells were divided into a normal group, a model group and 3 concentration treatment groups (100, 250, 500. Mu.g/mL) of the Chinese medicinal composition and a positive glutathione (625. Mu.M) group, 3 duplicate wells were used for each group, DMEM complete medium was added to the normal group and the model group as a control, 2mL of the corresponding drug-containing complete medium was added to each treatment group, 37 ℃ and 5% CO 2 was incubated for 24 hours, 2mL of the DMEM medium containing ethanol concentration 275mM was added to the model group and each treatment group for modeling, DMEM medium control was added to the normal group, 37 ℃ and 5% CO 2 was incubated for 24 hours, the medium was aspirated, PBS was washed twice, and cells were collected and examined CAT, MDA, GSH according to the kit instructions.
1.6 Effect of Chinese medicinal composition on ethanol-damaged BRL cell ROS
The logarithmic BRL cells were seeded at a density of 3.0X10 4/well in 24-well plates, and after addition of complete medium 37℃and 5% CO 2, the medium was aspirated. The cells were divided into a normal group, a model group and a Chinese medicinal composition group, 3 concentration treatment groups (100, 250, 500. Mu.g/mL) and positive glutathione (625. Mu.M) groups, 4 compound wells each, the normal group and the model group were added with DMEM complete culture medium as a control, 1mL of the corresponding drug-containing complete culture medium was added to each treatment group, 1mL of the culture medium was aspirated after incubation for 24 hours at 37℃and 5% CO 2, 1mL of the model was molded, the model group and each treatment group were added with DMEM culture medium control, the culture medium was aspirated after incubation for 24 hours at 37℃and 5% CO 2, the operation was performed according to the instruction of ROS detection kit, the cell conditions were observed under a fluorescence inversion microscope, and the average fluorescence intensity was calculated using Nis-Elements image shooting software.
2. Data processing
Experimental data were statistically processed by GRAPHPAD PRISM 9.2.2 biometric software: data are expressed in mean±sd and are analyzed using One-way ANOVA for group analysis.
3. Experimental results
3.1 Alcohol-induced BRL cell oxidative damage model establishment
As shown in FIG. 1, compared with the normal group, ethanol with the concentration of 250-450 mM has a certain degree of damage to the normal liver cells of the BRL rats, and shows a dose-dependent trend, and the calculation result shows that: under the experimental conditions, the inhibition rate of 275mM ethanol on BRL rat normal liver cell BRL proliferation is 48.3%, about half inhibition rate, and the concentration is selected as the concentration of ethanol for subsequent induction of BRL cell oxidative damage.
3.2 Effect of Chinese medicinal composition on proliferation of Normal BRL cells
As shown in fig. 2 (compared with the normal group, P < 0.001), the cell viability of the group at 10000 μg/mL concentration of the traditional Chinese medicine composition is significantly reduced (P < 0.001) compared with the normal group, the cell viability at other concentrations is not statistically different (P > 0.05), and the subsequent traditional Chinese medicine composition is selected for preliminary screening at a concentration of 1-500 μg/mL for hepatocyte injury protection efficacy experiment.
3.3 Effect of Chinese medicinal composition on growth of ethanol-damaged BRL cells
The results are shown in fig. 3 (P <0.05, P <0.001 compared to the model group), where the model group cell viability is significantly reduced (P < 0.001) compared to the normal control group, and the positive glutathione group cell viability is significantly increased (P < 0.001) compared to the model group, suggesting: ethanol-induced oxidative damage cell models were successful. The cell viability of the 100, 250, 500 μg/mL concentration group of the traditional Chinese medicine composition was significantly increased (P <0.05 or P < 0.001) compared to the model group. The subsequent experiments of the influence of the traditional Chinese medicine composition on the oxidation index of the ethanol-induced hepatic cell injury select the concentrations of 100, 250 and 500 mug/mL for further verification.
3.4 Effect of Chinese medicinal composition on ethanol-damaged BRL cells CAT
The results are shown in fig. 4 (P <0.05 compared to model group: P <0.01, P < 0.001) that model group cells significantly decreased CAT activity (P < 0.001) compared to normal control group, and positive glutathione group CAT activity significantly increased (P < 0.01) compared to model group, suggesting: oxidative damage cell models were successful. Compared with the model group, the cell CAT activity of the group with the concentration of 500 and 250 mug/mL of the traditional Chinese medicine composition is obviously improved (P <0.05 or P < 0.01).
3.5 Effect of Chinese medicinal composition on ethanol-damaged BRL cell MDA
The results are shown in fig. 5 (P <0.05 compared to model group: P <0.01, P < 0.001) that the MDA content of the cells of model group is significantly increased (P < 0.01) compared to the normal control group, and the MDA content of positive glutathione group is significantly decreased (P < 0.05) compared to the model group, suggesting that: oxidative damage cell models were successful. Compared with the model group, the MDA content of the cells in the group with the concentration of 500 mug/mL of the traditional Chinese medicine composition is obviously reduced (P is less than 0.01), and the MDA content of the cells in the group with the concentration of 250 mug/mL has a tendency of reduction (P is more than 0.05).
3.6 Effect of Chinese medicinal composition on ethanol-damaged BRL cell GSH
The results are shown in fig. 6 (comparison to model group: P <0.05,: P < 0.01), where the GSH content of the cells of the model group is significantly reduced (P < 0.01) compared to the normal control group, and the GSH content of the cells of the positive glutathione group is significantly increased (P < 0.05) compared to the model group, suggesting: oxidative damage cell models were successful. Compared with the model group, the cell GSH content of the traditional Chinese medicine composition in the group with the concentration of 500 mug/mL is obviously increased (P < 0.05).
3.7 Effect of Chinese medicinal composition on ethanol-damaged BRL cells ROS (A.Con. (Normal group), B.model group (Mod.), C.Chinese medicinal composition 500. Mu.g/mL group, D.Chinese medicinal composition 250. Mu.g/mL group, E.Chinese medicinal composition 100. Mu.g/mL group, F.glutathione group (625. Mu.M))
The results are shown in fig. 7 (P <0.05, P <0.01, P <0.001 compared to the model group), where the model group showed significantly increased ROS (P < 0.01) compared to the normal control group, and the positive glutathione group showed significantly decreased ROS (P < 0.01) compared to the model group, suggesting: oxidative damage cell models were successful. Compared with the model group, the concentration group of the traditional Chinese medicine composition of 500 mug/mL and 250 mug/mL has remarkably reduced ROS content (P <0.05 or P < 0.01).
The experimental result proves that the composition has obvious regulation effects on the reduction of cell viability caused by ethanol-induced BRL rat liver cell oxidative damage, the increase of cell MDA content, CAT activity reduction, GSH content reduction and ROS increase, and has good application prospect in medicines for treating or preventing alcoholic fatty liver.
Example 2: medicine effect experiment II
1. Experimental method
1.1. Sample dosage and route of administration
The human dosage of the traditional Chinese medicine composition is 1 bag (10 g) for 1 time, and the equivalent dosage of the rat is 1g/kg according to the body surface area. And 2 times and 4 times of experimental doses are added, namely, the low, medium and high doses are respectively 1g/kg,2g/kg and 4g/kg, the administration volume is 10mL/kg, and the stomach is irrigated for 1 time every day.
1.2. Glutathione tablet dosage and administration route of positive drug
The maximum clinical oral dosage of the glutathione sheet is 1200 mg/day, the dosage of the rat according to the body surface area conversion is 1200 mg/60 kg=120 mg/kg, the administration volume is 12mL/kg, and the stomach is irrigated 1 time a day.
1.3. Preparation of test samples
High dose: weighing 20g of the traditional Chinese medicine composition, adding distilled water to 50mL, and uniformly mixing to prepare 4g/10mL of suspension.
Medium dose: 10g of the traditional Chinese medicine composition is weighed, distilled water is added to 50mL, and the mixture is uniformly mixed to prepare 2g/10mL of suspension.
Low dose: weighing 5g of the traditional Chinese medicine composition, adding distilled water to 50mL, and uniformly mixing to prepare 1g/10mL of suspension.
1.4. Preparation of positive medicine glutathione tablet
Taking 5 pieces of 100mg glutathione, grinding, adding distilled water to dilute to 50mL, and preparing 100mg/mL positive medicine suspension.
1.5.30% Alcohol preparation
100ML of 60-degree red star Erguotou white spirit is added with distilled water to 200mL.
1.6. Grouping and handling of animals
After being adaptively fed in quarantine period, 70 SPF-class male SD rats are fed with normal feed except 10 normal control groups, and the rest rats are fed with high-fat feed (8 mL/kg of ethanol with the energy of 4.5kacl/g, the fat energy supply ratio of 37 percent) and the stomach of 30 percent and with 20 percent of sucrose, 15 percent of lard, 1.2 percent of cholesterol, 0.2 percent of sodium cholate and the like are added on the basis of the normal feed to mould. Blood lipid index was measured by orbital blood sampling after two weeks of feeding with alcohol + high fat diet, and model-successfully-modeled rats were randomly divided into 5 groups according to total Cholesterol (CHO) content: B. model control group (physiological saline administration), c.low dose group of Chinese medicinal composition (1 g/kg), d.medium dose group of Chinese medicinal composition (2 g/kg), e.high dose group of Chinese medicinal composition (4 g/kg) and f.positive control glutathione group, 10 animals per group, normal rats fed normally before as a.normal control group (distilled water administration, 10 mL/kg).
After grouping, the normal control group is continuously fed with normal feed, distilled water is drunk, and the distilled water is fed by pouring stomach when the distilled water is fed, and the concentration is 10mL/kg; the other groups except the normal control group are fed by high-fat feed, 30% alcohol is uniformly fed by stomach infusion in the morning, 10mL/kg of distilled water is fed by stomach infusion in the model control group in the afternoon, corresponding drugs are respectively fed by stomach infusion in the groups C, D, E and F, 1 time a day, 8w are continuously fed, fasting is carried out after the last feeding, and the indexes are detected by weighing, blood taking and liver tissue taking.
1.7. Observation of body weight and general State
The state of animals (appearance signs, behavioral activities, fecal properties, feeding conditions, etc.) was observed daily; body weight was weighed once a week.
1.8. Liver weight and liver coefficient
Liver was weighed for 12h after the last dose and liver coefficient = liver g/100g body weight.
1.9. Blood fat four index detection
Administration of 0w, 4w, blood collection through the orbit, centrifugation of the supernatant, and measurement of total Cholesterol (CHO), triglyceride (TG), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) levels in serum. The rats administered with 8w were anesthetized and blood was collected from the abdominal vein, and the supernatants were centrifuged to detect the total Cholesterol (CHO), triglyceride (TG), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) levels in the serum.
1.10. Liver function index detection
Administration 4w blood was collected through the orbit, and the supernatant was collected by centrifugation, and serum glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) activities were measured. After the administration of 8w of the rat anesthesia, blood is collected from abdominal veins, and the supernatant is collected by centrifugation, and the activity of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) in serum is detected.
1.11. Oxidation resistance index detection
After the last administration, the patients are fasted for 12 hours, anesthetized, blood is taken from the inferior vena cava, the supernatant is centrifugally taken, and the SOD activity and MDA content in serum are detected according to the instruction book of a superoxide dismutase (SOD) and Malondialdehyde (MDA) kit. The liver of the rat is weighed, homogenized, protein content is detected by BCA protein quantitative kit, and SOD activity and MDA content in liver tissue are detected by SOD and MDA kit.
1.12. Liver histopathological examination
After liver tissue is weighed, right liver leaves are uniformly taken and fixed by 4% paraformaldehyde, paraffin embedding is carried out, 5 mu m sections are cut, HE staining is carried out, the degree of loosening and edema of small She Bao pulp of the liver is observed under a light microscope, and the degree is scored according to the lesion light and heavy degree: no lesions "0 grade" score of 0, mild "grade I" score of 1, moderate "grade II" score of 2, severe "grade III" score of 3, and severe "grade IV" score of 4; another 5 fields were randomly taken for each slice and scored according to the steatosis scoring criteria: grade 0 indicates that the lipid drops in the liver cells are scattered, rare and basically normally counted for 0 points; grade I indicates that the number of hepatic cells containing lipid droplets is not more than 1/4 of 1 minute; grade II indicates that the number of hepatic cells containing lipid droplets is not more than 1/2 of 2 minutes; grade III indicates that the number of lipid-containing hepatocytes is no more than 3/4 of 3 minutes; grade IV indicates that liver tissue was almost replaced with lipid droplets for score 4.
2. Data processing
Experimental data were statistically processed by GRAPHPAD PRISM biometrics software: the metric data are expressed in mean±sd and analyzed using analysis of variance combined with Dunnett's multiplex comparison and the hepatopathology scores are analyzed using Kruskal-Wallis rank sum test combined with Dunnett's multiplex comparison.
3. Experimental results
3.1 Influence of the Chinese medicinal composition on the weight and general State of alcoholic fatty liver rats
The animal groups have good mental state, smooth fur color, free movement, uniform breathing, no obvious abnormality of food intake and feces, and no abnormal secretion in the mouth and nose during the experimental period. As can be seen from table 1, the weight of each group of animals was normally increased during each period of administration, and the weight of the normal control group and each of the administration groups were not statistically different from each other as compared with the model control group.
Table 1. Influence on body weight of alcohol + high fat diet rats (mean±sd, n=10)
Note that: there were no statistical differences between the groups compared to the model control group.
3.2 Effect of Chinese medicinal composition on liver coefficient of alcoholic fatty liver rat
As shown in table 2, the liver weight and liver coefficient of the model control rats increased significantly (P < 0.01) compared to the normal control group after 60d of administration. Compared with the model control group, the liver weight and liver coefficient of rats in the dosage group and the positive control group in the traditional Chinese medicine composition are obviously reduced (P < 0.05).
Table 2. Influence on liver coefficients of Alcoholic+high fat diet rats (mean+ -SD, n=10)
Note that: comparison to model control group,: p <0.01.
3.3 Effects of Chinese medicinal composition on blood lipid of alcoholic fatty liver rat
As shown in table 3, prior to dosing, the lipid index of rats in the model control group, in which the alcohol + high fat diet was modeled for two weeks, was compared to the normal control group: CHO, TG, LDL-C content is increased significantly (P < 0.01), HDL-C content is decreased significantly (P < 0.05). Compared with the model control group, the blood lipid indexes of the treatment groups have no obvious difference (P > 0.05).
As shown in table 4, 30d of administration was compared with the normal control group, and the model control group of the alcohol + high fat diet model was used for rat blood lipid index: CHO, TG, LDL-C content is increased significantly (P < 0.01), HDL-C content is decreased significantly (P < 0.01). Compared with the model control group, the content of serum CHO and LDL-C of rats in the dosage group in the traditional Chinese medicine composition is reduced (P > 0.05), and the blood lipid indexes of animals in the other groups have no statistical difference (P > 0.05).
As shown in table 5, the administration was performed for 60d, compared with the normal control group, the model control group rat blood lipid index: CHO, TG, LDL-C content is increased significantly (P < 0.01), HDL-C content is decreased significantly (P < 0.01). Compared with a model control group, the serum CHO and LDL-C contents of the high-dose group of the traditional Chinese medicine composition are obviously reduced (P < 0.05), the serum TG contents of the high-dose group of the traditional Chinese medicine composition and the positive control group of the traditional Chinese medicine composition are obviously reduced (P < 0.05).
Table 3. Effects on blood lipid in pre-dose alcohol+high fat diet rats (mean.+ -. SD, n=10)
Note that: compared to model control group,: p <0.05,: p <0.01.
Table 4. Effects on blood lipid in rats dosed with 30d alcohol + high fat diet (mean+ -SD, n=10)
Note that: comparison to model control group,: p <0.01.
Table 5. Effects on blood lipid in rats dosed with 60d alcohol + high fat diet (mean+ -SD, n=10)
Note that: compared to model control group,: p <0.05,: p <0.01.
3.4 Effect of Chinese medicinal composition on liver function index of alcoholic fatty liver rat
As shown in table 6, the liver function index of the model control rats was compared to the normal control after 30d of administration: serum ALT, AST activities were not statistically different (P > 0.05), ALP activity was significantly increased (P < 0.01). There was no statistical difference in serum ALT, AST, ALP activity (P > 0.05) for each group compared to the model control group.
As shown in table 7, the liver function index of the model control rats was compared to the normal control by 60 d: serum ALT, AST activity, ALP activity was significantly increased (P < 0.01). Compared with the model control group, the activity of the serum ALT, AST, ALP of the rat in the dosage group in the traditional Chinese medicine composition is obviously reduced (P <0.05 or P < 0.01), the activity of the serum AST and ALP of the rat in the high dosage group in the traditional Chinese medicine composition is obviously reduced (P <0.05 or P < 0.01), and the activity of the serum ALT, AST, ALP of the rat in the positive control group is obviously reduced (P <0.05 or P < 0.01).
Table 6. Effects on liver function index of rats dosed with 30d alcohol + high fat diet (mean+ -SD, n=10)
| ALT(U/L) | AST(U/L) | ALP(U/L) | |
| Normal control group | 114.03±19.56 | 173.26±63.13 | 512.01±74.07** |
| Model control group | 139.95±45.86 | 209.39±55.18 | 681.20±134.68 |
| Low dose group | 135.66±38.40 | 201.88±46.42 | 766.80±85.81 |
| Medium dose group | 127.34±28.38 | 170.41±59.10 | 723.90±150.76 |
| High dose group | 152.93±32.41 | 218.90±34.11 | 603.92±62.37 |
| Positive control group | 113.68±14.64 | 199.45±32.58 | 625.83±143.15 |
Note that: comparison to model control group,: p <0.01.
Table 7. Effects on liver function index of rats administered 60d alcohol + high fat diet (mean+ -SD, n=10)
| ALT(U/L) | AST(U/L) | ALP(U/L) | |
| Normal control group | 126.64±23.15** | 218.06±84.27** | 138.13±31.19** |
| Model control group | 315.41±114.29 | 453.30±169.20 | 211.13±56.55 |
| Low dose group | 371.40±211.43 | 457.52±123.58 | 169.60±37.65 |
| Medium dose group | 183.12±66.64* | 261.29±61.63** | 161.00±37.62* |
| High dose group | 243.36±72.38 | 321.66±103.23* | 153.54±29.18** |
| Positive control group | 189.45±52.13* | 271.18±55.02** | 163.18±34.55* |
Note that: compared to model control group,: p <0.05,: p <0.01.
3.5 Influence of the Chinese medicinal composition on the Oxidation index of alcoholic fatty liver rats
As shown in table 8, after 60d dosing, the serum SOD activity was significantly reduced (P < 0.01) and MDA content was significantly increased (P < 0.01) in the model control group compared to the normal control group. Compared with the model control group, the serum SOD activity of rats in the dosage group and the positive control group in the traditional Chinese medicine composition is obviously increased (P <0.05 or P < 0.01), and the serum MDA content of rats in the traditional Chinese medicine composition, the high dosage group and the positive control group is obviously reduced (P < 0.05).
As shown in table 9, after 60d dosing, the liver tissue SOD activity was significantly reduced (P < 0.01) and MDA content was significantly increased (P < 0.01) in the model control group compared to the normal control group. Compared with the model control group, the dose group and the positive control group in the traditional Chinese medicine composition have the advantages that the SOD activity of rat liver tissues is obviously increased (P < 0.05), and the MDA content is obviously reduced (P < 0.01).
Table 8. Influence on serum Oxidation index of Alcoholic+high fat diet rats (mean+ -SD, n=10)
Note that: compared to model control group,: p <0.05,: p <0.01.
Table 9. Influence on liver tissue Oxidation index of Alcoholic+high fat diet rats (mean+ -SD, n=10)
| SOD(U/mg) | MDA(μM/mg) | |
| Normal control group | 147.37±25.09** | 13.55±4.11** |
| Model control group | 103.09±21.24 | 24.41±5.35 |
| Low dose group | 109.38±27.75 | 21.88±4.11 |
| Medium dose group | 138.41±29.49* | 18.66±5.37* |
| High dose group | 126.31±20.12 | 19.68±6.23 |
| Positive control group | 132.21±24.91* | 17.05±4.15** |
Note that: compared to model control group,: p <0.05,: p <0.01.
3.6 Effects of Chinese medicinal composition on liver histopathology of alcoholic fatty liver rat
After 60d of administration, the naked eye is generally observed: the liver of the normal control group has ruddy color and normal size; the liver color of the model control group is obviously white and lacks blood color; compared with the model control group, the liver color of rats in the traditional Chinese medicine composition, the high-dose group and the positive control group is improved, and the volume is reduced to a certain extent.
The observation under HE staining microscope of liver tissue is shown in fig. 8 (magnification 200×, a. Normal control group, b. Model control group, c. Low dose group, d. Medium dose group, e. High dose group, f. Positive control group.): the liver cells of the normal control group are orderly arranged, clear in structure and low in fat content; the liver cells of the model control group have loose arrangement disorder structure, the cell volume is obviously increased, more lipid drops exist in liver lobules, and part of cytoplasm and nuclei of cells disappear to form lipid drop cavities. Compared with the model control group, the liver cell structure of rats in the traditional Chinese medicine composition, the high-dose group and the positive control group is obviously improved, the volume is obviously reduced, and the lipid drop quantity is obviously reduced.
The cytoplasmic loosening and edema score is shown in table 10, and the cytoplasmic loosening and edema score was significantly increased (P < 0.01) around the hepatic lobules of the model control rats compared to the normal control. Compared with the model control group, the degree of cytokinesis and edema around the liver lobules of rats in each dosage group of the traditional Chinese medicine composition is not statistically different (P > 0.05). The positive control group rats had a tendency to have a reduced cytoplasmic loosening around the hepatic lobules and a reduced edema level score (P > 0.05).
The steatosis score is shown in table 11, and the liver tissue steatosis score of the model control rats is significantly increased (P < 0.01) compared to the normal control. Compared with a model control group, the degree of hepatic steatosis of rats in a dosage group in the traditional Chinese medicine composition is obviously reduced (P < 0.05), and the degree of hepatic steatosis of rats in a high dosage group in the traditional Chinese medicine composition has a tendency of reducing (P > 0.05). The positive control group rats had significantly reduced liver tissue steatosis level scores (P < 0.01).
Table 10. Influence of score on the degree of cytoplasmic loosening and edema around hepatic lobules in alcohol + high fat diet rats (mean+ -SD, n=10)
Note that: comparison to model control group,: p <0.01.
Table 11. Influence on the degree of steatosis score of Alcoholic+high fat diet rats (mean+ -SD, n=10)
Note that: comparison to model control group,: p <0.05,: p <0.01.
In conclusion, the traditional Chinese medicine composition used in the invention can obviously reduce the increase of liver tissue weight and liver coefficient caused by the combination of alcohol and high-fat diet, reduce the contents of CHO, TG and LDL-C, reduce the activities of liver function indexes ALT, AST and ALP, and improve pathological conditions of liver tissues, wherein the pharmacodynamic effect of the traditional Chinese medicine composition on the rats with alcoholic fatty liver induced by the combination of alcohol and high-fat diet is possibly related to reducing the MDA contents in serum and liver and increasing SOD activities.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (6)
1. The application of a traditional Chinese medicine composition in preparing a medicine for treating or preventing alcoholic fatty liver is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 1-30 parts of astragalus membranaceus, 1-20 parts of kudzuvine root, 1-20 parts of radix bupleuri, 1-10 parts of lucid ganoderma, 1-20 parts of red paeony root, 1-20 parts of turtle shell, 1-20 parts of radix salviae miltiorrhizae, 1-10 parts of pseudo-ginseng, 1-20 parts of abrus herb and 1-30 parts of phyllanthus urinaria.
2. The use according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-15 parts of astragalus membranaceus, 1-10 parts of kudzuvine root, 1-10 parts of radix bupleuri, 1-6 parts of lucid ganoderma, 1-10 parts of red paeony root, 1-10 parts of turtle shell, 1-10 parts of radix salviae miltiorrhizae, 1-6 parts of pseudo-ginseng, 1-10 parts of abrus herb and 5-15 parts of phyllanthus urinaria.
3. The use according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5 parts of astragalus, 3 parts of kudzuvine root, 3 parts of bupleurum, 1.5 parts of lucid ganoderma, 3 parts of red paeony root, 3 parts of turtle shell, 3 parts of red sage root, 1.5 parts of pseudo-ginseng, 3 parts of abrus herb and 5 parts of phyllanthus urinaria.
4. The use according to claim 1, wherein the Chinese medicinal composition is a normal tablet, a dispersible tablet, an effervescent tablet, an orally disintegrating tablet, a buccal tablet, a chewable tablet, a capsule, a soft capsule, a microcapsule, a granule, a pill, a powder, a drop pill, a sustained release preparation, a controlled release preparation, an oral liquid preparation or an injection.
5. The use according to claim 1, wherein the preparation method of the traditional Chinese medicine composition is as follows:
Extracting carapax Trionycis and Ganoderma with water; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae, and Notoginseng radix with ethanol, and recovering ethanol under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and herba Phyllanthi Urinariae with water; mixing the filtrates, concentrating, drying, pulverizing into fine powder, adding adjuvants, and making into any pharmaceutically acceptable dosage forms.
6. The use according to claim 1, wherein the preparation method of the traditional Chinese medicine composition is as follows:
extracting carapax Trionycis and Ganoderma with 6-10 times of water for 2-3 times each for 3-4 hr, filtering, and mixing filtrates; extracting radix astragali, saviae Miltiorrhizae radix, radix Puerariae and Notoginseng radix with 6-10 times of 70% ethanol for 2-3 times each for 1-2 hr, filtering, mixing filtrates, and recovering ethanol at 50-70deg.C under reduced pressure; extracting bupleuri radix, herba abri, radix Paeoniae Rubra, and phyllanthus urinaria with 6-10 times of water for 2-3 times (each for 1-2 hr), filtering, mixing the filtrate with the above two extractive solutions, concentrating to obtain soft extract with relative density of 1.30-1.35 at 50deg.C, drying, pulverizing into fine powder, adding adjuvants, and making into any pharmaceutically acceptable dosage.
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