CN116115724B - Traditional Chinese medicine composition, pharmaceutical preparation, preparation method and application thereof - Google Patents
Traditional Chinese medicine composition, pharmaceutical preparation, preparation method and application thereof Download PDFInfo
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- CN116115724B CN116115724B CN202310324279.8A CN202310324279A CN116115724B CN 116115724 B CN116115724 B CN 116115724B CN 202310324279 A CN202310324279 A CN 202310324279A CN 116115724 B CN116115724 B CN 116115724B
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/537—Salvia (sage)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/282—Artemisia, e.g. wormwood or sagebrush
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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- Engineering & Computer Science (AREA)
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- Medical Informatics (AREA)
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- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a traditional Chinese medicine composition, a pharmaceutical preparation, a preparation method and application thereof. The pharmaceutical composition is prepared from the following raw material medicines: 8-20 parts of red sage root, 5-15 parts of lotus leaf, 5-15 parts of gynostemma pentaphylla, 5-15 parts of radix curcumae, 8-20 parts of bighead atractylodes rhizome, 8-20 parts of herba artemisiae capillaries, 8-20 parts of sedum sarmentosum and 5-15 parts of radix bupleuri. The traditional Chinese medicine composition has good effect on treating non-alcoholic steatohepatitis.
Description
Technical Field
The invention relates to a traditional Chinese medicine composition, a pharmaceutical preparation, a preparation method and application thereof, in particular to a traditional Chinese medicine composition and a pharmaceutical preparation for nonalcoholic steatohepatitis, and a preparation method and application thereof.
Background
Non-alcoholic steatohepatitis refers to a disease caused by excessive accumulation of liver fat due to other causes without excessive drinking. Dietary high calories, excessive free fatty acids, increased lipolysis in visceral adipose tissue, activation of liver neoadipogenesis, etc. can all lead to the accumulation of triglycerides in the liver. The early stage is simple fat accumulation, mainly exists in the form of triglyceride in lipid droplets, and further develops into a non-alcoholic steatohepatitis stage accompanied by hepatocyte injury, inflammatory infiltration and hepatic fibrosis. Some non-alcoholic steatohepatitis patients develop cirrhosis and further develop liver cancer.
The traditional Chinese medicine has no disease name of non-alcoholic steatohepatitis, and the modern traditional Chinese medicine belongs to the categories of liver nodule, accumulation, hypochondriac pain, turbid phlegm and the like according to clinical symptoms, etiology and pathogenesis of the non-alcoholic steatohepatitis.
CN113425788A discloses a traditional Chinese medicine composition for treating nonalcoholic steatohepatitis, which is prepared from 8-20 parts of bupleurum, 8-25 parts of bighead atractylodes rhizome, 8-20 parts of virgate wormwood herb, 8-20 parts of alisma rhizome, 20-35 parts of sedum sarmentosum, 8-15 parts of red sage root, 8-25 parts of red paeony root, 10-25 parts of hawthorn fruit, 8-20 parts of bitter orange and 0.05-0.3 part of bezoar. The prescription takes 'four-inverse powder' as a basic composition and is prepared by adding, subtracting and matching.
CN109692309a discloses a formula for treating nonalcoholic fatty liver disease, which comprises lotus leaf, poria cocos, bighead atractylodes rhizome, semen cassiae, coix seed, rhizoma alismatis, polyporus, gynostemma pentaphylla, rhizoma pinellinae praeparata, polygonum cuspidatum, herba artemisiae capillaris, astragalus membranaceus, liquorice, dried orange peel, radix bupleuri, radix paeoniae rubra, rhizoma curcumae zedoariae, radix salviae miltiorrhizae and rheum officinale.
CN113171415a discloses a traditional Chinese medicine composition for treating non-alcoholic fatty liver, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 8-20 parts of bupleurum, 13-33 parts of bighead atractylodes rhizome, 9-22 parts of poria cocos, 9-28 parts of raw hawthorn, 5-17 parts of rhizoma pinellinae praeparata, 5-17 parts of dried orange peel, 9-28 parts of red sage root, 9-22 parts of kudzuvine root, 5-17 parts of cassia seed, 8-17 parts of red paeony root, 8-17 parts of capillary artemisia, 8-17 parts of rhizoma alismatis, 9-33 parts of lotus leaf, 5-17 parts of dogbane leaf, 9-22 parts of gynostemma pentaphylla, 4-13 parts of sea buckthorn fruit and 5-13 parts of honey-fried licorice root.
The prescription for treating the non-alcoholic fatty liver disease has excessive medicine taste, multiple points and unknown main line, and is difficult to aim at the main cause and the key of the non-alcoholic fatty liver disease.
Disclosure of Invention
Aiming at the pathological factors of excessive dampness, qi stagnation and blood stasis in the early stage of the nonalcoholic steatohepatitis, the method uses eight traditional Chinese medicines of gynostemma pentaphylla, tarragon, sedum sarmentosum, bighead atractylodes rhizome, radix bupleuri, radix curcumae, radix salviae miltiorrhizae and lotus leaf according to the legislation of resolving dampness, promoting qi circulation and activating blood circulation, and aims at treating nonalcoholic steatohepatitis from the angles of liver qi discomfort, transverse adverse flow of spleen, spleen deficiency and dampness generation, spleen dampness, qi stagnation, unsmooth blood circulation, liver qi stagnation and malignant circulation.
On the basis, the invention aims to provide a traditional Chinese medicine composition which has good effect on treating nonalcoholic steatohepatitis.
The invention also aims at providing a preparation method of the traditional Chinese medicine composition.
It is a further object of the present invention to provide a pharmaceutical formulation.
It is a further object of the present invention to provide a pharmaceutical use of the pharmaceutical composition.
The invention provides a traditional Chinese medicine composition, which is prepared from the following raw material medicines: 8-20 parts of red sage root, 5-15 parts of lotus leaf, 5-15 parts of gynostemma pentaphylla, 5-15 parts of radix curcumae, 8-20 parts of bighead atractylodes rhizome, 8-20 parts of herba artemisiae capillaries, 8-20 parts of sedum sarmentosum and 5-15 parts of radix bupleuri.
According to the traditional Chinese medicine composition, the traditional Chinese medicine composition is preferably prepared from the following raw material medicines: 12-17 parts of red sage root, 8-12 parts of lotus leaf, 8-12 parts of gynostemma pentaphylla, 8-12 parts of radix curcumae, 12-17 parts of bighead atractylodes rhizome, 12-17 parts of herba artemisiae capillaris, 12-17 parts of sedum sarmentosum and 7-11 parts of radix bupleuri.
According to the traditional Chinese medicine composition, the traditional Chinese medicine composition is preferably prepared from the following raw material medicines: 15 parts of red sage root, 10 parts of lotus leaf, 10 parts of gynostemma pentaphylla, 10 parts of radix curcumae, 15 parts of bighead atractylodes rhizome, 15 parts of herba artemisiae scopariae, 15 parts of sedum sarmentosum and 9 parts of radix bupleuri.
According to the traditional Chinese medicine composition, preferably, the bighead atractylodes rhizome is stir-fried bighead atractylodes rhizome, and the radix bupleuri is vinegar-processed radix bupleuri.
According to the traditional Chinese medicine composition, preferably, the active components of the traditional Chinese medicine composition are only prepared from the raw materials.
On the other hand, the invention provides a pharmaceutical preparation, which comprises the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials.
The pharmaceutical preparation according to the present invention preferably, the pharmaceutically acceptable auxiliary material is selected from one or more of filler, flavoring agent, lubricant, binder, diluent, disintegrating agent, sustained release agent, glidant, and thickener.
The pharmaceutical preparation according to the present invention is preferably in a dosage form selected from one or more of a tablet, a pill, a capsule, a granule, a suspension, an ointment, an oral liquid or an injection.
In still another aspect, the invention provides a preparation method of the traditional Chinese medicine composition, and the traditional Chinese medicine composition is obtained by decocting all raw material medicines with water.
In still another aspect, the invention provides the use of the above-mentioned Chinese medicinal composition in preparing a medicament or health food for treating and/or preventing nonalcoholic steatohepatitis.
The traditional Chinese medicine composition disclosed by the invention has the advantages of capability of effectively treating nonalcoholic steatohepatitis, small toxic and side effects and remarkable curative effect due to mutual compatibility of the components.
Drawings
FIG. 1 is a graph of ALT levels in serum from mice in each group.
FIG. 2 is a graph showing AST content in serum of each group of mice.
FIG. 3 is a graph showing TC levels in serum of mice in each group.
FIG. 4 is a graph showing the TG content in serum of each group of mice.
FIG. 5 is a graph showing HDL-C content in serum of each group of mice.
FIG. 6 is a graph showing the LDL-C content in serum of each group of mice.
FIG. 7 is a graph showing TG content in liver homogenates of mice in each group.
FIG. 8 is a graph showing TC content in liver homogenates of mice in each group.
Fig. 9 is a graph showing MDA content in liver homogenates of mice in each group.
FIG. 10 is a gel electrophoresis diagram of related proteins.
Fig. 11 is a microscopic image of a mouse liver slice.
FIG. 12 shows NAS scores for each group of mice.
Wherein NC represents a blank control group, M represents a model group, TL represents a low-dose group of traditional Chinese medicine, TM represents a medium-dose group of traditional Chinese medicine, TH represents a high-dose group of traditional Chinese medicine and FEN represents a positive medicine group.
Detailed Description
The present invention will be further described with reference to specific examples, but the scope of the present invention is not limited thereto.
< Chinese medicinal composition >
The traditional Chinese medicine composition is prepared from the following raw material medicines: 8-20 parts of red sage root, 5-15 parts of lotus leaf, 5-15 parts of gynostemma pentaphylla, 5-15 parts of radix curcumae, 8-20 parts of bighead atractylodes rhizome, 8-20 parts of herba artemisiae capillaries, 8-20 parts of sedum sarmentosum and 5-15 parts of radix bupleuri.
Preferably, the pharmaceutical composition is prepared from the following raw material medicines: 12-17 parts of red sage root, 8-12 parts of lotus leaf, 8-12 parts of gynostemma pentaphylla, 8-12 parts of radix curcumae, 12-17 parts of bighead atractylodes rhizome, 12-17 parts of herba artemisiae capillaris, 12-17 parts of sedum sarmentosum and 7-11 parts of radix bupleuri.
According to a preferred embodiment of the present invention, the pharmaceutical composition is prepared from the following components including: 15 parts of red sage root, 10 parts of lotus leaf, 10 parts of gynostemma pentaphylla, 10 parts of radix curcumae, 15 parts of bighead atractylodes rhizome, 15 parts of herba artemisiae scopariae, 15 parts of sedum sarmentosum and 9 parts of radix bupleuri.
According to a preferred embodiment of the present invention, the pharmaceutical composition is made of only the above eight raw materials.
The applicant believes that the disease location of nonalcoholic steatohepatitis is in the liver, has close relationship with the spleen, and is caused by improper diet, unsmooth emotion, overstrain and the like, and is mainly caused by improper diet. If the people overeat the food are sweet and greasy, spleen is injured for a long time, spleen deficiency and excessive dampness are caused, damp evil is blocked, qi movement is not smooth, liver qi is not smooth, blood is not smooth, spleen is affected by the transverse adverse flow of qi for a long time, spleen is injured more, dampness is more concentrated, qi is stagnated, blood is healed, and malignant circulation is formed, so that the disease is caused.
Aiming at the pathological factors of excessive dampness, qi stagnation and blood stasis in the early stage of the nonalcoholic steatohepatitis, the invention uses eight traditional Chinese medicines of gynostemma pentaphylla, tarragon, sedum sarmentosum, bighead atractylodes rhizome, radix bupleuri, radix curcumae, radix salviae miltiorrhizae and lotus leaf according to the legislation of resolving dampness, promoting qi circulation and activating blood circulation, and aims at treating the nonalcoholic steatohepatitis from the angles of liver qi discomfort, transverse adverse flow of spleen, spleen deficiency and dampness generation, spleen dampness, qi stagnation, unsmooth blood circulation, liver qi depression and malignant circulation.
In the composition, the herba artemisiae scopariae has the effects of clearing liver and promoting bile flow, eliminating dampness and removing heat, the gynostemma pentaphylla has the effects of clearing heat and resolving phlegm, and invigorating spleen and replenishing qi, and the herba artemisiae scopariae are combined to be a monarch drug for removing damp-heat and phlegm stagnation, and assisting spleen and strengthening transportation. The radix bupleuri is used for soothing liver and relieving depression, the radix curcumae is used for promoting qi and activating blood, and the bighead atractylodes rhizome is used for strengthening earth and resolving dampness and also used for inhibiting wood. The red sage root is added to increase the blood circulation promoting and blood stasis removing effects of the radix curcumae; herba Sedi Gan Danli is wet, and has effects of relieving constipation, and folium Nelumbinis is light and clear, so that QING YANG can rise, and turbid blood stasis can be reduced, and can be used for treating damp evil. The method has the advantages of clear thought, accurate medicine selection, little medicine and special force, and can effectively improve the symptoms of the nonalcoholic steatohepatitis by taking the effects of dispelling the dampness, removing heat, promoting qi, activating blood and removing obstruction in collaterals.
< preparation method >
The Chinese medicinal composition of the present invention may be prepared by various preparation methods, such as water decoction or alcohol extraction. The components of the raw materials in the traditional Chinese medicine composition are as described above, and are not repeated here. Preferably, a water decoction method is used.
The method specifically comprises the following steps: (1) Decocting Saviae Miltiorrhizae radix, herba Gynostemmatis, radix Curcumae, atractylodis rhizoma, herba Artemisiae Scopariae, herba Sedi, bupleuri radix and water to obtain a first extract. (2) And (3) decocting the first extraction product and lotus leaves to obtain a first liquid medicine and first residues. (3) Decocting the first residue with water to obtain second medicinal liquid and second residue. And combining the first liquid medicine and the second liquid medicine to obtain the total liquid medicine. And optionally, (4) concentrating and drying the total medicinal solution.
In the step (1), the water consumption is 5-20 times of the total volume of the red sage root, the gynostemma pentaphylla, the radix curcumae, the white atractylodes rhizome, the herba artemisiae capillaries, the sedum sarmentosum and the radix bupleuri; preferably 7 to 15 times; more preferably 9 to 12 times. Preferably, the decoction is performed by medium fire. The decoction time can be 15-60 min; preferably 20 to 50 minutes; more preferably 30 to 40 minutes.
In the step (2), the decoction time can be 1-10 min after the water is boiled; preferably, the decoction time is 2-6 min after boiling water; more preferably, the decoction time is 3-4 min after boiling.
In the step (3), the water consumption is 0.5-2 times of the volume of the first medicine residue; preferably 0.8 to 1.5 times. Preferably, the decoction is performed by medium fire. The decoction time can be 10-50 min; preferably 15-40 min; more preferably 20 to 30 minutes.
< pharmaceutical preparation and use >
The invention also provides a pharmaceutical preparation, which comprises the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials. According to one embodiment of the present invention, the pharmaceutical formulation comprises only the Chinese medicinal composition as a pharmaceutically active ingredient, and no other pharmaceutically active ingredient. The formulation of the preparation is not limited, and for example, the preparation can be tablets, pills, capsules, granules, suspensions, ointments, oral liquids or injections and the like. Preferably, the preparation is an oral liquid or a granule.
The auxiliary materials can be filler, corrigent, lubricant, adhesive, diluent, disintegrating agent, slow release agent, glidant, thickener and other auxiliary materials known in the art, and are not particularly limited.
The invention also provides application of the pharmaceutical composition in preparing medicines or health-care foods for treating and/or preventing nonalcoholic steatohepatitis.
The traditional Chinese medicine composition can be used as the only active ingredient; can also be used in combination with other active ingredients for preventing or treating nonalcoholic steatohepatitis, or ingredients which do not have the effect of preventing or treating nonalcoholic steatohepatitis per se but can assist the Chinese medicinal composition in playing the role of preventing or treating nonalcoholic steatohepatitis. Preferably, the Chinese medicinal composition is used as the only active ingredient.
Example 1
15g of red sage root, 10g of gynostemma pentaphylla, 10g of radix curcumae, 15g of ground white atractylodes rhizome, 15g of herba artemisiae capillaries, 15g of sedum sarmentosum and 9g of vinegar radix bupleuri are mixed, 10 times of water is added, and then the mixture is decocted for 30min by medium fire, so as to obtain a first extract product.
Adding 10g of lotus leaves into the first extraction product, and then decocting until water is boiled for 3min to obtain a second extraction product. Separating the second extract to obtain a first liquid medicine and a first residue.
Decocting the first residue with equal volume of water for 20min to obtain third extract. And separating the third extraction product to obtain a second liquid medicine and second medicine residues. And combining the first liquid medicine and the second liquid medicine to obtain the total liquid medicine.
Concentrating the total medicinal liquid, and drying to obtain Chinese medicinal composition.
Example 2
15g of red sage root, 10g of gynostemma pentaphylla, 10g of radix curcumae, 15g of ground white atractylodes rhizome, 15g of herba artemisiae capillaries, 15g of sedum sarmentosum and 9g of vinegar radix bupleuri are mixed, 10 times of water is added, and then the mixture is decocted for 30min by medium fire, so as to obtain a first extract product. 10g of lotus leaf is added into the first extraction product, and then the mixture is decocted until water is boiled for 3min, so as to obtain a second extraction product. Separating the second extract to obtain a first liquid medicine and a first residue.
Decocting the first residue with equal volume of water for 20min to obtain third extract. And separating the third extraction product to obtain a second liquid medicine and second medicine residues. Combining the first liquid medicine and the second liquid medicine to obtain total liquid medicine
Concentrating the total liquid medicine to obtain concentrated liquid medicine.
Mixing the concentrated liquid medicine with dextrin, and granulating to obtain granule.
Experimental example
1. Feeding and sampling
SPF class 8 week old male C57 BL/6J mice (purchased from Beijing Veitwill laboratory animal technologies Co., ltd., license number: SCXK 2021-0006; raised in Beijing university animal houses, SPF class, license number: SYXK 2020-0036) were randomly divided into a blank control group, a model group, a low-dose group of traditional Chinese medicine, a medium-dose group of traditional Chinese medicine, a high-dose group of traditional Chinese medicine and a positive medicine group according to body weight after one week of adaptive feeding.
Each group was fed with CDAHF60 feed (purchased from tuina biotechnology (tin-free) limited) for 8 weeks, except for the blank group which was fed with normal maintenance feed; drinking water is normally provided. At the same time, the corresponding doses of drug were given to each group for intervention: the blank control group and the model group are simultaneously irrigated with 10mL/kg of physiological saline; the Chinese medicine low dose group is subjected to gastric lavage administration according to 7.425g/kg of the Chinese medicine composition of the example 1, which is equivalent to the crude drug amount; the traditional Chinese medicine dosage group is subjected to gastric lavage administration according to the traditional Chinese medicine composition of the example 1, wherein the dosage group corresponds to 14.85g/kg of crude drug; the high dose group of the traditional Chinese medicine is subjected to gastric lavage administration according to 29.7g/kg of the traditional Chinese medicine composition of the example 1, which is equivalent to the crude drug amount; the positive group was given by gavage at 40mg/kg fenofibrate. The gastric lavage dose was 10mL/kg. Each of the above groups was administered for 8 weeks.
Sampling was performed on the last day of the experiment at week 8, and fasting was performed for 12 hours on the day prior to sampling. The mice were anesthetized with 10% chloral hydrate (0.35 ml/100 g) and injected intraperitoneally when the material was obtained. The eyeball takes blood. The collected blood was placed in a 1.5mLEP tube, allowed to stand for 30min, and centrifuged at 3000rpm at 4℃for 15min to obtain a supernatant, which was used for subsequent experimental measurement.
Each group of liver tissues of mice was taken and crushed with clean scissors, placed in a clean EP tube, added with 200ul of a homogenization medium (0.86% cold physiological saline), homogenized on ice, centrifuged at 12000rpm for 10 minutes at 4 ℃, and the supernatant was sub-packaged to prepare liver homogenate samples. And freezing the rest fresh liver tissues for later use.
2. General status of mice
After the raising is finished, the hair of the mice in the blank control group is bright, and the body state is normal; the hair of the mice in the model group is gradually shiny and wet, and the mice in the model group are gradually in a state of being low. With the prolonged administration time, the hair of mice with low, medium and high dosage and positive drug groups is gradually improved, the state of the high dosage is better, the positive drug group is inferior, the medium dosage is slightly inferior, and the low dosage is slightly improved.
3. Liver and kidney function in mice
The content of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) in the serum of the mice is tested by using a full-automatic biochemical analyzer. The results obtained are shown in FIGS. 1 and 2.
As can be seen from fig. 1 and fig. 2, the medium-dose group and the high-dose group of the traditional Chinese medicine can obviously reduce the ALT and AST elevation of mice induced by CDAHF60, which indicates that the traditional Chinese medicine composition has an improving effect on liver cell injury.
4. Lipid levels in mice
The Total Cholesterol (TC), total Triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) levels in the serum of mice were tested using a fully automatic biochemical analyzer. The results obtained are shown in FIGS. 3 to 6.
As can be seen from fig. 3-6, the medium-dose group and the high-dose group of the traditional Chinese medicine can significantly reduce TG and LDL-C levels in blood and increase HDL-C levels in blood. Excessive decrease in HDL-C in mice due to modeling resulted in decrease in TC with decrease in HDL-C, but recovery to normal levels following administration.
The Total Cholesterol (TC) and total Triglyceride (TG) content in the liver homogenate of the mice were tested using a liver homogenate sample, a full-automatic biochemical analyzer. The results obtained are shown in FIGS. 7 to 8.
As can be seen from fig. 7-8, the medium and high dose groups of the traditional Chinese medicine were able to significantly reduce TC and TG levels in the liver. The results of figures 3-8 are combined to show that the traditional Chinese medicine composition has an improving effect on lipid metabolism disorder in mice.
5. MDA level in liver
The content of Malondialdehyde (MDA) in liver homogenate samples was tested using TBA method. The results are shown in FIG. 9.
As can be seen from fig. 9, the low-dose group, the medium-dose group and the high-dose group of the traditional Chinese medicine can significantly reduce the level of MDA in the liver, indicating that the traditional Chinese medicine composition has an improving effect on lipid peroxidation.
6. Expression of lipid metabolism-related proteins
Detection was performed using Western Blot: fresh liver tissue (50 mg) was placed in a 2mLEP tube, sheared, 1mL of RIPA lysate containing protease inhibitor and protein phosphatase inhibitor was added, homogenized on ice using a high-speed homogenizer, and then lysed on ice for 15min. Centrifuging at 12000rpm and 4deg.C for 15min, collecting supernatant, and measuring protein concentration. A small fraction (3. Mu.L) of the lysate was removed for protein quantification. To the remaining volume of cell lysate was added 5 x protein loading buffer. After heating at 95℃for 10min, the protein samples were stored at-20 ℃.
Equal amounts of protein and protein electrophoresis markers were loaded into SDS-PAGE gel wells, run for 20min at 80V, and then run for 1 hour at 120V.
The PVDF membrane was "activated" with methanol for 1 min and washed with transfer buffer prior to preparing the sandwich. The transfer stacks were prepared in the following stacking order: black plate-sponge-filter paper-SDS-PAGE gel-PVDF membrane-filter paper-sponge-transparent plate. Membranes were transferred at a constant flow of 300mA for 1 hour 15 minutes and the resulting membranes were used for antibody incubation.
The membranes were blocked with a 5% blocking milk solution for 1 hour at room temperature, after blocking was completed, the membranes were washed 1-2 times with 1 XTBST.
The membrane was then placed in an antibody diluted with an anti-dilution solution and incubated overnight at 4 ℃.
After the primary incubation was completed, the antibodies were recovered and the membranes were washed 3 times with 1XTBST for 5 minutes each. The membranes were incubated with the recommended dilution of labeled secondary antibody in TBST with 5% blocking buffer for 1 hour at room temperature. Membranes were washed 3 times for 5 minutes each with 1 XTBST. Development was performed using ECL luminescence. The results are shown in FIG. 10.
7. Pathological section of liver
The liver was subjected to H & E staining, oil red O staining and Masson staining, and the images were scanned and collected under a microscope, and the resulting images are shown in fig. 11.
The extent of modeling lesions was determined based on pathological diagnosis of NAFLD Activity Score (NAS) in the guidelines established by the national institutes of health, NASH clinical research network pathology committee, 2005. The scoring criteria are shown in the following table.
Wherein, the total integral is less than 3 minutes, and NASH can be eliminated; total points between 3-5 minutes, NASH is possible; the total integral is more than or equal to 5 minutes, and can diagnose NASH. The results are shown in FIG. 12.
The present invention is not limited to the above-described embodiments, and any modifications, improvements, substitutions, and the like, which may occur to those skilled in the art, fall within the scope of the present invention without departing from the spirit of the invention.
Claims (9)
1. The traditional Chinese medicine composition for treating the nonalcoholic steatohepatitis is characterized by being prepared from the following raw material medicines: 8-20 parts of red sage root, 5-15 parts of lotus leaf, 5-15 parts of gynostemma pentaphylla, 5-15 parts of radix curcumae, 8-20 parts of bighead atractylodes rhizome, 8-20 parts of herba artemisiae capillaries, 8-20 parts of sedum sarmentosum and 5-15 parts of radix bupleuri.
2. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines: 12-17 parts of red sage root, 8-12 parts of lotus leaf, 8-12 parts of gynostemma pentaphylla, 8-12 parts of radix curcumae, 12-17 parts of bighead atractylodes rhizome, 12-17 parts of herba artemisiae capillaris, 12-17 parts of sedum sarmentosum and 7-11 parts of radix bupleuri.
3. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines: 15 parts of red sage root, 10 parts of lotus leaf, 10 parts of gynostemma pentaphylla, 10 parts of radix curcumae, 15 parts of bighead atractylodes rhizome, 15 parts of herba artemisiae scopariae, 15 parts of sedum sarmentosum and 9 parts of radix bupleuri.
4. The traditional Chinese medicine composition according to claim 1, wherein the bighead atractylodes rhizome is earth-fried bighead atractylodes rhizome and the radix bupleuri is vinegar-processed radix bupleuri.
5. A pharmaceutical formulation comprising the traditional Chinese medicine composition of any one of claims 1-4 and pharmaceutically acceptable excipients.
6. The pharmaceutical formulation of claim 5, wherein the pharmaceutically acceptable excipients are selected from one or more of fillers, flavoring agents, lubricants, binders, diluents, disintegrants, sustained release agents, glidants, thickening agents.
7. The pharmaceutical formulation of claim 5, wherein the formulation of the pharmaceutical formulation is selected from one or more of a tablet, a pill, a capsule, a granule, a suspension, or an oral liquid.
8. The method for preparing a Chinese medicinal composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is obtained by decocting all raw materials with water.
9. Use of a traditional Chinese medicine composition according to any one of claims 1-4 for preparing a medicament for treating non-alcoholic steatohepatitis.
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