WO2020010664A1 - 一种中药组合物及其制备与应用 - Google Patents

一种中药组合物及其制备与应用 Download PDF

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WO2020010664A1
WO2020010664A1 PCT/CN2018/100211 CN2018100211W WO2020010664A1 WO 2020010664 A1 WO2020010664 A1 WO 2020010664A1 CN 2018100211 W CN2018100211 W CN 2018100211W WO 2020010664 A1 WO2020010664 A1 WO 2020010664A1
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extract
chinese medicine
traditional chinese
rhubarb
hawthorn
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PCT/CN2018/100211
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English (en)
French (fr)
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周俊杰
杨莉
詹常森
余婕婧
沈丹萍
丁丽丽
王峥涛
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上海和黄药业有限公司
上海中医药大学
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Priority to CA3050146A priority Critical patent/CA3050146C/en
Publication of WO2020010664A1 publication Critical patent/WO2020010664A1/zh

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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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • 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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/708—Rheum (rhubarb)
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/734—Crataegus (hawthorn)
    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH

Definitions

  • the invention belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition capable of protecting liver and biliary and its preparation and application.
  • Cholestasis is often clinically manifested as jaundice accompanied by an increase in serum bilirubin, bile salts, alkaline phosphatase and other levels, and histologically, bile components often accumulate in the liver. Cholestasis usually causes changes in the relevant liver parenchymal cells, in particular, the descaling effect of bile acids can cause feathery degeneration of liver cells.
  • Bile secretion is one of the most important functions of the liver. Bilirubin and bile acids are the main components of bile. The metabolic state of the two can reflect the liver's ability to transport organic anions. An increase in T-Bil with a significant increase in D-Bil indicates the presence of cholestatic jaundice. When cholestasis occurs, bile secretion decreases, and the distribution of bile acid storage is rapidly changed, causing the bile acid concentration in serum and urine to increase significantly.
  • a variety of organs contain ALT and AST. Liver ALT content is the highest, most of which are in the cytoplasm. Liver AST content is second only to the heart, mainly in hepatocyte mitochondria.
  • ALP is a group of enzymes that hydrolyze phosphate monoester compounds in alkaline environment. Serum ALP mainly comes from liver and bone. Cholestasis, hepatocyte damage, and hepatic occupying cause pathological conditions such as increased capillary bile duct pressure, which can induce increased ALP production and increased serum ALP activity. Elevated serum ALP is the most characteristic early manifestation of cholestasis.
  • the object of the present invention is to provide a traditional Chinese medicine composition capable of protecting liver and biliary and its preparation and application.
  • a Chinese medicinal composition is provided.
  • the raw materials of medicinal ingredients include: Polygonum cuspidatum, green peel, hawthorn and rhubarb.
  • the raw materials of the medicinal ingredients of the Chinese medicine composition are composed of Polygonum cuspidatum, green bark, hawthorn and rhubarb.
  • the weight ratio of Polygonum cuspidatum, green peel, hawthorn and rhubarb ranges from 1-30 parts of Polygonum cuspidatum, 1-12 parts of green peel, 1-30 parts of hawthorn and 0.1-10 parts of rhubarb.
  • the knotweed is 1-15 parts by weight.
  • Polygonum cuspidatum is 15-30 parts by weight.
  • Polygonum cuspidatum is 15 parts by weight.
  • the green peel is 1-6 parts by weight.
  • the green peel is 6-12 parts by weight.
  • the green peel is 6 parts by weight.
  • the hawthorn is 1-15 parts by weight.
  • hawthorn is 15-30 parts by weight.
  • hawthorn is 15 parts by weight.
  • rhubarb is from 0.1 to 1 part by weight.
  • rhubarb is 1-10 parts by weight.
  • rhubarb is 1 part by weight.
  • the weight ratio range between Polygonum cuspidatum, green peel, hawthorn and rhubarb is: 15 parts of Polygonum cuspidatum, 6 parts of green peel, 15 parts of hawthorn and 1 part of rhubarb.
  • a Chinese medicine extract is provided, which is prepared by extracting active ingredients with the Chinese medicine composition as a raw material.
  • each raw material and its formula in the traditional Chinese medicine composition are the most critical.
  • those skilled in the art can use various conventional methods for extracting active ingredients of traditional Chinese medicine to extract its effective ingredients, such as conventional decoction. , Alcohol extraction method, water extraction and alcohol precipitation method, alcohol extraction and water precipitation method, salting out method, etc., because the above extraction methods can obtain the main medicinal ingredients in the aforementioned traditional Chinese medicine formula, they can all have a certain curative effect on cholestasis .
  • the water extraction and alcohol precipitation method is as follows: firstly use water as a solvent to extract the active ingredients of the medicinal material, and then use different concentrations of ethanol to precipitate and remove impurities in the extract. Generally, when the ethanol content reaches 50 to 60% (weight / volume ratio, that is, g / 100mL), impurities such as starch can be removed. When the alcohol content exceeds 70%, except for a few ineffective ingredients such as tannins and water-soluble pigments, the rest are large. Some impurities can be removed by precipitation.
  • the alcohol extraction and water sedimentation method is a method of extracting medicinal material components with an appropriate concentration of ethanol, and then removing impurities in the extract with water.
  • the traditional Chinese medicine composition can be concentrated, purified or dried to obtain a traditional Chinese medicine extract after extracting the active ingredients.
  • concentration methods include, but are not limited to, atmospheric pressure evaporation, reduced pressure evaporation, thin film evaporation, and multi-effect evaporation.
  • the purification methods include, but are not limited to, salting out purification, macroporous resin adsorption purification, ethanol precipitation purification, and ion exchange resin purification.
  • drying methods include, but are not limited to: drying method, drum drying method, belt drying method, hygroscopic drying method, boiling drying method , Spray drying method, reduced pressure drying method, freeze drying method, infrared drying method, microwave drying method and the like.
  • the extraction uses an ethanol extraction method.
  • 60% to 80% ethanol weight / volume ratio, that is, g / 100mL
  • g / 100mL weight / volume ratio
  • the extraction uses a water extraction method.
  • a method for preparing the aforementioned Chinese medicine extract includes the following steps: the aforementioned Chinese medicine composition is used as a raw material to extract an active ingredient.
  • the preparation method includes the steps of taking Polygonum cuspidatum, green bark and hawthorn according to the mixing ratio, extracting with ethanol, concentrating and drying the extract, adding rhubarb powder after mixing to obtain a dry extract, and mixing.
  • the ethanol is selected from 60% to 80% ethanol. Preferably, 70% ethanol.
  • the rhubarb powder is obtained by crushing rhubarb.
  • the preparation method includes the steps of: taking Polygonum cuspidatum, green bark, hawthorn and rhubarb according to the mixing ratio, extracting with ethanol, concentrating and drying the extract to obtain a dry extract.
  • the ethanol is selected from 60% to 80% ethanol. Preferably, 70% ethanol.
  • the preparation method includes the steps of taking Polygonum cuspidatum, green bark, hawthorn and rhubarb according to the mixing ratio, extracting with water, filtering, concentrating, and drying.
  • a fourth aspect of the present invention there is provided the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a medicament for preventing and / or treating liver injury.
  • a fifth aspect of the present invention there is provided the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a drug for preventing and / or treating cholestasis.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can prevent and / or treat intrahepatic cholestasis.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can prevent and / or treat acute intrahepatic cholestasis.
  • a sixth aspect of the present invention there is provided the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing an ALT reducing agent.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce ALT levels in serum.
  • a seventh aspect of the present invention there is provided the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing an AST reducing agent.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the AST level in serum.
  • the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing an ALP reducing agent is provided.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the level of ALP in serum.
  • a ninth aspect of the present invention there is provided the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a T-Bil reducing agent.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the T-Bil level.
  • a tenth aspect of the present invention provides the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a D-Bil reducing agent.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce the D-Bil level.
  • the use of the aforementioned Chinese medicine composition or Chinese medicine extract for preparing a TBA reducing agent is provided.
  • the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract can be used to reduce TBA levels.
  • the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a medicament having any of the following effects: (1) reducing hepatocyte necrosis; (2) improving hepatocellular enlargement and cytoplasm Loose, balloon-like changes and feather-like changes; (3) reduce inflammatory cell infiltration.
  • a traditional Chinese medicine preparation comprising a therapeutically effective amount of the aforementioned traditional Chinese medicine extract.
  • the traditional Chinese medicine formulation preferably includes one or more conventional pharmaceutically acceptable excipients.
  • compositions include, but are not limited to, pharmaceutically acceptable carriers, diluents, fillers, binding agents, and other excipients.
  • inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyortho compounds such as polyethylene glycol, water , Sucrose, ethanol, glycerin, and the like, various preservatives, lubricants, dispersants, flavoring agents.
  • Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like can also be added, these substances are used as needed to help the stability of the formula or to help increase the activity or its biological effectiveness or Produces an acceptable mouthfeel or odor when taken orally.
  • Traditional Chinese medicine preparations such as granules and pastes can be prepared by conventional methods.
  • the pharmaceutical composition of the present invention can also be used with other therapeutic agents.
  • the traditional Chinese medicine preparation is an oral preparation, including but not limited to granules, pills, tablets, capsules, syrups, sprays and the like.
  • the effective therapeutic dose of the pharmaceutical composition of the present invention is that, based on the total weight of the raw materials, the oral safe and effective amount is usually 1.00-2.65g / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, and other factors, which are all within the skill of a skilled physician.
  • the present invention has the following beneficial effects:
  • the traditional Chinese medicine composition and traditional Chinese medicine extract provided by the present invention can significantly reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, significantly reduce hepatocyte necrosis, improve hepatocyte enlargement, cytoplasmic looseness, Balloon-like and feather-like changes can reduce inflammatory cell infiltration and prevent or treat cholestasis.
  • Figure 1 Effects of extract A, extract B, extract C, and UDCA on serum ALT, AST, ALP, T-Bil, D-Bil, and TBA in mice with cholestasis induced by ANIT; compared with Vehicle group, * P ⁇ 0.05; compared with the ANIT model group, # P ⁇ 0.05.
  • Figure 2 Analysis of liver pathological sections (H & E, ⁇ 400) in each group, A: normal control group; B: ANIT model group; C: extract A drug protection group; D: extract B drug protection group; E: extract C drug protection group; F: positive drug UDCA protection group.
  • ⁇ -naphthyl isothiocyanate (ANIT; Sigma, MO, USA); ursodeoxycholic acid (UDCA; TCI Japan, Japan); rhubarb powder (obtained from rhubarb crushing, 95% of which is passed through 100 mesh sieve, the remaining powder must pass through 80 mesh sieve), rhubarb, Polygonum cuspidatum, green peel, hawthorn are provided by Shanghai Hehuang Pharmaceutical Co., Ltd.
  • ALT kit AST kit, ALP kit, T-Bil kit, D-Bil kit, TBA kit (built in Nanjing, Shanghai, China); olive oil (Sinopec Group, Shanghai, China); carboxymethyl Cellulose sodium (CMC-Na; Sinopharm Group, Shanghai, China); physiological saline, neutral tissue fixation solution.
  • mice C57BL / 6 mice, weighing (20 ⁇ 2) g, male, clean, 60, purchased from Shanghai Slark Animal Experiment Co., Ltd. The animals were kept in an animal room at a temperature of (22 ⁇ 1) ° C, a relative humidity of (65 ⁇ 10)%, and a day and night alternation for 12 hours, and were adaptively fed for 3 days before the experiment.
  • ANIT Take an appropriate amount of powder, dissolve it in olive oil to make 10mg / mL, and sonicate until the powder is completely dissolved.
  • UDCA 0.5% sodium carboxymethyl cellulose (CMC-Na) was used to dissolve it to 9 mg / mL, and it was mixed thoroughly before gavage.
  • Extract A Take 360g of Polygonum cuspidatum, 144g of green peel, and 360g of hawthorn, extract with 70% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool. 24 g of rhubarb powder, mixed, and used as extract A.
  • Extract B Take 24g of rhubarb, 360g of Polygonum cuspidatum, 144g of green bark and 360g of hawthorn, extract with 70% ethanol, recover the solvent under reduced pressure to a thick paste, and dry at 100 ° C, -0.1MPa for 5h, let cool, and dry soak Cream as extract B.
  • Extract C Take 24g of rhubarb, 360g of Polygonum cuspidatum, 144g of green peel and 360g of hawthorn, and use water to cook and extract. The extract is recovered under reduced pressure to recover a thick paste, and dried at 100 ° C and -0.1MPa for 5h. Extract as extract C.
  • mice Sixty C57BL / 6 mice were randomly divided into the normal control group, the ANIT model group, the three extract drug protection groups, and the positive drug UDCA control group, with 10 mice in each group. All drugs were dissolved to the desired concentration with 0.5% sodium carboxymethyl cellulose (CMC-Na). The normal control group and the model group were given the corresponding CMC-Na solution. The remaining groups of mice were administered the same agent by gavage (i.g.) at a dose of 10 mL / kg daily for qd for 5 consecutive days.
  • gavage i.g.
  • the dose of the extract A drug-protected group was 2.69 g / kg
  • the extract B of the drug-protected group was 2.34 g / kg
  • the extract C of the drug-protected group was 2.13.
  • the dose of g / kg and the positive drug UDCA control group was 0.09 g / kg.
  • 10 mL / kg of olive oil was intragastrically administered to the mice in the other groups, and the ANIT olive oil solution was intragastrically administered at a dose of 10 mL / kg for modeling.
  • the administration was continued twice after 36h. Twelve hours before the end of the experiment, the animals were fasting and water.
  • mice were anesthetized with ether and blood was collected from the eyeballs. The whole blood was allowed to stand at room temperature for 3 hours, centrifuged at 3500 rpm / min, and centrifuged for 15 minutes. The upper serum was collected and the serum alanine aminotransferase (ALT) and aspartic acid were measured by a full-automatic biochemical analyzer Aminotransferase (AST) serum alkaline phosphatase (ALP) activity, serum total bilirubin (T-Bil), and direct bilirubin (D-Bil) content. The total bile acid (TBA) content was determined and calculated as described in the kit.
  • AST serum alkaline phosphatase
  • T-Bil serum total bilirubin
  • D-Bil direct bilirubin
  • tissue fixation After tissue fixation, tissue trimming, embedding in paraffin, cutting into 4 ⁇ m flakes, dewaxing xylene, gradient ethanol dehydration, hematoxylin-eosin staining (H & E staining), ethanol dehydration, xylene permeabilization, resin mounting, Observe under a light microscope.
  • Measurement data of experimental data are mean ⁇ SD It means that the comparison of two means is analyzed by one-way ANOVA in statistical software. P ⁇ 0.05 indicates that the difference is statistically significant.
  • the positive control drug UDCA can significantly reduce 5 serological indexes except AST (P ⁇ 0.05).
  • the above results show that extract A has a protective effect on liver injury in mice with cholestasis induced by ANIT, and is superior to UDCA in the degree of reducing ALT and AST indicators.
  • Extract A protection group compared with the model group, reduced hepatocyte necrosis, hepatocellular enlargement, cytoplasmic looseness, balloon-like and feather-like changes were improved, inflammatory cell infiltration was reduced, and occasionally normal-structured capillary bile ducts .
  • ANIT induces intrahepatic cholestasis in mice, which can also cause liver cell damage.
  • the pathophysiological manifestations of the hepatotoxicity matrix to animals are very similar to human drug-induced hepatitis, and are widely used to replicate intrahepatic cholestasis models.
  • the toxicity of ANIT is related to its metabolites. Under the action of liver P450 enzymes, the oxidation of sulfur in the structure of ANIT is a toxic product that attacks intrahepatic bile duct endothelial cells, leading to the occurrence of hyperbilirubinemia and reduced bile secretion. At the same time, it also causes membrane lipid peroxidation, which causes degeneration and necrosis of liver cells, and causes acute intrahepatic cholestasis.
  • the intrahepatic cholestasis model of mice induced by ANIT is used to evaluate the efficacy of rhubarb, Polygonum cuspidatum, green peel and hawthorn compound prescriptions for liver protection and gallbladder protection.
  • the experimental results show that extract A can significantly reduce the increase of serum ALT, AST, ALP, T-Bil, D-Bil and TBA in mice induced by ANIT. Compared with the positive drug UDCA, extract A can more effectively reduce the two serological indicators of ALT and AST.
  • Liver pathological sections showed that compared with the model group, hepatocyte necrosis was significantly reduced in the extract A-protected group, hepatocellular enlargement, cytoplasmic looseness, balloon-like changes and feather-like changes were improved, and inflammatory cell infiltration was reduced.
  • extract A has a good protective effect on the liver of model mice with cholestasis induced by ANIT.
  • Extract M-1 Take 24g of Polygonum cuspidatum, 144g of green bark and 360g of hawthorn, extract with 60% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract Then, 24 g of rhubarb powder was added and mixed to obtain extract M-1.
  • Extract M-2 Take 720g of Polygonum cuspidatum, 144g of green peel and 360g of hawthorn, extract with 80% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract Then, 24 g of rhubarb powder was added and mixed to obtain extract M-2.
  • Extract M-3 Take 360g of Polygonum cuspidatum, 24g of green peel and 360g of hawthorn, extract with 60% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract Then, 24 g of rhubarb powder was added and mixed, and used as an extract M-3.
  • Extract M-4 Take 360g of Polygonum cuspidatum, 288g of green peel and 360g of hawthorn, extract with 80% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract Then, 24 g of rhubarb powder was added and mixed to obtain M-4.
  • Extract M-5 Take 360g of Polygonum cuspidatum, 144g of green peel and 24g of hawthorn, extract with 60% ethanol, recover the solvent under reduced pressure to a thick paste, and dry at 100 ° C, -0.1MPa for 5h, let cool, and obtain a dry extract Then, 24 g of rhubarb powder was added and mixed to obtain M-5.
  • Extract M-6 Take 360g of Polygonum cuspidatum, 144g of green peel and 720g of hawthorn, extract with 80% ethanol, recover the solvent under reduced pressure to a thick paste, and dry at 100 ° C and -0.1MPa for 5h, let cool to obtain a dry extract Then, 24 g of rhubarb powder was added and mixed to obtain M-6.
  • Extract M-7 Take 360g of Polygonum cuspidatum, 144g of green peel and 360g of hawthorn, extract with 60% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract After that, 2.4 g of rhubarb powder was added and mixed to obtain M-7.
  • Extract M-8 Take 360g of Polygonum cuspidatum, 144g of green peel and 360g of hawthorn, extract with 80% ethanol, recover the solvent under reduced pressure to a thick paste, dry at 100 ° C, -0.1MPa for 5h, and let cool to obtain a dry extract Then, 240 g of rhubarb powder was added and mixed to obtain M-8.
  • the extract M-1 protection group was set up at a dose of 2.69g / kg; the extract M-2 protection group was dosed at 2.69g / kg; the extract M- 3 protection group, the dosage is 2.69g / kg; extract M-4 protection group, the dosage is 2.69g / kg; extract M-5 protection group, the dosage is 2.69g / kg; extract M -6 protection group, the dosage is 2.69g / kg; extract M-7 protection group, the dosage is 2.69g / kg; extract M-8 protection group, the dosage is 2.69g / kg, other operations Same as the animal experiment method in Section 2.2.
  • extract M-1 protection group extract M-2 protection group, extract M-3 protection group, extract M-4 protection group, extract M-5 protection group, extract M-6 protection group
  • the extract M-7 protection group and the extract M-8 protection group can significantly reduce the increase of serum indexes caused by ANIT, and the difference is statistically significant (P ⁇ 0.05). There was no significant difference between the normal control group (P> 0.05), but the effect of the extract A protection group was the best.
  • Extract M-1 protection group Liver pathological sections showed extract M-1 protection group, extract M-2 protection group, extract M-3 protection group, extract M-4 protection group, extract M-5 protection group, extract M-6
  • the protection group, the extract M-7 protection group, and the extract M-8 protection group have reduced hepatocyte necrosis, hepatocellular enlargement, cytoplasmic porosity, balloon-like changes and feather-like changes.
  • Infiltration of inflammatory cells was reduced, and capillary biliary ducts with normal structure were occasionally observed, but the effect of the extract A protection group was the best.

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Abstract

一种能够保肝利胆中药组合物及其制备与应用。该中药组合物药效成分原料包括：虎杖、青皮、山楂和大黄。该中药组合物及其提取物，能够显著降低血清ALT、AST、ALP、T-Bil、D-Bil和TBA，明显减少肝细胞坏死，改善肝细胞肿大、胞质疏松、呈气球样变及羽毛样变的情况，减轻炎性细胞浸润，进而预防或治疗胆汁淤积。

Description

一种中药组合物及其制备与应用 技术领域
本发明属于中医药领域，具体涉及一种能够保肝利胆中药组合物及其制备与应用。
背景技术
胆汁淤积临床上常表现为黄疸伴有血清胆红素、胆盐、碱性磷酸酶等水平的升高，组织学上常表现为胆汁成分在肝内聚集。胆汁淤积通常引起相关肝实质细胞的变化，尤其是胆汁酸去垢作用可引起肝细胞羽毛状变性。
胆汁分泌是肝脏最重要功能之一，其中胆红素和胆汁酸是胆汁主要成分，二者代谢状态可反映肝脏对有机阴离子转运能力。T-Bil增高伴D-Bil明显升高提示存在胆汁淤积性黄疸。发生胆汁淤积时，胆汁分泌下降，并迅速改变胆汁酸储存量的分布，使得血清和尿液中的胆汁酸浓度显著升高。多种器官含有ALT和AST，肝脏ALT含量为最高，大多数存在于细胞质中；而肝脏AST含量仅次于心脏，主要存在于肝细胞线粒体。这两种酶在胆汁淤积时一般不升高，仅当淤积引起肝细胞坏死或通透性改变时，ALT和AST进入血液，使血液中酶活性增加。ALP是一组碱性环境水解磷酸单酯化合物的酶，血清ALP主要来自肝脏和骨骼。胆汁淤积、肝细胞损伤和肝脏占位等病理条件使毛细胆管压力升高时，可诱发ALP产生增多，血清ALP活性增高。血清ALP升高是胆汁淤积最具有特征性的早期表现。
研究开发对于胆汁淤积具有良好治疗效果的药物，具有非常重要的意义。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种能够保肝利胆中药组合物及其制备与应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种中药组合物，其药效成分原料包括：虎杖、青皮、山楂和大黄。
一种实施方式中，所述中药组合物的药效成分原料由虎杖、青皮、山楂和大黄组成。
一种实施方式中，虎杖、青皮、山楂和大黄之间的重量份数比例范围是：虎杖1-30份、 青皮1-12份、山楂1-30份和大黄0.1-10份。
一种实施方式中，虎杖为1-15重量份。
一种实施方式中，虎杖为15-30重量份。
一种实施方式中，虎杖为15重量份。
一种实施方式中，青皮为1-6重量份。
一种实施方式中，青皮为6-12重量份。
一种实施方式中，青皮为6重量份。
一种实施方式中，山楂为1-15重量份。
一种实施方式中，山楂为15-30重量份。
一种实施方式中，山楂为15重量份。
一种实施方式中，大黄为0.1-1重量份。
一种实施方式中，大黄为1-10重量份。
一种实施方式中，大黄为1重量份。
一种实施方式中，虎杖、青皮、山楂和大黄之间的重量份数比例范围是：虎杖15份、青皮6份、山楂15份和大黄1份。
进一步地，本发明的第二方面，提供了一种中药提取物，为以上述中药组合物为原料提取有效成分制得。
本发明中，中药组合物中各原料及其配方最为关键，在获知中药配方后，本技术领域的技术人员可采用各种常规的中药有效成分提取方法来提取其有效成分，如常规的煎煮、醇提法、水提醇沉法、醇提水沉法、盐析法等，由于上述提取方法均能获得前述中药配方中的主要药效成分，因此均能对胆汁淤积具有一定的防治疗效。
其中，水提醇沉法为：先以水为溶剂提取药材有效成分，再用不同浓度的乙醇沉淀去除提取液中杂质的方法。一般含乙醇量达到50～60％(重量体积比即g/100mL)时，可去除淀粉等杂质，当含醇量达70％以上，除鞣质、水溶性色素等少数无效成分外，其余大部分杂质均可沉淀去除。
醇提水沉法为：先以适宜浓度的乙醇提取药材成分，再用水除去提取液中杂质的方法。
所述中药组合物提取有效成分后可再经常规的步骤浓缩、纯化或干燥获得中药提取物。其中浓缩的方法包括但不限于：常压蒸发、减压蒸发、薄膜蒸发、多效蒸发，纯化的方法包括但不限于：盐析纯化、大孔树脂吸附纯化、乙醇沉淀纯化、离子交换树脂纯化、絮凝沉淀纯化、膜分离纯化、聚酰胺吸附纯化、硅胶吸附层析纯化等，干燥的方法包括但不限于：烘 干法、鼓式干燥法、带式干燥法、吸湿干燥法、沸腾干燥法、喷雾干燥法、减压干燥法、冷冻干燥法、红外线干燥法、微波干燥法等。
一种实施方式中，所述提取采用乙醇提取法。例如，可采用60％～80％乙醇(重量体积比即g/100mL)提取。
一种实施方式中，所述提取采用水提取法。
本发明的第三方面，提供前述中药提取物的制备方法，包括如下步骤：取前述中药组合物为原料提取有效成分。
一种实施方式中，所述制备方法包括步骤：按配比取虎杖、青皮和山楂，用乙醇提取，提取液浓缩、干燥，得干浸膏后加入大黄粉，混匀，即可。
一种实施方式中，所述乙醇选自60％～80％乙醇。优选，70％乙醇。
所述大黄粉由大黄粉碎获得。
一种实施方式中，所述制备方法包括步骤：按配比取虎杖、青皮、山楂和大黄，用乙醇提取，提取液浓缩、干燥，得干浸膏。
一种实施方式中，所述乙醇选自60％～80％乙醇。优选，70％乙醇。
一种实施方式中，所述制备方法包括步骤：按配比取虎杖、青皮、山楂和大黄，用水提取，过滤，浓缩，干燥。
本发明的第四方面，提供前述中药组合物或中药提取物用于制备肝损伤预防和/或治疗药物的用途。
本发明的第五方面，提供前述中药组合物或中药提取物用于制备胆汁淤积预防和/或治疗药物的用途。
一种实施方式中，前述中药组合物或中药提取物可预防和/或治疗肝内胆汁淤积。
一种实施方式中，前述中药组合物或中药提取物可预防和/或治疗急性肝内胆汁淤积。
本发明的第六方面，提供前述中药组合物或中药提取物用于制备ALT降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低血清中ALT水平。
本发明的第七方面，提供前述中药组合物或中药提取物用于制备AST降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低血清中AST水平。
本发明的第八方面，提供前述中药组合物或中药提取物用于制备ALP降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低血清中ALP水平。
本发明的第九方面，提供前述中药组合物或中药提取物用于制备T-Bil降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低T-Bil水平。
本发明的第十方面，提供前述中药组合物或中药提取物用于制备D-Bil降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低D-Bil水平。
本发明的第十一方面，提供前述中药组合物或中药提取物用于制备TBA降低剂的用途。
一种实施方式中，前述中药组合物或中药提取物可用于降低TBA水平。
本发明的第十二方面，提供前述中药组合物或中药提取物用于制备具有如下之任一功效的药物的用途：(1)减少肝细胞坏死；(2)改善肝细胞肿大、胞质疏松、呈气球样变及羽毛样变的情况；(3)减轻炎性细胞浸润。
本发明的第十三方面，提供一种中药制剂，包括治疗有效量的前述中药提取物。
所述中药制剂好包括一种或多种常规的药学上可接受的辅料。
药学上可接受的辅料包括(但并不限于)：药物可接受的载体、稀释剂、填充剂、结合剂及其它赋形剂。本领域枝术人员已知的治疗惰性的无机或有机的载体包括(但不限于)乳糖、玉米淀粉或其衍生物、滑石、植物油、蜡、脂肪、多羚基化合物例如聚乙二醇、水、蔗糖、乙醇、甘油，诸如此类，各种防腐剂、润滑剂、分散剂、矫味剂。保湿剂、抗氧化剂、甜味剂、着色剂、稳定剂、盐、缓冲液诸如此类也可加入其中，这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。诸如颗粒剂和膏剂之类的中药制剂，可通过常规方法进行制备。本发明的药物组合物还可与其他治疗剂一起使用。
优选地，所述的中药制剂为口服制剂，包括但不限于颗粒剂、丸剂、片剂、胶囊剂、糖浆剂、喷雾剂等。
在提取出中药组合物中的有效成分后，可采用常规的药用辅料及添加剂制备前述药物组合物。
本发明的药物组合物的有效治疗剂量为，以原料药材的总重量计，口服安全有效量通常为1.00-2.65g/kg体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
与现有技术相比，本发明具有如下有益效果：
本发明所提供的中药组合物及中药提取物，能够显著降低血清ALT、AST、ALP、T-Bil、D-Bil和TBA，明显减少肝细胞坏死，改善肝细胞肿大、胞质疏松、呈气球样变及羽毛样变的情况，减轻炎性细胞浸润，进而预防或治疗胆汁淤积。
附图说明
图1：提取物A、提取物B、提取物C以及UDCA对ANIT所致胆汁淤积小鼠血清ALT、AST、ALP、T-Bil、D-Bil及TBA的影响；与Vehicle组比较， *P＜0.05；与ANIT模型组比较， #P＜0.05。
图2：各组肝脏病理切片分析(H&E，×400)，A：正常对照组；B：ANIT模型组；C：提取物A药物保护组；D：提取物B药物保护组；E：提取物C药物保护组；F：阳性药UDCA保护组。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规技术。
实施例1
一、实验材料
1.1、药品
α-萘异硫氰酸酯(α-naphthyl isothiocyanate，ANIT；Sigma,MO,USA)；熊去氧胆酸(UDCA；TCI Japan,Japan)；大黄粉(由大黄粉碎获得，其中95％药粉通过100目筛，余粉必须通过80目筛)、大黄、虎杖、青皮、山楂由上海和黄药业有限公司提供。
1.2、试剂
ALT试剂盒，AST试剂盒，ALP试剂盒，T-Bil试剂盒，D-Bil试剂盒，TBA试剂盒(南京建成，上海，中国)；橄榄油(国药集团，上海，中国)；羧甲基纤维素钠(CMC-Na；国药集团，上海，中国)；生理盐水、中性组织固定液。
1.3、主要仪器
全自动生化分析仪；光学显微镜；Thermo Scientific Varioskan Flash全波长扫描多功能读数仪(ThermoFisher Scientific,Germany)；Eppendorf 5424R低温高速离心机(Eppendorf,Germany)；SANYO制冰机(SANYO Electric Co.,Ltd.,Japan)；Millipore纯水仪(Millipore,Bedford,MA,USA)。
1.4、实验动物：C57BL/6小鼠，体重(20±2)g，雄性，清洁级，60只，购于上海斯莱克动物实验有限责任公司。动物饲养于温度(22±1)℃，相对湿度(65±10)％，12h小时昼夜更替的动物室内，实验前适应性喂养3d。
二、实验方法
2.1 药物配制
ANIT：取适量粉末，用橄榄油溶解配制成10mg/mL，超声至粉末完全溶解。
UDCA：用0.5％羧甲基纤维素钠(CMC-Na)溶解至9mg/mL，灌胃前充分混匀。
提取物A：取虎杖360g、青皮144g和山楂360g，使用70％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物A。
提取物B：取大黄24g、虎杖360g、青皮144g和山楂360g，使用70％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏，作为提取物B。
提取物C：取大黄24g、虎杖360g、青皮144g和山楂360g，使用水煎煮提取，提取液经减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏，作为提取物C。
2.2 动物实验方法
C57BL/6小鼠60只，随机分为正常对照组，ANIT模型组，三种提取物药物保护组，和阳性药UDCA对照组，每组10只。所有药物均用0.5％羧甲基纤维素钠(CMC-Na)溶解至所需浓度。正常对照组与模型组给予相应的CMC-Na溶液。其余各组小鼠每日同一试剂按10mL/kg剂量灌胃(i.g.)给药，qd，连续5天。具体地，每次，提取物A药物保护组的给药剂量为2.69g/kg、提取物B药物保护组的给药剂量为2.34g/kg、提取物C药物保护组的给药剂量为2.13g/kg、阳性药UDCA对照组的给药剂量为0.09g/kg。第5次给药12h后，除正常对照组灌胃给予橄榄油10mL/kg外，其余各组小鼠按照10mL/kg剂量灌胃给予 ANIT橄榄油溶液造模。造模12h，36h后继续给药两次。实验结束前12h，动物禁食不禁水。
所有动物在给予ANIT后48h(即末次给药12h后)，经乙醚麻醉后，摘眼球取血，供血清生化指标测定。切取相同部位(肝左叶)肝组织进行病理学检查，用10％中性福尔马林固定备用。
2.3 血清生化指标测定
小鼠经乙醚麻醉后摘眼球取血，全血室温静置3h，3500rpm/min，离心15min，取上层血清，全自动生化分析仪测定血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)血清碱性磷酸酶(ALP)活力，血清总胆红素(T-Bil)、直接胆红素(D-Bil)含量。按试剂盒所述方法测定并计算总胆汁酸(TBA)含量。
2.4、肝组织病理观察
组织固定后进行组织修剪，石蜡包埋，切成4μm薄片，二甲苯脱蜡，梯度乙醇脱水，苏木精-伊红染色(H&E染色)，乙醇脱水，二甲苯透化，树脂封固，在光学显微镜下观察。
2.5、统计分析
实验数据计量资料以均数±标准差
Figure PCTCN2018100211-appb-000001
表示,两均数的比较用统计软件中one-way ANOVA分析，P＜0.05表示差异有统计学意义。
三、实验结果
3.1、对ANIT所致小鼠胆汁淤积保护作用的血清生化指标评价
如表1，表2以及图1所示，ANIT模型组小鼠6项血清学指标较空白组小鼠显著升高(P＜0.05)，表明造模成功。与ANIT模型组比较，提取物A连续给药后能显著降低ANIT造成的血清各项指标升高情况，差异具有统计学意义(P＜0.05)，其中小鼠血清AST和ALP与正常对照组相比无显著性差异(P＞0.05)。提取物B能够显著降低小鼠血清中ALT、AST、T-Bil以及D-Bil水平(P＜0.05)，提取物C仅能显著降低ALT和AST水平(P＜0.05)。阳性对照药UDCA能够显著降低除AST以外其它5项血清学指标(P＜0.05)。以上结果表明，提取物A对ANIT所致胆汁淤积小鼠的肝损伤具有保护作用，且从降低ALT和AST指标的程度上优于UDCA。
表1 提取物A、提取物B、提取物C以及UDCA对ANIT所致胆汁淤积小鼠血清ALT、AST及ALP的影响
Figure PCTCN2018100211-appb-000002
Figure PCTCN2018100211-appb-000003
注：与Vehicle组比较， *P＜0.05；与模型组比较， #P＜0.05
表2 提取物A、提取物B、提取物C以及UDCA对ANIT所致胆汁淤积小鼠血清T-Bil、D-Bil及TBA的影响
Figure PCTCN2018100211-appb-000004
注：与Vehicle组比较， *P＜0.05；与模型组比较， #P＜0.05
3.2、对ANIT所致小鼠胆汁淤积保护作用的肝脏病理分析
如图2所示，光镜下观察可见，正常对照组中肝细胞形态正常，排列紧密，未见肝实质细胞病变，肝小叶结构完整，肝细胞索由终末小静脉向四周呈放射状排列，汇管区无明显炎症细胞浸润和纤维组织增生等改变，毛细胆管无淤胆。ANIT模型组，肝细胞肿大，胞质疏松，有的呈气球样变或羽毛样变，可见灶状坏死，肝小叶轮廓不清，汇管区有大量炎性细胞浸润，毛细胆管结构破坏或丢失。提取物A保护组，与模型组相比肝细胞坏死减少，肝细胞肿大，胞质疏松，呈气球样变及羽毛样变的情况改善，炎性细胞浸润减轻，偶见结构正常的毛细胆管。提取物B保护组，肝细胞坏死减少，肝细胞肿大，胞质疏松，呈气球样变及羽毛样变的情况改善，炎性细胞浸润减轻。提取物C保护组，肝细胞肿大，胞质疏松，气球样变及羽毛样变情况没有显著改善，可见灶状坏死面积减小，炎性细胞浸润减轻。经UDCA给药保护后，肝细胞坏死减少，肝细胞肿大，胞质疏松，呈气球样变及羽毛样变的情况改善，汇管区炎性细胞浸润减轻。
四、讨论
ANIT诱发小鼠肝内胆汁淤积，也可引起肝细胞损伤，其对动物肝毒性的治病基质在病 理生理学上的表现很像人类的药物性肝炎，被广泛应用于复制肝内胆汁淤积模型。ANIT的致毒与其代谢产物有关，在肝P450酶作用下，ANIT结构中的硫氧化代为为毒性产物攻击肝内胆管内皮细胞，导致高胆红素血症和胆汁分泌降低的出现。同时还引起膜脂质过氧化反应，致使肝细胞变性坏死，造成急性肝内胆汁淤积。
本发明的研究中，利用ANIT所致小鼠肝内胆汁淤积模型评价不同提取制备方式的大黄、虎杖、青皮和山楂复方保肝利胆的药效。实验结果表明，提取物A，能够显著降低ANIT所致小鼠血清ALT、AST、ALP、T-Bil、D-Bil和TBA升高。与阳性药UDCA相比，提取物A能更有效地降低ALT和AST两项血清学指标。肝脏病理切片显示，提取物A保护组，与模型组相比肝细胞坏死明显减少，肝细胞肿大，胞质疏松，呈气球样变及羽毛样变的情况改善，炎性细胞浸润减轻。
综上所述，提取物A对ANIT所致胆汁淤积模型小鼠的肝有良好保护作用。
此外，还参照提取物A的制备方法，分别制备了：
提取物M-1：取虎杖24g、青皮144g和山楂360g，使用60％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物M-1。
提取物M-2：取虎杖720g、青皮144g和山楂360g，使用80％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物M-2。
提取物M-3：取虎杖360g、青皮24g和山楂360g，使用60％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物M-3。
提取物M-4：取虎杖360g、青皮288g和山楂360g，使用80％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物M-4。
提取物M-5：取虎杖360g、青皮144g和山楂24g，使用60％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀，作为提取物M-5。
提取物M-6：取虎杖360g、青皮144g和山楂720g，使用80％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉24g，混匀， 作为提取物M-6。
提取物M-7：取虎杖360g、青皮144g和山楂360g，使用60％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉2.4g，混匀，作为提取物M-7。
提取物M-8：取虎杖360g、青皮144g和山楂360g，使用80％乙醇提取，提取液减压回收溶剂至稠膏，置100℃、-0.1MPa下干燥5h，放凉，得干浸膏后加入大黄粉240g，混匀，作为提取物M-8。
并参照第二部分2.2的动物实验方法，设置提取物M-1保护组，给药剂量为2.69g/kg；提取物M-2保护组，给药剂量为2.69g/kg；提取物M-3保护组，给药剂量为2.69g/kg；提取物M-4保护组，给药剂量为2.69g/kg；提取物M-5保护组，给药剂量为2.69g/kg；提取物M-6保护组，给药剂量为2.69g/kg；提取物M-7保护组，给药剂量为2.69g/kg；提取物M-8保护组，给药剂量为2.69g/kg，其他操作同第二部分2.2的动物实验方法。
结果发现：提取物M-1保护组、提取物M-2保护组、提取物M-3保护组、提取物M-4保护组、提取物M-5保护组、提取物M-6保护组、提取物M-7保护组、提取物M-8保护组均能显著降低ANIT造成的血清各项指标升高情况，差异具有统计学意义(P＜0.05)，其中小鼠血清AST和ALP与正常对照组相比无显著性差异(P＞0.05)，但是还是提取物A保护组的效果最好。肝脏病理切片显示，提取物M-1保护组、提取物M-2保护组、提取物M-3保护组、提取物M-4保护组、提取物M-5保护组、提取物M-6保护组、提取物M-7保护组、提取物M-8保护组，与模型组相比肝细胞坏死减少，肝细胞肿大，胞质疏松，呈气球样变及羽毛样变的情况改善，炎性细胞浸润减轻，偶见结构正常的毛细胆管，但是还是提取物A保护组的效果最好。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (11)

  1. 一种中药组合物，其药效成分原料包括：虎杖、青皮、山楂和大黄。
  2. 根据权利要求1所述的中药组合物，其特征在于，所述中药组合物的药效成分原料由虎杖、青皮、山楂和大黄组成。
  3. 根据权利要求1所述的中药组合物，其特征在于，虎杖、青皮、山楂和大黄之间的重量份数比例范围是：虎杖1-30份、青皮1-12份、山楂1-30份和大黄0.1-10份。
  4. 一种中药提取物，为以如权利要求1～3之任一项所述中药组合物为原料提取有效成分制得。
  5. 一种如权利要求4所述中药提取物的制备方法，包括如下步骤：取如权利要求1～3之任一项所述中药组合物为原料提取有效成分。
  6. 根据权利要求5所述的制备方法，其特征在于，所述制备方法选自以下一任一：(1)按配比取虎杖、青皮和山楂，用乙醇提取，提取液浓缩、干燥，得干浸膏后加入大黄粉，混匀，即可；(2)按配比取虎杖、青皮、山楂和大黄，用乙醇提取，提取液浓缩、干燥，得干浸膏；(3)按配比取虎杖、青皮、山楂和大黄，用水提取，过滤，浓缩，干燥。
  7. 根据权利要求6所述的制备方法，其特征在于，所述乙醇选自60％～80％乙醇；优选70％乙醇。
  8. 如权利要求1～3之任一项所述中药组合物或如权利要求4所述中药提取物用于制备肝损伤预防和/或治疗药物的用途。
  9. 如权利要求1～3之任一项所述中药组合物或如权利要求4所述中药提取物用于制备胆汁淤积预防和/或治疗药物的用途。
  10. 如权利要求1～3之任一项所述中药组合物或如权利要求4所述中药提取物用于制备具有如下之任一功效的药物的用途：(1)降低ALT水平；(2)降低AST水平；(3)降低ALP水平；(4)降低T-Bil水平；(5)降低D-Bil水平；(6)降低TBA水平；(7)减少肝细胞坏死；(8)改善肝细胞肿大、胞质疏松、呈气球样变及羽毛样变的情况；(9)减轻炎性细胞浸润。
  11. 一种中药制剂，包括治疗有效量的如权利要求1～3之任一项所述中药组合物或如权利要求4所述中药提取物。
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