CN108567843B - Traditional Chinese medicine composition and preparation and application thereof - Google Patents
Traditional Chinese medicine composition and preparation and application thereof Download PDFInfo
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- CN108567843B CN108567843B CN201810748766.6A CN201810748766A CN108567843B CN 108567843 B CN108567843 B CN 108567843B CN 201810748766 A CN201810748766 A CN 201810748766A CN 108567843 B CN108567843 B CN 108567843B
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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)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
- A61K36/704—Polygonum, e.g. knotweed
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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)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
- A61K36/708—Rheum (rhubarb)
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/734—Crataegus (hawthorn)
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
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- 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
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- 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 or decoction
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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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- 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
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition capable of protecting liver and benefiting gallbladder and a preparation method and application thereof. The traditional Chinese medicine composition comprises the following active ingredient raw materials: giant knotweed rhizome, green tangerine orange peel, hawthorn and rhubarb. The traditional Chinese medicine composition and the traditional Chinese medicine extract provided by the invention can obviously reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, obviously reduce hepatocyte necrosis, improve the conditions of hepatocyte swelling, cytopenia, ballooning and feather-like change, relieve inflammatory cell infiltration and further prevent or treat cholestasis.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition capable of protecting liver and benefiting gallbladder and a preparation method and application thereof.
Background
Cholestasis is clinically manifested by jaundice accompanied by elevated levels of serum bilirubin, bile salts, alkaline phosphatase, etc., and histologically by accumulation of bile components in the liver. Cholestasis usually causes changes in the relevant parenchymal hepatocytes, and especially bile acid detergency can cause feathering of hepatocytes.
Bile secretion is one of the most important functions of the liver, wherein bilirubin and bile acid are the main components of bile, and the metabolic states of the bilirubin and bile acid can reflect the transport capacity of the liver for organic anions. A high T-Bil level with a marked increase in D-Bil suggests the presence of cholestatic jaundice. When cholestasis occurs, bile secretion decreases and distribution of bile acid storage is rapidly changed, so that the concentration of bile acid in serum and urine is significantly increased. Many organs contain ALT and AST, with the highest hepatic ALT content, mostly present in the cytoplasm; the AST content of the liver is second to that of the heart and mainly exists in liver cell mitochondria. Both enzymes are not generally elevated during cholestasis, and ALT and AST enter the blood only when stasis causes hepatocyte necrosis or permeability changes, resulting in increased enzyme activity in the blood. ALP is a group of enzymes that hydrolyze phosphomonoester compounds in an alkaline environment, and serum ALP is mainly derived from liver and bone. When the pressure of the capillary bile duct is increased under pathological conditions such as cholestasis, hepatocyte injury, liver occupation and the like, the increase of ALP production and the increase of serum ALP activity can be induced. Elevated serum ALP is the most characteristic early manifestation of cholestasis.
The research and development of the medicine with good treatment effect on the cholestasis have very important significance.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a traditional Chinese medicine composition capable of protecting liver and benefiting gallbladder and preparation and application thereof.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
the invention provides a traditional Chinese medicine composition, which comprises the following active ingredient raw materials: giant knotweed rhizome, green tangerine orange peel, hawthorn and rhubarb.
In one embodiment, the raw materials of the effective components of the Chinese medicinal composition comprise giant knotweed rhizome, green tangerine orange peel, hawthorn and rhubarb.
In one embodiment, the weight parts of the giant knotweed rhizome, the green tangerine orange peel, the hawthorn and the rhubarb are within the following ranges: 1-30 parts of giant knotweed, 1-12 parts of green tangerine orange peel, 1-30 parts of hawthorn and 0.1-10 parts of rhubarb.
In one embodiment, the amount of the giant knotweed rhizome is 1 to 15 parts by weight.
In one embodiment, the amount of the giant knotweed rhizome is 15 to 30 parts by weight.
In one embodiment, the amount of the giant knotweed rhizome is 15 parts by weight.
In one embodiment, the green tangerine peel is 1-6 parts by weight.
In one embodiment, the green tangerine peel is 6-12 parts by weight.
In one embodiment, the green tangerine peel is 6 parts by weight.
In one embodiment, the amount of hawthorn is 1 to 15 parts by weight.
In one embodiment, the amount of hawthorn is 15 to 30 parts by weight.
In one embodiment, the amount of hawthorn is 15 parts by weight.
In one embodiment, the amount of rhubarb is 0.1 to 1 part by weight.
In one embodiment, the rhubarb is 1 to 10 parts by weight.
In one embodiment, the rhubarb is 1 part by weight.
In one embodiment, the weight parts of the giant knotweed rhizome, the green tangerine orange peel, the hawthorn and the rhubarb are within the following ranges: 15 parts of giant knotweed, 6 parts of green tangerine peel, 15 parts of hawthorn and 1 part of rhubarb.
Further, the second aspect of the present invention provides a Chinese medicine extract, which is prepared by extracting effective components from the above Chinese medicine composition.
In the invention, the raw materials and the formula of the traditional Chinese medicine composition are the most critical, after the traditional Chinese medicine formula is known, the active ingredients of the traditional Chinese medicine composition can be extracted by adopting various conventional traditional Chinese medicine active ingredient extraction methods, such as conventional decoction, an alcohol extraction method, a water extraction and alcohol precipitation method, an alcohol extraction and water precipitation method, a salting-out method and the like, and the main active ingredients in the traditional Chinese medicine formula can be obtained by the extraction methods, so that the traditional Chinese medicine composition has a certain prevention and treatment effect on cholestasis.
Wherein, the water extraction and alcohol precipitation method comprises the following steps: extracting the effective components of the medicinal materials by using water as a solvent, and then removing impurities in the extracting solution by using ethanol precipitation with different concentrations. Generally, when the ethanol content reaches 50-60% (weight to volume ratio, namely g/100mL), impurities such as starch and the like can be removed, and when the ethanol content reaches more than 70%, most of the impurities except a few ineffective components such as tannin, water-soluble pigment and the like can be removed by precipitation.
The alcohol extraction and water precipitation method comprises the following steps: extracting the medicinal materials with ethanol with appropriate concentration, and removing impurities from the extractive solution with water.
After the effective components of the traditional Chinese medicine composition are extracted, the traditional Chinese medicine extract can be obtained by conventional steps of concentration, purification or drying. Wherein the concentration method includes but is not limited to: atmospheric evaporation, reduced pressure evaporation, thin film evaporation, multiple effect evaporation, and purification methods include but are not limited to: salting-out purification, macroporous resin adsorption purification, ethanol precipitation purification, ion exchange resin purification, flocculation precipitation purification, membrane separation purification, polyamide adsorption purification, silica gel adsorption chromatography purification and the like, and drying methods include but are not limited to: drying, drum drying, belt drying, moisture absorption drying, boiling drying, spray drying, reduced pressure drying, freeze drying, infrared drying, microwave drying, etc.
In one embodiment, the extraction is by ethanol extraction. For example, 60% to 80% ethanol (weight to volume ratio, i.e., g/100mL) may be used for extraction.
In one embodiment, the extraction is by water extraction.
In a third aspect of the present invention, a preparation method of the aforementioned herbal extract is provided, which comprises the following steps: extracting effective components from the Chinese medicinal composition.
In one embodiment, the method of making comprises the steps of: proportionally extracting rhizoma Polygoni Cuspidati, pericarpium Citri Reticulatae viride and fructus crataegi with ethanol, concentrating the extractive solution, drying to obtain dry extract, adding radix et rhizoma Rhei powder, and mixing.
In one embodiment, the ethanol is selected from 60% to 80% ethanol. Preferably, 70% ethanol.
The radix et rhizoma Rhei powder is obtained by pulverizing radix et rhizoma Rhei.
In one embodiment, the method of making comprises the steps of: proportionally taking giant knotweed rhizome, green tangerine peel, hawthorn and rhubarb, extracting with ethanol, concentrating the extracting solution, and drying to obtain dry extract.
In one embodiment, the ethanol is selected from 60% to 80% ethanol. Preferably, 70% ethanol.
In one embodiment, the method of making comprises the steps of: proportionally taking giant knotweed rhizome, green tangerine peel, hawthorn and rhubarb, extracting with water, filtering, concentrating and drying.
In a fourth aspect of the present invention, there is provided a use of the aforementioned Chinese medicinal composition or Chinese medicinal extract for preparing a medicament for preventing and/or treating liver injury.
In a fifth aspect of the present invention, there is provided a use of the aforementioned Chinese medicinal composition or Chinese medicinal extract for preparing a medicament for preventing and/or treating cholestasis.
In one embodiment, the traditional Chinese medicine composition or the traditional Chinese medicine extract can prevent and/or treat intrahepatic cholestasis.
In one embodiment, the traditional Chinese medicine composition or the traditional Chinese medicine extract can prevent and/or treat acute intrahepatic cholestasis.
In a sixth aspect of the invention, the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing ALT-lowering agent is provided.
In one embodiment, the above-mentioned Chinese medicinal composition or Chinese medicinal extract can be used for reducing ALT level in serum.
In a seventh aspect of the present invention, there is provided a use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing an AST reducing agent.
In one embodiment, the above-mentioned Chinese medicinal composition or Chinese medicinal extract can be used for reducing the AST level in serum.
In an eighth aspect of the present invention, there is provided a use of the aforementioned Chinese medicinal composition or Chinese medicinal extract for the preparation of an ALP lowering agent.
In one embodiment, the above herbal composition or herbal extract is used to reduce serum ALP levels.
The ninth aspect of the invention provides the use of the traditional Chinese medicine composition or the traditional Chinese medicine extract for preparing the T-Bil reducing agent.
In one embodiment, the composition or extract can be used to reduce the level of T-Bil.
In the tenth aspect of the invention, the application of the traditional Chinese medicine composition or the traditional Chinese medicine extract in preparing the D-Bil reducing agent is provided.
In one embodiment, the composition or extract can be used to reduce the level of D-Bil.
In an eleventh aspect of the invention, the use of the aforementioned traditional Chinese medicine composition or traditional Chinese medicine extract for preparing a TBA reducing agent is provided.
In one embodiment, the composition or extract can be used for reducing TBA levels.
In a twelfth aspect of the present invention, there is provided a use of the aforementioned Chinese medicinal composition or Chinese medicinal extract for preparing a medicament having any one of the following effects: (1) reducing hepatocyte necrosis; (2) improving the conditions of hepatomegaly, cytopenia, ballooning and feather-like changes; (3) reducing inflammatory cell infiltration.
In a thirteenth aspect of the invention, a Chinese medicine preparation is provided, which comprises a therapeutically effective amount of the Chinese medicine extract.
The Chinese medicinal preparation preferably comprises one or more conventional pharmaceutically acceptable auxiliary materials.
Pharmaceutically acceptable excipients include (but are not limited to): pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients. Therapeutically 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, polyazulyl compounds such as polyethylene glycol, water, sucrose, ethanol, glycerol, and the like, various preservatives, lubricants, dispersants, flavoring agents. Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added to assist in the stability of the formulation or to aid in the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, as desired. The Chinese medicinal preparation such as granule and paste can be prepared by conventional method. The pharmaceutical compositions of the present invention may also be used with other therapeutic agents.
Preferably, the traditional Chinese medicine preparation is an oral preparation, including but not limited to granules, pills, tablets, capsules, syrups, sprays and the like.
After the effective components in the traditional Chinese medicine composition are extracted, the traditional Chinese medicine composition can be prepared by adopting conventional pharmaceutic adjuvants and additives.
The effective therapeutic dose of the pharmaceutical composition is that the oral administration safe and effective dose is 1.00-2.65g/kg body weight based on the total weight of the raw medicinal materials. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Compared with the prior art, the invention has the following beneficial effects:
the traditional Chinese medicine composition and the traditional Chinese medicine extract provided by the invention can obviously reduce serum ALT, AST, ALP, T-Bil, D-Bil and TBA, obviously reduce hepatocyte necrosis, improve the conditions of hepatocyte swelling, cytopenia, ballooning and feather-like change, relieve inflammatory cell infiltration and further prevent or treat cholestasis.
Drawings
FIG. 1: effects of extract A, extract B, extract C and UDCA on serum ALT, AST, ALP, T-Bil, D-Bil and TBA in ANIT-induced cholestatic mice; in comparison with the group of vehicles,*p is less than 0.05; in comparison to the ANIT model set,#P＜0.05。
FIG. 2: pathological section analysis of liver in each group (H & E, × 400), a: a normal control group; b: a group of ANIT models; c: extract a drug protected group; d: extract B drug protection group; e: extract C drug protected group; f: positive drug UDCA protected group.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods and preparation methods disclosed in the present invention all employ conventional techniques in the art.
Example 1
First, experimental material
1.1, medicine
Alpha-naphthalene isothiocyanate (ANIT; Sigma, MO, USA); ursodeoxycholic acid (UDCA; TCI Japan, Japan); radix et rhizoma Rhei powder (obtained by pulverizing radix et rhizoma Rhei, wherein 95% of medicinal powder passes through 100 mesh sieve, and the rest powder must pass through 80 mesh sieve), radix et rhizoma Rhei, rhizoma Polygoni Cuspidati, pericarpium Citri Reticulatae viride, and fructus crataegi are provided by Shanghai and radix et rhizoma Rhei pharmaceutical Co., Ltd.
1.2, reagents
ALT kit, AST kit, ALP kit, T-Bil kit, D-Bil kit, TBA kit (Nanjing Kangji, Shanghai, China); olive oil (national group, shanghai, china); sodium carboxymethylcellulose (CMC-Na; national group of pharmaceuticals, Shanghai, China); normal saline and neutral tissue fixing liquid.
1.3, Main Instrument
A full-automatic biochemical analyzer; an optical microscope; thermo Scientific Varioskan Flash full wavelength scanning multifunction reader (Thermo fisher Scientific, Germany); eppendorf 5424R low temperature high speed centrifuge (Eppendorf, Germany); SANYO ice makers (SANYO Electric co., ltd., Japan); millipore water purifier (Millipore, Bedford, MA, USA).
1.4, experimental animals: c57BL/6 mice, body weight (20. + -.2) g, male, clean grade, 60, purchased from Shanghai slyke animal laboratories, Inc. Animals are raised in an animal room with temperature (22 +/-1) DEG C, relative humidity (65 +/-10)%, and 12h day and night alternation, and are adaptively fed for 3d before experiments.
Second, Experimental methods
2.1 pharmaceutical formulation
ANIT: dissolving appropriate amount of powder with oleum Olivarum to obtain 10mg/mL powder, and ultrasonic treating to dissolve completely.
UDCA: dissolving with 0.5% sodium carboxymethylcellulose (CMC-Na) to 9mg/mL, and mixing well before intragastric administration.
And (3) extracting A: extracting 360g of giant knotweed rhizome, 144g of green tangerine peel and 360g of hawthorn fruit by using 70% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying the thick paste at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling the thick paste to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing the dry extract to obtain the extract A.
And (3) extracting B: extracting radix et rhizoma Rhei 24g, rhizoma Polygoni Cuspidati 360g, pericarpium Citri Reticulatae viride 144g and fructus crataegi 360g with 70% ethanol, recovering solvent under reduced pressure to obtain soft extract, drying at 100 deg.C and-0.1 MPa for 5 hr, and cooling to obtain dry extract as extract B.
And (3) extract C: decocting radix et rhizoma Rhei 24g, rhizoma Polygoni Cuspidati 360g, pericarpium Citri Reticulatae viride 144g and fructus crataegi 360g with water, recovering solvent under reduced pressure to obtain soft extract, drying at 100 deg.C and-0.1 MPa for 5 hr, and cooling to obtain dry extract as extract C.
2.2 animal Experimental methods
60C 57BL/6 mice were randomly assigned to a normal control group, ANIT model group, three extract drug protected group, and positive drug UDCA control group, 10 mice per group. All drugs were dissolved to the desired concentration with 0.5% sodium carboxymethylcellulose (CMC-Na). The normal control group and the model group were given the corresponding CMC-Na solution. The remaining groups of mice were gavaged (i.g.) with the same agent at a dose of 10mL/kg daily, qd, for 5 consecutive days. Specifically, the administration dose of the extract A drug-protected group was 2.69g/kg, the administration dose of the extract B drug-protected group was 2.34g/kg, the administration dose of the extract C drug-protected group was 2.13g/kg, and the administration dose of the positive drug UDCA control group was 0.09g/kg each time. After 12 hours of the 5 th administration, except for the normal control group which is administrated with 10mL/kg of olive oil by intragastric administration, the other groups of mice are administrated with ANIT olive oil solution by intragastric administration according to the dose of 10mL/kg for molding. The administration was continued twice after 12h, 36h molding. Animals were fasted without water deprivation 12h before the end of the experiment.
All animals were anesthetized with ether 48h after ANIT administration (i.e. 12h after the last administration), and then blood was collected from the eyeball and measured for blood supply clearance biochemical index. Liver tissues of the same site (left lobe of liver) were excised for pathological examination and fixed with 10% neutral formalin for use.
2.3 serum Biochemical index determination
After the mice are anesthetized by ether, the eyeballs are picked and blood is taken, the whole blood is kept stand for 3h at room temperature, 3500rpm/min and centrifuged for 15min, the upper serum is taken, and a full-automatic biochemical analyzer is used for measuring the activity of serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) serum alkaline phosphatase (ALP), the content of serum total bilirubin (T-Bil) and the content of direct bilirubin (D-Bil). The Total Bile Acid (TBA) content was determined and calculated as described in the kit.
2.4 pathological observation of liver tissue
After tissue fixation, tissue trimming, paraffin embedding, cutting into 4 μm slices, xylene deparaffinization, gradient ethanol dehydration, hematoxylin-eosin staining (H & E staining), ethanol dehydration, xylene permeabilization, resin sealing, and observation under an optical microscope.
2.5, statistical analysis
The experimental data are measured by mean + -SD
The comparison of the two averages was analyzed by one-way ANOVA in statistical software, with P < 0.05 indicating that the differences were statistically significant.
Third, experimental results
3.1 evaluation of serum Biochemical index for protection of mouse cholestasis caused by ANIT
As shown in Table 1, Table 2 and FIG. 1, 6 serological indexes of the ANIT model group mouse are obviously higher (P is less than 0.05) than those of the blank group mouse, which indicates that the modeling is successful. Compared with ANIT model group, the extract A can significantly reduce the elevation of each index of serum caused by ANIT after continuous administration, and the difference has statistical significance (P is less than 0.05), wherein the serum AST and ALP of mice have no significant difference (P is more than 0.05) compared with normal control group. The extract B can obviously reduce the levels of ALT, AST, T-Bil and D-Bil (P is less than 0.05) in mouse serum, and the extract C can only obviously reduce the levels of ALT and AST (P is less than 0.05). The positive control drug UDCA can obviously reduce other 5 serological indexes (P is less than 0.05) except AST. The results show that the extract A has a protective effect on liver injury of a cholestasis mouse caused by ANIT, and is superior to UDCA in the aspect of reducing ALT and AST indexes.
TABLE 1 Effect of extracts A, B, C and UDCA on serum ALT, AST and ALP in ANIT-induced cholestasis mice
Note: in comparison with the group of vehicles,*p is less than 0.05; in comparison with the set of models,#P＜0.05
TABLE 2 Effect of extract A, extract B, extract C and UDCA on ANIT induced cholestasis mouse serum T-Bil, D-Bil and TBA
Note: in comparison with the group of vehicles,*p is less than 0.05; in comparison with the set of models,#P＜0.05
3.2 pathological analysis of liver for protecting mouse cholestasis caused by ANIT
As shown in figure 2, it can be seen from the observation under a light microscope that the liver cells in the normal control group are normal in shape and closely arranged, liver parenchymal cell lesions are not seen, the liver lobule structure is complete, the liver cell cords are radially arranged from the terminal venules to the periphery, no obvious inflammatory cell infiltration, fibrous tissue hyperplasia and other changes exist in the region of the collector, and the capillary bile duct has no cholestasis. In the ANIT model group, hepatomegaly and cytoplasma are loose, some of the hepatomegaly and cytoplasma are changed like balloons or feathers, focal necrosis can be seen, hepatic lobular is unclear, a large amount of inflammatory cells infiltrate in a manifold area, and the structure of a capillary bile duct is damaged or lost. Compared with the model group, the extract A protects the group, reduces the necrosis of the liver cells, enlarges the liver cells, has loose cytoplasm, improves the balloon-like change and feather-like change, reduces the infiltration of inflammatory cells, and occasionally shows capillary bile ducts with normal structures. The extract B protects the group, reduces hepatocyte necrosis, hepatomegaly, cytopenia, and ballooning and feather-like changes, and reduces inflammatory cell infiltration. The extract C protected the group, and the hepatomegaly, cytopenia, ballooning and feathering conditions were not significantly improved, which was seen to reduce the area of focal necrosis and reduce inflammatory cell infiltration. After the administration of UDCA for protection, the conditions of liver cell necrosis reduction, liver cell swelling, cytopenia, ballooning and feather-like change are improved, and inflammatory cell infiltration in the junction area is reduced.
Fourth, discuss
ANIT induces intrahepatic cholestasis in mice and also causes hepatocyte damage, and its disease-treating substrate for animal hepatotoxicity behaves pathophysiologically much like human drug hepatitis, and is widely used to replicate intrahepatic cholestasis models. Toxicity of ANIT is related to its metabolites, and under the action of liver P450 enzymes, sulfur oxidation in ANIT structure is a toxic product attacking intrahepatic bile duct endothelial cells, resulting in hyperbilirubinemia and decreased bile secretion. Meanwhile, membrane lipid peroxidation is also caused, so that liver cells are degenerated and necrotized, and acute intrahepatic cholestasis is caused.
In the research of the invention, a mouse intrahepatic cholestasis model caused by ANIT is utilized to evaluate the effects of the rhubarb, the giant knotweed rhizome, the green tangerine peel and the hawthorn compound on protecting the liver and benefiting the gallbladder in different extraction and preparation modes. Experimental results show that the extract A can remarkably reduce the elevation of ALT, AST, ALP, T-Bil, D-Bil and TBA of mouse serum caused by ANIT. Compared with positive medicine UDCA, extract A can more effectively reduce two serological indexes of ALT and AST. The pathological section of the liver shows that the extract A in the protective group has obviously reduced hepatocyte necrosis, hepatomegaly, cytoplasma porosity, ballooning and feather-like changes and reduced inflammatory cell infiltration compared with the model group.
In conclusion, the extract A has a good protective effect on the liver of a cholestasis model mouse caused by ANIT.
In addition, referring to the preparation method of extract a, the following were prepared:
extract M-1: extracting 24g of giant knotweed, 144g of green tangerine peel and 360g of hawthorn with 60% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying at 100 ℃ and 0.1MPa for 5 hours, cooling to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing to obtain the extract M-1.
Extract M-2: extracting 720g of giant knotweed, 144g of green tangerine peel and 360g of hawthorn with 80% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing to obtain the extract M-2.
Extract M-3: extracting 360g of giant knotweed rhizome, 24g of green tangerine peel and 360g of hawthorn fruit by using 60% ethanol, recovering the solvent from the extracting solution under reduced pressure to form thick paste, drying the thick paste at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling the thick paste to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing the dry extract to obtain the extract M-3.
And (3) extraction M-4: extracting 360g of giant knotweed rhizome, 288g of green tangerine peel and 360g of hawthorn fruit by using 80% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying the thick paste at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling the thick paste to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing the dry extract to obtain the extract M-4.
And (3) extraction M-5: extracting 360g of giant knotweed rhizome, 144g of green tangerine peel and 24g of hawthorn with 60% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying at 100 ℃ and 0.1MPa for 5 hours, cooling to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing to obtain the extract M-5.
And (3) extraction M-6: extracting 360g of giant knotweed rhizome, 144g of green tangerine peel and 720g of hawthorn with 80% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling to obtain dry extract, adding 24g of rhubarb powder, and uniformly mixing to obtain the extract M-6.
And (3) extraction M-7: extracting 360g of giant knotweed rhizome, 144g of green tangerine peel and 360g of hawthorn fruit by using 60% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying the thick paste at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling the thick paste to obtain dry extract, adding 2.4g of rhubarb powder, and uniformly mixing the dry extract to obtain the extract M-7.
And (3) extraction M-8: extracting 360g of giant knotweed rhizome, 144g of green tangerine peel and 360g of hawthorn fruit by using 80% ethanol, recovering the solvent from the extracting solution under reduced pressure to obtain thick paste, drying the thick paste at 100 ℃ and under the pressure of-0.1 MPa for 5 hours, cooling the thick paste to obtain dry extract, adding 240g of rhubarb powder, and uniformly mixing the dry extract to obtain the extract M-8.
And referring to the animal experiment method of the second part 2.2, setting the extract M-1 protection group to administer 2.69 g/kg; the extract M-2 protected group is administered at a dose of 2.69 g/kg; the extract M-3 protected group is administered at a dose of 2.69 g/kg; the extract M-4 protected group is administered at a dose of 2.69 g/kg; the extract M-5 protected group is administered at a dose of 2.69 g/kg; the extract M-6 protective group is administered at a dose of 2.69 g/kg; the extract M-7 protected group is administered at a dose of 2.69 g/kg; extract M-8 protected group, administered at a dose of 2.69g/kg, and otherwise operated in the same manner as the animal experiments of section 2.2.
As a result, it was found that: the extract M-1 protection group, the extract M-2 protection group, the extract M-3 protection group, the extract M-4 protection group, the extract M-5 protection group, the extract M-6 protection group, the extract M-7 protection group and the extract M-8 protection group can obviously reduce the elevation of each index of serum caused by ANIT, and the difference has statistical significance (P is less than 0.05), wherein the AST and ALP of mouse serum have no significant difference (P is more than 0.05) compared with a normal control group, but the effect of the extract A protection group is the best. The pathological liver sections show that the extracts M-1 protection group, the extracts M-2 protection group, the extracts M-3 protection group, the extracts M-4 protection group, the extracts M-5 protection group, the extracts M-6 protection group, the extracts M-7 protection group and the extracts M-8 protection group have the best effect compared with the model group in that the conditions of hepatocyte necrosis, hepatocyte swelling, cytoplasmosis, ballooning and feathering are improved, inflammatory cell infiltration is reduced, and capillary bile ducts with normal structures are occasionally seen.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (7)
1. A Chinese medicinal composition for treating intrahepatic cholestasis comprises rhizoma Polygoni Cuspidati, pericarpium Citri Reticulatae viride, fructus crataegi and radix et rhizoma Rhei as effective component; the weight parts of the giant knotweed rhizome, the green tangerine orange peel, the hawthorn and the rhubarb are in the following proportion range: 1-30 parts of giant knotweed, 1-12 parts of green tangerine orange peel, 1-30 parts of hawthorn and 0.1-10 parts of rhubarb.
2. A Chinese medicinal extract for treating intrahepatic cholestasis is prepared by extracting effective components from the Chinese medicinal composition of claim 1.
3. A method for preparing the extract of the traditional Chinese medicine as claimed in claim 2, comprising the following steps: extracting effective components from the Chinese medicinal composition of claim 1.
4. The method of claim 3, wherein the method is selected from any one of: (1) extracting rhizoma Polygoni Cuspidati, pericarpium Citri Reticulatae viride and fructus crataegi with ethanol, concentrating the extractive solution, drying to obtain dry extract, adding radix et rhizoma Rhei powder, and mixing; (2) proportionally taking giant knotweed rhizome, green tangerine peel, hawthorn and rhubarb, extracting with ethanol, concentrating the extracting solution, and drying to obtain dry extract; (3) proportionally taking giant knotweed rhizome, green tangerine peel, hawthorn and rhubarb, extracting with water, filtering, concentrating and drying.
5. The method according to claim 4, wherein the ethanol is selected from 60 to 80% ethanol.
6. Use of the Chinese medicinal composition of claim 1 or the Chinese medicinal extract of claim 2 for the preparation of a medicament for the prevention and/or treatment of intrahepatic cholestasis.
7. A traditional Chinese medicine preparation for treating intrahepatic cholestasis, comprising a therapeutically effective amount of the traditional Chinese medicine composition of claim 1 or the traditional Chinese medicine extract of claim 2.
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| CN201810748766.6A CN108567843B (en) | 2018-07-10 | 2018-07-10 | Traditional Chinese medicine composition and preparation and application thereof |
| PCT/CN2018/100211 WO2020010664A1 (en) | 2018-07-10 | 2018-08-13 | Traditional chinese medicine composition, and preparation and use thereof |
| CA3050146A CA3050146C (en) | 2018-07-10 | 2018-08-13 | Traditional chinese medicine herb composition, making thereof, and application thereof |
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| CN201810748766.6A Active CN108567843B (en) | 2018-07-10 | 2018-07-10 | Traditional Chinese medicine composition and preparation and application thereof |
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| WO (1) | WO2020010664A1 (en) |
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| CN113521225A (en) * | 2020-04-21 | 2021-10-22 | 上海和黄药业有限公司 | Traditional Chinese medicine composition for treating liver injury and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557415A (en) * | 2004-02-03 | 2004-12-29 | 上海和黄药业有限公司 | Medicine preparation for treating liver and gallbladder disease and its preparing process |
| CN102085330A (en) * | 2009-12-02 | 2011-06-08 | 程兴顺 | Gallbladder-soothing capsules |
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2018
- 2018-07-10 CN CN201810748766.6A patent/CN108567843B/en active Active
- 2018-08-13 WO PCT/CN2018/100211 patent/WO2020010664A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557415A (en) * | 2004-02-03 | 2004-12-29 | 上海和黄药业有限公司 | Medicine preparation for treating liver and gallbladder disease and its preparing process |
| CN102085330A (en) * | 2009-12-02 | 2011-06-08 | 程兴顺 | Gallbladder-soothing capsules |
Non-Patent Citations (1)
| Title |
|---|
| 胆宁片对胆汁瘀积小鼠肝脏转运体及代谢酶基因表达的影响;王莉等;《中成药》;20130731;第35卷(第7期);第1385-1389页，尤其是第1385页摘要、第1388页表1和左栏3.2、第1389页左栏第2-9行 * |
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| WO2020010664A1 (en) | 2020-01-16 |
| CN108567843A (en) | 2018-09-25 |
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