CN113694104A - Traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function, preparation method and application thereof - Google Patents
Traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition with a protective effect on chemical liver injury and a function of promoting liver regeneration, and a preparation method and application thereof. The traditional Chinese medicine composition comprises the following components in parts by weight: 5-20 parts of kudzu root extract, 2-10 parts of astragalus extract, 2-8 parts of schisandra extract, 1.5-20 parts of hovenia dulcis thunb extract, 0.5-2 parts of refined flammulina velutipes polysaccharide and the auxiliary materials of salvia miltiorrhiza, vine tea, poria cocos, fructus amomi, moringa seeds and liquorice. Animal experiment research proves that the composition has protective effect on acute liver injury caused by alcohol and carbon tetrachloride, and can relieve liver injury. The composition can also induce hepatocyte proliferation and promote liver regeneration after hepatectomy injury.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine pharmacy, in particular to a traditional Chinese medicine composition with a protective effect on chemical liver injury and liver regeneration, a preparation method and application thereof.
Background
The liver is the largest and most functional digestive gland in the human body and participates in the processes of digestion, metabolism, excretion, detoxification, immunity and the like in the human body, wherein the metabolic function is the most important, and simultaneously participates in the hematopoietic process, stores and releases hematopoietic factors, synthesizes various enzymes and ensures the normal metabolic function of the human body. The liver has strong detoxification function to the foreign and the harmful substances generated by metabolism. However, many harmful factors can enter the liver through the portal vein of the gastrointestinal tract or systemic circulation to transform, and thus the liver is vulnerable to toxic substances. In the natural and human industrial production process, some substances with toxicity to liver exist, the toxicity is generally susceptible in people, the incubation period is short, the pathological process is directly related to the infection dosage, and the liver can be damaged to different degrees, such as hepatocyte necrosis, hepatic fibrosis, liver cirrhosis, liver cancer and the like. Chemical liver injury is liver injury caused by chemical hepatotoxic substances. These chemicals include alcohol, environmentally toxic chemicals and certain drugs.
At present, most of liver injuries are clinically caused by alcohol, and alcoholic liver injuries are a series of pathological changes such as liver damage caused by excessive alcohol intake. Alcoholic liver injury can be classified into acute alcoholic liver injury and chronic alcoholic liver injury. Acute alcoholic liver injury is liver function damage caused by excessive alcohol intake at one time, and is manifested by acute hepatitis symptoms such as inappetence, nausea, liver discomfort, jaundice, and the like. Chronic alcoholic liver injury is characterized by fibrosis and necrosis of liver cells and destruction of immune function of the liver due to long-term alcohol intake, and is often manifested as alcoholic fatty liver, alcoholic hepatitis and alcoholic cirrhosis. The pathogenesis of alcoholic liver injury is mainly (1) direct hepatotoxicity of alcohol and acetaldehyde, a metabolite of the alcohol; (2) oxidative stress and reactive oxygen radical damage; (3) cytokine and endotoxin mediated immune inflammation mechanism; (4) others, such as local tissue hypoxia, endoplasmic reticulum stress, increased iron load, apoptosis, and the like. Among them, oxidative stress caused by alcohol and a large amount of active oxygen radicals generated during the metabolism thereof is considered to be the most important early pathological mechanism of alcoholic liver disease. In the liver, ethanol is first oxidized by Alcohol Dehydrogenase (ADH) to form acetaldehyde, and then oxidized by acetaldehyde dehydrogenase (ALDH)To acetic acid and finally to CO in peripheral tissues2And water. In the alcohol metabolism process, the NAD +/NADH ratio is reduced along with the conversion of oxidized nicotinamide adenine dinucleotide (NAD +) to reduced NADH, so that NAD + -dependent biochemical reactions such as tricarboxylic acid cycle, fatty acid beta oxidation and oxidative phosphorylation, gluconeogenesis and the like are inhibited, glycolipid metabolism is disturbed, and the liver is subjected to steatosis. At the same time, the metabolic process can generate superoxide anion, H2O2The free radicals activate the peroxidation of intracellular lipid, destroy the structural function of protein, and change the permeability and flowability of cell membrane. Patients who drink excessive alcohol for a long time are easy to suffer from fatty liver, alcoholic hepatitis, hepatic fibrosis, cirrhosis and even liver cancer. In addition, overdosing of some drugs, such as acetaminophen, can also cause liver damage.
Modern medicine has no specificity in the treatment of liver injury, and the traditional Chinese medicine is developed for thousands of years, and is formulated according to syndrome differentiation through the compatibility of the medication rules of a prescription, so that the effect of the traditional Chinese medicine is better than that of western medicines, and the toxicity of the traditional Chinese medicine is obviously lower than that of the western medicines. Most products with liver protection function in the current market adopt cordyceps, pine pollen, lucid ganoderma and the like as medicines, and have the problems of high cost and unstable raw material supply, so that the application of related liver protection products is limited. Therefore, the traditional Chinese medicine formula for relieving the chemical liver injury, which has the advantages of novel concept, simple process, high extraction efficiency, low production cost and good drug effect, is found, and has great significance.
The traditional Chinese medicine accumulates abundant medicinal resources for protecting the liver in long-term clinical practice. Active ingredients with liver protection effects are actively searched by modern people from natural plants, and traditional Chinese medicinal materials such as flos puerariae lobatae, radix puerariae, semen hoveniae, radix salviae miltiorrhizae, radix notoginseng, liquorice, schisandra chinensis, needle mushroom and the like are proved to have good liver protection effects. The action mechanism of the natural liver-protecting medicine is mainly to play a role in protecting the liver through ways of eliminating free radicals, resisting lipid peroxidation, reducing transaminase and the like. For example: flos Puerariae Lobatae can be used in gastrointestinal absorption and metabolism of ethanol, and its water extract can reduce ethanol concentration by activating ethanol dehydrogenase activity, thereby effectively reducing ethanol contentThe concentration of ethanol in blood has protective effect on liver cell damage caused by ethanol (see: Lujie, Liwentaimen, Niyamin, preliminary study on the influence of water extract of Chinese medicinal materials on the activity of alcohol dehydrogenase [ J]The university of Tongji school newspaper medicine edition 2002,23(1): 23-25.). Studies such as the King et al find that the raisin tree seed polysaccharide can obviously reduce the concentration of ethanol in the blood of mice, activate ethanol dehydrogenase and reduce the activity of alanine aminotransferase, and show that the raisin tree seed polysaccharide has better function of relieving alcoholism and protecting liver (see: the extraction process and the research on the function of relieving alcoholism of the active polysaccharide of raisin tree seed, Cao Xiao Gao, Kun et al [ J]2007,10: 3-4.) of Guangxi light industry. Salvia miltiorrhiza can inhibit ALT and AST content increase in rat serum with liver injury induced by carbon tetrachloride, improve liver tissue SOD activity, and reduce MDA content. Histological observation shows that the salvia miltiorrhiza has obvious repairing effect on the damaged liver tissue (see: Wubailing. salvia miltiorrhiza for CCl)4Study of protective action against acute liver injury [ J]The journal of traditional Chinese medicine and pharmacology, 2002,20(3): 363.). Radix astragali has antiinflammatory, antioxidant and immunoregulatory effects, and can promote DNA synthesis of liver cell, and in vitro liver cell test proves that radix astragali total extract can obviously reduce CCl4And H2O2The increase of the MDA content of the liver cells (see the protection effect of the Qiu's season, Guishuangying and Astragalus extract on the acute chemical liver injury of mice [ J]Anhui journal of medicine, 2009,13(6).
Moringa oleifera (Moringa oleifera Lam.) is a plant of Moringa genus of Moringaceae family, is a tree with perennial tropical fallen leaves, is a novel health food, has great development potential, has rich nutritional value, and also shows good activity in the aspects of oxidation resistance, blood sugar reduction, blood fat reduction and fungi resistance. Nilanjan Das et al found that the extract of Moringa oleifera leaves was effective in reducing liver damage caused by mice fed a high fat diet. The liver damage of mice in the moringa group is effectively reduced, the endogenous antioxidant activity index is obviously increased, and the lipid peroxidation condition is inhibited (see: Nilangan Das, Kunal Sikker, Santianth Ghosh, et al, Moringa oleifera lam. leaf extract expression stages in microorganism fed with high-fat di [ J ]. Indian Journal pf Experiment Biology,2012,50:404 and 412.). The protective effect of Moringa oleifera flowers and leaf alcohol extracts such as Sharifudin SA on acetaminophen (APAP) induced liver injury is reduced, and the liver marker enzyme activity is reduced in Moringa oleifera extract administration groups, and the liver injury degree is remarkably reduced in pathological tissue observation (see: Sharifudin S A, Fakurazi S, Hidayat M T, et al.
Flammulinavelipes (Curt.: Fr.) Sing.) is one of edible fungi, is rich in nutrition, and contains various bioactive substances such as polysaccharide, immunomodulatory protein, and policosanol, and these substances have wide pharmacological activities of resisting tumor, virus, insect, fungus, Human Immunodeficiency Virus (HIV), reducing serum cholesterol, lowering blood pressure, and improving immunity. Polysaccharide components extracted from needle mushrooms serving as raw materials have a protective effect on liver injury, and attract attention in recent years. For example: found that the flammulina velutipes polysaccharide can obviously reduce CCl4Serum ALT and AST activity of mice with acute liver injury, liver homogenate SOD activity improvement, MDA content reduction, CCl4Has certain protective effect on the induced stress liver injury of mice (see: Li Yi Fang, Zai Wei, Wang Min, etc.. Flammulina velutipes polysaccharide has protective effect on the acute liver injury of mice [ J]The journal of Guangdong institute of medicine, 2010,26(2): 162-165.).
Disclosure of Invention
The invention takes the traditional Chinese medicine theory as guidance, develops and develops the traditional Chinese medicine composition with auxiliary treatment effect on chemical liver injury by applying the raw material formulation which is published by the Ministry of health and can be used for medicine and food or used for health care, and evaluates the liver protection effect of the traditional Chinese medicine composition through the pharmacodynamics experiment of mice.
At present, the formula and the preparation method of the anti-alcoholism liver-protecting product prepared by taking kudzuvine root, astragalus, schisandra chinensis, hovenia dulcis thunb, salvia miltiorrhiza, poria cocos, fructus amomi and moringa seeds as main raw materials and adding refined flammulina velutipes polysaccharide with the liver-protecting effect are not reported.
The invention aims at providing a traditional Chinese medicine composition, and aims at providing a preparation method of the traditional Chinese medicine composition and an application of the traditional Chinese medicine composition in preparing a liver protection medicine aiming at chemical liver injury or a medicine for promoting liver regeneration.
In order to achieve the purpose, the invention adopts the following technical scheme: a traditional Chinese medicine composition with a protective effect on chemical liver injury and a function of promoting liver regeneration comprises the following components in parts by mass: 5-20 parts of kudzu root extract, 2-10 parts of astragalus extract, 2-8 parts of schisandra extract, 1.5-20 parts of hovenia dulcis thunb extract and 0.5-2 parts of refined flammulina velutipes polysaccharide.
Further, the feed also comprises the following components in parts by mass: 1-10 parts of salvia miltiorrhiza, 1-10 parts of vine tea, 1-2 parts of poria cocos, 1-4 parts of fructus amomi, 5-10 parts of moringa seeds and 1-10 parts of liquorice.
3. A preparation method of a traditional Chinese medicine composition with a protective effect on chemical liver injury and a function of promoting liver regeneration comprises the following steps:
step 1: pulverizing radix Puerariae, radix astragali, fructus Schisandrae chinensis, and semen Hoveniae respectively to coarse powder, adding polar organic solvent water solution, extracting, mixing extractive solutions, filtering, concentrating under reduced pressure, and drying to obtain Chinese medicinal extract powder;
step 2: pulverizing needle mushroom, microwave extracting with water as solvent to obtain needle mushroom crude polysaccharide, and purifying with macroporous resin and cellulose column to obtain refined needle mushroom polysaccharide;
and step 3: the components obtained in the steps 1 and 2 are uniformly mixed according to the proportion of claim 1 to prepare the traditional Chinese medicine composition which has the effects of protecting chemical liver injury and promoting liver regeneration.
Further, the specific steps of preparing the traditional Chinese medicine extract powder in the step 1 are as follows: respectively crushing kudzu vine root, astragalus, schisandra chinensis and hovenia dulcis thunb to 100 meshes, extracting for 1-3 times at 50-90 ℃ by using a polar organic solvent aqueous solution with the mass concentration of 40-80% and the weight of 8-16 times of the medicinal materials, wherein the mass concentration of the polar organic solvent aqueous solution is 1-3 hours each time, combining the extracting solutions, filtering, concentrating under reduced pressure, and drying to prepare traditional Chinese medicine extract powder; the drying mode is spray drying, freeze drying, microwave drying or vacuum drying; the polar organic solution is one or more than two mixed solvents of methanol, ethanol or acetone.
Further, the method also comprises the steps of crushing the salvia miltiorrhiza, the vine tea, the poria cocos, the fructus amomi, the moringa seeds and the liquorice into coarse powder, mixing the coarse powder according to the proportion, carrying out superfine grinding, adding the mixture into the components prepared in the steps 1 and 2, and mixing the mixture uniformly according to the proportion.
Further, the specific steps for preparing the refined flammulina velutipes polysaccharide in the step 2 are as follows:
step 2.1: drying needle mushrooms, crushing to 100 meshes, putting the needle mushrooms into a microwave extractor with water as a solvent, wherein the water is 12-20 times of the mass of medicinal materials, and extracting for 30-60 min at the microwave power of 400-600W and the extraction temperature of 50-90 ℃; collecting ultrasonic microwave extractive solution, filtering, concentrating under reduced pressure to one tenth of original volume, precipitating the concentrated solution with 95% ethanol until the final concentration of ethanol is 80%, standing overnight, centrifuging, collecting precipitate, washing the precipitate with anhydrous ethanol, diethyl ether and acetone, and drying to obtain crude Flammulina velutipes polysaccharide;
step 2.2: removing protein from the crude polysaccharide by trichloroacetic acid method, adding 5% trichloroacetic acid five times the volume of the crude polysaccharide under ice bath condition, stirring for 10min, standing overnight, adjusting pH to 7, filtering, concentrating, and lyophilizing to obtain refined crude polysaccharide;
step 2.3: and further purifying the refined crude polysaccharide through D101 macroporous resin and DEAE-52 cellulose to obtain the refined flammulina velutipes polysaccharide.
Further, step 2.3 purification of the refined crude polysaccharide specifically comprises the following steps:
(1) dissolving refined crude polysaccharide with water 8-15 times of the weight of the medicinal materials, adding the obtained solution into a D101 macroporous resin chromatographic column, carrying out equilibrium adsorption for 20-40 min, eluting with a certain volume of purified water and 0.1, 0.3 and 0.5mol/L sodium chloride aqueous solution in sequence, and quantitatively collecting; detecting polysaccharide content in the eluate by using a trace phenol-sulfuric acid method, drawing an elution curve, combining the same components, concentrating under reduced pressure, dialyzing, and freeze-drying;
(2) dissolving main components separated from the D101 macroporous resin in water, adding the main components into a DEAE-52 cellulose chromatographic column, sequentially eluting with a certain volume of purified water and 0.1, 0.2 and 0.3mol/L sodium chloride aqueous solution, quantitatively collecting, detecting the polysaccharide content in an eluent by adopting a trace phenol-sulfuric acid method, drawing an elution curve, combining the same components, concentrating under reduced pressure, dialyzing and freeze-drying to obtain the refined flammulina velutipes polysaccharide with relatively uniform molecular weight.
Further, the molecular weight of the refined flammulina velutipes polysaccharide prepared in the step 2 is 31.7kDa by a high performance liquid chromatography-evaporative light scattering method, and the peak-off time is 13.3 minutes.
Application of a Chinese medicinal composition in preparing liver protecting medicine or liver regeneration promoting medicine for treating chemical liver injury is provided.
Furthermore, the traditional Chinese medicine composition also comprises a medically allowable carrier, and the traditional Chinese medicine composition is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquid, mixture, buccal agents, granules, medicinal granules, pills, powder, ointment, pellets, suspension, solutions, injections, powder injections, freeze-dried powder injections, suppositories, ointments, plaster, creams, sprays, drops, dropping pills or patches.
The traditional Chinese medicine composition with the protective effect on chemical liver injury provided by the invention has obvious effects of dispelling the effects of alcohol and preventing drunkenness, obviously reduces the drunkenness rate of mice and prolongs the tolerance time. The traditional Chinese medicine composition can remarkably relieve acute liver injury of mice caused by alcohol, remarkably improve the contents of SOD and GSH in liver tissues, reduce the MDA level and reduce the TG level in serum. The traditional Chinese medicine composition can also remarkably relieve acute liver injury of mice caused by carbon tetrachloride, remarkably reduce ALT and AST levels of serum and MDA level of liver tissues, and can be used for preparing liver-protecting medicines.
The invention also provides application of the traditional Chinese medicine composition in preparing a liver protection and regeneration medicine, and the traditional Chinese medicine composition has the effects of promoting liver regeneration after liver partial resection, improving liver weight ratio recovery capability, promoting hepatocyte proliferation, effectively stimulating the regeneration potential of residual hepatocytes and can be used for preparing liver protection medicines.
The invention has the beneficial effects that:
1. the invention has the advantages that the refined flammulina velutipes polysaccharide with the liver protection effect is added on the basis of the traditional liver protection prescription, a new generation product with the liver protection effect is developed, the application of the flammulina velutipes is expanded, the flammulina velutipes is developed into a new raw material of a novel liver protection product from common food, the added value of the flammulina velutipes can be obviously improved, the product quality can be greatly improved, and the invention has obvious innovation.
2. According to the invention, the acute alcoholism test of mice proves that the traditional Chinese medicine composition has a remarkable anti-alcoholism effect, remarkably reduces the alcoholism rate, prolongs the tolerance time and shortens the sobering-up time.
3. According to the invention, the mouse acute alcoholic liver injury model verifies that the traditional Chinese medicine composition has a remarkable liver protection effect, can resist acute liver injury caused by ethanol, and effectively improves pathological changes caused by alcoholic liver injury. The traditional Chinese medicine composition can improve the activities of SOD and GSH of liver tissues, reduce the contents of MDA and TG, enhance the capability of organisms on resisting free radicals and have obvious liver protection effect on acute alcoholic liver injury.
4. The invention also verifies that the traditional Chinese medicine composition has obvious liver protection effect through a mouse acute carbon tetrachloride liver injury model, can resist acute liver injury caused by carbon tetrachloride, and effectively improves pathological changes caused by the carbon tetrachloride liver injury. The traditional Chinese medicine composition can obviously reduce ALT and AST activities and MDA levels of mice acute liver injury induced by carbon tetrachloride, and obviously improve hepatic histology pathological changes of the mice with liver injury.
5. The traditional Chinese medicine composition can promote liver regeneration after liver partial resection, improve liver weight ratio recovery capability, promote hepatocyte proliferation and effectively stimulate regeneration potential of residual hepatocytes through a mouse 70% hepatectomy model.
Drawings
FIG. 1 is a flow chart of the preparation process of the Chinese medicinal composition in the example.
FIG. 2 is an HPLC-ELSD chromatogram of the purified Flammulina velutipes polysaccharide of the example.
FIG. 3 is a pathological section of liver tissue of a mouse as an alcoholic liver injury model in example 1 (A: normal group; B: model group; C: administration group)
FIG. 4 is a pathological section of liver tissue of mouse as a model of liver injury by carbon tetrachloride in example 2 (A: normal group; B: model group; C: administration group)
FIG. 5 is a graph showing the results of the liver/body weight ratio of the mice after the hepatectomy of example 70% (P <0.01, compared with the model control; the results show that the liver regeneration rate of the Chinese medicinal composition is significantly higher on days 1 and 3 after the operation than the model control)
FIG. 6 shows the results of the ALT and AST values in the serum of the mice after the hepatectomy of 70% in example (P <0.01, compared with the model control; the results show that the liver index ALT and AST values of the Chinese medicinal composition are recovered on days 1 and 3 after the operation)
FIG. 7 is a graph showing the results of the proliferation of hepatocytes in mice after 70% hepatectomy in example (DAPI staining of nuclei, EDU labeling of proliferating nuclei; the results show that the Chinese medicinal composition significantly improves hepatocyte proliferation)
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of this application and the above-described drawings, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The following examples use the main instruments and materials:
the medicinal materials are as follows: radix puerariae, radix astragali, fructus schizandrae (Xian Sanjiang bioengineering Co., Ltd.), semen hoveniae (Shanxi Tiandi Yuan Biotech Co., Ltd.), needle mushroom (Zhengdong ecological agriculture development center in Dangery region in Zhenjiang City), radix salviae miltiorrhizae, poria cocos, fructus amomi, moringa seeds and liquorice.
An experimental instrument: XL-20B pulverizer (Guangzhou Xulan mechanical equipment Co., Ltd.), microwave extractor (Zhengzhou century Shuxike laboratory instruments Co., Ltd.), rotary evaporator (Shanghai Yarong Biochemical instruments Co., Ltd.), constant temperature water bath (Jiangsu Jintan medical instruments Co., Ltd.), vacuum drying oven (Shanghai Hengshi instruments Co., Ltd.), KQM-X4(/ B) type planetary ball mill, HPLC-ELSD (Agilent Co., Ltd.).
Preparation of refined flammulina velutipes polysaccharide
In the embodiment, the needle mushroom polysaccharide is extracted by a microwave extraction method, and then purified and separated by D101 macroporous resin and DEAE-52 cellulose to obtain the refined needle mushroom polysaccharide with high purity and uniform molecular weight, which comprises the following steps:
microwave extraction of needle mushroom crude polysaccharide: drying needle mushroom, pulverizing to 100 mesh, extracting with 15 times of water (W/W) as solvent, placing in microwave extractor with microwave power of 500W, extraction temperature of 70 deg.C, and extraction time of 40 min; collecting ultrasonic microwave extractive solution, filtering, concentrating under reduced pressure to one tenth of original volume, precipitating the concentrated solution with 95% ethanol until the final concentration of ethanol is 80%, standing overnight, centrifuging, collecting precipitate, washing the precipitate with anhydrous ethanol, diethyl ether and acetone, and vacuum drying to obtain crude Flammulina velutipes polysaccharide.
② removing protein from the needle mushroom crude polysaccharide by trichloroacetic acid method: under the ice bath condition, adding five times of volume of 5% trichloroacetic acid, stirring for 10min, standing overnight, adjusting pH to 7, filtering, concentrating, and lyophilizing to obtain refined needle mushroom crude polysaccharide.
③ purifying the D101 macroporous resin: d101 macroporous resin is pretreated, ultrasonic degasified, slowly filled into a chromatographic column (40 x 500mm), compacted by a constant flow pump, filled into 2/3 with the height of the column being equal to the column length, and fully eluted by distilled water for later use. Dissolving refined needle mushroom crude polysaccharide 2g in 20mL of water, centrifuging, collecting supernatant, slowly adding into chromatographic column, performing equilibrium adsorption for 20min, sequentially eluting with a certain volume of purified water and 0.1, 0.3 and 0.5M sodium chloride water solution at flow rate of 1mL/min, and collecting one tube per 10 mL. And (3) detecting the polysaccharide content in the eluent by a trace phenol-sulfuric acid method, and drawing an elution curve by taking the absorbance value as a vertical coordinate and the tube number as a horizontal coordinate. The same fractions were pooled according to the elution profile, concentrated, dialyzed and lyophilized.
Purifying DEAE-52 cellulose: after DEAE-52 pretreatment, the column is packed. Dissolving 500mg of main component separated from D101 macroporous resin in 10mL of water, slowly adding into DEAE-52 cellulose chromatographic column, and performing equilibrium adsorption for 20 min. Eluting with a certain volume of purified water and 0.1, 0.2 and 0.3M aqueous solution of sodium chloride sequentially at a flow rate of 1mL/min, and collecting one tube per 10 mL. And detecting the polysaccharide content in the eluent by a trace phenol-sulfuric acid method, drawing an elution curve, combining the same components, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain the refined flammulina velutipes polysaccharide with relatively uniform molecular weight.
Molecular weight determination of refined flammulina velutipes polysaccharide
And (3) measuring the molecular weight of the refined flammulina velutipes polysaccharide by a high performance liquid chromatography-evaporative light scattering (HPLC-ELSD) method.
Analyzing parameters: the instrument comprises the following steps: agilent 1260, ELSD detector (evaporation temperature 30 ℃, drift tube temperature 30 ℃, nitrogen flow rate 1.59L/min); mobile phase: 20mmol/L ammonium acetate aqueous solution (0.6 mL/min); a chromatographic column: TSK-gel column G4000PW (7.5 mm. times.300 mm), TSK-guard column PWH (7.5 mm. times.75 mm); column temperature: 30 ℃; the amount of sample was 20. mu.L.
Establishing a standard curve: respectively and precisely weighing 10mg of dextran standard substances (molecular weight of 5kDa, 25kDa, 80kDa, 270kDa and 670kDa) with different molecular weights to prepare standard substance solution of 1mg/mL, filtering through a 0.45-micrometer filter membrane, and injecting sample for analysis. Linear regression calculations were performed on retention times (T) and the natural logarithms (log (mw)) of the corresponding molecular weights for the different molecular weight standards to obtain a standard equation for standard molecular weight versus retention time of-1.21 × log (mw) + 28.35.
③ measuring the molecular weight of the refined flammulina velutipes polysaccharide: precisely weighing the refined flammulina velutipes polysaccharide, preparing into a standard solution of 1mg/mL, filtering through a 0.45-micron filter membrane, and carrying out sample injection analysis. The result is shown in figure 2, the refined Flammulina velutipes polysaccharide has single symmetric peak, the peak-off time is 13.3 minutes, the molecular weight of the polysaccharide is 31.7kDa when calculated by substituting into a standard curve.
Example 1
The preparation method of the traditional Chinese medicine composition with liver protection and regeneration effects is a mixture of traditional Chinese medicine extract and superfine powder, and comprises the following steps: respectively crushing kudzu vine root, astragalus membranaceus, schisandra chinensis and hovenia dulcis thunb into coarse powder, extracting for 2 times at 90 ℃ by adopting ethanol aqueous solution with the concentration of 70% in 10 times of the amount (w/w) of medicinal materials, 2 hours each time, combining extracting solutions, filtering, concentrating under reduced pressure, drying to prepare traditional Chinese medicine extract powder, and taking 5 parts of kudzu vine root extract, 2 parts of astragalus membranaceus extract, 2 parts of schisandra chinensis extract and 4 parts of hovenia dulcis thunb extract according to a formula; ② taking 1 part of the prepared high-purity and uniform molecular weight refined flammulina velutipes polysaccharide; pulverizing 1 part of poria cocos, 1 part of fructus amomi, 2 parts of moringa seeds and 1 part of liquorice into coarse powder, mixing the coarse powder of the traditional Chinese medicine in proportion, putting the mixture into a planetary ball mill, and carrying out dry grinding for 30 minutes without adding any anticaking agent and grinding aid to prepare traditional Chinese medicine superfine powder; the components obtained from the third step are mixed evenly to prepare the traditional Chinese medicine composition with the protective effect on chemical liver injury.
Example 2
The preparation method of the traditional Chinese medicine composition with liver protection and regeneration effects is a mixture of traditional Chinese medicine extract and superfine powder, and comprises the following steps: respectively crushing the kudzu root, the astragalus, the schisandra and the hovenia dulcis thunb into coarse powder, respectively extracting for 2 times at 90 ℃ by adopting ethanol aqueous solution with the concentration of 80 percent by weight (w/w) of 8 times of medicinal materials, 2 hours each time, combining extracting solutions, filtering, concentrating under reduced pressure, drying to prepare traditional Chinese medicine extract powder, and taking 8 parts of the kudzu root extract, 3 parts of the astragalus extract, 3 parts of the schisandra extract and 3 parts of the hovenia dulcis thunb extract according to a formula; 2 parts of the prepared high-purity and uniform-molecular-weight refined flammulina velutipes polysaccharide; ③ 1 part of liquorice is crushed into coarse powder, the coarse powder is put into a planetary ball mill after being mixed according to the proportion, and dry crushing is carried out for 45 minutes without adding any anticaking agent and grinding aid, thus preparing the traditional Chinese medicine superfine powder; the components obtained from the third step are mixed evenly to prepare the traditional Chinese medicine composition with the protective effect on chemical liver injury.
Example 3
The preparation method of the traditional Chinese medicine composition with liver protection and regeneration effects is a mixture of traditional Chinese medicine extract and superfine powder, and comprises the following steps: respectively crushing kudzu vine root, astragalus membranaceus, schisandra chinensis and hovenia dulcis thunb into coarse powder, extracting for 2 times at 95 ℃ by adopting ethanol water solution with the concentration of 70% in 10 times of the amount (w/w) of medicinal materials, extracting for 3 hours each time, combining the extracting solutions, filtering, concentrating under reduced pressure, drying to prepare traditional Chinese medicine extract powder, and taking 5 parts of kudzu vine root extract, 2 parts of astragalus membranaceus extract, 2 parts of schisandra chinensis extract and 4 parts of hovenia dulcis thunb extract according to a formula; ② taking 1 part of the prepared high-purity and uniform molecular weight refined flammulina velutipes polysaccharide; pulverizing 3 parts of vine tea, 1 part of poria cocos, 1 part of fructus amomi, 2 parts of moringa seed and 1 part of liquorice into coarse powder, mixing the coarse powder of the traditional Chinese medicine in proportion, putting the mixture into a planetary ball mill, and carrying out dry grinding for 30 minutes without adding any anticaking agent and grinding aid to prepare traditional Chinese medicine superfine powder; the components obtained from the third step are mixed evenly to prepare the traditional Chinese medicine composition with the protective effect on chemical liver injury.
Evaluation experiment for relieving or neutralizing the effect of alcohol of Chinese medicinal composition
The 38% ethanol solution is determined as the optimal dose for giving the alcohol, and the anti-inebriation effect of the traditional Chinese medicine composition in the example 1 is evaluated by observing the influence of the traditional Chinese medicine composition on the intoxication rate, tolerance time and sobering-up time of the acute alcoholism mice.
Animals: the ordinary-grade Kunming mouse is full-male, has the weight of 20 +/-2 g and is provided by a Qinglongshan animal breeding farm in Jiangning district of Nanjing City of Jiangsu province. The room temperature is 22 +/-2 ℃, and the humidity is 50-75%.
Group and medicine: mice were randomly divided into 3 groups: model control group, Chinese medicinal composition group, and positive control group, each group contains 15 animals. The traditional Chinese medicine composition comprises the following components: 10g/kg (body weight of mouse, the same applies hereinafter) was prepared with distilled water and used. Positive control group: 5g/kg of Hei Wang Jinzun (Shenzhen Shang health science and technology development Limited company) prepared with distilled water for later use.
Third, reagent: anhydrous ethanol: chemical reagent of national drug group, Inc., analytically pure. Distilled water is used for preparing a solution with the content of 38 percent for standby.
Influence of the traditional Chinese medicine composition on tolerance time and intoxication rate of acute alcoholism mice: after the mice are fasted for 12 hours, the traditional Chinese medicine composition group and the positive control group are both administrated once, the intragastric administration volume is 20mL/kg, and the model control group is administrated with distilled water with the same volume. After 30min, the 3 groups were gavaged with 38% ethanol solution at 20mL/kg, and the mice were observed and recorded for tolerance time (time from gavage with 38% ethanol solution to disappearance of mouse righting reflex) to calculate the intoxication rate within 4 h.
The influence of the traditional Chinese medicine composition on the sobering-up time of the acute alcoholism mice is as follows: the animals were divided into groups and administered as above, and each group had 12 animals. After fasting for 12h, the mice of each group are intragastrically filled with 38% ethanol solution 20mL/kg, after 30min, each intervention group is administered once, and the model control group is infused with distilled water with the same volume. The time to sober-up of the mice (time from after dosing until the mice righting reflex was recovered) was observed and recorded.
Sixthly, statistical method: data are expressed in mean ± SD and processed using the SPSS for Windows 13.0 statistical software package. The measured data are analyzed by unpaired sample t test, and the counted data are analyzed by chi test2And (5) checking, wherein the checking level alpha is 0.05.
The experimental result is that:
the traditional Chinese medicine composition can obviously reduce the drunkenness rate of acute alcoholism mice, prolong the tolerance time and shorten the sobering-up time: compared with the model control group, the traditional Chinese medicine composition group and the positive control group can obviously reduce the drunkenness rate of mice, obviously prolong the tolerance time of the mice and shorten the sobering time of the mice (P <0.01 or P < 0.05). The drunkenness rate, the tolerance time and the sobering-up time of the mice of the traditional Chinese medicine composition group are all better than those of a positive control group, and the results are shown in table 1.
TABLE 13 comparison of intoxication rate, duration of tolerance and time to sober-up between groups
Note: compared with the model control group,*P<0.05，**P<0.01；
protective effect of traditional Chinese medicine composition on alcoholic liver injury
The liver protection effect of the traditional Chinese medicine composition is evaluated by establishing an acute alcoholic liver injury model. The composition of example 1 was selected and administered prophylactically for 12 d. 13d, the mice are subjected to one-time intragastric administration of 50% ethanol solution (12mL/kg) to form an acute alcoholic liver injury mouse model. After 12h of damage, the liver protection effect of the traditional Chinese medicine composition is further evaluated by measuring indexes such as Malondialdehyde (MDA), superoxide dismutase (SOD), reduced Glutathione (GSH), serum Triglyceride (TG) and the like in liver tissues of mice.
Animals: the ordinary-grade Kunming mouse is full-male, has the weight of 20 +/-2 g and is provided by a Qinglongshan animal breeding farm in Jiangning district of Nanjing City of Jiangsu province. The room temperature is 22 +/-2 ℃, and the humidity is 50-75%.
Grouping and administration: 84 mice were randomly divided into 7 groups: blank control group, model control group, high (low) dosage group of Chinese medicinal composition, semen Hoveniae polysaccharide group, pollen powder group, and positive control group, each group contains 12 animals. The specific administration dose is as follows, the traditional Chinese medicine composition is used in a high dose group, and the dosage is 0.7g/kg (the body weight of a mouse, the same below); the traditional Chinese medicine composition is used in a low-dose group, and the dosage is 0.35 g/kg; 0.5g/kg of raisin tree seed polysaccharide group; 0.5g/kg of pueraria pollen powder; positive control group, 0.5 g/kg; the traditional Chinese medicine composition is prepared according to example 1. The positive control group is prepared with Hewang Jinzun (Shenzhen Shanwang health science and technology development Co., Ltd.) and distilled water. The test sample is orally gavaged once every 9: 00-10: 00 days, and the blank control group and the model control group are given distilled water (20mL/kg) with the same volume for 12 days.
Reagent and instrument: anhydrous ethanol: chemical reagent of national drug group, Inc., analytically pure; SOD kit, GSH kit, MDA kit: nanjing is built into a bioengineering institute; TG kit: the other reagents are analytically pure, as determined by Wenzhou Jinma Biotechnology Ltd; UV-2401PC ultraviolet spectrophotometer (Shimadzu, Japan); biofuge bench high speed refrigerated centrifuge (Heraeus, germany); fluko FA25 high speed shears (shanghai frank fluid machines manufacturing ltd); a constant temperature oscillation water bath kettle (medical instrument factory in Jintan city, Jiangsu province).
Establishing and obtaining a model: and after the last administration, performing primary intragastric lavage on the model control group and the test group, administering ethanol (12mL/kg) with the volume fraction of 50%, administering equal amount of distilled water to the blank control group, killing animals after fasting for 12h, taking blood to separate serum, refrigerating TG to be tested, performing laparotomy, taking out the liver, immediately fixing a part of the liver by 10% formalin, embedding the part in conventional paraffin, slicing the part, performing normal HE staining, and observing the tissue morphology of the liver under a light microscope. Washing another part of liver with ice-cold physiological saline, absorbing moisture with filter paper, weighing about 0.3g of wet weight, preparing 10% liver tissue homogenate in ice bath, centrifuging at 3000rpm for 15min, collecting supernatant, storing at below 4 deg.C, and detecting SOD, GSH, and MDA.
The statistical method comprises the following steps: data are expressed in mean ± SD and processed using the SPSS for Windows 13.0 statistical software package. The measurement data is analyzed by single-factor variance, and the inspection level alpha is 0.05.
Sixthly, the experimental result is as follows:
a. the traditional Chinese medicine composition can improve the activities of SOD and GSH of liver tissues: compared with the blank control group, the SOD content and the GSH content of the model control group are obviously reduced (P < 0.05). Compared with the model control group, the traditional Chinese medicine composition high and low dose group and the positive control group can obviously improve the SOD and GSH content (P <0.05 or P <0.01) in the body of the mouse. In addition, the single component of hovenia dulcis thunb polysaccharide and pueraria lobata powder can also improve SOD and GSH values, but the effect is not as good as that of the traditional Chinese medicine composition group, and the results are shown in table 2.
b. The traditional Chinese medicine composition can reduce the contents of liver tissue MDA and serum TG: the model control group had significantly higher MDA and TG levels (P <0.05) compared to the blank control group. Compared with a model control group, the traditional Chinese medicine composition high and low dose group and the positive control group can obviously reduce the MDA content (P <0.05 or P <0.01) in the mouse body, the traditional Chinese medicine composition high and low dose group can obviously reduce the TG content (P <0.05) in the mouse body, and the positive control group has no statistical significance. Similarly, the single component of hovenia dulcis thunb polysaccharide and pueraria lobata powder group can also improve the MDA and TG levels, but the effect is not as good as that of the traditional Chinese medicine composition group, and the results are shown in Table 2.
c. Pathological section of liver tissue: pathological sections are carried out on liver tissues of mice in a normal group, a model group and a traditional Chinese medicine composition group, as shown in figure 3, the liver lobule structure of the normal group is clear, liver cells have no obvious pathological changes, cytoplasm is rich, and the nuclear structure is clear. The normal tissue structure of the liver of the model group disappears, the liver sinuses disappear, and the cytoplasm of the liver cells is loose, so that balloon-like degeneration can be seen. The traditional Chinese medicine composition has clear liver lobule structure, no obvious abnormality of liver funicle and liver sinus, and uniform cytoplasm staining.
The experiment further proves that the traditional Chinese medicine composition can effectively relieve acute liver injury caused by ethanol and improve the pathological change degree of alcoholic liver injury. Can remarkably reduce the inhibiting effect of alcohol on SOD and GSH, improve the SOD activity in a body, increase the GSH content, and further effectively eliminate the attack of free radicals generated by excessive drinking on biomacromolecules, thereby realizing the protective effect on the liver. In addition, the composition can also accelerate the removal of free radicals generated in the ethanol metabolism process, reduce the lipid peroxidation level and reduce the generation of TG in serum. In addition, the observation result of a liver histopathological section by a light microscope shows that the traditional Chinese medicine composition can obviously improve the pathological changes of liver histology of a liver injury model mouse.
Comparison of acute alcoholic liver injury SOD, GSH, MDA, TG between groups in Table 27
Note: compared with the model control group,*P<0.05，**P<0.01; compared with the positive control group, the composition has the advantages that,▲P<0.05。
protective effect of traditional Chinese medicine composition on carbon tetrachloride liver injury
Establishing an acute carbon tetrachloride liver injury model, and evaluating the liver protection effect of the traditional Chinese medicine composition. The Chinese medicinal composition of example 2 was selected for prophylactic administration for 18 days. On day 19, mice were gavaged once with a 0.5% carbon tetrachloride solution (10mL/kg) to form an acute carbon tetrachloride liver injury mouse model. The liver protection effect of the traditional Chinese medicine composition is further evaluated by measuring three indexes of ALT, AST and liver tissue MDA in the serum of a mouse.
Animals: the ordinary-grade Kunming mouse is full-male, has the weight of 20 +/-2 g and is provided by a Qinglongshan animal breeding farm in Jiangning district of Nanjing City of Jiangsu province. The room temperature is 22 +/-2 ℃, and the humidity is 50-75%.
Grouping and administration: the 84 mice were divided into 7 groups at random, a blank control group, a model control group, a high (low) dose group of the traditional Chinese medicine composition, a high (low) dose group of the positive control, and a needle mushroom polysaccharide group, wherein each group contains 12 mice. The specific administration dose is as follows, the traditional Chinese medicine composition is used in a high dose group, and the dosage is 0.7g/kg (the body weight of a mouse, the same below); the traditional Chinese medicine composition is used in a low-dose group, and the dosage is 0.35 g/kg; positive control high dose group, 0.7 g/kg; positive control low dose group, 0.35 g/kg; 0.5g/kg of flammulina velutipes polysaccharide group. The traditional Chinese medicine composition is prepared according to example 3. The positive control group is prepared with Hewang Jinzun (Shenzhen Shanwang health science and technology development Co., Ltd.) and distilled water. The test sample is orally gavaged once every 9: 00-10: 00 days, and the blank control group and the model control group are given distilled water (20mL/kg) with the same volume for 18 days.
Reagent and instrument: carbon tetrachloride: and 5, analyzing and purifying by Jiangsu Xuzhou reagent II factory. Preparing a solution with the content of 0.5% by using sesame oil for later use; ALT, AST kit: biosun corporation, usa; SOD kit, GSH kit, MDA kit: nanjing is built into a bioengineering institute; the other reagents are analytically pure; UV-2401PC ultraviolet spectrophotometer (Shimadzu, Japan); biofuge bench high speed refrigerated centrifuge (Heraeus, germany); fluko FA25 high speed shears (shanghai frank fluid machines manufacturing ltd); a constant temperature oscillation water bath kettle (medical instrument factory in Jintan city, Jiangsu province).
Establishing and obtaining a model: after the administration on the 18 th day of the experiment, animals in each group are fasted for 16h, 10mL/kg of 0.5% carbon tetrachloride sesame oil solution is administered to the model group and each test group through oral gavage at one time, the same amount of sesame oil is administered to the blank control group, and the test group continues to administer the test sample until the end of the experiment (the interval between the test sample and the carbon tetrachloride gavage is more than 4 h). Killing the animals 24h after contamination, taking blood to separate serum, and refrigerating AST and ALT to be detected; the liver was removed by rapid laparotomy, and the portion was immediately fixed with 10% formalin, embedded in normal paraffin, sectioned, stained with normal HE, and the morphology of the liver tissue was observed under light. Another part of liver is washed by normal saline, filter paper absorbs moisture, the wet weight is weighed to be about 0.2g, 10% liver tissue homogenate is prepared under ice bath, centrifugation is carried out for 15min at 3000rpm, supernatant is taken, and the supernatant is stored at 4 ℃ and is reserved for MDA detection.
The statistical method comprises the following steps: data are expressed in mean ± SD and processed using the SPSS for Windows 13.0 statistical software package. The measurement data was measured by a rank sum test (Mann-Whitney U), with a test level α of 0.05.
Sixthly, the experimental result is as follows:
a. the traditional Chinese medicine composition can reduce the contents of ALT and AST in serum: compared with a blank control group, the serum ALT and AST content of the model control group mice is obviously increased (P <0.01 or P < 0.05). Compared with the model control group, the high-dose group and the positive control group of the traditional Chinese medicine composition can reduce ALT and AST (P is less than 0.01 or P is less than 0.05) of the serum of mice, and in addition, the single component of the flammulina velutipes polysaccharide can also reduce ALT and AST, but the improvement effect is not as good as that of the traditional Chinese medicine composition, and the results are shown in Table 3.
b. The traditional Chinese medicine composition can reduce the MDA content of liver tissues: compared with a blank control group, the liver tissue of the mouse of the model control group has obviously increased MDA content (P < 0.05). Compared with a model control group, the high dose of the traditional Chinese medicine composition can reduce the MDA content in liver tissues of mice (P <0.05), and the rest groups have no statistical significance (P > 0.05); the high-dose group of the traditional Chinese medicine composition has a remarkably reduced MDA effect (P is less than 0.05) compared with the positive high-dose group, and the results are shown in a table 3.
TABLE 37 interclass CCl4Comparison of ALT, AST and MDA for inducing acute liver injury of mice
Note: compared with the model control group,*P<0.05，**P<0.01; compared with the positive high-dose group,▲P<0.05。
c. pathological section of liver tissue: the normal group has clear hepatic lobule structure, no obvious pathological changes of hepatic cells, rich cytoplasm and clear nuclear structure. The normal tissue structure of the liver of the model group disappears, the liver sinuses disappear, and the cytoplasm of the liver cells is loose, so that balloon-like degeneration can be seen. The traditional Chinese medicine composition has clear liver lobule structure, no obvious abnormality of liver funicle and liver sinus, and uniform cytoplasm staining.
The pharmacodynamic experiments show that the traditional Chinese medicine composition has a protective effect on the mouse acute liver injury induced by carbon tetrachloride and improves the pathological changes of liver histology of a liver injury model mouse. After the high-dose group of the traditional Chinese medicine composition is continuously perfused for 18 days, ALT, AST, activity and MDA level of mice can be obviously reduced, and the damage of carbon tetrachloride to the liver is reduced. The observation result of a liver histopathological section by a light microscope shows that the traditional Chinese medicine composition can obviously improve the pathological changes of liver histology of a liver injury model mouse.
Protection effect of traditional Chinese medicine composition on 70% liver resection injury
And establishing a 70% hepatic resection injury model, and evaluating the hepatic regeneration effect of the traditional Chinese medicine composition. After hepatectomy, the Chinese medicinal composition of example 3 was administered for 5 consecutive days. The liver regeneration effect of the traditional Chinese medicine composition is further evaluated by measuring ALT and AST indexes in mouse serum, a liver/body weight ratio and an EDU proliferation test.
Animals: common grade ICR mice, male, weighing 20 + -2 g, were provided by the Experimental animals center of Jiangsu university. The room temperature is 22 +/-2 ℃, and the humidity is 50-75%.
Reagent and instrument: ALT and AST kits, Nanjing, to build a bioengineering institute; EdU cell proliferation assay kit, department of biotechnology, sharp bo, guangzhou; the other reagents are analytically pure; microplate reader (BioTek, usa); biofuge bench high speed refrigerated centrifuge (Heraeus, germany); analytical electronic balance (beijing sidoris instrument systems ltd).
Establishing a model: mice were divided randomly into 3 groups, sham operated control, hepatectomy, and hepatectomy/Chinese medicinal composition high, 15 mice per group. The classical 70% hepatectomy operation is performed as follows: performing abdominal injection anesthesia with 10% chloral hydrate, sterilizing the abdomen, taking a vertical incision in the middle of the lower abdomen of the xiphoid process, wherein the length of the incision is 2.5cm, the abdominal organ tissues are not damaged when the abdomen is opened, sequentially cutting the epidermis, the muscular layer and the peritoneum to find the liver, pulling open and fixing the abdominal muscles at two sides by using forceps, and fully exposing the liver; the ophthalmology cuts and dissociates ligaments related to the liver, ligates the liver middle leaf and the left outer leaf in sequence and excises the liver middle leaf and the left outer leaf, and pays attention to the preservation of the gallbladder; the rest liver tissue and the abdominal contents are collected, the abdomen is closed by suturing in sequence, and the incision is disinfected again after suturing. And fasting and water prohibition are carried out for 6h after operation. Subcutaneously injecting 5% glucose injection to replace evaporation loss in operation, and placing in a 37 deg.C incubator for 6 h; the anesthesia, abdominal opening, abdominal closing and postoperative treatment of the sham operation group are the same as those of the hepatectomy group, and the operation time is kept consistent as much as possible.
Fourthly, administration and material drawing: traditional Chinese medicine composition group, 0.5g/kg, the traditional Chinese medicine composition was prepared according to example 4. After surgery, the test sample is orally gavaged once at a rate of 9: 00-10: 00 every day, and distilled water (20mL/kg) with the same volume is given to a sham-operated control group and a liver resection group for 5 days. Mice were intraperitoneally injected with EDU (50mg/kg) for 4h prior to sacrifice to mark newly proliferating nuclei. Taking the operation end as 0h, killing the animals at 1d, 3d and 5d after the operation according to the grouping time point requirements, taking blood, separating serum, and refrigerating AST and ALT to be detected. Weighing and recording the weight of each group of regenerated livers, and calculating the liver/body weight ratio by the following formula: liver/body weight ratio (regenerated liver weight)/postoperative body weight × 100%. Another part of the liver was immediately fixed with 10% formalin, dehydrated with sucrose, frozen and sectioned, EDU stained, and observed under a fluorescence inverted microscope.
The statistical method comprises the following steps: data are expressed in mean ± SD and processed using the SPSS for Windows 13.0 statistical software package. The measurement data was measured by a rank sum test (Mann-Whitney U), with a test level α of 0.05.
Sixthly, the experimental result is as follows:
a. the traditional Chinese medicine composition can obviously improve the liver/body weight ratio: the results are shown in fig. 5, and the liver weight ratio of the mice gradually increased with time after hepatectomy. The traditional Chinese medicine composition group can remarkably accelerate the recovery of the liver weight ratio of a hepatectomized mouse (P is less than 0.01).
b. The traditional Chinese medicine composition can reduce the contents of ALT and AST in serum: compared with a blank control group, the serum ALT and AST content of the mice of the liver resection control group is obviously increased (P is less than 0.01) 1 and 3 days after operation. Compared with the hepatectomy group, the traditional Chinese medicine composition group can obviously reduce the serum ALT and AST (P <0.01) of the mice, and the result is shown in figure 6.
c. The traditional Chinese medicine composition can promote the proliferation of mouse liver cells: the results are shown in FIG. 7, where the mouse hepatocytes were partially proliferated but less abundant after hepatectomy. The traditional Chinese medicine composition group obviously improves the number of EDU positive cell nucleuses in the liver cells, and shows that the traditional Chinese medicine composition group improves the proliferation of the liver cells after the hepatectomy and has obvious promotion effect on the regeneration of the liver cells after the hepatectomy.
The invention fully utilizes the traditional Chinese medicine theory, has novel concept, simple process, high extraction efficiency, low production cost and obvious effect of protecting the liver, and can be used for preparing the liver-protecting medicine.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (10)
1. A traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function is characterized in that: the traditional Chinese medicine composition comprises the following components in parts by mass: 5-20 parts of kudzu root extract, 2-10 parts of astragalus extract, 2-8 parts of schisandra extract, 1.5-20 parts of hovenia dulcis thunb extract and 0.5-2 parts of refined flammulina velutipes polysaccharide.
2. The traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function according to claim 1, is characterized in that: the paint also comprises the following components in parts by mass: 1-10 parts of salvia miltiorrhiza, 1-10 parts of vine tea, 1-2 parts of poria cocos, 1-4 parts of fructus amomi, 5-10 parts of moringa seeds and 1-10 parts of liquorice.
3. A method for preparing a Chinese medicinal composition for protecting against chemical liver injury and promoting liver regeneration according to claim 1 or 2, which comprises the following steps:
step 1: pulverizing radix Puerariae, radix astragali, fructus Schisandrae chinensis, and semen Hoveniae respectively to coarse powder, adding polar organic solvent water solution, extracting, mixing extractive solutions, filtering, concentrating under reduced pressure, and drying to obtain Chinese medicinal extract powder;
step 2: pulverizing needle mushroom, microwave extracting with water as solvent to obtain needle mushroom crude polysaccharide, and purifying with macroporous resin and cellulose column to obtain refined needle mushroom polysaccharide;
and step 3: the components obtained in the steps 1 and 2 are uniformly mixed according to the proportion of claim 1 to prepare the traditional Chinese medicine composition which has the effects of protecting chemical liver injury and promoting liver regeneration.
4. The production method according to claim 3, characterized in that: the specific steps for preparing the traditional Chinese medicine extract powder in the step 1 are as follows: respectively crushing kudzu vine root, astragalus, schisandra chinensis and hovenia dulcis thunb to 100 meshes, extracting for 1-3 times at 50-90 ℃ by using a polar organic solvent aqueous solution with the mass concentration of 40-80% and the weight of 8-16 times of the medicinal materials, wherein the mass concentration of the polar organic solvent aqueous solution is 1-3 hours each time, combining the extracting solutions, filtering, concentrating under reduced pressure, and drying to prepare traditional Chinese medicine extract powder; the drying mode is spray drying, freeze drying, microwave drying or vacuum drying; the polar organic solution is one or more than two mixed solvents of methanol, ethanol or acetone.
5. The production method according to claim 3, characterized in that: the method also comprises the steps of crushing the salvia miltiorrhiza, the vine tea, the poria cocos, the fructus amomi, the moringa seeds and the liquorice into coarse powder, mixing the coarse powder in proportion, carrying out superfine grinding, adding the mixture into the components prepared in the steps 1 and 2, and mixing the mixture uniformly in proportion.
6. The production method according to claim 3, characterized in that: the preparation of the refined flammulina velutipes polysaccharide in the step 2 comprises the following specific steps:
step 2.1: drying needle mushrooms, crushing to 100 meshes, putting the needle mushrooms into a microwave extractor with water as a solvent, wherein the water is 12-20 times of the mass of medicinal materials, and extracting for 30-60 min at the microwave power of 400-600W and the extraction temperature of 50-90 ℃; collecting ultrasonic microwave extractive solution, filtering, concentrating under reduced pressure to one tenth of original volume, precipitating the concentrated solution with 95% ethanol until the final concentration of ethanol is 80%, standing overnight, centrifuging, collecting precipitate, washing the precipitate with anhydrous ethanol, diethyl ether and acetone, and drying to obtain crude Flammulina velutipes polysaccharide;
step 2.2: removing protein from the crude polysaccharide by trichloroacetic acid method, adding 5% trichloroacetic acid five times the volume of the crude polysaccharide under ice bath condition, stirring for 10min, standing overnight, adjusting pH to 7, filtering, concentrating, and lyophilizing to obtain refined crude polysaccharide;
step 2.3: and further purifying the refined crude polysaccharide through D101 macroporous resin and DEAE-52 cellulose to obtain the refined flammulina velutipes polysaccharide.
7. The method of claim 6, wherein: the purification of the refined crude polysaccharide in the step 2.3 specifically adopts the following steps:
(1) dissolving refined crude polysaccharide with water 8-15 times of the weight of the medicinal materials, adding the obtained solution into a D101 macroporous resin chromatographic column, carrying out equilibrium adsorption for 20-40 min, eluting with a certain volume of purified water and 0.1, 0.3 and 0.5mol/L sodium chloride aqueous solution in sequence, and quantitatively collecting; detecting polysaccharide content in the eluate by using a trace phenol-sulfuric acid method, drawing an elution curve, combining the same components, concentrating under reduced pressure, dialyzing, and freeze-drying;
(2) dissolving main components separated from the D101 macroporous resin in water, adding the main components into a DEAE-52 cellulose chromatographic column, sequentially eluting with a certain volume of purified water and 0.1, 0.2 and 0.3mol/L sodium chloride aqueous solution, quantitatively collecting, detecting the polysaccharide content in an eluent by adopting a trace phenol-sulfuric acid method, drawing an elution curve, combining the same components, concentrating under reduced pressure, dialyzing and freeze-drying to obtain the refined flammulina velutipes polysaccharide with relatively uniform molecular weight.
8. The production method according to claim 3, characterized in that: the molecular weight of the refined flammulina velutipes polysaccharide prepared in the step 2 is 31.7kDa by a high performance liquid chromatography-evaporative light scattering method, and the peak-off time is 13.3 minutes.
9. Use of the composition of claim 1 or 2 in the preparation of a medicament for protecting liver against chemical liver injury or promoting liver regeneration.
10. Use according to claim 9, characterized in that: the traditional Chinese medicine composition also comprises a medically allowable carrier, and the traditional Chinese medicine composition is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, capsules, hard capsules, soft capsules, sustained-release capsules, oral liquid, mixtures, buccal agents, granules, medicinal granules, pills, powder, ointment, pellets, suspensions, solutions, injections, powder injections, freeze-dried powder injections, suppositories, ointments, plasters, creams, sprays, drops, dripping pills or patches.
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