CN115245533A - Application of south mountain flower root extract in pulmonary fibrosis treatment - Google Patents

Application of south mountain flower root extract in pulmonary fibrosis treatment Download PDF

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CN115245533A
CN115245533A CN202110459959.1A CN202110459959A CN115245533A CN 115245533 A CN115245533 A CN 115245533A CN 202110459959 A CN202110459959 A CN 202110459959A CN 115245533 A CN115245533 A CN 115245533A
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pulmonary fibrosis
flower root
mountain flower
south mountain
extracting
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CN115245533B (en
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靳洪涛
张金兰
胡广
生宁
李思铮
李梦林
江海燕
王冬梅
王喆
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Institute of Materia Medica of CAMS
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Abstract

The invention belongs to the field of medicines, and relates to application of a south mountain flower root extract in preparation of a medicine for treating pulmonary fibrosis. The effective part for resisting pulmonary fibrosis is discovered for the first time, has clear active ingredients and comprehensive effect of treating pulmonary fibrosis, has the effect of treating pulmonary fibrosis obviously superior to that of the prior art, and can generate synergistic interaction with chemical drugs for resisting pulmonary fibrosis. According to the specific effective compounds in the extracts, the synergistic effect between the compounds is found, and the corresponding pharmaceutical composition for treating pulmonary fibrosis is disclosed.

Description

Application of south mountain flower root extract in pulmonary fibrosis treatment
Technical Field
The invention belongs to the field of medicines, and particularly relates to an extraction part extracted from a traditional Chinese medicine, namely, a south mountain flower root and application of a compound in preventing and treating pulmonary fibrosis diseases.
Background
Pulmonary Fibrosis (PF) is a disease with diffuse exudation, infiltration and fibrosis of pulmonary interstitium as main pathological changes, and its main symptoms include dyspnea, shortness of breath, dry cough, dyspnea and suffocation, and in severe cases, the patient can die due to exhaustion of breath. The etiology of most patients with pulmonary fibrosis is unknown (idiopathic), and this group of diseases is called Idiopathic Interstitial Pneumonia (IIP), which is a large group of interstitial lung diseases. The most common disease type in idiopathic interstitial pneumonia, which has pulmonary fibrosis as a main manifestation, is Idiopathic Pulmonary Fibrosis (IPF), which is a serious interstitial lung disease that can cause progressive loss of lung function. The pathogenesis is still unclear at present, and only two drugs specialized for the treatment of idiopathic pulmonary fibrosis are on the market worldwide. Pirfenidone (Pirfenidone) was first used in japan for IPF treatment in 2008, and Pirfenidone and Nintedanib (Nintedanib) were approved by the U.S. FDA for specific treatment of IPF in 2014. However, these two drugs also do not reverse or prevent the progression of IPF, are expensive, and place a significant burden on the patient and the family.
Pulmonary fibrosis can be divided into partial manifestations of primary lung disease and systemic disease. (1) pulmonary fibrosis as types of primary lung diseases are: radiation fibrosis (i.e., fibrosis caused by radioactivity), fibrosis as a result of an injurious inflammatory lung disease (e.g., sclerosing tuberculosis, alveolar lung, cystic lung fibrosis), fibrosis as a result of anaphylactic reactions, interstitial fibrosis due to drugs, terminal manifestations of fibrosis as interstitial inflammation, etc.; (2) pulmonary fibrosis is a partial manifestation of systemic diseases: granulomatosis (such as sarcoidosis, amyloidosis, nevus hamartoma disease, and xanthomatosis), collagen diseases, and rheumatic diseases (such as scleroderma, lupus erythematosus, periarteritis nodosa, dermatomyositis, and rheumatoid arthritis).
In recent years, chinese medical workers find effective drugs for treating and delaying pulmonary fibrosis from traditional Chinese medicines to achieve great achievements, and searching the drugs for treating pulmonary fibrosis from natural sources is considered to be an effective way for developing the drugs.
The south mountain flower is also called Pistacia chinensis. Is root of south mountain flower (Prismatomeris tetrandra (Roxb.) K.Schum) of Rubiaceae, and is one of special medicinal materials in Guangxi province. Has the effects of strengthening root and bone, promoting diuresis, removing jaundice, removing blood stasis, promoting tissue regeneration, cooling blood and stopping bleeding.
In recent years, researchers find that the south mountain flower root extract can inhibit liver fibrosis by inducing apoptosis of rat astrocytes. However, there is no report on the use of the extract of the root of south mountain flower or the compound derived from the root of south mountain flower for pulmonary fibrosis resistant activity and purposes. The prior art (China new drug journal, volume 25 and phase 24 in 2016, pages 2868-2869) discloses that an ethyl acetate extraction part and a water part of an ethanol extract of south mountain flower roots can be active parts for resisting hepatic fibrosis, and stigmast-4-en-3-one, nordamnacanthal and other compounds can be obtained by separation and purification from the ethyl acetate extraction part and the water part, but no evidence indicates that the compounds identified by separation and purification are active components and the application of the compounds is for resisting hepatic fibrosis. The prior art (journal of Chinese comparative medicine, volume 30, stage 2, pages 9-14) discloses that scopoletin can protect liver of a rat suffering from hepatic fibrosis, positively regulate and control the expression of cytokines related to hepatic fibrosis, reduce the level of an extracellular matrix of the liver, improve the degree of pathological changes of the hepatic fibrosis of the rat, and has the effect of resisting the hepatic fibrosis. The prior art (journal of Beijing university of traditional Chinese medicine, 32 vol. No. 1, page 42-44) discloses that the south mountain flower root alcohol extract can obviously reduce the proliferation and deposition of collagen fibers in rat liver and reduce the area of fibrotic tissues, and has certain development value in the aspect of hepatic fibrosis treatment. However, the above uses are against liver fibrosis, and the mechanism of fibrosis occurrence in different organs is different. Clinically, liver fibrosis is fibrosis caused by bacterial virus infection, toxin damage, metabolic abnormality and the like, and the liver fibrosis is considered to be a reversible process at present. The pulmonary fibrosis is considered to be an irreversible process, the lung is directly communicated with the outside, the cause of the pulmonary fibrosis is more complex, and the etiology of most patients is unknown. In view of the prior art, many drugs have the effect of preventing and treating liver fibrosis, but the drugs with the effect of preventing and treating lung fibrosis are very rare, as mentioned above, only pirfenidone and nintedanib are known to be used for lung fibrosis at present, that is, the drugs for preventing and treating liver fibrosis cannot be generally used for preventing and treating lung fibrosis.
Guangxi Yingkang pharmaceutical industry Limited liability company has developed tablets prepared from south mountain flowers for treating silicosis, and the indications are 'treating silicosis', and the preparation method comprises the steps of heating and refluxing with 60% ethanol for three times, each time for 2 hours, combining the extracting solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating into thick paste, stirring with 10 times of warm water for three times, standing, filtering, concentrating the filtrate under reduced pressure, vacuum drying, crushing, adding an appropriate amount of auxiliary materials, granulating, pressing into 1000 tablets, and coating sugar coatings. However, silicosis is known to be a lung disease caused by inhaled silicon dioxide or silica and its crystalline forms such as quartz and other less common forms such as cristobalite and fossilizid, and its rapidly progressive form behaves similarly to interstitial pneumonia, not equally to pulmonary fibrosis. Next, it is considered in the prior art that the main active ingredient of the tablet is a polymer formed by a plurality of metal elements mainly including organic aluminum, and the action mechanism is that aluminum forms insoluble aluminum silicate on the surface of silicon dioxide, so that the aluminum silicate loses the toxic effect on macrophages, thereby playing a role in antagonizing the quartz cell toxic effect (see Li Yu, a master edition, mine dust and occupational hazard prevention and control technology, metallurgical industry press, 2017, 10 months, page 211). However, the inventors have unexpectedly found that the effective ingredients in the prevention and treatment of pulmonary fibrosis are different from those in the treatment of silicosis, and found that the effective ingredients for the prevention and treatment of pulmonary fibrosis are preferable through a large number of studies and experiments.
The prior art (Chinese herbal medicine, vol. 51, no. 4, p. 1031-1036) discloses that polysaccharides from south mountain root can reduce inflammation level by inhibiting inflammatory cell infiltration, down-regulate expression level of alpha-SMA and Vimentin in lung tissue, and up-regulate expression level of E-cadherin, which suggests that the xanthorrhiza polysaccharides can effectively improve immune balance, inhibit development of inflammatory response and EMT, thereby improving fibrosis degree of silicosis tissue and having better therapeutic effect on silicosis fibrosis. The polysaccharide is prepared by pulverizing decoction pieces 10kg, ultrasonic extracting with hot water, concentrating under reduced pressure, and precipitating with 80% ethanol to obtain crude polysaccharide 905g. The crude polysaccharide was redissolved in distilled water, protein removed by the Savage method, passed through filter membrane U at high pressure, concentrated by ultrafiltration membrane a and removed of low relative molecular mass components, and the concentrate dried to yield 560g of polysaccharide. The above contents suggest that the polysaccharide component is an active component of south mountain flower root for preventing and treating pulmonary fibrosis. However, the above experiments did not compare the effectiveness of the polysaccharide and the non-polysaccharide components in the south mountain flower root, and the inventors found that the polysaccharide component is not the main effective component of the south mountain flower root in preventing and treating pulmonary fibrosis after intensive research.
The invention provides a method for finding an effective part with definite effect on preventing and treating pulmonary fibrosis from south mountain flower roots and providing pharmaceutical application thereof.
Disclosure of Invention
The invention aims to provide a new pharmaceutical application of a south mountain flower root extract, namely the application of the south mountain flower root extract in preparing a medicament for treating pulmonary fibrosis. The effective part of the extract for resisting pulmonary fibrosis is discovered for the first time, and the extract has the advantages of simple preparation method, clear effective part, clear active ingredients, comprehensive effect of treating pulmonary fibrosis, obvious effect of treating pulmonary fibrosis superior to that of the prior art, capability of generating synergistic interaction with chemical drugs for resisting pulmonary fibrosis and the like.
In the present invention, the "effective part" refers to an extract with relatively definite ingredients composed of one or more kinds of ingredients extracted from a single plant, and may also be referred to as an effective ingredient or an effective intermediate (the guidelines (trial) on the quality research techniques of new drugs in traditional Chinese medicine, 1 month in 2021). The term "anthraquinone, anthraquinone glycoside and iridoid effective fraction" as used herein means that the compounds of anthraquinone, anthraquinone glycoside and iridoid components in the extract are the chemical components that are mainly used in the composition structure of the extract and exert the main therapeutic action, and the extract may contain other components derived from the same plant, but these other components are usually not chemical components that can exert the therapeutic action in the extract, because their content is lower than the threshold value for exerting the therapeutic action or they do not have the desired therapeutic effect.
Furthermore, the invention aims to provide a preparation method of the effective part of the south mountain flower root.
Further, the invention aims to provide a pharmaceutical composition for treating pulmonary fibrosis, which consists of active ingredients of anthraquinone, anthraquinone glycoside and iridoid of southern mountain flower root.
Still further, it is an object of the present invention to provide a pharmaceutical composition for the treatment of pulmonary fibrosis, which consists of scopoletin and methylisoidin-1-methyl ether.
Furthermore, the present invention aims at providing a pharmaceutical composition for treating pulmonary fibrosis, which combines the south mountain flower root extract with the chemical drug pirfenidone or nintedanib for resisting pulmonary fibrosis, and has obvious synergistic effect.
In order to achieve the purpose, the invention provides the following specific technical scheme:
the application of the south mountain flower root extract in preparing the medicine for preventing and treating pulmonary fibrosis is characterized in that: the extract of the south mountain flower root is effective parts of anthraquinone, anthraquinone glycoside and iridoid. Preferably, the content of the active ingredients of anthraquinone, anthraquinone glycoside and iridoid in the extract accounts for more than 60 percent, more preferably more than 70 percent and most preferably 100 percent of the content of the extract by weight.
Preferably, the anthraquinone effective components comprise 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methyl isoalizarin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy-3-hydroxymethylanthraquinone, desmethyl damnacanthal, methyl isoalizarin-1-methyl ether, damnacanthal, 1,3-dihydroxy-2-methoxymethylanthraquinone, 3-hydroxy-1-methoxy-2-hydroxymethylanthraquinone, 1,3-dihydroxy-2-ethoxymethylanthraquinone, 1-hydroxy-2,3-dimethoxy-7-methylanthraquinone, 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5,6-dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, 1,3-dihydroxy-6-methoxy-2-methoxymethylanthraquinone, scopoletin, 3,5-dihydroxy-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 3-hydroxy-1,5,6-trimethoxy-2-methylanthraquinone, 2-hydroxy-4,6,7-trimethoxy-3-methylanthraquinone, 4-hydroxy-4984 zxft-7-trimethoxy-methylanthraquinone, 5272-zxft 527945-methoxy-7945-dimethoxymethyl-anthraquinone.
Preferably, the anthraquinone glycoside active ingredient comprises one or more of lucidin 3-O-beta-primeveride, damnacanthol 3-O-beta-primeveride and rubiadin 3-O-beta-primeveride.
Preferably, the iridoid active ingredient comprises one or more of prism, deacetylasperuloside, deacetylasperulosidic acid and asperulosidic acid.
Preferably, the extract of the root of Kanshizua contains 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisoindole-1-methyl ether, methylisoindole and scopoletin.
More preferably, the extract of the root of Kansbill contains at least scopoletin and 1-methyl ether of methyliodipines. The scopoletin and the methylisoid-1-methyl ether are matched and combined to generate a synergistic interaction effect, and the effect is obviously better than the simple superposition of the respective effects of the scopoletin and the methylisoid-1-methyl ether on preventing and treating the pulmonary fibrosis.
Furthermore, the invention also provides application of scopoletin in preparing a medicament for preventing and treating pulmonary fibrosis.
Furthermore, the invention also provides application of the methyl isoalizarin-1-methyl ether in preparation of a medicament for preventing and treating pulmonary fibrosis.
More preferably, the extract has comprehensive effects of preventing and treating pulmonary fibrosis, can improve pulmonary fibrosis caused by various factors, can improve lung function, reduce collagen deposition and fibroblast proliferation and activation of lung tissues, and can inhibit pulmonary fibrosis by inhibiting TGF-beta and TNF-alpha of lung tissues. The ethyl acetate extract of the south mountain flower roots can reduce the increase of COL1A1, COL3A1 and alpha-SMA protein expression caused by pulmonary fibrosis of mice. The action route is not through forming insoluble aluminum silicate on the surface of silicon dioxide, so that the aluminum silicate loses toxic effect on macrophages.
Further, the pulmonary fibrosis is not caused by silicosis.
The invention also provides a preparation method of the south mountain flower root extract, namely extracting the south mountain flower root by ethanol, removing impurities by using a preparation chromatography, and refining and purifying to obtain active ingredients of anthraquinone, anthraquinone glycoside and iridoid; or extracting root of south mountain flower with ethyl acetate solvent; or extracting root of south mountain flower with solvent, concentrating the extractive solution, and extracting with ethyl acetate. Preferably, the solvent is one or a combination of more than two of water, methanol, ethanol, propanol, butanol and the like.
Preferably, the root of the south mountain flower is taken and extracted by petroleum ether, the extracting solution is discarded, then the root of the south mountain flower is extracted by ethyl acetate, and the ethyl acetate extract is obtained by decompression and concentration.
Preferably, the preparation method can also comprise the following steps: extracting the root of south mountain flower with water, extracting with 95% ethanol, mixing the water and ethanol extractive solutions, concentrating the extractive solution under reduced pressure, extracting with ethyl acetate, and removing solvent by rotary evaporation to obtain the extract.
Preferably, the preparation method can also comprise the following steps: reflux-extracting rhizoma Dioscoreae Hypoglaucae with 95% ethanol for three times, concentrating the extractive solution under reduced pressure, removing impurities by preparative chromatography, and refining and purifying to obtain anthraquinone, anthraquinone glycoside and iridoid effective components, wherein the chromatography can be one or more of macroporous resin method, molecular sieve chromatography, silica gel adsorption chromatography, polyamide adsorption chromatography, etc. Preferably silica gel column chromatography, with a gradient of water and acetonitrile.
Optionally, the preparation method may further include: extracting the south mountain flower root with 95 percent ethanol for three times under reflux, extracting with ethyl acetate, and then performing rotary evaporation to remove the solvent to obtain the south mountain flower root extract.
The invention also provides a pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that: comprises anthraquinone, anthraquinone glycoside and iridoid effective components in the root of the southern mountain. Preferably, the weight ratio of the three effective extraction parts is 1-10:1-10:1-5.
Further, the pharmaceutical composition for treating pulmonary fibrosis consists of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisoindole-1-methyl ether, methylisoindole and scopoletin. Preferably, the weight ratio of the four compounds is 1-10:1-10:1-10:1-10.
Further, the pharmaceutical composition for treating pulmonary fibrosis consists of scopoletin and methyl isoalizarin-1-methyl ether. Preferably, the weight ratio of the two compounds is 1-10:1-10.
The present invention also preferably provides a pharmaceutical composition for the treatment of pulmonary fibrosis, characterized in that: the medicine consists of the south mountain flower root extract and a chemical medicine for resisting pulmonary fibrosis, wherein the chemical medicine for resisting pulmonary fibrosis is pirfenidone or nintedanib. The south mountain flower root extract and the chemical medicine combination for resisting pulmonary fibrosis have a synergistic interaction effect, and the effect is obviously better than the simple superposition of the respective effects of preventing and treating pulmonary fibrosis of the south mountain flower root extract and the chemical medicine. Preferably, the weight ratio of the south mountain flower root extract to the chemical medicine for resisting pulmonary fibrosis is 2-20:1-10.
The medicine for preventing and treating pulmonary fibrosis can prevent and/or treat pulmonary fibrosis, and comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is any one of a southern mountain root extract, scopoletin, methylisoidin-1-methyl ether and the medicine composition.
In the medicine, the content of the active ingredient accounts for 0.1-99.9% (w/w), and the content of the pharmaceutically acceptable auxiliary materials accounts for 0.1-99.9% (w/w), and the total is 100%.
The medicament of the present invention may be in any pharmaceutically acceptable dosage form including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, buccal agents, granules, electuary, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, aerosols, drops, patches.
The medicament of the invention can use proper medicinal auxiliary materials, and specific examples of the auxiliary materials comprise: fillers, diluents, carriers, excipients, disintegrants, binders, lubricants, flavoring agents, surfactants, colorants, coating agents, propellants, stabilizers, and the like.
Optionally, a suitable pharmaceutically acceptable carrier may be added during the preparation of the medicament, said pharmaceutically acceptable carrier being selected from the group consisting of: starch, sucrose, lactose, mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, EDTA disodium, calcium sodium EDTA, carbonates of monovalent alkali metals, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, silicon derivatives, cellulose and derivatives thereof, alginates, gelatin, polyvinylpyrrolidone, glycerol, tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.
The invention has the advantages and beneficial effects that: the effective part of the extract for resisting pulmonary fibrosis is discovered for the first time, and the extract has the advantages of simple preparation method, definite effective part, clear active ingredients, comprehensive effect of treating pulmonary fibrosis, obvious effect of treating pulmonary fibrosis superior to the prior art, synergistic effect of resisting pulmonary fibrosis between two specific compounds, synergistic effect of the extract and chemical drugs for resisting pulmonary fibrosis and the like.
Drawings
FIG. 1 shows the effect of different extraction sites of south mountain flower root on the pulmonary function of bleomycin-induced pulmonary fibrosis mice.
FIG. 2 shows the effect of different extraction sites of south mountain flower root on the degree of fibrosis of mice with pulmonary fibrosis induced by bleomycin.
FIG. 3 shows the effect of different extraction sites of south mountain flower root on the content of hydroxyproline in lung tissue of bleomycin-induced pulmonary fibrosis mice.
FIG. 4 shows the effect of different preparation processes of south mountain flower root on the pulmonary function of bleomycin-induced pulmonary fibrosis mice.
FIG. 5 shows the effect of different preparation processes of south mountain flower root on the degree of fibrosis of mice with pulmonary fibrosis induced by bleomycin.
FIG. 6 shows the effect of different preparation processes of south mountain flower root on the content of hydroxyproline in lung tissue of bleomycin-induced pulmonary fibrosis mice.
FIG. 7 shows the effect of different preparation processes of south mountain flower root on the expression of TGF-beta and TNF-alpha in lung tissue of mice with pulmonary fibrosis induced by bleomycin.
FIG. 8 shows the effect of different preparation processes of south mountain flower root on the expression of the proteins COL1A1, COL3A1 and alpha-SMA in the lung tissues of mice with pulmonary fibrosis induced by bleomycin.
FIG. 9 Effect of scopoletin and methylisoidin-1-methyl ether on α -SMA protein expression following TGF- β 1-induced fibroblast activation.
FIG. 10 Effect of the drug combinations of the present invention on α -SMA protein expression following TGF-. Beta.1-induced fibroblast activation.
Detailed Description
Example 1 preparation of extract of south mountain flower root
Taking 1kg of south mountain flower root, carrying out reflux extraction for 3 times by using 8 times of 95% ethanol, merging extracting solutions, concentrating under reduced pressure, extracting for 3 times by using 100ml of ethyl acetate, merging extracting solutions, concentrating under reduced pressure to remove a solvent, drying to obtain 3g of south mountain flower root extract, and detecting by chromatography, wherein the content of active ingredients of anthraquinone, anthraquinone glycoside and iridoid accounts for 64% of the total amount of the extract.
Example 2 preparation of extract of south mountain flower root
Taking 1kg of south mountain flower root, extracting with 8 times of water for 2 times, extracting with 5 times of 95% ethanol for 2 times, combining water and ethanol extracting solutions, concentrating the extracting solution under reduced pressure, extracting with 200ml of petroleum ether, discarding the extracting solution, extracting with 100ml of ethyl acetate for 3 times, combining the extracting solutions, concentrating under reduced pressure to remove the solvent, drying to obtain 8g of south mountain flower root extract, and detecting by chromatography, wherein the content of active ingredients of anthraquinone, anthraquinone glycoside and iridoid accounts for 71% of the total amount of the extract.
Example 3 preparation of extract of south African mountain flower root
Taking 1kg of south mountain flower root, crushing to 50 meshes, extracting with appropriate amount of petroleum ether for 1 time, discarding the petroleum ether extract, extracting with 5 times of ethyl acetate for 3 times, combining the extracts, concentrating under reduced pressure and drying to obtain 11g of ethyl acetate extract, and detecting by chromatography, wherein the content of anthraquinone, anthraquinone glycoside and iridoid active ingredients accounts for 79% of the total amount of the extract.
Example 4 preparation of extract of south mountain flower root
Taking 1kg of south mountain flower root, performing reflux extraction for 3 times by using 95% ethanol in an amount which is 8 times that of the south mountain flower root, combining extracting solutions, performing reduced pressure concentration, and removing impurities by using a preparative chromatographic column, wherein the adopted chromatographic column is a C18 column, and a mobile phase is a mobile phase A: water (0.1% formic acid, 1mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1mmol/L ammonium acetate), gradient elution conditions: 0-10min,20% by weight of B;10-60min,20-100% B, flow rate: 0.3mL/min, column temperature: at 30 ℃. Collecting three effective component parts of anthraquinone, anthraquinone glycoside and iridoid in sections respectively, concentrating under reduced pressure, and drying to obtain three effective component parts of anthraquinone, anthraquinone glycoside and iridoid. Or mixing the above three effective components uniformly to obtain extract, and detecting by chromatography, wherein the content of anthraquinone, anthraquinone glycoside and iridoid effective components accounts for 100% of the total extract.
EXAMPLE 5 preparation of pharmaceutical composition
Three effective parts of anthraquinone, anthraquinone glycoside and iridoid prepared in the embodiment 4 are taken, and the effective parts are respectively weighed according to the weight ratio of 8:1:1 by weight ratio.
EXAMPLE 6 preparation of pharmaceutical composition
Taking 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methyl isoalizarin-1-methyl ether, methyl isoalizarin and scopoletin according to the weight ratio of 1:5:5:1 by weight ratio.
EXAMPLE 7 preparation of pharmaceutical composition
Taking scopoletin lactone and methylisoidin-1-methyl ether, and mixing the raw materials according to the weight ratio of 1:1 by weight ratio.
EXAMPLE 8 preparation of pharmaceutical composition
The south mountain flower root extract of example 1 and pirfenidone were taken at a 10:1 by weight ratio.
EXAMPLE 9 preparation of pharmaceutical composition
The extract of south mountain flower root of example 1 and nintedanib were taken as 8:1 by weight ratio.
EXAMPLE 10 preparation of capsules
Taking 10 parts by weight of the south mountain flower root extract of example 2, adding 12 parts by weight of lactose and 2 parts by weight of microcrystalline cellulose, dry granulating, sieving, grading, and encapsulating to obtain capsules.
EXAMPLE 11 preparation of inhalation spray
The pharmaceutical composition of example 6 was taken, and an appropriate amount of surfactant, propellant and stabilizer were added to prepare an inhalation spray.
Experimental example 1: therapeutic effect of different extraction parts of south mountain flower root on bleomycin-induced pulmonary fibrosis of mice
1. Experimental Material
1.1 Experimental animals
Specific pathogen-free (SPF) grade C57BL/6N mice (male, 18-22g in body weight) purchased from experimental animal technology ltd, viton, beijing, and having a production license number: SCXK- (Jing) 2016-0011, issuing agency: the scientific and technical committee of Beijing City, the certification number: 11400700300671.
1.2 drugs and Primary Agents
Figure BDA0003041899750000081
1.3 Main Instrument of experiment
Figure BDA0003041899750000082
2. Experimental methods
2.1 Molding and grouping
About 80 SPF male C57BL/6N mice were selected, 10 mice were used as a blank control group, and the rest mice were subjected to pulmonary fibrosis caused by tracheal instillation of bleomycin. Pentobarbital sodium 80mg/kg is injected into the abdominal cavity to anaesthetize the mouse, the upper incisors of the mouse are fixed on a tracheal instillation operating table, LED light penetrates through the skin at the neck, and the trachea opening of the mouse is observed inwards from the oral cavity. The trocar was inserted into the mouse trachea and connected to HRH-MAG4 lung liquid quantitative nebulizer to push 50 μ L of sterile PBS dissolved bleomycin solution rapidly with a molding dose of 0.04U/vial. And (3) keeping the upright gentle swirling mice after administration to ensure that the solution is uniformly distributed in the lung, and after the mice revive, supplying water and feed to complete the establishment of the pulmonary fibrosis mouse model. The blank control group was run the same as the tube except that sterile PBS solution was administered.
After 14 days, randomly grouping into a model group, a nintedanib positive medicine group, a radix astragali group, a south mountain flower root ethyl acetate extract group, a south mountain flower root n-butyl alcohol extract group and a south mountain flower root water extract group according to the weight of the surviving model animals.
2.2 methods of administration
After 14 days of model building, the mice of each group were administered the corresponding drug by gavage daily for 14 days, and the blank and model groups were administered 0.5% CMC-Na aqueous solution; the Nintedanib positive drug is used for feeding Nintedanib aqueous solution, and the feeding dose is 50mg/kg; the radix astragali tablet group is administered with radix astragali tablet suspension, and the administration dose is 200mg/kg; the group for administering the extract of the root of south mountain flower is administered with ethyl acetate extract of the root of south mountain flower, n-butanol extract of the root of south mountain flower, water extract suspension of the root of south mountain flower at a dose of 100mg/kg.
2.3 Observation index
Mouse body weights were recorded weekly and mice deaths were recorded daily after self-molding. After 14 days of administration, 90mg/kg of pentobarbital sodium is injected into the abdominal cavity of the mouse for sufficient anesthesia, the trachea is exposed, and the lung function of the mouse is detected by connecting with Flexitent. The chest cavity of the mouse is opened, the lung blood is flushed through heart perfusion, the lung is taken out, the left large leaf of the lung of the mouse is fixed in 4% paraformaldehyde solution and is embedded in normal paraffin for subsequent Masson staining and immunohistochemical observation of lung tissue pathological change and pulmonary fibrosis degree. Washing each leaf of the right lung in normal saline, drying surface water by filter paper, storing in a refrigerator at-80 deg.C, and detecting Hydroxyproline (HYP) content of lung tissue.
2.3.1 mouse weight and cage side observations
Cage-side observations include, but are not limited to, the following: the death of the mice, whether there are wet rale, nasal spray, arch back, pili, etc., and changes in the activity and behavior pattern of the limbs. The time of occurrence, degree and duration, etc. are recorded. The weights of the mice before and after molding and before death were recorded.
2.3.2 respiratory function detection
The administration is carried out for 14 days, pentobarbital sodium is intraperitoneally injected within 24 hours after the last administration for sufficient anesthesia, and the patient is placed on a mouse dissection board in a supine position. The neck skin of the mice was wiped clean with alcohol, the skin was cut open, the neck muscles were separated blunt, the trachea was exposed, a small incision was transected under the thyroid cartilage, a tracheal cannula was inserted and ligated with suture. The trachea cannula and the Flexitent instrument are connected, the mouse inspiration total volume (IC) is detected through the Deep inspiration module, the Snapshot-150 module detects the resistance (Rrs), the compliance (Crs) and the elasticity (Ers) of a respiratory system, the Quick Prime-3 module detects indexes such as main airway resistance (Rn), tissue damping (G) and tissue elasticity (H), and PVs-P draws the mouse respiration P-V loop.
2.3.3Masson staining
The left lung of the mouse was fixed in 4% paraformaldehyde for preparation of pathological sections. Fixing the tissue for at least 72 hours, washing the tissue with water to obtain a fixing solution, performing gradient dehydration with alcohol, and embedding the xylene-transparent tissue. After tissue embedding, slicing was performed by a semi-automatic rotary microtome, the thickness of the slices was 4 μm, and the slices were baked at 60 ℃ for 1 hour. The rapid Masson dyeing method using Nanjing is characterized by comprising the following specific steps: (1) Paraffin section dewaxing into water: xylene twice for 10min, 100% ethanol, 95% ethanol, 75% ethanol, 30% ethanol twice each for two min, wen Zhengliu water rinse twice for 60 s. (2) nuclear staining: regaud's hematoxylin stain 60s,0.1% acetic acid wash rinse 30s. (3) sizing and dyeing: ponceau dye solution dyeing 60s,0.1% acetic acid wash rinsing for 30s. (4) differentiation: differentiating the 1% phosphomolybdic acid aqueous solution for 6-8min, observing the fiber part to be light pink under a mirror, and discarding the color separation solution without washing with water. (5) collagen counterstaining: re-dyeing with aniline blue water solution for 5min, discarding, and washing with anhydrous ethanol. (6) sealing: and sealing the sheet by using neutral resin after blow-drying. (7) microscopic examination: collagen fibers, mucus and cartilage are blue; cytoplasm, muscle, cellulose, glia are red; bluish purple nucleus. Staining was quantified using ImageJ v1.50c image software. Each section randomly selects 5 different visual fields, a blue collagen area is positive, and the ratio of the positive area to the total area of tissues in the whole visual field is used as a lung fibrosis index.
2.3.4 Hydroxyproline (HYP) assay
The method is characterized in that a Nanjing constructed hydroxyproline detection kit is used for detecting the hydroxyproline content of right lung tissues of mice according to an instruction, and the method comprises the following specific steps: (1) hydrolysis of the sample: accurately weighing 20-40mg of tissue wet weight in a 15mL centrifuge tube, adding 1mL of hydrolysate, and uniformly mixing. Adding cover to boiling water bath for 10min, shaking, and continuing boiling water bath for 10min to hydrolyze completely. (2) adjusting the pH: after the centrifuge tube is cooled by flowing water, 10 mul of indicator is added, the mixture is shaken up, and the pH value is adjusted to be about 6.0-6.8. Adding double distilled water to 10mL, and mixing uniformly. Adding about 30mg of active carbon into 3ml of diluted hydrolysate to make the solution clear and colorless, mixing uniformly, centrifuging at 3500rpm for 10min, and carefully taking the supernatant for detection. And (3) sample detection: the operation is the same except that 1mL of double distilled water is added into the blank tube, 1mL of standard application liquid with the concentration of 5 mug/mL is added into the standard tube, and 1mL of sample to be detected is added into the sample tube. Adding 0.5mL of reagent I (chloramine T solution and citric acid buffer solution), uniformly mixing and standing for 10min; adding 0.5mL of reagent II (perchloric acid solution), uniformly mixing and standing for 10min; adding 0.5mL of reagent tris (dimethylaminobenzaldehyde solution), mixing, carrying out water bath at 60 ℃ for 15min, cooling, and centrifuging at 3500rpm for 10min. (4) detecting the absorbance: and taking the supernatant, detecting the absorbance at the wavelength of 550nm, and determining the absorbance value of each tube. And calculating the content of hydroxyproline in the tissues according to a calculation formula.
3. Results of the experiment
3.1 the effect of different extraction parts of the south mountain flower root on the respiratory function of mice.
The body weights of the mice before and after molding were recorded, and the results are shown in table 1: compared with a blank control group, the weight of the mice in the model group is greatly reduced, and the Nintedanib and the ethyl acetate extract group of the south mountain flower root have the weight improvement effect. The respiratory function results are shown in table 2 and figure 1: compared with a blank control group, the total volume of the respiratory system of the mice in the model group is reduced, and the compliance of the respiratory system is reduced; the south mountain root ethyl acetate extract can effectively improve the total capacity and respiratory compliance of a mouse respiratory system, the effect is superior to that of a radix astragali group, and the result has statistical difference. The south mountain flower root ethyl acetate extract can improve the respiratory function of mice.
Figure BDA0003041899750000101
Table 1 mouse body weight recordings (in g, compared to the blank group, # p<0.05， ## p<0.01, comparison with model group,. About.p<0.05)
Figure BDA0003041899750000102
Table 2 mouse respiratory system function (compared to the blank group, ## p<0.01, comparison with model group, p<0.05,**p<0.01)
3.2 the influence of different extraction parts of the south mountain flower root on the degree of pulmonary fibrosis of mice caused by bleomycin.
Masson staining (FIG. 2) shows that the blank control group only has positive staining of blue collagen around the bronchial wall and the blood vessel wall, and the morphological structure is basically normal. The mice in the model group have pulmonary fibrosis areas, and collagen fiber tissues are proliferated and thickened. The positive medicines of nintedanib and the ethyl acetate extract of south mountain flower root have better improvement effect. The positive area fraction is shown in the bar graph of fig. 2.
3.3 influence of different extraction parts of south mountain flower root on hydroxyproline content of mouse lung tissue.
Hydroxyproline accounts for 13.4% of collagen, very little of elastin and none of the other proteins. In the case of pulmonary fibrosis, the main added component is collagen fiber, the content of hydroxyproline in lung tissue is measured, and the content can be converted into the content of lung collagen to reflect the degree of pulmonary fibrosis. As shown in Table 3 and FIG. 3, the lung of the model mice showed a statistically different increase in hydroxyproline content (p < 0.01). The south mountain flower root ethyl acetate extracts are all statistically different (p < 0.01) compared with the model group. The results show that the ethyl acetate extract of the south mountain flower root can reduce the degree of collagen deposition and pulmonary fibrosis of the lung of the mouse caused by bleomycin.
Figure BDA0003041899750000111
Table 3 hydroxyproline content in mouse lung tissue (compared to the blank, ## p<0.01, comparison with model group,. Star.p<0.01)
Experimental example 2: therapeutic action of extracts of south mountain flower root prepared by different methods on pulmonary fibrosis of mice caused by bleomycin 1. Experimental materials
1.1 Experimental animals
Specific pathogen-free (SPF) grade C57BL/6N mice (male, 18-22g in body weight) purchased from experimental animal technology ltd, viton, beijing, and having a production license number: SCXK- (Jing) 2016-0011, issuing agency: the scientific and technical committee of Beijing City, the certification number: 11400700300671.
1.2 drugs with Primary Agents
Figure BDA0003041899750000112
Figure BDA0003041899750000121
1.3 Main Instrument of experiment
Figure BDA0003041899750000122
2. Experimental methods
2.1 Molding and grouping
About 80 SPF male C57BL/6N mice were selected, 10 mice were used as a blank control group, and the rest mice were subjected to pulmonary fibrosis caused by tracheal instillation of bleomycin. Pentobarbital sodium is injected into the abdominal cavity, 80mg/kg of the mouse is anesthetized, the upper incisors of the mouse are fixed on a tracheal instillation operating table, LED light penetrates through the skin of the neck, and the trachea opening of the mouse is observed inwards from the oral cavity. The trocar was inserted into the mouse trachea and connected to HRH-MAG4 lung liquid quantitative nebulizer to push 50 μ L of sterile PBS dissolved bleomycin solution rapidly with a molding dose of 0.04U/vial. And (3) keeping the upright gentle swirling mice after administration to ensure that the solution is uniformly distributed in the lung, and after the mice revive, supplying water and feed to complete the establishment of the pulmonary fibrosis mouse model. The blank control group was run the same as the tube except that sterile PBS solution was administered.
After 14 days, the animals were randomly grouped into 10 animals each, based on the weight of the surviving model animals, into the model group, the nintedanib-positive drug group, the example 1 group, the example 2 group, the example 3 group, and the example 4 group.
2.2 methods of administration
After 14 days of model building, the mice of each group were administered the corresponding drug by gavage daily for 14 days, and the blank and model groups were administered 0.5% CMC-Na aqueous solution; the Nintedanib positive drug is administrated with a Nintedanib aqueous solution, and the administration dose is 50mg/kg; examples 1-4 the south mountain flower root extract suspension prepared by 0.5% CMC-Na aqueous solution was administered in an amount of 100mg/kg, respectively.
2.3 Observation index
Mouse body weights were recorded weekly and mice deaths were recorded daily after self-molding. After 14 days of administration, 90mg/kg of pentobarbital sodium is injected into the abdominal cavity of the mouse for sufficient anesthesia, the trachea is exposed, and the lung function of the mouse is detected by connecting with Flexitent. The chest cavity of the mouse is opened, the lung blood is flushed through heart perfusion, the lung is taken out, the left large leaf of the lung of the mouse is fixed in 4% paraformaldehyde solution and is embedded in normal paraffin for subsequent Masson staining and immunohistochemical observation of lung tissue pathological change and pulmonary fibrosis degree. After each leaf of the right lung is fully rinsed in physiological saline, surface water is absorbed by filter paper and stored in a refrigerator at the temperature of minus 80 ℃ for the subsequent Hydroxyproline (HYP) content detection of lung tissues, the detection of TGF-beta and TNF-alpha content and the expression of related protein of the pulmonary fibrosis by Western Blot detection.
2.3.1 mouse weight and cage side observations
The same as in experimental example 1.
2.3.2 respiratory function detection
The same as in experimental example 1.
2.3.3Masson staining
The same as in experimental example 1.
2.3.4 Hydroxyproline (HYP) assay
The same as in experimental example 1.
2.3.5 Immunohistochemical (IHC) staining
The left lung of the mouse was fixed in 4% paraformaldehyde for preparation of pathological sections. Fixing the tissue for at least 72 hours, washing the tissue with water to obtain a fixing solution, performing gradient dehydration with alcohol, and embedding the xylene-transparent tissue. After tissue embedding, the tissue was sliced with a semi-automatic rotary microtome, the thickness of the slices was 4 μm, and the slices were baked at 60 ℃. Dewaxing with xylene, hydrating with gradient ethanol, and repairing antigen (0.01M citrate buffer, pH6.0, repairing under high pressure for 2 min); blocking of endogenous catalase with 3% hydrogen peroxide, 5% BSA blocking at room temperature. Sections were incubated overnight at 4 ℃ with alpha-SMA primary antibody. After washing the sections with PBS buffer, the corresponding secondary antibodies were incubated for 1h at room temperature. Dripping a DAB color developing agent which is prepared freshly, counterstaining with hematoxylin, differentiating with hydrochloric acid and alcohol, dehydrating and drying with gradient ethanol, and sealing the piece, and observing the piece under a microscope to obtain a brown positive expression. Staining was quantified using ImageJ v1.50c image software. Each section was randomly selected from 5 different fields and analyzed for relative area of positive expression.
2.3.6 enzyme-linked immunosorbent assay (ELISA) assay
Samples of lung tissue from each group of mice were collected and the tissue samples were frozen at-80 ℃ until detection. A small piece of lung tissue was weighed, added with a certain amount of PBS buffer, and homogenized thoroughly using a homogenizer. Centrifuge at 3500rpm for 20min and collect the supernatant for ELISA testing. Taking the supernatant, and detecting the contents of TNF-alpha and TGF-beta in lung tissues according to the method of the specifications of a rat TNF-alpha and TGF-beta kit of Jiangsu Jingmei Biotechnology Co. The total protein concentration of the tissues is detected by using a BCA protein quantitative kit, and the result is expressed by the ratio of the content of TNF-alpha or TGF-beta to the total protein.
2.3.7Western blot detection of expression of related proteins
Samples of lung tissue from each group of mice were collected and the tissue samples were frozen at-80 ℃ until detection. A small piece of lung tissue was weighed, added with a certain amount of RIPA lysate containing 1% protease inhibitor and 1% protein phosphatase inhibitor, ground and lysed on ice for 45min. The lysate was centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant was collected and the protein concentration was measured using the BCA protein assay kit. 50 microgram protein samples were diluted with 5 Xloading buffer and heated at 100 ℃ for 5 minutes, electrophoresed in SDS-PAGE gels, subsequently transferred to PVDF membranes, blocked with 5% skim milk for 1h, incubated overnight at 4 ℃ for the first antibody, washed with TBST buffer for 3 times, incubated with the corresponding second antibody for 1h at room temperature, and developed with ECL chemiluminescence. After recording by chemiluminescence exposure, the bands were analyzed using ImageJ.
2.4 statistical methods
The experimental results are expressed as mean. + -. Standard deviation
Figure BDA0003041899750000131
Representing that statistical software SPSS 20 is used for analysis, student's t test is adopted for comparing a model group with a blank group, single-factor analysis of variance (One Way ANOVA) and Homogeneity test are adopted for each administration group and the model group, and least significant method (LSD) test is adopted for testing if the variances are uniform; if the variance is uneven, a test of Tamhane's T2 is adopted. With p<A difference of 0.05 was significant.
3. Results of the experiment
3.1 influence of extracts of south mountain flower root prepared by different methods on the respiratory function of mice.
The body weight, mortality and respiratory function of the mice were recorded after molding and after treatment with the south mountain flower root extract. Body weight results are shown in table 4: compared with a blank control group, the weight of the mice in the model group is greatly reduced, the south mountain flower root extracts prepared by different preparation methods are recovered to different degrees after treatment, and the mortality rate of the mice in the administration group is lower than that of the mice in the model group. The respiratory function results are shown in table 5 and fig. 4: compared with a blank control group, the total volume of the respiratory system of the mice in the model group is reduced, and the compliance of the respiratory system is reduced; the groups of examples 1-4 were able to effectively increase the total respiratory volume and respiratory compliance of mice with statistical differences in the results. It was shown that the groups of examples 1 to 4 were able to improve the respiratory function of the mice.
Figure BDA0003041899750000141
Table 4 mice body weight records (in g) and final mortality (compared to the blank group, # p<0.05)
Figure BDA0003041899750000142
table 5 mouse respiratory system function (compared to blank, ## p<0.01, comparison with model group,. About.p<0.05,**p<0.01)
3.2 the effect of extracts of south mountain flower root prepared by different methods on the degree of pulmonary fibrosis of mice caused by bleomycin.
Masson staining (FIG. 5) shows that the blank control group only has positive staining of blue collagen around the bronchial wall and the blood vessel wall, and the morphological structure is basically normal. The mice in the model group have pulmonary fibrosis areas, and collagen fiber tissues are proliferated and thickened. The groups were given different degrees of improvement, with the positive drug nintedanib group and the example 4 group having the best improvement. The positive area fraction is shown in the bar graph of fig. 2.
3.3 influence of extracts of south mountain flower root prepared by different methods on hydroxyproline content in mouse lung tissue.
As shown in Table 6 and FIG. 6, the lung of the model mice showed a statistically different increase in hydroxyproline content (p < 0.01). The lung tissue hydroxyproline content course of different concentration administration groups of the south mountain flower roots is reduced in a dose-dependent manner, and each example group is statistically different from the model group (p < 0.01). The results show that the south mountain flower root extract can reduce the degree of collagen deposition and pulmonary fibrosis of the mouse lung caused by bleomycin, wherein the effect of the group of example 4 is best.
Figure BDA0003041899750000151
Table 6 hydroxyproline content in mouse lung tissue (p # p <0.01 compared to blank group, p # p <0.01 compared to model group)
3.4 influence of extracts from different preparation methods of south mountain flower root on the expression of mouse lung tissue TGF-beta and TNF-alpha.
TGF-beta is the most important regulator of extracellular matrix synthesis and deposition dysregulation leading to tissue fibrosis, and is associated with fibrosis, sclerosis and atherosclerosis of various organs. TNF-alpha is a cytotoxic factor and plays a key role in the pathogenesis of pulmonary fibrosis. Therefore, we tested the TGF-. Beta.and TNF-. Alpha.content in mouse lung tissue by ELISA. The results are shown in Table 7 and FIG. 7, and the levels of TGF-beta and TNF-alpha in the lung tissues of the mice in the model group are increased and have statistical difference; the administration group reduced the levels of TGF-beta and TNF-alpha in lung tissue. The results show that the corresponding extract of the south mountain flower root can inhibit the progression of pulmonary fibrosis by influencing the expression of TGF-beta and TNF-alpha.
Figure BDA0003041899750000152
Table 7 mouse lung tissue TGF- β and TNF- α content (compared to blank, # p <0.01, compared to model, # p <0.05, # p < 0.01)
3.5 influence of extracts of south mountain flower root prepared by different methods on the expression of COL1A1, COL3A1 and alpha-SMA proteins in mouse lung tissues.
After pulmonary fibrosis occurs, fibroblasts are activated to release a large amount of extracellular matrix, the main components of the extracellular matrix are COL1A1 and COL3A1, and alpha-SMA is the main mark of fibroblast activation. We examined the expression of COL1A1 and COL3A1 proteins in mouse lung tissues by Western Blot. Meanwhile, we also examined the distribution of the alpha-SMA in mouse lung tissue by immunohistochemistry. The results (table 8 and figure 8) show that mouse lung tissue collagen type I and type III release was increased and α -SMA expression was increased and widely distributed following bleomycin stimulation, statistically different (p < 0.01) compared to the blank group. The extracts of the south mountain flower roots prepared by different methods have obvious improvement effect after treatment, and have statistical difference (p is less than 0.01) compared with a model group. The results show that the south mountain flower root extract can reduce the increase of COL1A1, COL3A1 and alpha-SMA protein expression caused by pulmonary fibrosis of mice, and the effect of example 4 is most remarkable.
Figure BDA0003041899750000161
TABLE 8 influence of different preparation methods of south mountain flower root extracts on bleomycin-induced lung tissue protein expression in mice
Experimental example 3: effective compounds in south mountain flower root extract for inhibiting fibroblast activation
1. Background of the experiment
Fibroblasts do not express alpha actin (alpha-SMA) in an inactivated state, and after being activated by TGF-beta 1, the fibroblasts are activated into myofibroblasts and stop proliferating, express the alpha-SMA and release extracellular matrix-related proteins. We obtained from literature and experiments that the extraction site of the anthoxanthin from south mountain flower contains two known compounds, namely scopoletin and methylisoidin-1-methyl ether, and we designed fibroblast activation experiments and treated the compounds contained in the extract of the anthoxanthin from south mountain flower to test the anti-fibroblast activation activity of scopoletin and methylisoidin-1-methyl ether.
2. Experimental Material
HPF cells of human lung fibroblast cell line were purchased from Shanghai enzyme-linked Biotech, inc., human recombinant TGF-. Beta.1 was purchased from Peprotech, and scopoletin and methyliodiaxin-1-methyl ether were purchased from Beijing Biyate Biotechnology, inc.
3. Experimental methods
Spreading HPF cells on a 6-well plate, growing to about 70%, discarding the culture medium, adding 1mL of serum-free culture medium (containing 0.1% DMSO) into a negative control group, adding 1mL of serum-free culture medium containing 10ng/mL TGF-beta 1 into a dosing group and a mould group, respectively adding scopoletin or methylisoid-1-methyl ether into the dosing group to make the final concentration reach 5 μ g/mL, and horizontally and rotationally shaking the culture plate to uniformly mix the liquid medicine. Standing at 37 deg.C, 5% CO 2 Incubate in incubator for 48 hours. The medium was then discarded, 100. Mu.L of RIPA lysate (containing 1% protease, phosphatase inhibitor) was added to lyse the cells, and the cells were harvested using a spatula, centrifuged at 13000rpm,4 ℃ for 10min. And taking the supernatant, transferring the supernatant into a new EP tube, and carrying out Western blot experiment to detect the expression condition of the alpha-SMA protein.
4. Results of the experiment
As a result, as shown in Table 9 and FIG. 9, the expression of α -SMA by fibroblasts was increased after TGF- β 1 induction. And scopoletin and methylisoid-1-methyl ether both have the effects of inhibiting the increase of alpha-SMA expression and inhibiting the activation of fibroblasts, and the results have statistical difference (p is less than 0.01).
Figure BDA0003041899750000171
TABLE 9 inhibitory Activity of scopoletin and methylisoidin-1-methyl ether on fibroblasts
Experimental example 4: the pharmaceutical composition of the invention inhibits fibroblast activation
The test materials and test methods were the same as in example 3, and the administration groups were each a nintedanib group, and example 1,5,6, 7, 8, and 9 groups. The dose of the nintedanib group, example 8 and example 9 group was 2. Mu.M, and the dose of the remaining example groups was 5. Mu.g/mL. Example 1 is ethyl acetate extract of south mountain flower root; example 5 the pharmaceutical composition is an anthraquinone, an anthraquinone glycoside, and an iridoid in an amount of 8:1:1 by weight ratio; example 6 pharmaceutical combination 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisoindole-1-methyl ether, methylisoindole and scopoletin according to 1:5:5:1, mixing: example 7 the drug combination is scopoletin and methylisoidin-1-methyl ether, as per 1:1, mixing; example 8 pharmaceutical combination of south mountain flower root extract and pirfenidone of example 1 as per 10:1, mixing; example 9 pharmaceutical combination of south mountain flower root extract of example 1 and nintedanib as per 8:1 by weight ratio.
Results of the experiment
As shown in table 10 and fig. 10, when compared with the data in table 9 in experimental example 3, it can be seen that the value of the scopoletin group is 5.10, the value of the methylisoixadain-1-methyl ether group is 4.22, and the value of example 7 is 3.10 when the scopoletin and the methylisoidin-1-methyl ether are combined under the same dosage, which shows that the results in the example 7 group are significantly better than the results when the scopoletin and the methylisoidin-1-methyl ether are used alone, that is, the combination of the scopoletin and the methylisoidin-1-methyl ether has a synergistic effect. In addition, as can be seen from the results of comparing the nintedanib group, the example 1 group and the example 9, the example 9 group has significantly better technical effects under the condition of the same dosage as the nintedanib group and lower dosage than the example 1 group, and the specification shows that the combination of the nanshan mountain flower root extract and the chemical drugs such as pirfenidone or nintedanib has synergistic effect.
Figure BDA0003041899750000172
TABLE 10 inhibitory Activity of different drug combinations on fibroblasts
Experimental example 5: radix astragali extract traditional Chinese medicine component analysis based on liquid chromatography-mass spectrometry technology
1. Chromatographic and mass spectral conditions
Agilent 1290 hplc chromatography coupled with agilent 6550 triple quadrupole time-of-flight mass spectrometry. Column was Waters BEH C18 (2.1 x 100mm,1.7 μm), mobile phase a: water (0.1% formic acid, 1mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1mmol/L ammonium acetate), gradient elution conditions: 0-5min,20% by weight of B;5-30min,20-100% B, flow rate: 0.3mL/min, column temperature: at 30 ℃.
Adopting a first-level High Resolution (HRMS) and second-level high resolution (HRMS/MS) mass spectrum negative ion acquisition mode, wherein the temperature of the drying gas is as follows: 280 ℃, flow rate of drying gas: 11L/min, sheath gas temperature: 325 ℃, sheath gas flow rate: 12L/min, primary mass spectrum scanning range: 150-1200m/z, secondary mass spectrum scanning range: 50-1200m/z, secondary impact voltage: 10/20/30/40/50V.
2. Results of the experiment
Identification and identification of traditional Chinese medicine components in south mountain flower root extract based on liquid chromatography high-resolution mass spectrometry technology
Based on the optimized chromatographic mass spectrometry conditions, first-level high-resolution mass spectrometry (HRMS) data is acquired. Comparing the HRMS data with a self-established database, identifying the traditional Chinese medicine components in the southern mountain flower root extract, further collecting second-level high-resolution mass spectrum (HRMS/MS) data of the traditional Chinese medicine components in the southern mountain flower root extract under different collision voltages, and identifying 30 traditional Chinese medicine components in the southern mountain flower root extract, wherein the 30 traditional Chinese medicine components comprise 23 anthraquinone components, 4 iridoid components and 3 anthraquinone glycoside components, and part of compounds are isomer compounds. The basic information for each identified component is shown in the table below.
Figure BDA0003041899750000181
TABLE 11 radix astragali extract Chinese medicinal components identified based on liquid chromatography high resolution mass spectrometry
Conclusion of the experimental examples: the south mountain flower root extract can improve pulmonary fibrosis caused by different factors, can improve lung function, reduce collagen deposition and fibroblast proliferation and activation of lung tissues, and can inhibit pulmonary fibrosis by inhibiting TGF-beta and TNF-alpha of the lung tissues. The extracted parts of the south mountain flower roots are anthraquinone, iridoid and anthraquinone glycoside. Two specific compounds of scopoletin and methyl isoalizarin-1-methyl ether definitely have the function of inhibiting the activation of fibroblasts. The south radix sophorae flavescentis extract has a definite effect of resisting pulmonary fibrosis, scopoletin and methylisoidin-1-methyl ether prove that the south radix sophorae flavescentis extract has a potential of resisting pulmonary fibrosis on a cellular level, and the south radix sophorae flavescentis extract has a synergistic effect after being combined. Meanwhile, the south mountain flower root extract has a synergistic effect after being combined with chemical drugs such as pirfenidone or nintedanib for resisting pulmonary fibrosis. In conclusion, the south mountain flower root extract and the components thereof can be used for preparing the medicine for resisting the lung fibrosis.

Claims (16)

1. The application of the south mountain flower root extract in preparing the medicine for treating pulmonary fibrosis is characterized in that: the extract of the south mountain flower root is effective parts of anthraquinone, anthraquinone glycoside and iridoid; the preparation method of the south radix hill flower root extract comprises the steps of extracting the south radix hill flower root with ethanol, removing impurities by using a preparation chromatography method, and refining and purifying to obtain anthraquinone, anthraquinone glycoside and iridoid effective components; or extracting root of south mountain flower with ethyl acetate; or extracting root of south mountain flower with solvent, concentrating the extractive solution, and extracting with ethyl acetate.
2. Use according to claim 1, characterized in that: the preparation method of the south radix sophorae flavescentis extract comprises the steps of carrying out reflux extraction on the south radix sophorae flavescentis with 95% ethanol for three times, carrying out reduced pressure concentration on an extracting solution, removing impurities by adopting a preparation chromatography, and refining and purifying to obtain anthraquinone, anthraquinone glycoside and iridoid effective components, wherein the chromatography is one or more of a macroporous resin method, a molecular sieve chromatography, a silica gel column chromatography and a polyamide column chromatography.
3. Use according to claim 2, characterized in that: the chromatography is silica gel column chromatography, and water and acetonitrile are adopted for gradient elution.
4. Use according to claim 1, characterized in that: the preparation method of the south mountain flower root extract comprises the steps of taking the south mountain flower root, extracting with petroleum ether, removing an extracting solution, then extracting with ethyl acetate, and concentrating under reduced pressure to obtain the ethyl acetate extract.
5. Use according to claim 1, characterized in that: the preparation method of the south mountain flower root extract comprises the steps of extracting the south mountain flower root with a solvent, concentrating an extracting solution, and extracting with ethyl acetate to obtain the south mountain flower root extract, wherein the solvent is one or a combination of more than two of water, methanol, ethanol, propanol, butanol and the like.
6. Use according to claim 5, characterized in that: the preparation method of the south mountain flower root extract comprises the steps of extracting the south mountain flower root with water, extracting with 95% ethanol, combining water and ethanol extracting solutions, concentrating the extracting solution under reduced pressure, extracting with ethyl acetate, and performing rotary evaporation to remove a solvent to obtain the south mountain flower root extract.
7. Use according to claim 5, characterized in that: the preparation method of the south mountain flower root extract comprises the steps of carrying out reflux extraction on the south mountain flower root by 95% ethanol for three times, extracting by ethyl acetate, and then carrying out rotary evaporation to remove a solvent to obtain the south mountain flower root extract.
8. Use according to claim 1, characterized in that: the pulmonary fibrosis is not caused by silicosis.
9. Use according to claim 2, characterized in that: the treatment of pulmonary fibrosis is to improve the lung function, reduce the collagen deposition and fibroblast proliferation and activation of lung tissues, inhibit TGF-beta and TNF-alpha of lung tissues and inhibit pulmonary fibrosis.
10. Use according to claim 1, characterized in that: the anthraquinone effective components comprise 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methyl isoalizarin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy-3-hydroxymethylanthraquinone, desmethyl damnacanthal, methyl isoalizarin-1-methyl ether, damnacanthal, 1,3-dihydroxy-2-methoxymethylanthraquinone, 3-hydroxy-1-methoxy-2-hydroxymethylanthraquinone, 1,3-dihydroxy-2-ethoxymethylanthraquinone, 1-hydroxy-2,3-dimethoxy-7-methylanthraquinone, 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5,6-dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, 1,3-dihydroxy-6-methoxy-2-methoxymethylanthraquinone, scopoletin, 3,5-dihydroxy-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 3-hydroxy-1,5,6-trimethoxy-2-methylanthraquinone, 2-hydroxy-4,6,7-trimethoxy-3-methylanthraquinone, 4-hydroxy-4984 zxft-7-hydroxymethyl anthraquinone, 5272-dihydroxy-zft 527945-methoxy-7945-dimethoxy-2-methylanthraquinone;
the anthraquinone glycoside active ingredient comprises one or more of lucidin 3-O-beta-primeveride, damnacanthol 3-O-beta-primeveride and rubiadin 3-O-beta-primeveride;
the iridoid active ingredient comprises one or more of prism, deacetylasperuloside, deacetylasperulosidic acid and asperulosidic acid.
11. The application of the south mountain flower root extract in preparing the medicine for treating pulmonary fibrosis is characterized in that: the extract of the root of the south mountain flower is one or two of scopoletin and methyl isoalizarin-1-methyl ether.
12. A pharmaceutical composition for the treatment of pulmonary fibrosis, comprising: the composition consists of effective extraction parts of anthraquinone, anthraquinone glycoside and iridoid in the southern mountain root, wherein the weight ratio of the three effective extraction parts is 1-10:1-10:1-5.
13. A pharmaceutical composition for the treatment of pulmonary fibrosis, comprising: consists of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methyl isoalizarin-1-methyl ether, methyl isoalizarin and scopoletin, wherein the weight ratio of the four compounds is 1-10:1-10:1-10:1-10.
14. A pharmaceutical composition for the treatment of pulmonary fibrosis, comprising: consists of scopoletin and methyl isoalizarin-1-methyl ether, and the weight ratio of the two compounds is 1-10:1-10.
15. A pharmaceutical composition for the treatment of pulmonary fibrosis, comprising: the medicine comprises a south mountain flower root extract and chemical medicines for resisting pulmonary fibrosis, wherein the chemical medicines for resisting pulmonary fibrosis are pirfenidone or nintedanib, and the weight ratio of the south mountain flower root extract to the chemical medicines for resisting pulmonary fibrosis is 2-20:1-10.
16. Use of a composition according to any one of claims 12 to 15 in the manufacture of a medicament for the treatment of pulmonary fibrosis.
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