CN115245533B - Application of Nanshan flower root extract in treating pulmonary fibrosis - Google Patents
Application of Nanshan flower root extract in treating pulmonary fibrosis Download PDFInfo
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- CN115245533B CN115245533B CN202110459959.1A CN202110459959A CN115245533B CN 115245533 B CN115245533 B CN 115245533B CN 202110459959 A CN202110459959 A CN 202110459959A CN 115245533 B CN115245533 B CN 115245533B
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- Prior art keywords
- anthraquinone
- pulmonary fibrosis
- extract
- methylanthraquinone
- hydroxy
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Abstract
The invention belongs to the field of medicines, and relates to application of a rhizoma arisaematis root extract in preparation of a medicine for treating pulmonary fibrosis, wherein the rhizoma arisaematis root extract is an anthraquinone, anthraquinone glycoside and iridoid effective part. The effective part for resisting the pulmonary fibrosis is discovered for the first time, the active ingredients are clear, the effect of treating the pulmonary fibrosis is comprehensive, the effect of treating the pulmonary fibrosis is obviously better than that of the prior art, and the effective part can produce a synergistic effect with chemical medicines for resisting the pulmonary fibrosis. Based on the specific effective compounds in the above extracts, a synergistic effect between the compounds is found, and corresponding pharmaceutical compositions for treating pulmonary fibrosis are disclosed.
Description
Technical Field
The invention belongs to the field of medicines, and in particular relates to an extraction part extracted from a traditional Chinese medicine Nanshanhua root and application of a compound in preventing and treating pulmonary fibrosis diseases.
Background
Pulmonary Fibrosis (PF) is a disease of the type of diffuse exudation, infiltration and fibrosis of the pulmonary interstitium into major lesions, the major symptoms of which include dyspnea, shortness of breath, dry cough, asthma and suffocation, and severe cases of which lead to respiratory failure and death of the patient. The etiology of the vast majority of patients with pulmonary fibrosis is unknown (idiopathic), and this group of diseases is called Idiopathic Interstitial Pneumonia (IIP), a major class of interstitial lung diseases. The most common type of disease with pulmonary fibrosis lesions as the main manifestation in idiopathic interstitial pneumonia is Idiopathic Pulmonary Fibrosis (IPF), which is a serious interstitial lung disease that can lead to progressive loss of pulmonary function. The pathogenesis of this is currently still not well understood, and only two drugs are marketed worldwide that are specifically used to treat idiopathic pulmonary fibrosis. Pirfenidone (Pirfenidone) was first used in japan for IPF treatment, and Pirfenidone and nindani (Nintedanib) were approved by the us FDA for specific treatment of IPF in 2014. However, these two drugs cannot reverse or prevent the development of IPF, and are expensive, thus bringing a large burden to patients and families.
Pulmonary fibrosis can be divided into partial manifestations of primary pulmonary disease and systemic disease. (1) pulmonary fibrosis as a type of primary pulmonary disease are: radiation fibrosis (i.e., fibrosis due to radioactivity), fibrosis to the result of damaging inflammatory lung lesions (e.g., sclerotic tuberculosis, alveolus lung, small saccular pulmonary fibrosis), fibrosis to the result of allergic reaction, interstitial fibrosis due to drugs, fibrosis as an end-stage manifestation of interstitial inflammation, etc.; (2) pulmonary fibrosis is manifested in part by systemic disease: granulomatosis (such as sarcoidosis, amyloidosis, mole-type hamartoma, xanthomatosis), collagenous diseases, and rheumatic diseases (such as scleroderma, lupus erythematosus, perinodular arteritis, dermatomyositis, and rheumatoid arthritis).
In recent years, medical workers in China have achieved great achievements by finding effective medicines for treating and delaying pulmonary fibrosis from traditional Chinese medicines, and searching medicines for treating pulmonary fibrosis from natural sources is considered as an effective way for developing the medicines.
Nanshanhua is also known as dog bone wood. Is root of Rubiaceae plant Nanshanhua [ Prismatomeris connata Y.Z.Ruan ], and is one of the characteristic medicinal materials in Guangxi. 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, scholars have found that the extract of the root of the Sonchus arvensis can inhibit liver fibrosis by inducing apoptosis of rat astrocytes. However, there is no report on the use of the extract of the roots of the Nanshanhua or the compounds derived from the roots of the Nanshanhua for the anti-pulmonary fibrosis activity and the use. The prior art (J.Inula in China, vol.25 and 24 in 2016, pages 2868-2869) discloses that the ethyl acetate extraction part and the water part of the ethanol extract of the roots of the Sonchus arvensis are possibly active sites for resisting hepatic fibrosis, and several compounds such as stigmast-4-en-3-one, nortiger thorn aldehyde and the like can be obtained through separation and purification, but no evidence indicates that the compounds identified through separation and purification are active ingredients and the application of the compounds is anti-hepatic fibrosis. Scopoletin is disclosed in the prior art (J.China comparative medicine, volume 30, phase 2, pages 9-14) to protect liver of liver fibrosis rats, positively regulate and control expression of cytokines related to liver fibers, reduce the level of extracellular matrix of liver, improve the pathological changes of liver fibrosis of rats and have the function of resisting liver fibrosis. The prior art (university of Beijing, vol.32, no. 1, pages 42-44) discloses that the alcohol extract of the roots of Nanshanhua can obviously reduce the deposition of collagen fiber hyperplasia in the liver of rats and the area of fibrotic tissue, and has a certain development value in the aspect of liver fibrosis treatment. However, the above uses are anti-hepatic fibrosis, and the fibrosis occurrence mechanisms of different organs are different. Clinically liver fibrosis is mostly fibrosis caused by bacterial and viral infection, toxin damage, metabolic abnormality and the like, and is currently considered to be a reversible process. While pulmonary fibrosis is considered an irreversible process, the lungs are in direct communication with the outside world, the cause of pulmonary fibrosis is more complex and the etiology of most patients is unknown. From the prior art, many drugs have an effect of preventing and treating liver fibrosis, but it is clear that drugs having an effect of preventing and treating lung fibrosis are very rare, and as described above, only pirfenidone and nilamide are known to be used for the prevention and treatment of lung fibrosis so far, that is, drugs for preventing and treating liver fibrosis are generally not used for the prevention and treatment of lung fibrosis.
The tablet prepared from the south mountain flower for treating silicosis is developed by Guangxi Yingkang pharmaceutical industry Limited liability company, and is suitable for treating silicosis, and the preparation method comprises the steps of heating and reflux extracting with 60% ethanol for three times, each time for 2 hours, merging extracting solutions, filtering, recovering ethanol from filtrate under reduced pressure, concentrating into thick paste, uniformly stirring with 10 times of water at a temperature, standing, filtering, concentrating the filtrate under reduced pressure, vacuum drying, crushing, adding a proper amount of auxiliary materials, granulating, pressing into 1000 tablets, and coating sugar. However, silicosis is known to be a disease of the lungs caused by inhalation of silica or silica and its crystalline forms such as quartz and other less common forms such as cristobalite and phosphoquartz, and its rapid progression has a similar appearance to interstitial pneumonia and is not equivalent to pulmonary fibrosis. Secondly, in the prior art, the main active ingredient of the tablet is a polymer formed by a plurality of metal elements mainly comprising organic aluminum, and the action mechanism is that aluminum forms insoluble aluminum silicate on the surface of silicon dioxide, so that the aluminum loses toxic action on macrophages, thereby antagonizing quartz cell toxic effect (see main editions of Li, etc., 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 active ingredient in the prevention and treatment of pulmonary fibrosis is not the same as that in silicosis, and have found that the active ingredient for the prevention and treatment of pulmonary fibrosis is preferable through a great deal of research and experiments.
The prior art (Chinese herbal medicine, volume 51, stage 4, pages 1031-1036) discloses that polysaccharide from the roots of Sonchus arvensis can reduce the inflammatory level by inhibiting inflammatory cell infiltration, down regulate the alpha-SMA and Vimentin expression level in lung tissues, up regulate the E-cadherin expression level, and suggest that the polysaccharide from the roots of Sonchus arvensis can effectively improve the immune balance, inhibit the development of inflammatory reaction and EMT, thereby improving the fibrosis degree of silicosis tissues and having better therapeutic effect on silicosis fibrosis. The polysaccharide is prepared by pulverizing decoction pieces 10kg, extracting with water under ultrasonic heating, concentrating under reduced pressure, and precipitating with 80% ethanol to obtain 905g crude polysaccharide. After the crude polysaccharide is redissolved in distilled water, protein is removed by a Savage method, the crude polysaccharide passes through a filter membrane U at high pressure, then is concentrated by an ultrafiltration membrane A, low relative molecular mass components are removed, and 560g of polysaccharide is obtained by drying the concentrate. The above suggests that the polysaccharide component is an active component of the roots of Sonchus arvensis for preventing and treating pulmonary fibrosis. However, in the above experiments, the effectiveness of polysaccharide and non-polysaccharide components in the roots of the nanshan flowers was not compared, and the inventors have found that polysaccharide components are not main effective components of the roots of the nanshan flowers for preventing and treating pulmonary fibrosis after intensive researches.
The invention provides an effective part with definite effect on preventing and treating pulmonary fibrosis from the root of the Nanshanhua and provides the pharmaceutical application thereof.
Disclosure of Invention
The invention aims to provide a novel pharmaceutical application of a south mountain flower root extract, namely an 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 has the advantages of simple preparation method, clear effective part, clear active ingredient, comprehensive effect of treating pulmonary fibrosis, obviously better effect of treating pulmonary fibrosis than the prior art, capability of generating synergistic effect with chemical medicines for resisting pulmonary fibrosis and the like.
Furthermore, the invention aims to provide a preparation method of the effective part of the south mountain flower root.
Still further, the present invention aims to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of anthraquinone, anthraquinone glycoside and iridoid active ingredients of the roots of the nanshan flowers.
Still further, it is an object of the present invention to provide a pharmaceutical composition for treating pulmonary fibrosis, which consists of scopoletin and methyl alizarin-1-methyl ether.
Furthermore, the invention aims to provide a pharmaceutical composition for treating pulmonary fibrosis, which combines the extract of the root of the Sonchus arvensis with the chemical drug pirfenidone or nidanib for resisting pulmonary fibrosis, and has obvious synergistic effect.
In order to achieve the above object, the present invention provides the following specific technical solutions:
an application of a rhizoma arisaematis root extract in preparing a medicament for preventing and treating pulmonary fibrosis is characterized in that: the extract of the root of the Sonchus arvensis is anthraquinone, anthraquinone glycoside and iridoid active ingredients. Preferably, the active ingredient content is more than 60%, more preferably more than 70%, most preferably 100% of the extract content.
Preferably, the method comprises the steps of, the anthraquinone active ingredients comprise 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylimadin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy-3-hydroxymethylanthraquinone, normethylanthraquinone, methylimadin-1-methyl ether, tiger-aldehyde, 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-methylanthraquinone, scopolide, 3, 6-dimethoxy-2, 3-dimethoxy-anthraquinone, 7-methylanthraquinone, 7-hydroxy-1, 2, 3-trimethoxy-2-methylanthraquinone, 3-hydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5, 6-methylanthraquinone, 3-hydroxy-2-methoxy-methylanthraquinone, 3-hydroxy-2-methylanthraquinone, 3-hydroxy-2-methyl anthraquinone, one or more of 1, 3-dihydroxy-5, 6-dimethoxy-2-methoxymethylanthraquinone.
Preferably, the anthraquinone glycoside active ingredient comprises one or more of lucidin 3-O-beta-primeveroside, damnacanthol 3-O-beta-primeveroside, rubiadin-O-beta-primeveroside.
Preferably, the iridoid active ingredient comprises one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, asperulosidic acid.
Preferably, the extract of the roots of the Sonchus arvensis contains at least 1, 3-dihydroxy-5, 6-dimethoxy-2-methylanthraquinone, methylimadder-1-methyl ether, methylimadder and scopoletin.
More preferably, the extract of the roots of the Sonchus arvensis contains at least scopoletin and methyl alizarin-1-methyl ether. Wherein, the synergistic effect is generated by the collocation and combination of scopoletin and methyl alizarin-1-methyl ether, and the effect is obviously better than the simple superposition of the respective effects of scopoletin and methyl alizarin-1-methyl ether for preventing and treating pulmonary fibrosis.
Furthermore, the invention also provides application of scopoletin in preparing medicines for preventing and treating pulmonary fibrosis.
Furthermore, the invention also provides an application of the methyl alizarin-1-methyl ether in preparing medicines for preventing and treating pulmonary fibrosis.
More preferably, the extract of the present invention has an overall effect of preventing and treating pulmonary fibrosis, can improve pulmonary fibrosis caused by various factors, can improve pulmonary function, can reduce collagen deposition and fibroblast proliferation and activation of pulmonary tissues, and can inhibit TGF-beta and TNF-alpha of pulmonary tissues to inhibit pulmonary fibrosis. The ethyl acetate extract of the roots of the Arnica herb can reduce the increase of COL1A1, COL3A1 and alpha-SMA protein expression caused by pulmonary fibrosis of mice. The action route is not to form insoluble aluminum silicate on the surface of silicon dioxide, so that the insoluble aluminum silicate loses toxic action on macrophages.
Further, the pulmonary fibrosis is not caused by silicosis.
The invention also provides a preparation method of the extract of the roots of the arisaema with ethanol, and then the impurities are removed by using a preparation chromatography, and anthraquinone, anthraquinone glycoside and iridoid active ingredients are obtained by refining and purifying; or extracting root of Arnica herb with ethyl acetate solvent; or extracting root of Arnica herb with solvent, concentrating the extractive solution, and extracting with ethyl acetate. Preferably, the solvent is one or more of water, methanol, ethanol, propanol, butanol, etc.
Preferably, the roots of the mountain flower are firstly extracted by petroleum ether, the extracting solution is discarded, then the extracting solution is extracted by ethyl acetate, and the ethyl acetate extract is obtained by decompression concentration.
Preferably, the preparation method may further be: extracting radix Ardisiae Japonicae with water, extracting with 95% ethanol, mixing water and ethanol extractive solutions, concentrating under reduced pressure, extracting with ethyl acetate, and removing solvent by rotary evaporation.
Preferably, the preparation method may further be: reflux-extracting radix Arnicae with 95% ethanol for three times, concentrating the extractive solution under reduced pressure, removing impurities by preparative chromatography, and refining to obtain anthraquinone, anthraquinone glycoside and iridoid effective components, wherein the chromatography can be one or more of macroporous resin method, molecular sieve chromatography, adsorption chromatography such as silica gel and polyamide, preferably silica gel column chromatography.
Optionally, the preparation method may further be: reflux-extracting the root of the mountain flower with 95% ethanol for three times, extracting with ethyl acetate, and removing the solvent by rotary evaporation to obtain the mountain flower root extract.
The invention also provides a pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that: is prepared from anthraquinone, anthraquinone glycoside and iridoid as effective components. 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, methylimadder-1-methyl ether, methylimadder 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 alizarin-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 treating pulmonary fibrosis, characterized in that: the composition comprises the extract of the root of the Sonchus arvensis and a chemical medicine for resisting pulmonary fibrosis, wherein the chemical medicine for resisting pulmonary fibrosis is pirfenidone or nidanib. The combination of the Nanshan flower root extract and the chemical medicine for resisting pulmonary fibrosis has a synergistic effect, and the effect is obviously better than the simple superposition of the effect of the Nanshan flower root extract and the chemical medicine for respectively preventing and treating pulmonary fibrosis. Preferably, the weight ratio of the extract of the roots of the Sonchus arvensis and 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 consists of an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is any one of an extract of the roots of the Chinese alpine yarrow, scopoletin, methyl alizarin-1-methyl ether and a pharmaceutical composition.
In the medicine, the content of the active ingredients is 0.1-99.9% (w/w), and the content of the pharmaceutically acceptable auxiliary materials is 0.1-99.9% (w/w), which is 100%.
The medicament of the 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, electuaries, pills, powders, ointments, pellets, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, aerosols, drops and patches.
The medicine of the invention can use proper auxiliary materials of the medicine, and specific examples of the auxiliary materials include: fillers, diluents, carriers, excipients, disintegrants, binders, lubricants, flavoring agents, surfactants, colorants, coating agents, propellants, stabilizers, and the like.
Optionally, a suitable pharmaceutically acceptable carrier selected from the group consisting of: starch, sucrose, lactose, mannitol, sorbitol, sodium metabisulfite, sodium bisulphite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, disodium vitamin C, EDTA, calcium sodium EDTA, monovalent alkali metal carbonates, 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 the pulmonary fibrosis is discovered for the first time, and the extract has the advantages of simple preparation method, clear effective part, clear active ingredient, comprehensive effect of treating the pulmonary fibrosis, obviously better effect of treating the pulmonary fibrosis than the prior art, wherein two specific compounds have synergistic effect of resisting the pulmonary fibrosis, and the extract can also generate synergistic effect with chemical medicines for resisting the pulmonary fibrosis.
Drawings
FIG. 1 effect of different extraction sites of Nanshanhua root on the pulmonary function of bleomycin-induced pulmonary fibrosis mice.
FIG. 2 effect of different extraction sites of Nanshan flower root on the degree of fibrosis of bleomycin-induced pulmonary fibrosis mice.
FIG. 3 effect of different extraction sites of Nanshanhua root on hydroxyproline content of lung tissue of bleomycin-induced pulmonary fibrosis mice.
FIG. 4 effect of different preparation processes of Nanshan flower root on bleomycin-induced pulmonary fibrosis mouse pulmonary function.
FIG. 5 effect of different preparation processes of Nanshan flower root on the degree of fibrosis of bleomycin-induced pulmonary fibrosis mice.
FIG. 6 effect of different preparation processes of Nanshan flower root on the hydroxyproline content of the lung tissue of bleomycin-induced pulmonary fibrosis mice.
FIG. 7 effect of different preparation processes of Nanshanhua root on the expression of TGF-beta and TNF-alpha in lung tissue of bleomycin-induced pulmonary fibrosis mice.
FIG. 8 effects of different preparation processes of Nanshanhua root on bleomycin-induced pulmonary fibrosis mouse lung tissue COL1A1, COL3A1, α -SMA protein expression.
FIG. 9 effect of scopoletin and methylimadder-1-methyl ether on expression of alpha-SMA protein following TGF-beta 1 induced fibroblast activation.
FIG. 10 effect of the pharmaceutical combination of the invention on expression of alpha-SMA protein following TGF-beta 1 induced fibroblast activation.
Detailed Description
Example 1 preparation of Nanshan flower root extract
Reflux-extracting 1kg of radix Ardisiae Japonicae with 8 times of 95% ethanol for 3 times, mixing extractive solutions, concentrating under reduced pressure, extracting with 100ml ethyl acetate for 3 times, mixing extractive solutions, concentrating under reduced pressure to remove solvent, drying to obtain 3g of radix Ardisiae Japonicae extract, and detecting by chromatography, wherein the content of anthraquinone, anthraquinone glycoside and iridoid active ingredients is 64% of the total amount of extract.
Example 2 preparation of Nanshan flower root extract
Taking 1kg of Arnica root, extracting with 8 times of water for 2 times, extracting with 5 times of 95% ethanol for 2 times, mixing water and ethanol extractive solutions, concentrating under reduced pressure, extracting with 200ml of petroleum ether, discarding extractive solution, extracting with 100ml of ethyl acetate for 3 times, mixing extractive solutions, concentrating under reduced pressure to remove solvent, drying to obtain 8g of Arnica root extract, and detecting by chromatography, wherein the content of anthraquinone, anthraquinone glycoside and iridoid active ingredients is 71% of the total amount of the extract.
Example 3 preparation of Nanshan flower root extract
Taking 1kg of mountain flower root, crushing to about 50 meshes, extracting with a proper amount of petroleum ether for 1 time, discarding 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 Nanshan flower root extract
Reflux-extracting radix Aristolochiae Kaempferi 1kg with 8 times of 95% ethanol for 3 times, mixing the extractive solutions, concentrating under reduced pressure, removing impurities with preparative chromatographic column (C18 column) with mobile phase (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% B;10-60min,20-100% b, flow rate: 0.3mL/min, column temperature: 30 ℃. Respectively collecting the three effective component parts of anthraquinone, anthraquinone glycoside and iridoid in segments, concentrating under reduced pressure, and drying to obtain purified three effective component parts of anthraquinone, anthraquinone glycoside and iridoid. Or mixing the above three effective components uniformly to obtain extract of radix Ardisiae Japonicae, and detecting by chromatography, wherein the content of anthraquinone, anthraquinone glycoside and iridoid effective components is 100% of the total amount of the extract.
EXAMPLE 5 preparation of pharmaceutical compositions
Three effective parts of anthraquinone, anthraquinone glycoside and iridoid prepared in example 4 are taken and respectively calculated according to the weight of 8:1:1 by weight ratio.
EXAMPLE 6 preparation of pharmaceutical compositions
Taking 1, 3-dihydroxyl-5, 6-dimethoxy-2-methylanthraquinone, methyl alizarin-1-methyl ether, methyl alizarin and scopoletin according to the following weight ratio of 1:5:5:1 by weight ratio.
EXAMPLE 7 preparation of pharmaceutical compositions
Scopoletin and methyl alizarin-1-methyl ether are taken according to the following formula 1:1 by weight ratio.
EXAMPLE 8 preparation of pharmaceutical compositions
Taking the extract of the roots of the Inulae flos of the example 1 and pirfenidone according to the following weight ratio of 10:1 by weight ratio.
EXAMPLE 9 preparation of pharmaceutical compositions
The extract of the roots of the Nanshanhua and the Nidamib of example 1 were taken according to 8:1 by weight ratio.
Example 10 preparation of capsules
10 parts by weight of the extract of the roots of the Arnica herb of example 2 are taken, 12 parts by weight of lactose and 2 parts by weight of microcrystalline cellulose are added, and the mixture is subjected to dry granulation, sieving, granulating and encapsulating to obtain capsules.
EXAMPLE 11 preparation of inhalation spray
The pharmaceutical composition of example 6 was taken and formulated into an inhalation spray by adding appropriate amounts of surfactant, propellant and stabilizer.
Experimental example 1: treatment effect of different extraction parts of Nanshanhua root on bleomycin-induced pulmonary fibrosis of mice
1. Experimental materials
1.1 laboratory animals
Specific pathogen free (Specific pathogen-free, SPF) grade C57BL/6N mice (Male, body weight 18-22 g), purchased from Peking Vitre Lihua laboratory animal technologies Co., ltd., production license number: SCXK- (jing) 2016-0011, certification authority: beijing scientific and technical Committee, pass number: 11400700300671.
1.2 drugs and Primary Agents
1.3 Main experiment instrument
2. Experimental method
2.1 modeling and grouping
About 80 SPF-class male C57BL/6N mice are taken, 10 mice are taken as blank control groups, and the rest mice are subjected to tracheal instillation of bleomycin to cause pulmonary fibrosis. The pentobarbital sodium 80mg/kg is injected into the abdominal cavity to anesthetize the mice, the upper incisors of the mice are fixed on the tracheal instillation operation table, the LED light penetrates the neck skin, and the mouth of the mice is observed from the oral cavity inwards. The trocar was inserted into the trachea of the mice and a HRH-MAG4 pulmonary liquid quantitative nebulizer was connected to rapidly push 50 μl of sterile PBS-dissolved bleomycin solution with a molding dose of 0.04U/piece. The mice are kept upright and light after administration, so that the solution is uniformly distributed in the lung, water and feed are administered after the mice are awakened, and the pulmonary fibrosis mice model is built. The blank was operated in the same manner except that the sterile PBS solution was administered to the trachea.
After 14 days, the animals are randomly grouped into a model group, a Nidamib positive medicine group, a yellow root tablet 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 establishment, mice of each group were given the corresponding drug intervention by daily gavage for 14 days, and the blank group and model group were given 0.5% cmc-Na aqueous solution; the Nidaminib positive medicine group is used for dosing a Nidaminib water distribution solution with the dosage of 50mg/kg; administering a suspension of the Huangguang tablets into the Huangguang tablet group, wherein the administration dosage is 200mg/kg; the administration amount of the extract of the roots of the mountain flower is 100mg/kg by respectively administering the ethyl acetate extract of the roots of the mountain flower, the n-butanol extract of the roots of the mountain flower and the aqueous extract suspension of the roots of the mountain flower.
2.3 observations index
The body weight of the mice was recorded weekly from the molding, and the death of the mice was recorded daily. Mice were anesthetized intraperitoneally with 90mg/kg of pentobarbital sodium 14 days after administration, the trachea was exposed, and flexvent was connected to detect pulmonary function in the mice. After the chest of the mice was opened and the heart perfused to flush out the lung blood, the lungs were removed and the left rat lung was fixed in 4% paraformaldehyde solution, and conventionally paraffin embedded for subsequent Masson staining and immunohistochemistry to observe lung tissue pathology and degree of lung fibrosis. Taking each leaf of the right lung, fully rinsing in physiological saline, sucking the surface moisture by filter paper, and preserving in a refrigerator at-80 ℃ for subsequent detection of Hydroxyproline (HYP) content of lung tissues.
2.3.1 mice body weight and cage side observations
Cage side observations include, but are not limited to, the following: the death of mice is whether there are wet royalties, nasal spray, bow back, vertical hair, etc., and changes in limb activity and behavior patterns. The time of occurrence, extent and duration, etc. are recorded. The body weight of mice before and after molding was recorded weekly.
2.3.2 respiratory function detection
For 14 days, 90mg/kg of pentobarbital sodium was injected intraperitoneally for full anesthesia 24 hours after the last administration, and the supine position was placed on the dissected plate of the mice. The skin of the neck of the mouse was rubbed with alcohol, the skin was cut off, the neck muscle was blunt-separated, the trachea was exposed, a small opening was transected under the thyroid cartilage, a tracheal cannula was inserted and ligated with a suture. Connecting the tracheal intubation and a Flexvent instrument, detecting the total volume (IC) of the inspiration of the mouse through a Deep information module, detecting respiratory system resistance (Rrs), compliance (Crs) and elasticity (Ers) by a Snapshot-150 module, detecting indexes such as main airway resistance (Rn), tissue damping (G) and tissue elasticity (H) by a Quick Prime-3 module, and drawing the respiratory P-V loop of the mouse by PVs-P.
2.3.3Masson staining
The left lung of the mouse was fixed in 4% paraformaldehyde and used to prepare pathological sections. Fixing the tissue for at least 72 hours, washing the tissue with water, dehydrating the tissue by alcohol gradient, and embedding the tissue into xylene transparent tissue. After tissue embedding, slicing is carried out by adopting a semi-automatic rotary slicing machine, the slicing thickness is 4 mu m, and the slices are baked for 1h at 60 ℃. The method uses Nanjing to build rapid Masson dyeing, and comprises the following specific steps: (1) dewaxing paraffin sections into water: xylene is rinsed twice for 10min, 100% ethanol, 95% ethanol, 75% ethanol, 30% ethanol for two min each, and warm distilled water for 60s twice. (2) Nuclear staining: regaud's hematoxylin stain 60s,0.1% acetic acid wash rinse 30s. (3) size staining: ponceau dye liquor dyeing for 60s,0.1% acetic acid wash rinse for 30s. (4) differentiation: the 1% phosphomolybdic acid aqueous solution is differentiated for 6-8min, the fiber part is observed to be light pink under a mirror, and the color separation liquid is discarded without washing. (5) collagen counterstaining: counterstaining with aniline blue water solution for 5min, pouring, and washing with absolute ethanol. (6) sealing piece: and (5) sealing the sheet by using neutral resin after blow-drying. (7) microscopic examination: collagen fibers, mucus and cartilage are blue; cytosol, muscle, cellulose, glia are red; the nucleus is bluish violet. Staining was quantitatively analyzed using ImageJ v1.50c image software. Each slice randomly selects 5 different visual fields, and the blue collagen area is positive, and the ratio of the positive area to the total area of the tissue in the whole visual field is taken as the lung fibrosis index.
2.3.4 Hydroxyproline (HYP) detection
The detection kit for detecting hydroxyproline in right lung tissue of mice by using Nanjing is prepared according to the specification, and comprises the following specific steps: (1) sample hydrolysis: accurately weighing the tissue wet weight of 20-40mg in a 15mL centrifuge tube, adding 1mL of hydrolysate, and uniformly mixing. Covering with boiling water bath for 10min, shaking, and continuing boiling water bath for 10min to allow hydrolysis. (2) adjusting the pH: after the centrifuge tube is cooled by running water, 10 mu L of indicator is added, the mixture is shaken uniformly, and the pH is regulated to be about 6.0-6.8. Double distilled water was added to 10mL and mixed well. About 30mg of activated carbon was added to 3ml of the diluted hydrolysate to clarify and colorless the solution, and the mixture was centrifuged at 3500rpm for 10min, and the supernatant was carefully taken for detection. (3) sample detection: the method comprises the steps of adding 1mL of double distilled water into a blank tube, adding 1mL of standard application liquid with the concentration of 5 mug/mL into a standard tube, adding 1mL of sample to be detected into a sample tube, and operating the same. 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; 0.5mL of reagent tris (dimethylaminobenzaldehyde solution) was added, mixed well, water-bath at 60℃for 15min, cooled and centrifuged at 3500rpm for 10min. (4) detecting absorbance: the absorbance at 550nm was measured from the supernatant, and the absorbance value of each tube was measured. And calculating the content of hydroxyproline in the tissue according to a calculation formula.
3. Experimental results
3.1 influence of different extraction parts of the root of the Nanshan mountain on the respiratory function of mice.
The body weight of the mice before and after molding was recorded and the results are shown in table 1: compared with the blank control group, the weight of the mice in the model group is greatly reduced, and the Nidamib and the ethyl acetate extract of the Nanshan flower root have weight improvement effect. The respiratory function results are shown in table 2 and fig. 1: compared with the blank control group, the total capacity of the respiratory system of the mice in the model group is reduced, and the compliance of the respiratory system is reduced; the ethyl acetate extract of the roots of the rhizoma polygonati can effectively improve the total respiratory system capacity and the respiratory system compliance of mice, and has better effect than that of a radix polygoni multiflori tablet group and statistically different results. Indicating that the ethyl acetate extract of the roots of the mountain flower can improve the respiratory function of mice.
Table 1 mice body weight was recorded (unit g, compared to the blank, # p<0.05, ## p<0.01, p compared to model group<0.05)
Table 2 mouse respiratory function (compared to the blank, ## p<0.01, p compared to model group<0.05,**p<0.01)
3.2 influence of different extraction sites of the roots of the Nanshanhua on the pulmonary fibrosis degree of mice caused by bleomycin.
Masson staining (fig. 2) showed that the blank group had positive staining of blue collagen only around the bronchial wall, the vessel wall, and the morphological structure was substantially normal. Model mice present areas of pulmonary fibrosis, with proliferation and thickening of collagenous fibrous tissue. The positive medicine, namely the nintedanib and the ethyl acetate extract of the roots of the mountain flowers, have better improvement effect. The positive area ratio is shown in the bar graph of fig. 2.
3.3 influence of different extraction sites of the roots of the Nanshanhua on the hydroxyproline content of the lung tissue of the mice.
Hydroxyproline is 13.4% in collagen, very little in elastin, and none in other proteins. When the lung is fibrosed, the main component to be increased is collagen fiber, and the content of hydroxyproline in lung tissue is measured and can be converted into the content of collagen in lung so as to reflect the degree of pulmonary fibrosis. The results are shown in Table 3 and FIG. 3, with statistically different (p < 0.01) levels of hydroxyproline in the lungs of mice in the model group. Statistical differences (p < 0.01) occurred in the ethyl acetate extract of the roots of the nanshan flowers compared to the model group. The result shows that the ethyl acetate extract of the rhizoma arisaematis root can reduce the pulmonary collagen deposition and the pulmonary fibrosis degree of mice caused by bleomycin.
Table 3 mouse lung tissue hydroxyproline content (compared to the blank, ## p<0.01, p compared to model group<0.01)
Experimental example 2: therapeutic effect of extracts of different preparation methods of Nanshanhua root on bleomycin-induced pulmonary fibrosis of mice
1. Experimental materials
1.1 laboratory animals
Specific pathogen free (Specific pathogen-free, SPF) grade C57BL/6N mice (Male, body weight 18-22 g), purchased from Peking Vitre Lihua laboratory animal technologies Co., ltd., production license number: SCXK- (jing) 2016-0011, certification authority: beijing scientific and technical Committee, pass number: 11400700300671.
1.2 drugs and Primary Agents
1.3 Main experiment instrument
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2. Experimental method
2.1 modeling and grouping
About 80 SPF-class male C57BL/6N mice are taken, 10 mice are taken as blank control groups, and the rest mice are subjected to tracheal instillation of bleomycin to cause pulmonary fibrosis. The pentobarbital sodium 80mg/kg is injected into the abdominal cavity to anesthetize the mice, the upper incisors of the mice are fixed on the tracheal instillation operation table, the LED light penetrates the neck skin, and the mouth of the mice is observed from the oral cavity inwards. The trocar was inserted into the trachea of the mice and a HRH-MAG4 pulmonary liquid quantitative nebulizer was connected to rapidly push 50 μl of sterile PBS-dissolved bleomycin solution with a molding dose of 0.04U/piece. The mice are kept upright and light after administration, so that the solution is uniformly distributed in the lung, water and feed are administered after the mice are awakened, and the pulmonary fibrosis mice model is built. The blank was operated in the same manner except that the sterile PBS solution was administered to the trachea.
After 14 days, animals were randomly grouped into model groups, the nilanide positive drug group, the example 1 group, the example 2 group, the example 3 group, the example 4 group, 10 animals per group, based on the weight of the surviving model groups.
2.2 methods of administration
After 14 days of model establishment, mice of each group were given the corresponding drug intervention by daily gavage for 14 days, and the blank group and model group were given 0.5% cmc-Na aqueous solution; the Nidaminib positive medicine group is used for dosing a Nidaminib water distribution solution with the dosage of 50mg/kg; example 1-example 4 groups were each dosed with 100mg/kg of suspension of extract of the roots of the Sonchus arvensis in 0.5% CMC-Na aqueous solution.
2.3 observations index
The body weight of the mice was recorded weekly from the molding, and the death of the mice was recorded daily. Mice were anesthetized intraperitoneally with 90mg/kg of pentobarbital sodium 14 days after administration, the trachea was exposed, and flexvent was connected to detect pulmonary function in the mice. After the chest of the mice was opened and the heart perfused to flush out the lung blood, the lungs were removed and the left rat lung was fixed in 4% paraformaldehyde solution, and conventionally paraffin embedded for subsequent Masson staining and immunohistochemistry to observe lung tissue pathology and degree of lung fibrosis. After the right lung leaves are fully rinsed in physiological saline, the filter paper absorbs surface moisture, and the filter paper is preserved in a refrigerator at the temperature of minus 80 ℃ for subsequent detection of Hydroxyproline (HYP) content, TGF-beta and TNF-alpha content of lung tissues and Western Blot detection of the expression of pulmonary fibrosis related proteins.
2.3.1 mice body 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) detection
The same as in experimental example 1.
2.3.5 Immunohistochemical (IHC) staining
The left lung of the mouse was fixed in 4% paraformaldehyde and used to prepare pathological sections. Fixing the tissue for at least 72 hours, washing the tissue with water, dehydrating the tissue by alcohol gradient, and embedding the tissue into xylene transparent tissue. After tissue embedding, slicing is carried out by a semi-automatic rotary slicing machine, the slicing thickness is 4 mu m, and the slice is baked at 60 ℃. Dewaxing by xylene, hydrating by gradient ethanol, and repairing by antigen (0.01M citrate buffer solution, pH6.0, high pressure for 2 min); endogenous catalase was blocked with 3% hydrogen peroxide, and 5% bsa was blocked at room temperature. Sections were incubated overnight with a-SMA primary antibody at 4 ℃. After washing the sections with PBS buffer, the corresponding secondary antibodies were incubated for 1h at room temperature. Dripping freshly prepared DAB color-developing agent, counterstaining with hematoxylin, differentiating with alcohol hydrochloride, dehydrating with gradient alcohol, drying, transparent xylene, and observing the tan as positive expression under microscope. Staining was quantified using ImageJ v1.50c image software. Each slice was randomly selected from 5 different fields and analyzed for the relative area of positive expression.
2.3.6 enzyme-linked immunosorbent assay (ELISA) detection
The lung tissue samples of 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 thoroughly homogenized using a homogenizer. Centrifugation at 3500rpm for 20min, the supernatant was collected for ELISA assay. The supernatant was used to detect the levels of TNF- α and TGF- β in lung tissue according to the instructions of the Jiangsu Jingmei Biotech Co., ltd. Rat TNF- α and TGF- β kits. The BCA protein quantification kit was used to measure the total protein concentration of the tissue, and the results were expressed as the ratio of TNF- α or TGF- β content to total protein.
2.3.7Western blot detection of expression of related proteins
The lung tissue samples of each group of mice were collected and the tissue samples were frozen at-80 ℃ until detection. A small piece of lung tissue is taken, weighed, added with a certain amount of RIPA lysate containing 1% protease inhibitor and 1% protein phosphatase inhibitor, ground and cracked 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. Mu.g of protein samples were diluted with 5 Xloading buffer and heated at 100℃for 5 minutes, run on SDS-PAGE gels, subsequently transferred to PVDF membranes, blocked with 5% nonfat milk powder for 1h, incubated overnight at 4℃and the corresponding secondary antibodies were incubated at room temperature for 1h after 3 washes with TBST buffer, and developed with ECL chemiluminescent solution after washing. After exposure recording by a chemiluminescent instrument, the bands were analyzed using ImageJ.
2.4 statistical methods
Experimental results are expressed as mean ± standard deviationThe analysis was performed using statistical software SPSS 20, the model group was tested against the blank group using Student's t, each group dosed and model group was tested for variance alignment using One Way ANOVA, the Homogeneity test for variance alignment, and the least significant method (LSD) test if the variances were aligned; if the variance is not the sameThe alignment was checked using Tamhane's T2. At p<0.05 is significant.
3. Experimental results
3.1 influence of extracts of different preparation methods of Nanshan flower root on respiratory function of mice.
Body weight, mortality, and respiratory function of mice after molding and after treatment with the nanshan flower root extract were recorded. The body weight results are shown in table 4: compared with the blank control group, the weight of the mice in the model group is greatly reduced, the recovery of different degrees occurs after the treatment of the Nanshanhua root extract of different preparation methods, and the death 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 the blank control group, the total capacity 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 system capacity and respiratory system compliance of mice, with statistical differences. It is shown that the groups of examples 1-4 are able to improve the respiratory function of mice.
Table 4 mice body weight record (unit g) and final mortality (compared to the blank, # p<0.05)
table 5 mouse respiratory function (compared to the blank, ## p<0.01, p compared to model group<0.05,**p<0.01)
3.2 effects of extracts of different preparation methods of Nanshanhua root on the pulmonary fibrosis degree of mice caused by bleomycin.
Masson staining (fig. 5) showed that the blank group had positive staining of blue collagen only around the bronchial wall, the vessel wall, and the morphological structure was substantially normal. Model mice present areas of pulmonary fibrosis, with proliferation and thickening of collagenous fibrous tissue. There was a different degree of improvement in each group administered, with the positive drug, the nilamide group, and the example 4 group having the best improvement. The positive area ratio is shown in the bar graph of fig. 2.
3.3 effects of extracts of different preparation methods of Nanshan flower root on hydroxyproline content of mouse lung tissue.
The results are shown in table 6 and fig. 6, with a statistical difference (p < 0.01) in the increase of hydroxyproline content in the lungs of mice in the model group. Lung tissue hydroxyproline content range dose-dependent decline in different concentration dosing groups of nanshan flower root, statistical differences (p < 0.01) appear for each example group compared to the model group. The results show that the extract of the roots of the Nanshanhua can reduce the pulmonary collagen deposition and pulmonary fibrosis degree of mice caused by bleomycin, wherein the effect of the example 4 group is optimal.
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Table 6 hydroxyproline content in lung tissue of mice (# p <0.01 compared to the blank group, # p <0.01 compared to the model group)
3.4 Effect of extracts of different preparations of Nanshanhua root on expression of TGF-beta and TNF-alpha in mouse lung tissue.
TGF-beta is the most important regulator of tissue fibrosis due to deregulation of extracellular matrix synthesis and deposition, and is associated with a variety of organ fibrosis, cirrhosis, and atherosclerosis. TNF- α is a cytokine that plays a critical role in the pathogenesis of pulmonary fibrosis. Thus, we detected TGF- β and TNF- α content in mouse lung tissue by ELISA. The results are shown in Table 7 and FIG. 7, and there were statistical differences in the rise of TGF-beta and TNF-alpha levels in the lung tissue of mice in the model group; the administration group reduced the TGF-beta and TNF-alpha content in lung tissue. The results show that the corresponding extract of the root of the Sonchus arvensis might inhibit the progression of pulmonary fibrosis by affecting the expression of TGF-beta and TNF-alpha.
Table 7 mouse lung tissue TGF-beta and TNF-alpha content (compared to blank, #p <0.01, compared to model, #p <0.05, #p < 0.01)
3.5 Effect of extracts of different preparation methods of Nanshan flower root on mouse lung tissue COL1A1, COL3A1 and alpha-SMA protein expression.
After pulmonary fibrosis occurs, fibroblasts are activated to release a large amount of extracellular matrix, the main components of which are COL1A1, COL3A1, and α -SMA is the main marker of fibroblast activation. We examined COL1A1 and COL3A1 protein expression in mouse lung tissue by Western Blot. Meanwhile, we also detected the distribution of α -SMA in mouse lung tissue by immunohistochemistry. The results (table 8 and fig. 8) show that the release of type I and type III collagen from mouse lung tissue after bleomycin stimulation is increased, α -SMA expression is increased and widely distributed, statistically different (p < 0.01) compared to the blank group. The extract of different preparation methods of the south mountain flower root has obvious improvement effect after treatment, and has statistical difference (p < 0.01) compared with a model group. The results show that the extract of the roots of the Nanshanhua can reduce the increase of COL1A1, COL3A1 and alpha-SMA protein expression caused by pulmonary fibrosis of mice, and the effect of the example 4 is the most remarkable.
TABLE 8 influence of extracts of different preparation methods of Nanshanhua root on bleomycin-induced expression of mouse lung tissue protein
Experimental example 3: effective compounds in Nanshan flower root extract inhibit fibroblast activation
1. Experimental background
Fibroblasts do not express alpha actin (alpha-SMA) in the unactivated state, and after activation by TGF- β1, the fibroblasts activate to myofibroblasts and stop proliferation, express alpha-SMA and release extracellular matrix related proteins. We obtained from literature and experiments that the anthraquinone extraction site of the Sonchifolia root contains two known compounds of scopoletin and methyliapigenin-1-methyl ether, designed into a fibroblast activation experiment and given the treatment of the compounds contained in the Sonchifolia root extract, examined the anti-fibroblast activation activity of scopoletin and methyliapigenin-1-methyl ether.
2. Experimental materials
Human lung fibroblast line HPF cells were purchased from Shanghai enzyme-linked biotechnology Co., ltd, human recombinant TGF-beta 1 was purchased from Peprotech Co., scopoletin and methylimadder-1-methyl ether were purchased from Beijing-Bettrey biological medicine Co., ltd.
3. Experimental method
HPF cells are spread on a 6-well plate, the culture medium is discarded until the HPF cells grow to about 70%, 1mL of serum-free culture medium (containing 0.1% DMSO) is added to a negative control group, 1mL of serum-free culture medium containing 10ng/mL TGF-beta 1 is added to a preparation group and a dosing group, scopoletin or methyl alizarin-1-methyl ether is respectively added to the dosing group, the final concentration reaches 5 mug/mL, and the liquid medicine is uniformly mixed by horizontally shaking the culture plate. Placed at 37 ℃ and 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 collected using a spatula and centrifuged at 13000rpm at 4℃for 10min. And taking the supernatant, transferring the supernatant to a new EP tube, and carrying out Western blot experiment to detect the expression condition of the alpha-SMA protein.
4. Experimental results
The results are shown in Table 9 and FIG. 9, where fibroblast expression was increased after TGF-. Beta.1 induction. While scopoletin and methyl alizarin-1-methyl ether both have the effect of inhibiting the increase of alpha-SMA expression, have the effect of inhibiting the activation of fibroblasts, and have statistical differences (p < 0.01).
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Table 9 scopoletin and methyl alizarin-1-methyl ether inhibiting Activity on fibroblasts
Experimental example 4: the pharmaceutical composition of the invention inhibits fibroblast activation
The experimental materials and experimental methods were the same as in experimental example 3, and the administration groups were respectively the nilamide groups, examples 1, 5,6, 7, 8, and 9. The doses of Nidamib, example 8 and example 9 were 2. Mu.M, and the other example groups were 5. Mu.g/mL. Example 1 is ethyl acetate extract of the root of the mountain flower; example 5 pharmaceutical combinations of anthraquinones, anthraquinone glycosides and iridoids according to weight 8:1:1 weight ratio; example 6 pharmaceutical combination of 1, 3-dihydroxy-5, 6-dimethoxy-2-methylanthraquinone, methyliapigenin-1-methyl ether, methyliapigenin, and scopoletin according to 1:5:5:1 weight ratio mixing: example 7 the pharmaceutical combination is scopoletin and methylimadder-1-methyl ether according to 1:1 weight ratio; example 8 pharmaceutical combination of the extract of the root of the south mountain flower of example 1 and pirfenidone according to 10:1 weight ratio; example 9 pharmaceutical combination of the extract of the roots of the nanshan flower of example 1 and nilamide cloth according to 8:1 by weight ratio.
Experimental results
As shown in table 10 and fig. 10, comparing the data in table 9 of experimental example 3, the scopoletin group had a value of 5.10 and the methylimadder-1-methyl ether group had a value of 4.22, and example 7, which was a combination of scopoletin and methylimadder-1-methyl ether, had a value of 3.10, and the result in example 7 was significantly better than the result of scopoletin and methylimadder-1-methyl ether alone, that is, scopoletin and methylimadder-1-methyl ether, which were combined, and had a synergistic effect. In addition, as is clear from comparing the results of the nilamide group, the example 1 group and the example 9, the example 9 group has a significantly better technical effect in the same dose as the nilamide group and in a lower dose than the example 1 group, and the specification has a synergistic effect after combining the nanshan flower root extract with chemicals such as pirfenidone or nilamide.
TABLE 10 inhibition of fibroblast inhibitory Activity by different combinations of drugs
Experimental example 5: analysis of traditional Chinese medicine components of radix scutellariae extract based on liquid chromatography-mass spectrometry technology
1. Chromatographic and mass spectral conditions
Agilent 1290 ultra-high performance liquid chromatography combined with Agilent 6550 triple quadrupole time-of-flight mass spectrometry. The column was Waters BEH C18 (2.1 x 100mm,1.7 μm), mobile phase a phase: 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% B;5-30min,20-100% B, flow rate: 0.3mL/min, column temperature: 30 ℃.
Adopting a primary High Resolution (HRMS) and secondary high resolution (HRMS/MS) mass spectrum anion acquisition mode, and drying gas temperature: 280 ℃, drying gas flow rate: 11L/min, sheath air temperature: 325 ℃, sheath air flow rate: 12L/min, primary mass spectrum scan range: 150-1200m/z, secondary mass spectrum scan range: 50-1200m/z, secondary collision voltage: 10/20/30/40/50V.
2. Experimental results
Identification and authentication of traditional Chinese medicine components in Nanshanhua root extract based on liquid chromatography high-resolution mass spectrometry technology
Primary High Resolution Mass Spectrometry (HRMS) data is collected based on optimized chromatographic mass spectrometry conditions. Comparing HRMS data with a self-built database, identifying traditional Chinese medicine components in the extract of the root of the Arnica, further collecting secondary high resolution mass spectrum (HRMS/MS) data of the traditional Chinese medicine components of the extract of the root of the Arnica under different collision voltages, and identifying 30 traditional Chinese medicine components in the extract of the root of the Arnica, wherein the 30 traditional Chinese medicine components comprise 23 anthraquinone components, 4 iridoid components and 3 anthraquinone glycoside components, and partial compounds are isomer compounds. The basic information of each identified component is shown in the following table.
Table 11 Chinese herbal medicine composition of Huangguan extract identified based on liquid chromatography high resolution mass spectrometry
Conclusion of experimental example: the extract of the roots of the Sonchus arvensis of the invention can improve pulmonary fibrosis caused by different factors, can improve pulmonary function, reduce collagen deposition and fibroblast proliferation and activation of pulmonary tissues, and can inhibit TGF-beta and TNF-alpha of the pulmonary tissues to inhibit pulmonary fibrosis. The extraction part of the south mountain flower root is anthraquinone, iridoid and anthraquinone glycoside. Two specific compounds, scopoletin and methyl alizarin-1-methyl ether, clearly have the effect of inhibiting fibroblast activation. The extract of the roots of the Sonchus arvensis has definite anti-pulmonary fibrosis effect, and scopoletin and methyl alizarin-1-methyl ether prove to have the anti-pulmonary fibrosis potential on the cellular level, and have synergistic effect after combination. Meanwhile, after the Nanshan flower root extract is combined with chemicals such as pirfenidone or nindani which are chemicals for resisting pulmonary fibrosis, the Nanshan flower root extract has a synergistic effect. In summary, the extract of the roots of the Sonchus arvensis and the components thereof can be used for preparing medicines for resisting pulmonary fibrosis.
Claims (7)
1. Use of a nanshan flower root extract in the preparation of a medicament for treating pulmonary fibrosis, characterized in that: the extract of the root of the Sonchus arvensis is an anthraquinone, anthraquinone glycoside and iridoid effective part; wherein the content of anthraquinone, anthraquinone glycoside and iridoid active ingredients accounts for more than 64% of the total amount of the extract;
the preparation method of the nanshan flower root extract comprises the following steps:
reflux extracting radix Arnicae with 95% ethanol for three times, concentrating the extractive solution under reduced pressure, removing impurities by preparative chromatography, and purifying to obtain anthraquinone, anthraquinone glycoside and iridoid effective components, wherein the chromatography is silica gel column chromatography, and gradient eluting with water and acetonitrile;
or extracting radix Ardisiae Japonicae with petroleum ether, discarding extractive solution, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate extract;
or extracting radix Ardisiae Japonicae with water, extracting with 95% ethanol, mixing water and ethanol extractive solutions, concentrating under reduced pressure, extracting with ethyl acetate, and rotary evaporating to remove solvent.
2. Use according to claim 1, characterized in that: the content of the anthraquinone, anthraquinone glycoside and iridoid active ingredients is more than 71% of the total extract.
3. Use according to claim 1, characterized in that: the content of the anthraquinone, anthraquinone glycoside and iridoid active ingredients accounts for more than 79% of the total extract.
4. Use according to claim 1, characterized in that: the content of the anthraquinone, anthraquinone glycoside and iridoid active ingredients accounts for 100% of the total amount of the extract.
5. Use according to claims 1-4, characterized in that: the treatment of pulmonary fibrosis is to improve pulmonary function, reduce collagen deposition and fibroblast proliferation and activation of pulmonary tissue, inhibit TGF-beta and TNF-alpha of pulmonary tissue and inhibit pulmonary fibrosis.
6. Use according to claim 1, characterized in that: the anthraquinone active ingredients comprise 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylimadin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy-3-hydroxymethylanthraquinone, normethylanthraquinone, methylimadin-1-methyl ether, tiger-aldehyde, 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-methylanthraquinone, scopolide, 3, 6-dimethoxy-2, 3-dimethoxy-anthraquinone, 7-methylanthraquinone, 7-hydroxy-1, 2, 3-trimethoxy-2-methylanthraquinone, 3-hydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5, 6-methylanthraquinone, 3-hydroxy-2-methoxy-methylanthraquinone, 3-hydroxy-2-methylanthraquinone, 3-hydroxy-2-methyl anthraquinone, one or more of 1, 3-dihydroxy-5, 6-dimethoxy-2-methoxymethylanthraquinone;
The anthraquinone glycoside active ingredient comprises one or more of lucidin 3-O-beta-primeveroside, damnacanthol 3-O-beta-primeveroside, rubiadin 3-O-beta-primeveroside;
the iridoid active ingredient comprises one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidicacid and asperulosidic acid.
7. A pharmaceutical composition for treating pulmonary fibrosis, characterized by: the extract consists of effective extraction parts of anthraquinone, anthraquinone glycoside and iridoid in the root of the Arnica herb, wherein the weight ratio of the three effective extraction parts is 8:1:1, a step of;
the preparation method of the effective extraction parts of the anthraquinones, anthraquinone glycosides and iridoids in the arisaema root comprises the following steps: reflux-extracting radix Arnicae with 95% ethanol for three times, concentrating the extractive solution under reduced pressure, removing impurities by preparative chromatography, refining and purifying to obtain anthraquinone, anthraquinone glycoside and iridoid effective components, wherein the chromatography is silica gel column chromatography, gradient eluting with water and acetonitrile, collecting three effective component parts of anthraquinone, anthraquinone glycoside and iridoid respectively, concentrating under reduced pressure, and drying to obtain purified anthraquinone, anthraquinone glycoside and iridoid effective component parts.
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