WO2022228095A1

WO2022228095A1 - Application of prismatomeris connata y. z. ruan root extract in preparation of medicine for treating pulmonary fibrosis

Info

Publication number: WO2022228095A1
Application number: PCT/CN2022/086065
Authority: WO
Inventors: 靳洪涛; 张金兰; 胡广; 生宁; 李思铮; 李梦林; 江海燕; 王冬梅; 王喆
Original assignee: 中国医学科学院药物研究所
Priority date: 2021-04-27
Filing date: 2022-04-11
Publication date: 2022-11-03
Also published as: CN116585295A; CN115245533A; CN115245533B

Abstract

A use of Prismatomeris connata Y. Z. Ruan root extract in the preparation of medicine for treating pulmonary fibrosis. The extract has effective parts of anthraquinones, anthraquinone glycosides and iridoids. The extract can be used alone or in combination with a chemical agent capable of resisting pulmonary fibrosis, and is prepared into a pharmaceutical composition for treating pulmonary fibrosis.

Description

Application of Nanshan Flower Root Extract in the Treatment of Pulmonary Fibrosis

technical field

The invention belongs to the field of medicine, and in particular relates to the application of extraction parts and compounds obtained by extracting the root of the traditional Chinese medicine Nanshan flower in preventing and treating pulmonary fibrosis diseases.

Background technique

Pulmonary fibrosis (PF) is a type of disease in which diffuse exudation, infiltration and fibrosis of the pulmonary interstitium are the main lesions. Its main symptoms include dyspnea, shortness of breath, dry cough, and wheezing. die. The vast majority of patients with pulmonary fibrosis are of unknown etiology (idiopathic). This group of diseases is called idiopathic interstitial pneumonia (IIP), which is a large category of interstitial lung diseases. Idiopathic pulmonary fibrosis (IPF) is the most common type of disease with pulmonary fibrosis as the main manifestation in idiopathic interstitial pneumonia. Sexual lung disease. At present, its pathogenesis is still unclear, and only two drugs are marketed specifically for the treatment of idiopathic pulmonary fibrosis worldwide. Pirfenidone was first used in Japan for the treatment of IPF in 2008, and in 2014, pirfenidone and nintedanib were approved by the US FDA for the specific treatment of IPF. However, neither of these two drugs can reverse or prevent the progression of IPF, and they are expensive, placing a greater burden on patients and their families.

Pulmonary fibrosis can be divided into primary pulmonary disease and partial manifestations of systemic disease. (1) The types of pulmonary fibrosis as primary lung diseases include: radiation fibrosis (ie, fibrosis caused by radiation), fibrosis as a result of damaging inflammatory lung lesions (such as sclerosing tuberculosis, honeycomb lung, Small cystic pulmonary fibrosis), fibrosis as the result of allergic reaction, drug-induced interstitial fibrosis, fibrosis as the end-stage manifestation of interstitial inflammation, etc.; (2) pulmonary fibrosis is the result of systemic disease Some manifestations include: granulomatous disease (such as sarcoidosis, amyloidosis, nevus hamartoma, xanthoma), collagen disease and rheumatic disease (such as scleroderma, lupus erythematosus, nodular disease) periarteritis, dermatomyositis, rheumatoid arthritis), etc.

In recent years, Chinese medical workers have achieved fruitful results in discovering effective drugs for treating and delaying pulmonary fibrosis in traditional Chinese medicine. Finding drugs for the treatment of pulmonary fibrosis from natural sources is considered an effective way to develop such drugs.

Nanshan flower is also known as dog bone wood. It is the root of Prismatomeris connata Y.Z. Ruan, a Rubiaceae plant, and is one of the characteristic medicinal materials in Guangxi. There are strong roots and bones, dampness and yellowing, blood stasis and regeneration, cooling blood and hemostasis.

In recent years, some scholars have found that Nanshan flower root extract can inhibit liver fibrosis by inducing apoptosis of rat astrocytes. However, there is no report on the anti-pulmonary fibrosis activity and use of Nanshan flower root extract or compounds derived from Nanshan flower root. In the prior art (Chinese Journal of New Drugs, Vol. 25 and No. 24 in 2016, pages 2868-2869), it was disclosed that the ethyl acetate extraction site and the water site of the ethanolic extract of Nanshan flower root may be active against liver fibrosis Several compounds such as stigma-4-en-3-one and norsine can be obtained by separation and purification, but there is no evidence that the compounds identified by separation and purification are active ingredients, and their use is anti-hepatic fibrosis. . The prior art (Chinese Journal of Comparative Medicine, Vol. 30, Issue 2, Pages 9-14) discloses that scopolide can protect the liver of hepatic fibrosis rats, positively regulates the expression of cytokines related to hepatic fibrosis, It can reduce the level of extracellular matrix in liver, improve the degree of hepatic fibrosis in rats, and has anti-hepatic fibrosis effect. The prior art (Journal of Beijing University of Traditional Chinese Medicine, Vol. 32, No. 1, Pages 42-44) discloses that the alcohol extract of Nanshan flower root can significantly reduce the proliferation and deposition of collagen fibers in the liver of rats, and reduce the area of fibrotic tissue. It has certain development value in the treatment of liver fibrosis. However, the above-mentioned uses are anti-hepatic fibrosis, and the mechanisms of fibrosis in different organs are different. Clinically, liver fibrosis is mostly fibrosis caused by bacterial and viral infection, toxin damage, and abnormal metabolism. It is currently believed that liver fibrosis is a reversible process. Pulmonary fibrosis is considered to be an irreversible process, and the lungs are directly connected to the outside world. The causes of pulmonary fibrosis are more complicated, and the etiology of most patients is unknown. From the prior art, many drugs have the effect of preventing and treating liver fibrosis, but the drugs that clearly have the effect of preventing and treating pulmonary fibrosis are very rare. As mentioned above, so far, only pirfenidone and nintedanib are known to be For pulmonary fibrosis, that is to say, drugs used to prevent and treat liver fibrosis are generally not used to prevent and treat pulmonary fibrosis.

Guangxi Yingkang Pharmaceutical Co., Ltd. has developed a tablet prepared from Nanshan flower for the treatment of silicosis, and its indication is "treatment of silicosis". liquid, filtered, the filtrate was decompressed to recover ethanol, concentrated to a thick paste, stirred three times with 10 times the amount of warm water, allowed to stand, filtered, the filtrate was concentrated under reduced pressure, vacuum-dried, pulverized, added with appropriate amount of auxiliary materials, made into granules, Compressed into 1,000 tablets, coated with sugar, and obtained. However, silicosis is known to be a lung disease 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 rapidly progressive manifestations are similar to interstitial pneumonia. performance, and pulmonary fibrosis is not the same. Secondly, it is considered in the prior art that the main active ingredient of the above-mentioned tablet is a polymer formed by a variety of metal elements mainly composed of organoaluminum, and the mechanism of action is that aluminum forms insoluble aluminum silicate on the surface of silicon dioxide, which makes it difficult to dissolve. Macrophages lose their toxic effect, thus antagonizing the cytotoxic effect of quartz (see Li Yu et al., Editor-in-Chief, "Mine Dust and Occupational Hazard Prevention and Control Technology", Metallurgical Industry Press, October 2017, p. 211). However, the inventors unexpectedly found that the active ingredients in the prevention and treatment of pulmonary fibrosis are not the same as those in the treatment of silicosis, and through a large number of studies and experiments, they found the preferred active ingredients for prevention and treatment of pulmonary fibrosis.

The prior art (Chinese Herbal Medicine, Vol. 51, No. 4, pp. 1031-1036) discloses that polysaccharides from Nanshan flower root can reduce the level of inflammation by inhibiting the infiltration of inflammatory cells, and down-regulate the expression levels of α-SMA and Vimentin in lung tissue, Up-regulation of the expression level of E-cadherin suggests that the polysaccharide of yellow root can effectively improve the immune balance, inhibit the development of inflammatory response and EMT, thereby improving the degree of fibrosis in silicosis tissue, and has a good therapeutic effect on silicosis fibrosis. The preparation method of the polysaccharide is as follows: after crushing 10 kg of decoction pieces, extracting with ultrasonic heating water, concentrating under reduced pressure, and precipitating with 80% ethanol to obtain 905 g of crude polysaccharide. After the crude polysaccharide was redissolved in distilled water, the protein was removed by the Savage method, passed through the filter membrane U under high pressure, and then concentrated through the ultrafiltration membrane A to remove low molecular weight components, and the concentrate was dried to obtain 560 g of polysaccharide. The above content suggests that polysaccharide components are the active components of Nanshan flower root in preventing and treating pulmonary fibrosis. However, the above experiments did not compare the effectiveness of polysaccharide and non-polysaccharide components in Nanshan flower root. After in-depth research, the inventor found that polysaccharide components are not the main effective components of Nanshan flower root for preventing and treating pulmonary fibrosis.

The present invention is proposed in order to find an effective part having a definite effect on prevention and treatment of pulmonary fibrosis from Nanshan flower root, and to provide its pharmaceutical use.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new pharmaceutical use of the Nanshan flower root extract, that is, the use of the Nanshan flower root extract in the preparation of a medicine for treating pulmonary fibrosis. The anti-pulmonary fibrosis effective parts of the extract are discovered for the first time, and the preparation method is simple, the effective parts are clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is obviously better than the prior art, and can be combined with the existing technology. Anti-pulmonary fibrosis chemicals have the advantages of synergistic effect.

In the present invention, "effective part" refers to an extract composed of one or several types of components extracted from a single plant with relatively clear components, which can also be called effective components or effective intermediates ("Quality of New Chinese Medicines"). Research Technical Guiding Principles (for Trial Implementation, January 2021). "Anthraquinones, Anthraquinone Glycosides and Iridoids Effective Parts" means that the compounds of anthraquinones, anthraquinone glycosides and iridoids in the extract are the constituents of the extract. Although the extract may also contain other components derived from the same plant, these other components are usually not chemical components that can effectively play a therapeutic role in the extract. Because its content is lower than the threshold for exerting a therapeutically effective effect or itself does not have a desired therapeutically effective effect, etc.

Further, the object of the present invention is to provide a kind of preparation method of the above-mentioned effective parts of Nanshan flower root.

Further, the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of anthraquinones, anthraquinone glycosides and iridoids active ingredients of Radix Nanshanii.

Further, the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of scopolide and methylisorubidin-1-methyl ether.

Further, the object of the present invention is to provide a pharmaceutical composition for the treatment of pulmonary fibrosis, which combines the above-mentioned Nanshan flower root extract with anti-pulmonary fibrosis chemical drug pirfenidone or nintedanib, and has Obvious synergistic effect.

In order to achieve the above object, the present invention provides the following specific technical solutions:

A use of Nanshan flower root extract in preparing a medicine for preventing and treating pulmonary fibrosis, characterized in that the Nanshan flower root extract is anthraquinones, anthraquinone glycosides and iridoids effective parts. Preferably, in the above extract, the active ingredients of anthraquinones, anthraquinone glycosides and iridoids account for more than 60% of the content of the extract, more preferably more than 70%, and most preferably 100% by weight.

Preferably, the anthraquinone active ingredients include 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylisorubicin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy anthraquinone -3-Hydroxymethyl anthraquinone, desmethyl oxal, methyl isoalizarin-1-methyl ether, ox aldehyde, 1,3-dihydroxy-2-methoxymethyl anthraquinone, 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, scopolactone, 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, A kind of 4-hydroxy-1,2,3-trimethoxy-7-hydroxymethylanthraquinone and 1,3-dihydroxy-5,6-dimethoxy-2-methoxymethylanthraquinone or more.

Preferably, the anthraquinone glycoside active ingredients include one or more of lucidin 3-O-β-primeveroside, damnacanthol 3-O-β-primeveroside, and rubiadin 3-O-β-primerveroside.

Preferably, the iridoid active ingredients include one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, and asperulosidic acid.

Preferably, the Nanshan flower root extract contains at least 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin Alizarin and scopolactone.

More preferably, the Nanshan flower root extract contains at least scopolactone and methylisorubidin-1-methyl ether. Among them, the combination of scopolactone and methylisorubidin-1-methyl ether produces a synergistic effect, and its effect is obviously better than that of scopolide and methylisorubidin-1-methyl ether. Simple superposition of pulmonary fibrosis effects.

Further, the present invention also provides the use of scopolide in preparing a medicine for preventing and treating pulmonary fibrosis.

Further, the present invention also provides the use of methylisorubicin-1-methyl ether in preparing a medicine for preventing and treating pulmonary fibrosis.

More preferably, the extract of the present invention has a comprehensive prevention and treatment effect on pulmonary fibrosis, can improve pulmonary fibrosis caused by a variety of different factors, can improve lung function, and reduce collagen deposition in lung tissue and fibroblast proliferation and activation. , can inhibit lung tissue TGF-β and TNF-α and inhibit pulmonary fibrosis. The ethyl acetate extract of Nanshan flower root can reduce the elevated expression of COL1A1, COL3A1 and α-SMA proteins caused by pulmonary fibrosis in mice. Its action pathway is not through the formation of insoluble aluminum silicate on the surface of silica, which makes it lose its toxic effect on macrophages.

Still further, the pulmonary fibrosis is not caused by silicosis.

The present invention also provides the preparation method of the Nanshan flower root extract, namely extracting Nanshan flower root with ethanol, then using preparative chromatography to remove impurities, and refining and purifying to obtain anthraquinones, anthraquinone glycosides and iridoids The active ingredient is obtained by extracting Nanshan flower root with ethyl acetate solvent; or after extracting Nanshan flower root with solvent, concentrating the extract, and then extracting with ethyl acetate. Preferably, the solvent is one or a combination of two or more of water, methanol, ethanol, propanol, butanol and the like.

Preferably, the Nanshan flower root is extracted with petroleum ether first, the extract is discarded, then extracted with ethyl acetate, and concentrated under reduced pressure to obtain the ethyl acetate extract.

Preferably, the preparation method can also be as follows: extracting Nanshan flower root with water, then extracting with 95% ethanol, combining water and ethanol extracts, concentrating the extract under reduced pressure, extracting with ethyl acetate and then rotary evaporation to remove the solvent obtained from Nanshan Flower root extract.

Preferably, the preparation method can also be as follows: 95% ethanol reflux extraction of Nanshan flower root three times, the extract is concentrated under reduced pressure, the impurities are removed by preparative chromatography, and the anthraquinones, anthraquinone glycosides and iridoids are obtained by refining and purification The chromatographic method can be one or more of macroporous resin method, molecular sieve chromatography, silica gel adsorption chromatography, polyamide adsorption chromatography, etc. Preferred is silica gel column chromatography, eluting with a gradient of water and acetonitrile.

Optionally, the preparation method can also be: the Nanshan flower root extract obtained by refluxing extraction with 95% ethanol for three times, ethyl acetate extraction, and rotary evaporation to remove the solvent.

The present invention also provides a pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that it is composed of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids in Nanshan flower root. Preferably, the weight ratio of the above three effective extraction parts is 1-10:1-10:1-5.

Further, the pharmaceutical composition for treating pulmonary fibrosis is composed of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methyl Composed of isolarin and scopolactone. Preferably, the weight ratio of the above four compounds is 1-10:1-10:1-10:1-10.

Further, the pharmaceutical composition for treating pulmonary fibrosis is composed of scopolactone and methylisorubicin-1-methyl ether. Preferably, the weight ratio of the above two compounds is 1-10:1-10.

The present invention also preferably provides a pharmaceutical composition for the treatment of pulmonary fibrosis, which is characterized in that it is composed of the above-mentioned Nanshan flower root extract and an anti-pulmonary fibrosis chemical, and the anti-pulmonary fibrosis chemical is pyridine Nintedone or nintedanib. The combination of the Nanshan flower root extract and the chemical drug against pulmonary fibrosis has a synergistic effect, and its effect is obviously better than the simple superposition of the respective effects of the Nanshan flower root extract and the chemical drug for preventing and treating pulmonary fibrosis. Preferably, the weight ratio of Nanshan flower root extract and anti-pulmonary fibrosis chemical is 2-20:1-10.

The medicine for preventing and treating pulmonary fibrosis according to the present invention can prevent and/or treat pulmonary fibrosis, and the medicine is composed of active ingredients and pharmaceutically acceptable excipients, and the active ingredients are Nanshan flower root extract, scopolamine Any one of lactone, methylisorubicin-1-methyl ether, and the pharmaceutical composition.

In the medicine, the content of the active ingredient is 0.1-99.9% (w/w), and the content of pharmaceutically acceptable excipients is 0.1-99.9% (w/w), and the total is 100% .

The medicament of the present invention can be in any pharmaceutically acceptable dosage form, and these dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, buccal preparations, granules, granules, Pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, aerosols, drops, patches.

Appropriate pharmaceutical excipients can be used for the medicine of the present invention, and specific examples of excipients include: fillers, diluents, carriers, excipients, disintegrants, binders, lubricants, flavoring agents, surfactants, colorants , coating agent, propellant, stabilizer, etc.

A suitable pharmaceutically acceptable carrier can be optionally added when preparing a medicament, and the pharmaceutically acceptable carrier is selected from: starch, sucrose, lactose, mannitol, mannitol, sorbitol, sodium metabisulfite, hydrogen sulfite Sodium, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, Acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, silicon derivatives, cellulose and its derivatives, alginate, gelatin , polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, stearin Calcium acid, magnesium stearate, etc.

The advantages and beneficial effects of the present invention are as follows: the effective anti-pulmonary fibrosis part of the extract is discovered for the first time, the preparation method is simple, the effective part is clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is comprehensive. It is obviously superior to the prior art, wherein the two specific compounds have a synergistic effect against pulmonary fibrosis, and the extract can also have the advantages of synergistic effect with anti-pulmonary fibrosis chemical drugs.

Description of drawings

Figure 1 Effects of different extraction parts of Nanshan flower root on lung function in mice with bleomycin-induced pulmonary fibrosis.

Figure 2 The effect of different extraction parts of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.

Fig. 3 Effects of different extraction parts of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.

Figure 4 Effects of different preparation techniques of Nanshan flower root on lung function of mice with bleomycin-induced pulmonary fibrosis.

Figure 5. Effects of different preparation processes of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.

Figure 6. Effects of different preparation techniques of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.

Figure 7 Effects of different preparation techniques of Nanshan flower root on the expression of TGF-β and TNF-α in lung tissue of mice with bleomycin-induced pulmonary fibrosis.

Figure 8 Effects of different preparation techniques of Nanshan flower root on the expression of COL1A1, COL3A1 and α-SMA proteins in lung tissue of mice with bleomycin-induced pulmonary fibrosis.

Fig. 9 Effects of scopolactone and methylisorubicin-1-methyl ether on the expression of α-SMA protein after TGF-β1-induced fibroblast activation.

Figure 10 The effect of the pharmaceutical combination of the present invention on the expression of α-SMA protein after TGF-β1-induced fibroblast activation.

Detailed ways

Example 1 Preparation of Nanshan Flower Root Extract

Take 1 kg of Nanshan flower root, extract 3 times with 8 times the amount of 95% ethanol, combine the extracts, concentrate under reduced pressure, extract 3 times with 100 ml of ethyl acetate, combine the extracts, concentrate under reduced pressure to remove the solvent, and then dry to obtain the obtained 3 g of Nanshan flower root extract was detected by chromatography, and the content of anthraquinones, anthraquinone glycosides and iridoids accounted for 64% of the total extract.

Example 2 Preparation of Nanshan Flower Root Extract

Take 1kg of Nanshan flower root, extract twice with 8 times the amount of water, and then extract twice with 5 times the amount of 95% ethanol, combine the water and ethanol extracts, concentrate the extract under reduced pressure, extract with 200ml of petroleum ether, and discard the extract , and then extracted 3 times with 100ml of ethyl acetate, combined the extracts, concentrated under reduced pressure to remove the solvent and then dried. The obtained Nanshan flower root extract 8g was detected by chromatography, among which anthraquinones, anthraquinone glycosides and cycloalkenyl ethers The content of terpenoid active ingredients accounted for 71% of the total extract.

Example 3 Preparation of Nanshan Flower Root Extract

Take 1kg of Nanshan flower root, pulverize it to a fineness of about 50 mesh, first extract it once with an appropriate amount of petroleum ether, discard the petroleum ether extract, and then extract three times with 5 times the amount of ethyl acetate, combine the extracts, and concentrate and dry under reduced pressure. 11 g of the ethyl acetate extract was obtained, and the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounted for 79% of the total amount of the extracts through chromatographic detection.

Example 4 Preparation of Nanshan Flower Root Extract

Take 1kg of Nanshan flower root, extract 3 times with 8 times the amount of 95% ethanol, the extracts are combined, concentrated under reduced pressure, and a preparative chromatographic column is used to remove impurities. The adopted chromatographic column is a C18 column, and the mobile phase is the mobile phase A phase: Water (0.1% formic acid, 1 mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1 mmol/L ammonium acetate), gradient elution conditions: 0-10min, 20%B; 10-60min, 20- 100% B, flow rate: 0.3 mL/min, column temperature: 30°C. The three active components of anthraquinones, anthraquinone glycosides and iridoids were collected in sections, and concentrated and dried under reduced pressure to obtain the purified three active components of anthraquinones, anthraquinone glycosides and iridoids. part. The above three kinds of effective parts can also be mixed evenly to obtain the Nanshan flower root extract. After chromatographic detection, the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounts for 100% of the total amount of the extract. %.

The preparation of embodiment 5 pharmaceutical composition

Take three effective parts of anthraquinones, anthraquinone glycosides and iridoids prepared in Example 4, and mix them according to the weight ratio of 8:1:1 respectively.

The preparation of embodiment 6 pharmaceutical composition

Take 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin and scopolactone in a ratio of 1:5 : 5:1 weight ratio mixing.

The preparation of embodiment 7 pharmaceutical composition

Take scopolactone and methylisorubicin-1-methyl ether and mix them according to the weight ratio of 1:1.

The preparation of embodiment 8 pharmaceutical composition

Take the Nanshan flower root extract of Example 1 and pirfenidone, and mix them in a weight ratio of 10:1.

The preparation of embodiment 9 pharmaceutical composition

Take the Nanshan flower root extract of Example 1 and nintedanib, and mix them in a weight ratio of 8:1.

Example 10 Preparation of capsules

Take 10 parts by weight of Nanshan flower root extract in Example 2, add 12 parts by weight of lactose and 2 parts by weight of microcrystalline cellulose, dry granulation, sieve, granulate, put into capsules, and obtain capsules.

Example 11 Preparation of Inhalation Spray

Take the pharmaceutical composition of Example 6, add appropriate amount of surfactant, propellant and stabilizer to prepare an inhalation spray.

Experimental Example 1: Therapeutic effect of different extraction parts of Nanshan flower root on bleomycin-induced pulmonary fibrosis in mice

1. Experimental materials

1.1 Experimental animals

Specific pathogen-free (SPF) grade C57BL/6N mice (male, body weight 18-22g), purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK-(Beijing) 2016 -0011, issuing authority: Beijing Municipal Science and Technology Commission, certificate number: 11400700300671.

1.2 Drugs and main reagents

1.3 The main instruments of the experiment

2. Experimental method

2.1 Modeling and grouping

About 80 SPF grade male C57BL/6N mice were selected, 10 were used as blank control group, and the rest were treated with tracheal instillation of bleomycin to cause pulmonary fibrosis. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital 80 mg/kg, and the upper incisors of the mice were fixed on the tracheal instillation console. A trocar was inserted into the mouse trachea, connected to an HRH-MAG4 pulmonary liquid quantitative nebulizer, and quickly pushed into 50 μL of sterile PBS-dissolved bleomycin solution at a dose of 0.04 U per mouse. After administration, the mice were kept upright and swirled to make the solution evenly distributed in the lungs, and water and feed were given to the mice after they recovered, and the pulmonary fibrosis mouse model was established. In the blank control group, other operations were the same except that sterile PBS solution was administered to the trachea.

After 14 days, the animals in the surviving modeling group were randomly divided into model group, nintedanib positive drug group, Huanggen tablet group, Nanshan flower root ethyl acetate extract group, and Nanshan flower root n-butanol extract group. , Nanshan flower root water extract group.

2.2 Method of administration

14 days after the establishment of the model, the mice in each group were given the corresponding drug intervention by gavage every day for 14 days. The blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution, The administration dose was 50 mg/kg; the Huanggen tablet group was administered with Huanggen tablet suspension, the administration dose was 200 mg/kg; the Nanshan flower root extract administration group was given Nanshan flower root ethyl acetate extract, Nanshan flower root extract Butanol extract, Nanshan flower root water extract suspension, dosage 100mg/kg.

2.3 Observation indicators

The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis. Each lobe of the right lung was taken and rinsed in normal saline, and then the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content in lung tissue.

2.3.1 Mice body weight and observation around the cage

The observation by the cage includes but is not limited to the following indicators: the death of the mice, whether there are wet rales, nasal sprays, arched back, erect hair, etc., as well as changes in limb activity and behavior. Record the occurrence time, extent and duration, etc. The body weight of the mice before and after the modeling, and before the mice were sacrificed, were recorded.

2.3.2 Respiratory function test

On the 14th day of administration, the mice were fully anesthetized by intraperitoneal injection of pentobarbital sodium 90 mg/kg within 24 hours after the last administration, and the mice were placed in the supine position on the dissection board. The skin of the mouse neck was wiped with alcohol, the skin was cut open, the neck muscles were bluntly separated, the trachea was exposed, a small incision was made transversely under the thyroid cartilage, a tracheal cannula was inserted and ligated with sutures. Connect tracheal intubation and Flexivent instrument, detect mouse total inspiratory volume (IC) by Deep Inflation module, detect respiratory system resistance (Rrs), compliance (Crs), elasticity (Ers) by SnapShot-150 module, Quick Prime-3 The module detects the main airway resistance (Rn), tissue damping (G) and tissue elasticity (H) and other indicators, and PVs-P draws the P-V loop of mouse breathing.

2.3.3 Masson staining

The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 μm and baked at 60°C for 1 h. Using Nanjing Jiancheng rapid Masson staining, the specific steps are: (1) Paraffin sections are dewaxed and put into water: xylene for 10 min twice, 100% ethanol, 100% ethanol, 95% ethanol, 75% ethanol, 30% ethanol for two minutes each, Rinse twice with warm distilled water for 60 s. (2) Nuclear staining: Regaud's hematoxylin staining for 60s, 0.1% acetic acid washing solution for 30s. (3) Pulp dyeing: Ponceau red dyeing solution for 60s, 0.1% acetic acid washing solution for 30s rinsing. (4) Differentiation: 1% phosphomolybdic acid aqueous solution was differentiated for 6-8 minutes, and the fiber part was light pink when observed under a microscope, and the color separation solution was discarded without washing. (5) Collagen counterstaining: counterstaining with aniline blue aqueous solution for 5 min, discarded, and rinsed with absolute ethanol. (6) Sealing: After drying, seal the film with neutral resin. (7) Microscopic examination: collagen fibers, mucus, and cartilage are blue; cytoplasm, muscle, cellulose, and glia are red; nucleus is blue-purple. Staining was quantitatively analyzed using ImageJ v1.50C image software. Five different fields of view were randomly selected for each section, and the blue collagen area was positive, and the ratio of the positive area to the total area of the entire field of view was used as the pulmonary fibrosis index.

2.3.4 Hydroxyproline (HYP) detection

The Nanjing Jiancheng Hydroxyproline Detection Kit was used to detect the content of hydroxyproline in the right lung tissue of mice according to the instructions. The specific steps are as follows: (1) Sample hydrolysis: Accurately weigh 20-40 mg of tissue wet weight into a 15 mL centrifuge tube, Add 1 mL of the hydrolyzate and mix well. Cover with a boiling water bath for 10 minutes, shake well, and continue the boiling water bath for 10 minutes to fully hydrolyze. (2) Adjust pH: add 10 μL of indicator after cooling down the centrifuge tube, shake well, and adjust the pH to about 6.0-6.8. Add double distilled water to 10mL and mix well. Take 3ml of the diluted hydrolyzate and add about 30mg activated carbon to make the solution clear and colorless, mix well, centrifuge at 3500rpm for 10min, and carefully take the supernatant for detection. (3) Sample detection: It is divided into blank tube, standard product tube, and measuring tube, except that 1 mL of double-distilled water is added to the blank tube, 1 mL of standard application solution with a concentration of 5 μg/mL is added to the standard product tube, and 1 mL of the sample to be tested is added to the sample tube. , the operation is the same. Add 0.5mL reagent one (chloramine T solution and citric acid buffer), mix well and let stand for 10min; add 0.5mL reagent two (perchloric acid solution), mix well and let stand for 10min; add 0.5mL reagent three (dimethyl Aminobenzaldehyde solution), mix well, water bath at 60°C for 15min, after cooling, centrifuge at 3500rpm for 10min. (4) Detection of absorbance: Take the supernatant to detect the absorbance at a wavelength of 550 nm, and measure the absorbance value of each tube. The content of hydroxyproline in the tissue was calculated according to the calculation formula.

3. Experimental results

3.1 The effect of different extraction parts of Nanshan flower root on the respiratory function of mice.

The body weight of the mice before and after modeling was recorded, and the results are shown in Table 1: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the body weight of the nintedanib and Nanshan flower root ethyl acetate extract groups improved. effect. The results of respiratory function are shown in Table 2 and Figure 1: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the ethyl acetate extract of Nanshan flower root can effectively improve the respiratory system of the mice The total system capacity and respiratory system compliance were better than those of the Huanggen tablet group, and the results were statistically different. It shows that the ethyl acetate extract of Nanshan flower root can improve the respiratory function of mice.

Table 1 Records of body weight of mice (unit g, compared with blank group, ^# p<0.05, ^## p<0.01, compared with model group, *p<0.05)

Table 2. Mouse respiratory system function (compared with blank group, ^## p<0.01, compared with model group, *p<0.05, **p<0.01)

3.2 The effect of different extraction parts of Nanshan flower root on the degree of pulmonary fibrosis in mice induced by bleomycin.

Masson staining (Fig. 2) showed that in the blank control group, there was only positive staining of blue collagen in the bronchial wall and around the blood vessel wall, and the morphological structure was basically normal. The mice in the model group developed areas of pulmonary fibrosis, and collagen fibers proliferated and thickened. The positive drug nintedanib and ethyl acetate extract of Nanshan flower root had better improvement effect. The percentage of positive area is shown in the bar graph in Figure 2.

3.3 The effect of different extraction parts of Nanshan flower root on the content of hydroxyproline in mouse lung tissue.

Hydroxyproline accounts for 13.4% in collagen, a very small amount in elastin, and does not exist in other proteins. In pulmonary fibrosis, the main component that increases is collagen fibers, and the content of hydroxyproline in lung tissue can be converted into the content of lung collagen to reflect the degree of pulmonary fibrosis. The results are shown in Table 3 and Figure 3. The hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p<0.01). Compared with the model group, the ethyl acetate extract of Nanshan flower root showed statistical difference (p<0.01). The results showed that the ethyl acetate extract of Nanshan flower root could reduce the collagen deposition and pulmonary fibrosis in mice induced by bleomycin.

Table 3 Content of hydroxyproline in lung tissue of mice (compared with blank group, ^## p<0.01, compared with model group, **p<0.01)

Experimental Example 2: Therapeutic effect of extracts of Nanshan flower root with different preparation methods on bleomycin-induced pulmonary fibrosis in mice

1. Experimental materials

1.1 Experimental animals

1.2 Drugs and main reagents

1.3 The main instruments of the experiment

2. Experimental method

2.1 Modeling and grouping

After 14 days, according to the body weight of the surviving model animals, they were randomly divided into the model group, the nintedanib positive drug group, the embodiment 1 group, the embodiment 2 group, the embodiment 3 group, and the embodiment 4 group, with 10 animals in each group. .

2.2 Method of administration

14 days after the establishment of the model, the mice in each group were given the corresponding drug intervention by gavage every day for 14 days. The blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution, The administration dose was 50 mg/kg; Example 1-Example 4 groups were respectively administered the Nanshan flower root extract suspension prepared with 0.5% CMC-Na aqueous solution, and the administration dose was 100 mg/kg.

2.3 Observation indicators

The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis. The lobes of the right lung were taken and rinsed in normal saline, and the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content, TGF-β and TNF-α content in lung tissue. and Western Blot to detect the expression of pulmonary fibrosis-related proteins.

2.3.1 Mice body weight and observation around the cage

Same as experimental example 1.

2.3.2 Respiratory function test

Same as experimental example 1.

2.3.3 Masson staining

Same as experimental example 1.

2.3.4 Hydroxyproline (HYP) detection

Same as experimental example 1.

2.3.5 Immunohistochemical (IHC) staining

The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 μm and baked at 60°C. After dewaxing with xylene, hydration with gradient ethanol, antigen retrieval (0.01M citrate buffer, pH 6.0, high pressure repair for 2 min); blocking endogenous catalase with 3% hydrogen peroxide, 5% BSA at room temperature closed. Sections were incubated with α-SMA primary antibody overnight at 4°C. After the sections were washed with PBS buffer, the corresponding secondary antibodies were incubated at room temperature for 1 h. Freshly prepared DAB chromogenic reagent was added dropwise, hematoxylin counterstained, hydrochloric acid alcohol differentiation, dehydration and drying with gradient ethanol, and xylene was transparent. Staining was quantitatively analyzed using ImageJ v1.50C image software. Five different fields of view were randomly selected for each section, and the relative area of positive expression was analyzed.

2.3.6 Enzyme-linked immunosorbent assay (ELISA) detection

Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of PBS buffer after weighing, and use a homogenizer to fully homogenize. Centrifuge at 3500rpm for 20min, and collect the supernatant for ELISA test. The supernatant was collected and the contents of TNF-α and TGF-β in the lung tissue were detected according to the instructions of the rat TNF-α and TGF-β kit of Jiangsu Jingmei Biotechnology Co., Ltd. The total protein concentration of tissue was detected by BCA protein quantification kit, and the results were expressed as the ratio of the content of TNF-α or TGF-β to total protein.

2.3.7 Western blot to detect the expression of related proteins

Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of RIPA lysis buffer containing 1% protease inhibitor and 1% protein phosphatase inhibitor after weighing, grind and lyse on ice for 45 min. The lysate was centrifuged at 12,000 rpm at 4°C for 20 min, and the supernatant was taken, and the protein concentration was measured using the BCA protein assay kit. 50 μg protein sample was diluted with 5× loading buffer, heated at 100°C for 5 minutes, electrophoresed in SDS-PAGE gel, then transferred to PVDF membrane, blocked with 5% nonfat milk powder for 1h, and primary antibody at 4°C After incubation overnight, the membrane was washed three times with TBST buffer and incubated with the corresponding secondary antibody for 1 h at room temperature. After washing, the membrane was developed with ECL chemiluminescence solution. After recording by chemiluminometer exposure, bands were analyzed using ImageJ.

2.4 Statistical methods

The experimental results are expressed as mean ± standard deviation

Statistical software SPSS 20 was used for analysis, Student's t test was used to compare the model group with the blank group, One Way ANOVA was used between each administration group and the model group, and the homogeneity of variance was tested by Homogeneity. Least significant test (LSD) test; Tamhane's T2 test was used if the variance was unequal. The difference was considered significant at p<0.05.

3. Experimental results

3.1 The effects of different preparation methods of Nanshan flower root extracts on respiratory function of mice.

The body weight, mortality and respiratory function of mice were recorded after modeling and after treatment with Nanshan flower root extract. The body weight results are shown in Table 4: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the Nanshan flower root extracts with different preparation methods recovered to varying degrees after treatment, and the mortality rate of the mice in the administration group was lower than the model group. The results of respiratory function are shown in Table 5 and Figure 4: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the examples 1-4 groups can effectively improve the total respiratory system of the mice. Volume and respiratory compliance, the results were statistically different. It is shown that the groups of Examples 1-4 can improve the respiratory function of mice.

Table 4. Recording of body weight of mice (unit g) and the final number of deaths (compared with blank group, ^# p<0.05)

Table 5. Mouse respiratory system function (compared with blank group, ^## p<0.01, compared with model group, *p<0.05, **p<0.01)

3.2 The effects of different preparation methods of Nanshan flower root extracts on the degree of bleomycin-induced pulmonary fibrosis in mice.

Masson staining (Fig. 5) showed that in the blank control group, there was only positive staining of blue collagen around the bronchial wall and blood vessel wall, and the morphological structure was basically normal. The mice in the model group developed areas of pulmonary fibrosis, and collagen fibers proliferated and thickened. The administration groups had different degrees of improvement, among which the positive drug nintedanib group and the Example 4 group had the best improvement effect. The percentage of positive area is shown in the bar graph in Figure 2.

3.3 The effect of different preparation methods of Nanshan flower root extract on the content of hydroxyproline in mouse lung tissue.

The results are shown in Table 6 and Figure 6. The hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p<0.01). The hydroxyproline content in the lung tissue of the Nanshan flower root administration groups with different concentrations decreased in a dose-dependent manner, and there was a statistical difference between each example group and the model group (p<0.01). The results show that Nanshan flower root extract can reduce the collagen deposition and pulmonary fibrosis in mice caused by bleomycin, among which Example 4 has the best effect.

Table 6. The content of hydroxyproline in the lung tissue of mice (compared with the blank group, ##p<0.01, compared with the model group, **p<0.01)

3.4 The effects of different preparation methods of Nanshan flower root extracts on the expression of TGF-β and TNF-α in mouse lung tissue.

TGF-β is the most important regulator of tissue fibrosis caused by dysregulation of extracellular matrix synthesis and deposition, and is associated with various organ fibrosis, sclerosis, and atherosclerosis. TNF-α is a cytotoxic factor that plays a key role in the pathogenesis of pulmonary fibrosis. Therefore, we detected TGF-β and TNF-α content in mouse lung tissue by ELISA. The results are shown in Table 7 and Figure 7. The contents of TGF-β and TNF-α in the lung tissue of the mice in the model group increased, and there was a statistical difference; the administration group decreased the contents of TGF-β and TNF-α in the lung tissue. The results showed that the corresponding extracts of Nanshan flower root may inhibit the progression of pulmonary fibrosis by affecting the expression of TGF-β and TNF-α.

Table 7 Contents of TGF-β and TNF-α in lung tissue of mice (compared with blank group, ##p<0.01, compared with model group, *p<0.05, **p<0.01)

3.5 The effects of different preparation methods of Nanshan flower root extracts on the expression of COL1A1, COL3A1 and α-SMA proteins in mouse lung tissue.

After the occurrence of pulmonary fibrosis, fibroblasts are activated to release a large amount of extracellular matrix. The main components of extracellular matrix are COL1A1 and COL3A1, and α-SMA is the main marker of fibroblast activation. We examined the expression of COL1A1 and COL3A1 proteins in mouse lung tissue by Western Blot. At the same time, we also detected the distribution of α-SMA in mouse lung tissue by immunohistochemistry. The results (Table 8 and Figure 8) showed that after stimulation with bleomycin, the release of type I collagen and type III collagen in the lung tissue of mice increased, and the expression of α-SMA was increased and widely distributed, which was statistically different from the blank group (p <0.01). The extracts of Nanshan flower root with different preparation methods have obvious improvement effect after treatment, and there is a statistical difference compared with the model group (p<0.01). The results show that Nanshan flower root extract can reduce the elevated expression of COL1A1, COL3A1 and α-SMA proteins caused by pulmonary fibrosis in mice, and the effect of Example 4 is the most significant.

Table 8 Effects of different preparation methods of Nanshan flower root extract on bleomycin-induced mouse lung tissue protein expression

Experimental Example 3: Effective Compounds in Nanshan Flower Root Extract Inhibit Fibroblast Activation

1. Experimental background

Fibroblasts do not express α-actin (α-SMA) in the unactivated state, after being activated by TGF-β1, fibroblasts are activated to myofibroblasts and stop proliferating, express α-SMA and release extracellular matrix related proteins. We obtained from literature and experiments that the extract of Nanshan flower root anthraquinone contains two known compounds, scopolactone and methylisorubicin-1-methyl ether, we designed a fibroblast activation experiment and gave Nanshan flower root extract The anti-fibroblast activating activity of scopolactone and methylisorubicin-1-methyl ether was examined in the treatment of the compounds contained in the study.

2. Experimental materials

Human lung fibroblast cell line HPF cells were purchased from Shanghai Enzyme Link Biotechnology Co., Ltd., human recombinant TGF-β1 was purchased from Peprotech Company, and scopolide and methylisorubidin-1-methyl ether were purchased from Beijing Biterenkang Biotechnology Co., Ltd. Pharmaceutical Technology Co., Ltd.

3. Experimental method

The HPF cells were spread on a 6-well plate, and the medium was discarded after growing to about 70%. The negative control group was added with 1 mL of serum-free medium (containing 0.1% DMSO), and the modeling group and the administration group were added with 1 mL containing 10ng/ml. TGF-β1 serum-free medium, scopolactone or methylisorubicin-1-methyl ether were added to the administration group to make the final concentration reach 5 μg/mL, and the culture plate was shaken horizontally to mix the liquid. Place in a 37 °C, 5% _CO2 incubator for 48 h. Then, the medium was discarded, 100 μL of RIPA lysis solution was added to lyse the cells (containing 1% protease, phosphatase inhibitor), and the cells were collected using a scraper, centrifuged at 13000 rpm, 4° C. for 10 min. The supernatant was taken and transferred to a new EP tube for Western blot to detect the expression of α-SMA protein.

4. Experimental results

The results are shown in Table 9 and Figure 9. After induction of TGF-β1, fibroblasts express increased α-SMA. However, both scopolactone and methylisorubidin-1-methyl ether inhibited the increased expression of α-SMA and inhibited the activation of fibroblasts, and the results were statistically different (p<0.01).

Table 9 Fibroblast inhibitory activity of scopolactone and methylisorubicin-1-methyl ether

Experimental Example 4: The pharmaceutical composition of the present invention inhibits the activation of fibroblasts

The experimental materials and experimental methods were the same as those in Experimental Example 3, and the administration groups were the nintedanib group and the Examples 1, 5, 6, 7, 8, and 9 groups, respectively. The administration dose of nintedanib group, Example 8 and Example 9 group was 2 μM, and the administration dose of the other example groups was 5 μg/mL. Example 1 is the ethyl acetate extract of Nanshan flower root; Example 5 drug combination is anthraquinones, anthraquinone glycosides and iridoids are mixed in a weight ratio of 8:1:1; Example 6 drug combination For 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin and scopolactone according to 1:5: Mixing in a weight ratio of 5:1: the drug combination in Example 7 is scopolactone and alizarin-1-methyl ether, which are mixed in a weight ratio of 1:1; the drug combination in Example 8 is Nanshan in Example 1 The flower root extract and pirfenidone were mixed in a weight ratio of 10:1; the drug combination in Example 9 was that the Nanshan flower root extract and nintedanib of Example 1 were mixed in a weight ratio of 8:1.

Experimental results

The results are shown in Table 10 and Figure 10. Compared with the data in Table 9 in Experimental Example 3, it can be seen that under the same dose, the value of the scopolactone group is 5.10, and the methylisorubidin-1-methyl ether group It is 4.22, and the Example 7 after combining scopolactone and methylisorubicin-1-methyl ether is 3.10. It can be seen that the results of Example 7 group are obviously better than those of scopolactone and methylisorubidin-1-methyl ether. The result of methyl ether alone, that is, the combination of scopolactone and methylisorubicin-1-methyl ether has a synergistic effect. In addition, by comparing the results of the nintedanib group, the example 1 group and the example 9, it can be seen that in the case of the same dose as the nintedanib group and a lower dose than the example 1 group, the example 9 group has obvious Better technical effect, the instructions combine the Nanshan flower root extract with chemical drugs such as pirfenidone or nintedanib, which has a synergistic effect.

Table 10 Inhibitory activity of different drug combinations on fibroblasts

Experimental Example 5: Analysis of Chinese medicinal components in Huang root extract based on LC/MS technology

1. Chromatographic and Mass Spectrometry Conditions

Agilent 1290 UPLC coupled to Agilent 6550 Triple Quadrupole Time-of-Flight Mass Spectrometer. The chromatographic column is Waters BEH C18 (2.1*100mm, 1.7μm), 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°C.

Adopt the negative ion acquisition mode of primary high resolution (HRMS) and secondary high resolution (HRMS/MS) mass spectrometry, drying gas temperature: 280 °C, drying gas flow rate: 11L/min, sheath gas temperature: 325 °C, sheath gas flow rate: 12L /min, scanning range of primary mass spectrometry: 150-1200 m/z, scanning range of secondary mass spectrometry: 50-1200 m/z, secondary collision voltage: 10/20/30/40/50V.

2. Experimental results

Identification and identification of traditional Chinese medicine components in Nanshan flower root extract based on liquid chromatography and high-resolution mass spectrometry

Based on optimized chromatographic mass spectrometry conditions, first-order high-resolution mass spectrometry (HRMS) data were collected. The HRMS data was compared with the self-built database to identify the traditional Chinese medicine components in the extract of Nanshan flower root, and the secondary high-resolution mass spectrometry (HRMS/MS) data of the traditional Chinese medicine component in the Nanshan flower root extract was further collected under different collision voltages. Thirty traditional Chinese medicine components in Nanshan flower root extract were identified, including 23 anthraquinones, 4 iridoids and 3 anthraquinone glycosides, and some compounds were isomer compounds. The basic information of each component identified is shown in the table below.

Table 11 Identification of Chinese medicinal components of Huang Root Extract based on liquid chromatography high-resolution mass spectrometry

Experimental example conclusion: The Nanshan flower root extract of the present invention can improve pulmonary fibrosis caused by different factors, can improve lung function, reduce collagen deposition in lung tissue and fibroblast proliferation and activation, and can inhibit lung tissue TGF-β and TNF-α. α inhibits pulmonary fibrosis. The extracting parts of Nanshan flower root in the present invention are anthraquinones, iridoids and anthraquinone glycosides. Two of the specific compounds, scopolactone and methylisorubidin-1-methyl ether, clearly inhibit the activation of fibroblasts. The Nanshan flower root extract of the present invention has a definite anti-pulmonary fibrosis effect, and scopolactone and methylisorubidin-1-methyl ether have proved the anti-pulmonary fibrosis potential at the cellular level, and their combination has a synergistic increase effect. At the same time, after the Nanshan flower root extract of the present invention is combined with chemical drugs such as pirfenidone or nintedanib for anti-pulmonary fibrosis, it has a synergistic effect. In conclusion, the Nanshan flower root extract and its constituent components of the present invention can be used to prepare medicines against pulmonary fibrosis.

Claims

A use of Nanshan flower root extract in preparing medicine for treating pulmonary fibrosis, characterized in that: the Nanshan flower root extract is anthraquinones, anthraquinone glycosides and iridoids effective parts; The preparation method of Nanshan flower root extract is to take Nanshan flower root and extract it with ethanol, then use preparative chromatography to remove impurities, and refine and purify to obtain anthraquinones, anthraquinone glycosides and iridoids active components; It can be obtained by extracting with ethyl acetate; or after extracting Nanshan flower root with solvent, concentrating the extract, and then extracting with ethyl acetate.
The use according to claim 1, characterized in that: the preparation method of the Nanshan flower root extract is: 95% ethanol reflux extraction of Nanshan flower root three times, the extract is concentrated under reduced pressure, the impurities are removed by preparative chromatography, and the purification is carried out. The active ingredients of anthraquinones, anthraquinone glycosides and iridoids are obtained, and the chromatography is one or more of macroporous resin method, molecular sieve chromatography, silica gel column chromatography and polyamide column chromatography.
The use according to claim 2, wherein the chromatography is silica gel column chromatography, and gradient elution with water and acetonitrile is adopted.
Use according to claim 1, is characterized in that: the preparation method of described Nanshan flower root extract is: take Nanshan flower root and extract with petroleum ether first, discard the extract, then extract with ethyl acetate, and concentrate under reduced pressure An ethyl acetate extract was obtained.
The use according to claim 1, wherein: the preparation method of the Nanshan flower root extract is: after taking Nanshan flower root and extracting it with a solvent, concentrating the extract, and then extracting it with ethyl acetate to obtain the solvent. It is one or a combination of two or more of water, methanol, ethanol, propanol, butanol, etc.
The use according to claim 5, characterized in that: the preparation method of the Nanshan flower root extract is as follows: Nanshan flower root is extracted with water, then extracted with 95% ethanol, combined with water and ethanol extract, and the extract is concentrated under reduced pressure , using ethyl acetate extraction and rotary evaporation to remove the solvent of Nanshan flower root extract.
The use according to claim 5, characterized in that: the preparation method of the Nanshan flower root extract is: the Nanshan flower root is extracted three times by refluxing with 95% ethanol, and the Nanshan flower root is extracted by rotary evaporation to remove the solvent after ethyl acetate extraction. thing.
The use according to claim 1, wherein the pulmonary fibrosis is not caused by silicosis.
The use according to claim 2, characterized in that: the treatment of pulmonary fibrosis is to improve lung function, reduce collagen deposition in lung tissue and fibroblast proliferation and activation, and inhibit lung tissue TGF-β and TNF-α. Pulmonary Fibrosis.
The use according to claim 1, wherein the anthraquinone active ingredients include 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylisorubicin, 2-hydroxy-3 -Methoxyanthraquinone, 2-Hydroxy-3-hydroxymethylanthraquinone, Demethyl succinylaldehyde, Methylisorubicin-1-methyl ether, stigma aldehyde, 1,3-dihydroxy-2- Methoxymethyl anthraquinone, 3-hydroxy-1-methoxy-2-hydroxymethyl anthraquinone, 1,3-dihydroxy-2-ethoxymethyl anthraquinone, 1-hydroxy-2,3-dihydroxymethyl anthraquinone Methoxy-7-methylanthraquinone, 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methyl Anthraquinone, 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, scopolactone, 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-trimethyl Oxy-3-methylanthraquinone, 4-hydroxy-1,2,3-trimethoxy-7-hydroxymethylanthraquinone, 1,3-dihydroxy-5,6-dimethoxy-2- one or more of methoxymethylanthraquinone;

The anthraquinone glycoside active ingredients include one or more of lucidin 3-O-β-primeveroside, damnacanthol 3-O-β-primeveroside, and rubiadin 3-O-β-primerveroside;

The iridoid active ingredients include one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, and asperulosidic acid.
A use of Nanshan flower root extract in preparing medicine for treating pulmonary fibrosis, characterized in that: the Nanshan flower root extract is one or both of scopolactone and methylisorubidin-1-methyl ether. kind.
A medicinal composition for the treatment of pulmonary fibrosis, characterized in that: it is composed of the effective extraction parts of anthraquinones, anthraquinone glycosides and iridoids in the root of Nanshan flower, and the weight proportions of the above three effective extraction parts are 1-10:1-10:1-5.
A pharmaceutical composition for treating pulmonary fibrosis, characterized in that it is composed of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, It is composed of methylisorubicin and scopolactone, and the weight ratio of the above four compounds is 1-10:1-10:1-10:1-10.
A pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that: it is composed of scopolactone and methylisorubicin-1-methyl ether, and the weight ratio of the two compounds is 1-10:1-10.
A pharmaceutical composition for the treatment of pulmonary fibrosis, characterized in that: it is composed of an extract of Nanshan flower root and an anti-pulmonary fibrosis chemical, wherein the anti-pulmonary fibrosis chemical is pirfenidone or nintedanib , the weight ratio of Nanshan flower root extract and anti-pulmonary fibrosis chemical is 2-20:1-10.
Use of the composition of any one of claims 12-15 in the preparation of a medicament for treating pulmonary fibrosis.