A pharmaceutical composition for treating pulmonary fibrosis and a preparation method thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating pulmonary fibrosis and a preparation method thereof. The composition comprises the following raw materials in parts by weight: 12-18 parts of Astragalus membranaceus, 6-12 parts of Ophiopogon japonicus, 4-10 parts of Poria cocos, 1-3 parts of Radix Glycyrrhizae, 10-16 parts of Salvia miltiorrhiza, 6-12 parts of Scutellaria baicalensis, 4-10 parts of Morus alba, 2-6 parts of Pericarpium Citri Reticulatae Viride, 1-4 parts of Sophora flavescens and 1-4 parts of Fritillaria cirrhosa. The present application is a three-phase and fixed-ratio compound of polysaccharides, phenolic acids and flavonoids. The polysaccharides are obtained by water extraction and alcohol precipitation, and are used as a mucosal retention adhesion carrier. The phenolic acids and flavonoids are extracted by low-degree ethanol refluxing, so as to ensure the structural integrity and maintain stability. The alkaloids are extracted by ethanol and then mildly salified, so as to reduce irritation and adsorption loss. The polysaccharides are then subjected to selective light fermentation to build a slightly acidic environment. In the compounding process, a buffer system is used to adjust pH, and cyclodextrin is used to stably bind the components. The three phases synergistically intervene in oxidation-inflammation-fibrosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological drugs, and particularly relates to a pharmaceutical composition for treating pulmonary fibrosis and a preparation method thereof. BACKGROUND

[0002] In interstitial lung disease, pulmonary fibrosis is the main pathological change, which progresses insidiously and is irreversible. The core mechanism involves sustained oxidative stress and inflammation amplification after epithelial damage, TGF-beta and Smad-driven epithelial-mesenchymal transition and fibroblast activation, and imbalance of collagen deposition and matrix remodeling. The first-line clinical drugs mainly include pirfenidone and nintedanib, which can delay progression but are difficult to reverse scars. Adverse reactions and drug costs limit long-term accessibility. In this context, traditional Chinese medicine excels in multi-pathway regulation and is often used as an adjuvant therapy, but the prescription lacks standardization and repeatability, making it difficult to cover the entire chain of anti-oxidation-anti-inflammation-anti-fibrosis.

[0003] Existing traditional Chinese medicine preparations mostly use single decoction or single alcohol extraction. Hydrophilic polysaccharides and hydrophobic phenolic acids, flavonoids, and alkaloids are mutually restrained in the same solvent system: polyphenols easily form non-specific associations with polysaccharides and proteins, flavonoid ligands are difficult to absorb due to dissolution, alkaloids have poor dispersibility in the form of free bases, strong bitterness and strong irritation, and acid-base association and π-π stacking with polyphenols result in turbidity and sedimentation. Blind fermentation of the whole prescription can improve taste, but often accompanied by degradation of target components and increased batch-to-batch variability, making it difficult to obtain a stable slightly acidic environment and absorbable structure type ratio. In terms of dosage forms, decoctions and oral liquids are mainly used, which have problems such as slow early onset, poor clarity and shelf-life stability, and insufficient taste compliance. Quality control mostly stays at the level of total polysaccharides and total flavonoids, lacking process markers (such as fermentation organic acids and postbiotics) and structure type markers (such as free flavonoids and total flavonoids ratio), making it difficult to support large-scale expansion. To improve the above-mentioned pain points, some technical routes attempt high-end processes such as membrane separation, column chromatography purification, liposomes and nano delivery to improve bioavailability and stability, but the equipment investment and process complexity are high, making it difficult to match the existing production line of most traditional Chinese medicine enterprises. Traditional Chinese medicine injections can improve exposure, but require high control of sterility, pyrogen and allergic reaction, and safety and compliance pressure is significant. SUMMARY

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a pharmaceutical composition for treating pulmonary fibrosis and a preparation method thereof.

[0005] The technical effect of the present application is realized by the following technical scheme: a pharmaceutical composition for treating pulmonary fibrosis, which comprises the following raw materials by weight: 12-18 parts of Radix Astragali, 6-12 parts of Ophiopogon japonicus, 6-10 parts of Poria cocos, 1-3 parts of Radix Glycyrrhizae Preparata, 10-16 parts of Salvia miltiorrhiza, 6-12 parts of Scutellaria baicalensis, 4-10 parts of Morus alba, 2-6 parts of Pericarpium Citri Reticulatae Viride, 1-4 parts of Sophora flavescens and 1-4 parts of Fritillaria cirrhosa.

[0006] Preferably, the composition of the pharmaceutical composition comprises the following raw materials by weight parts: 15-18 parts of Huangqi, 8-12 parts of Maidong, 8-10 parts of Fuling, 2-3 parts of Zhigancao, 12-16 parts of Danshen, 8-12 parts of Huangqin, 6-10 parts of Sangbaipi, 3-6 parts of Chenpi, 2-4 parts of Kushen and 2-4 parts of Chuanbeimu;

[0007] Preferably, the composition of the pharmaceutical composition comprises the following raw materials by weight parts: 15-18 parts of Huangqi, 8-12 parts of Maidong, 8-10 parts of Fuling, 2-3 parts of Zhigancao, 12-16 parts of Danshen, 8-12 parts of Huangqin, 6-10 parts of Sangbaipi, 3-6 parts of Chenpi, 2-4 parts of Kushen and 2-4 parts of Chuanbeimu;

[0008] Preferably, in any of the above-mentioned pharmaceutical compositions for treating pulmonary fibrosis, the composition further comprises the following raw materials by weight parts: 4-8 parts of maltodextrin, 2-4 parts of β-cyclodextrin, 1-2.5 parts of glycerol and 0.02-0.05 parts of complex fermentation bacteria;

[0009] Preferably, the complex fermentation bacteria are composed of Lactobacillus plantarum and Lactobacillus paracasei at a mass ratio of 2:1; the activity of the Lactobacillus plantarum is 1-5 x 10 10 CFU / g; and the activity of the Lactobacillus paracasei is 5-10 x 10 10 CFU / g;

[0010] Preferably, another aspect of the present application provides a preparation method of a pharmaceutical composition for treating pulmonary fibrosis, comprising the following steps:

[0011] S1: Huangqi, Maidong, Fuling and Zhigancao are added into 10-12 times weight parts of deionized water, and water extraction treatment is carried out at 90-100°C for 60-90 min, filtration is carried out, water extraction treatment is repeated once, the two filtrates are combined, and the combined filtrate is concentrated to a relative density of 1.1-1.2 at 60°C to obtain a concentrated solution;

[0012] S2: 95% ethanol solution is added to the concentrated solution of step S1 to make the final volume fraction of the system 70-80%, and after stirring, the system is left to stand at room temperature for 8-12 h, the precipitate is collected and dissolved with deionized water at 60-65°C to obtain a polysaccharide phase concentrated solution;

[0013] S3: 60-70% of the polysaccharide phase concentrated solution of step S2 is taken as a fermentation part, the soluble solids are adjusted to 8-12°Bx, complex fermentation bacteria are inoculated, static fermentation is carried out at 35-37°C for 18-24 h, then 72-75°C treatment is carried out for 15-20 min, and then cooling to 35-40°C to obtain a fermentation liquor;

[0014] S4: Salvia miltiorrhiza, Scutellaria baicalensis, Morus alba and Citrus reticulata were refluxed with 50-60% ethanol for 60-90 min, and the refluxing was repeated once. The filtrates of the two refluxing were combined and concentrated under reduced pressure to 20-30% solid, and then de-alcoholized to ethanol residue <1% to obtain a condensed liquid of phenolic acid and flavonoid phase;

[0015] S5: Sophora flavescens and Fritillaria cirrhosa were refluxed with 60-70% ethanol for 60-90 min, and the filtrate was obtained. Citric acid was slowly added to adjust the pH to 3.5-4, and the mixture was left to stand for 30-60 min. The mixture was concentrated under reduced pressure to 20-30% solid to obtain a condensed liquid of alkaloid phase;

[0016] S6: The fermentation liquid of step S3 and the condensed liquid of unfermented polysaccharide phase reserved in step S2 were mixed. Then, the condensed liquid of phenolic acid and flavonoid phase of step S4 and the condensed liquid of alkaloid phase of step S5 were mixed. The pH was adjusted to 5.5-6 with a buffer, and maltodextrin, β-cyclodextrin and glycerol were sequentially added. The mixture was stirred at room temperature at 200-400 rpm for 30-60 min to obtain a compounded liquid;

[0017] S7: The compounded liquid of step S6 was vacuum degassed at -0.06 to -0.08 MPa for 10-20 min. Then, the solid content was adjusted to 28-32%, and the mixture was spray dried. The powder was collected and sieved through an 80-mesh sieve to obtain a pharmaceutical composition;

[0018] Preferably, in step S4, the liquid-solid ratio of the ethanol refluxing was 8-10 mL / g, and the refluxing temperature was 78-82°C.

[0019] Preferably, in step S5, the liquid-solid ratio of the ethanol refluxing was 6-10 mL / g, and the refluxing temperature was 75-80°C.

[0020] Preferably, in step S6, the buffer was composed of citric acid and sodium citrate at a molar ratio of 1:1.

[0021] Preferably, in step S7, the spray drying parameters were as follows: feed temperature 35-45°C, inlet air temperature 145-158°C, outlet air temperature 72-80°C, and atomization pressure 0.2-0.35 MPa.

[0022] The beneficial effects of the present application are as follows:

[0023] Compared with the prior art, the polysaccharide phase with the functions of benefiting qi and resisting oxidation, the phenolic acid flavonoid phase with the function of inhibiting fibrosis signals, and the alkaloid phase with the functions of inhibiting inflammation and activating fibroblasts are separated and proportionally compounded to form a complex oral system carried by a hydrophilic matrix. First, a polysaccharide phase rich in arabinose and glucose residues is obtained by water extraction and hot alcohol precipitation, and is used as a mucosal retention and adhesion carrier to construct a basic skeleton; then, Danshensu and flavonoids of Scutellaria are extracted by low-degree ethanol reflux extraction, so that the integrity of aromatic hydroxyl and carboxyl structures is ensured to facilitate subsequent dissociation balance and chemical stability in a weak acid environment; and the alkaloids of Sophora and Fritillaria are ethanol extracted and mildly salted, so that they exist in the form of salts in the water phase, reduce irritation, and avoid non-specific adsorption loss of polyphenols by alkaline components. When the three phases are compounded, the polysaccharide is used as a continuous phase, and the phenolic acid flavonoids and the salted alkaloids are used as dispersed phases to be uniformly embedded, so that the carrier retention, pathway inhibition and inflammation control work cooperatively in the same system, and the mutual restraint of active components and poor taste caused by traditional whole prescription decoction are avoided.

[0024] On the basis of phase separation extraction, only the polysaccharide phase is subjected to selective light fermentation and low-temperature inactivation, and the lactic acid and postbiotics produced by fermentation construct a slightly acidic environment while maintaining the polysaccharide skeleton, which not only improves the chemical stability of phenolic acids and flavonoids in the intestinal segment and the ability to diffuse across the membrane, but also forms a persistent hydrated layer on the mucosal surface with exopolysaccharides, prolongs the local exposure time, and realizes the coupling and amplification of carrier retention, slightly acidic stability, and passive diffusion. The fermentation window is limited to mild conditions to avoid non-selective degradation of phenolic acid skeletons and alkaloid salt types, while providing a mild buffer for subsequent short-time acid catalytic deglucosylation, allowing partial conversion of flavonoid glycosides to free forms, improving the permeation and first-pass loss of poorly soluble flavonoids, and making up for the shortcomings of simple water-alcohol extraction at the oral absorption end. In the compounding stage, the prescription pH is stabilized in the weak acid range by a citric acid and sodium citrate buffer system, allowing phenolic acids to coexist in ionic-molecular form to obtain more balanced solubility and membrane permeability, and alkaloids to remain in salt form to reduce bitterness and local irritation; supplemented by a small amount of malt dextrin and cyclodextrin packaging, the hydrophobic aggregation and π-π stacking between polyphenols and alkaloids are inhibited, and heat-induced polymerization during spray drying or pasteurization is reduced. The polysaccharide phase and phenolic acid flavonoid phase are dominant in the compounding ratio, providing an antioxidant and microcirculation-improving chassis, and the alkaloid phase is embedded at a low dose to play a directional role in anti-inflammatory and inhibition of activation, avoiding the oral taste and safety risks brought by high alkaline load. The polysaccharide phase improves mucosal adhesion and free radical scavenging capacity, reduces oxidative stress and epithelial damage, and improves the inflammatory microenvironment; the phenolic acid flavonoid phase maintains effective exposure under weak acid and carrier protection, inhibits TGF-β and Smad-mediated transcriptional activation and corrects the epithelial-mesenchymal transition process, while improving microcirculation and matrix metalloproteinase imbalance to reduce collagen deposition tendency; the alkaloid phase is released stably under the support of salinization and packaging, inhibits the inflammatory cascade and upregulation of fibroblasts α-SMA, and cooperatively weakens the abnormal expression of collagen I and fibronectin. The three phases realize the step-by-step cooperation of the carrier layer, the chemical stability layer, and the pathway intervention layer in the same drug delivery system, which is more likely to obtain simultaneous control of the oxidative-inflammation-fibrosis three pathological links than single water decoction or single fermentation system, and reduces the rebound of single target intervention. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Cumulative dissolution results of the pharmaceutical compositions prepared for Examples 1-3 and Comparative Examples 1-3 of the present application;

[0026] Figure 2 Absorbance change results of the pharmaceutical compositions prepared for Examples 1-3 and Comparative Examples 1-3 of the present application;

[0027] Figure 3 α-SMA inhibition rate results of the pharmaceutical compositions prepared for Examples 1-3 and Comparative Examples 1-3 of the present application;

[0028] Figure 4The results show the gel shrinkage rates of the pharmaceutical compositions prepared in Examples 1-3 and Comparative Examples 1-3 of this invention. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0030] Example 1: A pharmaceutical composition for treating pulmonary fibrosis, comprising the following raw materials in parts by weight: 15 parts Astragalus membranaceus, 10 parts Ophiopogon japonicus, 8 parts Poria cocos, 2 parts prepared Glycyrrhiza uralensis, 14 parts Salvia miltiorrhiza, 10 parts Scutellaria baicalensis, 8 parts Morus alba root bark, 4 parts Citrus reticulata peel, 3 parts Sophora flavescens and 3 parts Fritillaria cirrhosa.

[0031] The pharmaceutical composition further comprises the following raw materials in parts by weight: 6 parts maltodextrin, 3 parts β-cyclodextrin, 2 parts glycerol and 0.04 parts compound fermentation bacteria;

[0032] The compound fermentation bacteria consist of *Lactobacillus plantarum* and *Lactobacillus paracasei* in a 2:1 mass ratio; the activity of *Lactobacillus plantarum* is 3 × 10⁻⁶. 10 CFU / g; the activity of the *Lactobacillus paracasei* was 8 × 10⁻⁶. 10 CFU / g;

[0033] The preparation of the pharmaceutical composition for treating pulmonary fibrosis includes the following steps:

[0034] S1: Add Astragalus membranaceus, Ophiopogon japonicus, Poria cocos and prepared licorice root to 11 times the weight of deionized water, extract at 98℃ for 75 min, filter, repeat the water extraction once, combine the two filtrates, concentrate at 60℃ to a relative density of 1.2 to obtain a concentrated solution.

[0035] S2: Add 95% ethanol solution to the concentrate in step S1 to make the final volume fraction of the system 75%. After stirring, let it stand at room temperature for 10 hours, collect the precipitate, dissolve it in deionized water at 62°C, and obtain the polysaccharide phase concentrate.

[0036] S3: Take 66% of the polysaccharide phase concentrate from step S2 as the fermentation fraction, adjust the soluble solids to 10°Bx, inoculate with compound fermentation bacteria, statically ferment at 36℃ for 20h, treat at 73℃ for 18min, and then cool to 37℃ to obtain the fermentation broth.

[0037] S4: Refluxing Danshen, Huangqi, Sangbaipi and Chenpi with 55% ethanol for 80 min, the ratio of refluxing liquid to solid is 9 mL / g, the refluxing temperature is 80°C, repeating refluxing once, combining the two times of filtrate, concentrating to 25% solid under reduced pressure, de-ethanolizing to ethanol residual <1%, obtaining phenolic acid flavonoid phase concentrate;

[0038] S5: Refluxing Kushen and Chuanbeimu with 65% ethanol for 80 min, the ratio of refluxing liquid to solid is 8 mL / g, the refluxing temperature is 78°C, taking the filtrate, slowly adding citric acid to adjust pH to 3.7, standing for 45 min, concentrating to 25% solid under reduced pressure, obtaining alkaloid phase concentrate;

[0039] S6: Mixing the fermentation liquid of step S3 and the un-fermented polysaccharide phase concentrate reserved in step S2, then mixing with the phenolic acid flavonoid phase concentrate of step S4 and the alkaloid phase concentrate of step S5, adjusting pH to 5.7 with buffer composed of citric acid and sodium citrate in 1:1 molar ratio, sequentially adding malt dextrin, β-cyclodextrin and glycerol, stirring at room temperature for 50 min at 300 rpm, obtaining compound liquid;

[0040] S7: Vacuum de-aerating the compound liquid of step S6 at -0.07 MPa for 15 min, then spray drying with solid content adjusted to 30%, feeding temperature 40°C, inlet air 152°C, outlet air 75°C, atomization pressure 0.28 MPa, collecting powder, sieving through 80 mesh, obtaining pharmaceutical composition.

[0041] Example 2: A pharmaceutical composition for treating pulmonary fibrosis, the composition comprising the following raw materials by weight: 18 parts of Huangqi, 12 parts of Maidong, 10 parts of Fuling, 3 parts of Zhigancao, 16 parts of Danshen, 12 parts of Huangqi, 10 parts of Sangbaipi, 6 parts of Chenpi, 4 parts of Kushen and 4 parts of Chuanbeimu.

[0042] The composition of the pharmaceutical composition further comprises the following raw materials by weight: 8 parts of malt dextrin, 4 parts of β-cyclodextrin, 2.5 parts of glycerol and 0.05 parts of compound fermentation bacteria;

[0043] The compound fermentation bacteria are composed of Lactobacillus plantarum and Lactobacillus paracasei in a mass ratio of 2:1; the activity of the Lactobacillus plantarum is 5×10 10 CFU / g; the activity of the Lactobacillus paracasei is 10×10 10 CFU / g;

[0044] The preparation of the pharmaceutical composition for treating pulmonary fibrosis comprises the following steps:

[0045] S1: Astragalus, Ophiopogon, Poria cocos and licorice were added into 12 times weight of deionized water, and water extraction was carried out at 100°C for 90 min. After filtration, water extraction was repeated once. The two filtrates were combined and concentrated at 60°C to a relative density of 1.1 to obtain a concentrated solution;

[0046] S2: 95% ethanol solution was added to the concentrated solution of step S1 to make the final volume fraction 80%. After stirring, it was left to stand at room temperature for 12 h. The precipitate was collected and dissolved in deionized water at 65°C to obtain a polysaccharide phase concentrated solution;

[0047] S3: 60% of the polysaccharide phase concentrated solution of step S2 was taken as the fermentation portion, and the soluble solids were adjusted to 8°Bx. After inoculation with a complex fermentation bacteria and static fermentation at 35°C for 18 h, it was treated at 72°C for 15 min, and then cooled to 35°C to obtain a fermentation broth;

[0048] S4: Salvia, Scutellaria, White Mulberry Bark and Pericarpium Citri Reticulatae were refluxed with 50% ethanol for 60 min. The ethanol reflux liquid-solid ratio was 8 mL / g, and the reflux temperature was 78°C. Reflux treatment was repeated once. The two filtrates were combined, concentrated under reduced pressure to a solid content of 20%, and de-ethanized to ethanol residue <1% to obtain a phenolic acid flavonoid phase concentrated solution;

[0049] S5: Sophora and Fritillaria were refluxed with 60% ethanol for 60 min. The ethanol reflux liquid-solid ratio was 6 mL / g, and the reflux temperature was 75°C. The filtrate was slowly added with citric acid to adjust the pH to 3.5, and left to stand for 30 min. It was concentrated under reduced pressure to a solid content of 20% to obtain an alkaloid phase concentrated solution;

[0050] S6: The fermentation broth of step S3 and the unfermented polysaccharide phase concentrated solution retained in step S2 were mixed evenly. Then, the phenolic acid flavonoid phase concentrated solution of step S4, the alkaloid phase concentrated solution of step S5 were compounded and mixed evenly. The pH was adjusted to 5.5 with a buffer composed of citric acid and sodium citrate at a molar ratio of 1:1. Maltodextrin, β-cyclodextrin and glycerol were added in turn. After stirring at room temperature at 200 rpm for 30 min, a compounded solution was obtained;

[0051] S7: The compounded solution of step S6 was vacuum degassed at -0.06 MPa for 10 min. Then the solid content was adjusted to 28%, and spray drying was carried out. The feeding temperature was 35°C, the inlet air temperature was 145°C, the outlet air temperature was 80°C, the atomization pressure was 0.2 MPa. The powder was collected and sieved through an 80 mesh sieve to obtain a pharmaceutical composition.

[0052] Example 3: A pharmaceutical composition for treating pulmonary fibrosis, which consists of the following raw materials by weight: 12 parts of Astragalus, 6 parts of Ophiopogon, 6 parts of Poria cocos, 1 part of licorice, 10 parts of Salvia, 6 parts of Scutellaria, 4 parts of White Mulberry Bark, 2 parts of Pericarpium Citri Reticulatae, 1 part of Sophora and 1 part of Fritillaria.

[0053] The composition of the pharmaceutical composition also includes the following raw materials by weight parts: 4 parts of malt dextrin, 2 parts of β-cyclodextrin, 1 part of glycerol and 0.02 parts of complex fermentation bacteria;

[0054] The complex fermentation bacteria are composed of Lactobacillus plantarum and Paracasei lactobacillus in a mass ratio of 2:1; the activity of the Lactobacillus plantarum is 1×10 10 CFU / g; and the activity of the Paracasei lactobacillus is 5×10 10 CFU / g;

[0055] The preparation of the pharmaceutical composition for treating pulmonary fibrosis includes the following steps:

[0056] S1: Add Astragalus, Ophiopogon, Poria cocos and Radix Glycyrrhizae to 10 times the weight of deionized water, and treat with water extraction at 90°C for 60 min, filter, repeat the water extraction treatment once, combine the two filtrates, concentrate to a relative density of 1.1 at 60°C, and obtain a concentrated solution;

[0057] S2: Add 95% ethanol solution to the concentrated solution of step S1 to make the final volume fraction of the system 70%, stir and then stand at room temperature for 8 h, collect the precipitate, dissolve it with deionized water at 60°C, and obtain a polysaccharide phase concentrated solution;

[0058] S3: Take 70% of the polysaccharide phase concentrated solution of step S2 as the fermentation part, adjust the soluble solids to 12°Bx, inoculate the complex fermentation bacteria, and after static fermentation at 37°C for 24 h, treat at 75°C for 20 min, then cool to 40°C, and obtain a fermentation liquor;

[0059] S4: Treat Salvia, Scutellaria, White Mulberry Bark and Pericarpium Citri Reticulatae with 60% ethanol reflux for 90 min, the liquid-solid ratio of the ethanol reflux treatment is 10 mL / g, the reflux temperature is 82°C, repeat the reflux treatment once, combine the two filtrates, concentrate under reduced pressure to a solid content of 30%, and de-ethanolize to ethanol residue <1%, and obtain a phenolic acid flavonoid phase concentrated solution;

[0060] S5: Treat Sophora and Fritillaria with 70% ethanol reflux for 90 min, the liquid-solid ratio of the ethanol reflux treatment is 10 mL / g, the reflux temperature is 80°C, take the filtrate, slowly add citric acid to adjust the pH to 4, stand for 60 min, and concentrate under reduced pressure to a solid content of 30%, and obtain an alkaloid phase concentrated solution;

[0061] S6: Mix the fermentation liquor of step S3 and the unfermented polysaccharide phase concentrated solution reserved in step S2; and then mix and uniformly compound the phenolic acid flavonoid phase concentrated solution of step S4 and the alkaloid phase concentrated solution of step S5, adjust the pH to 6 with a buffer composed of citric acid and sodium citrate in a molar ratio of 1:1, and then add malt dextrin, β-cyclodextrin and glycerol, stir at room temperature at 400 rpm for 60 min, and obtain a compounded solution;

[0062] S7: The compounded solution of step S6 was vacuum degassed at -0.08 MPa for 20 min; then the solid content was adjusted to 32%, and spray dried with a feed temperature of 45°C, an inlet temperature of 158°C, an outlet temperature of 72°C, and an atomization pressure of 0.35 MPa. The powder was collected and sieved through an 80-mesh screen to obtain the pharmaceutical composition.

[0063] Comparative Example 1: The operation procedure of Comparative Example 1 was basically the same as that of Example 1, with the main difference being that Comparative Example 1 omitted the selective light fermentation of step S3 and directly used the unfermented polysaccharide phase concentrate obtained in step S2 to participate in the compounding of step S6; the remaining operation parameters were kept consistent.

[0064] Comparative Example 2: The operation procedure of Comparative Example 2 was basically the same as that of Example 1, with the main difference being that Comparative Example 2 did not perform acidification and salting treatment in step S5, i.e., did not use citric acid to adjust the pH, and the obtained alkaloid phase concentrate was directly used in step S6 for compounding; the remaining operation parameters were kept consistent.

[0065] Comparative Example 3: The operation procedure of Comparative Example 3 was basically the same as that of Example 1, with the main difference being that Comparative Example 3 removed Bitter and Fritillaria cirrhosa from the prescription, did not prepare an alkaloid phase concentrate, and used the polysaccharide phase to make up for the missing solid mass during compounding; the remaining operation parameters were kept consistent.

[0066] Performance test:

[0067] Physicochemical stability test: 0.5 g of the pharmaceutical composition prepared in Examples 1-3 and Comparative Examples 1-3 was taken and added to 100 mL of buffer medium (10 mM citric acid / sodium citrate, pH 5.8, 25°C) and uniformly stirred by magnetic stirring. The time when the sample was added was defined as 0 min. At 0.5 min, 2 min, 5 min, 10 min, 20 min, and 30 min, 2 mL of supernatant was taken, filtered through a 0.22 μm filter membrane, and immediately supplemented with an equal volume of fresh medium of the same composition. HPLC (UV 286 nm) was used to quantitatively determine Danshensu, and the cumulative dissolution amount (mg / g) was calculated. The results are shown in Table 1. Figure 1

[0068] 1% (w / v) solutions of the pharmaceutical compositions prepared in Examples 1-3 and Comparative Examples 1-3 were prepared, and the medium was the same as above (10 mM citric acid and sodium citrate, pH 5.8, 25°C). After being uniformly mixed and made up to volume, the solutions were placed in a 40°C environment. The time when the sample was added was defined as 0 h. At 0 h and 24 h, the supernatant was taken and placed in a 1 cm cuvette, and the absorbance A600 was measured at 600 nm. The absorbance change ΔA = A600(24h) - A600(0h) was recorded. The results are shown in Table 2. Figure 2

[0069] Based on the results of the performance test, the physicochemical stability test, and the light stability test, it can be seen that the pharmaceutical composition prepared in Example 1 has good performance, stability, and light stability. Figure 1 and Figure 2 ​​Based on the result analysis of Comparative Example 1 and Example 1, the early and overall dissolution of Comparative Example 1 is significantly lagged, and the turbidity increases faster; this can be due to the fact that the lactic acid dominated slightly acidic environment and the postbiotic matrix are not formed, the ratio of free flavonoids and total flavonoids is maintained at a low level, the non-specific association and aggregation between phenolic acid flavonoids and polysaccharides are more likely to occur, and the equivalent diffusion coefficient decreases; at the same time, the lack of extracellular polysaccharides produced by fermentation to support the interface wetting and stable dispersion leads to the decrease of effective contact area and the increase of micro-aggregation, which is manifested as the slow dissolution curve and the rising absorbance; based on the result analysis of Comparative Example 2 and Example 1, Comparative Example 2 shows the slowest dissolution and the largest absorbance increase; this can be due to the fact that the alkaloid in the form of free base is easy to self-aggregate and to form acid-base association and π-π stacking with phenolic acid after compounding, thereby forming a hydrophobic microzone and a coarse colloid, which produces obvious turbidity and occupies the active surface in the early stage, thereby inhibiting the rapid release of phenolic acid flavonoids; even if the final pH of the preparation is unified to weak acidity, the non-uniform aggregates formed in the early stage are still difficult to completely reverse, so that the stability and dissolution are both damaged; based on the result analysis of Comparative Example 3 and Example 1, the dissolution and absorbance change of Comparative Example 3 are at a medium level, which is better than those of Comparative Examples 1 and 2 but lower than those of Examples; this can be due to the fact that although the risk of aggregation caused by unsalted alkaloids is avoided, the hydrophilic microdomain and carrier dispersion effect brought by the salted alkaloids and cyclodextrin are also lost, and the compounding system is inferior to the examples in terms of wetting, interface diffusion and micro-uniformity.

[0070] Cell function test: human embryonic lung fibroblasts MRC-5 were selected, and the groups were blank control, positive control group (model control, TGF-β1 induction), Example 1-3 groups and Comparative Example 1-3 groups; all samples were prepared into 10 mg / mL sterile aqueous solution according to equal solid, equal drug volume and final pH = 5.5, and were diluted to a final concentration of 50 μg / mL (CCK-8 cell survival rate test has been done, and the cell survival rate is all > 90%) using DMEM working solution containing 1% FBS; MRC-5 was diluted to 1.5×10 4Cells were seeded into 96-well plates and incubated overnight at 37°C with 5% CO2. The culture medium was discarded, and the samples were pretreated with fresh medium containing 1% FBS for 2 hours. The blank control group received no TGF-β1 or sample. The positive control group received TGF-β1 to 5 ng / mL. The treatment groups received TGF-β1 (5 ng / mL) and their respective samples (50 μg / mL). Incubation continued for 48 hours. After incubation, the cells were gently washed with PBS, and the lysis and dilution system provided by the ELISA kit was added. An α-SMA sandwich ELISA was performed according to the instructions. OD was read at 450 nm, and the concentration was calculated from the standard curve. The α-SMA inhibition rate was calculated as: (mean of positive control group - mean of each treatment group) / (mean of positive control group - mean of blank control group) × 100%. Each group had n=3 wells, and the results were independently replicated twice. Figure 3 As shown.

[0071] based on Figure 3 Results analysis showed that the pharmaceutical compositions prepared in the embodiments of the present invention generally exhibited excellent α-SMA inhibition rates. However, Example 3 showed the worst inhibition effect, which may be due to its significantly reduced ratio of alkaloids in the three-phase formulation, weaker microacid stability and interfacial dispersion, resulting in a lower inhibition amplitude, significantly inferior to Examples 1 and 2. Based on the results of Example 1 and Comparative Example 1, the α-SMA inhibition rate of Comparative Example 1 decreased significantly. This may be due to the lack of a microacidic environment and post-biotic matrix from fermentation, resulting in a lower ratio of free flavonoids and total flavonoids. Phenolic acid flavonoids and polysaccharides easily associate and aggregate, leading to a decrease in equivalent exposure and a significant decrease in α-SMA inhibition. Low; based on the results of Example 1 and Comparative Example 2, Comparative Example 2 showed the worst α-SMA inhibition effect. This may be because the alkaloids, in the form of free bases, undergo acid-base association and π-π stacking with phenolic acids, forming hydrophobic microclusters and increasing turbidity. The effective concentration of the active component and cell accessibility decreased synchronously, resulting in the worst functional endpoint. Based on the results of Example 1 and Comparative Example 3, the α-SMA inhibition of Comparative Example 3 was between that of Comparative Example 1 and Example 3. This may be because although the risk of unsalted aggregation was avoided, the targeted inhibition of fibroblast activation by alkaloids was lost, the three-phase coupling was weakened, and it was difficult to achieve the level of Example 3 by relying solely on polysaccharides and phenolic acid flavonoids.

[0072] Collagen gel shrinkage assay: Human lung fibroblasts MRC-5 were collected and divided into blank control, positive control (model control, TGF-β1 induced), Examples 1-3, and Comparative Examples 1-3. All samples were prepared with equal solids, equal drug volume, and final pH=5.5 to prepare a 10 mg / mL sterile aqueous solution, which was diluted to a final concentration of 50 μg / mL using DMEM working solution containing 1% FBS. Collagen gel was prepared in 24-well plates: Type I collagen mixture (final collagen 2 mg / mL, isotonic with 1×DMEM, pH adjusted to 7.4 with a small amount of NaOH) was prepared on ice, and MRC-5 suspension was added to a final concentration of 2×10⁻⁶.5 cells / mL, 0.5 mL per well, 37℃ incubation for 30 min gelation; along the wall of the hole, a circle of light is drawn to release the gel edge, add 0.5 mL medium: blank control group without TGF-β1 and sample; positive control group with TGF-β1 to 5 ng / mL; treatment group with TGF-β1 (5 ng / mL) and each add corresponding sample (50 μg / mL), place in 37℃, 5% CO2 incubator and record 0h and 48h gel diameter (area) from the top distance, measure area A0 (0h) and A48 (48h); calculate the gel contraction rate (%) = (A0-A48) / A0x100%, the results are shown in Figure 4 .

[0073] Based on Figure 4 The results analysis, the drug composition prepared in the embodiments of the present application has a relatively excellent gel contraction rate as a whole, wherein the performance of Example 3 is relatively worse than that of Example 1 and Example 2, which may be due to the fact that the three-phase ratio of Example 3 is significantly different from that of Example 1 and Example 2, and the alkaloid dosage is significantly reduced; based on the results analysis of Example 1 and Comparative Example 1, the gel contraction rate of Comparative Example 1 is higher than that of Example 1; this may be due to the fact that the lack of micro-acid brought by fermentation and the postbiotic matrix, the low ratio of free flavonoids and total flavonoids, the easy association and aggregation of phenolic acid flavonoids with polysaccharides, the decrease of equivalent exposure and cell accessibility, and the insufficient inhibition of myofibroblast phenotype, resulting in the increase of contraction rate; based on the results analysis of Example 1 and Comparative Example 2, Comparative Example 2 has the worst performance; this may be due to the fact that the alkaloid in the form of free base is easy to produce acid-base association and π-π stacking with phenolic acid, forming hydrophobic micelles and causing poor dispersion, and the effective concentration and accessibility of active components are reduced; the three-phase synergy is destroyed, and the ability to inhibit stretch and stress fiber formation is the lowest; based on the results analysis of Example 1 and Comparative Example 3, the performance of Comparative Example 3 is between Comparative Example 1 and Example; this may be due to the fact that although the risk of unsalified aggregation is avoided, the directional inhibition of alkaloid relative fibroblast activation and stretch signal is missing, the three-phase coupling is weakened, and it is difficult for polysaccharides and phenolic acid flavonoids to fully inhibit contraction.

[0074] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that, It is composed of the following ingredients by weight: 12-18 parts Astragalus membranaceus, 6-12 parts Ophiopogon japonicus, 6-10 parts Poria cocos, 1-3 parts prepared Glycyrrhiza uralensis, 10-16 parts Salvia miltiorrhiza, 6-12 parts Scutellaria baicalensis, 4-10 parts Morus alba root bark, 2-6 parts Citrus reticulata peel, 1-4 parts Sophora flavescens and 1-4 parts Fritillaria cirrhosa.

2. The pharmaceutical composition for treating pulmonary fibrosis according to claim 1, characterized in that, It is composed of the following ingredients by weight: 15 parts Astragalus membranaceus, 10 parts Ophiopogon japonicus, 8 parts Poria cocos, 2 parts prepared Glycyrrhiza uralensis, 14 parts Salvia miltiorrhiza, 10 parts Scutellaria baicalensis, 8 parts Morus alba root bark, 4 parts Citrus reticulata peel, 3 parts Sophora flavescens and 3 parts Fritillaria cirrhosa.

3. A pharmaceutical composition for treating pulmonary fibrosis, characterized in that, It is composed of the following raw materials in parts by weight: 12-18 parts Astragalus membranaceus, 6-12 parts Ophiopogon japonicus, 6-10 parts Poria cocos, 1-3 parts prepared Glycyrrhiza uralensis, 10-16 parts Salvia miltiorrhiza, 6-12 parts Scutellaria baicalensis, 4-10 parts Morus alba root bark, 2-6 parts Citrus reticulata peel, 1-4 parts Sophora flavescens, 1-4 parts Fritillaria cirrhosa, 4-8 parts maltodextrin, 2-4 parts β-cyclodextrin, 1-2.5 parts glycerol and 0.02-0.05 parts compound fermentation bacteria.

4. The pharmaceutical composition for treating pulmonary fibrosis according to claim 3, characterized in that, The compound fermentation bacteria consist of *Lactobacillus plantarum* and *Lactobacillus paracasei* in a 2:1 mass ratio; the activity of *Lactobacillus plantarum* is 1–5 × 10⁻⁶. 10 CFU / g; the activity of the *Lactobacillus paracasei* is 5–10 × 10⁻⁶ CFU / g. 10 CFU / g.

5. A method for preparing a pharmaceutical composition for treating pulmonary fibrosis according to any one of claims 3-4, characterized in that, Includes the following steps: S1: Add Astragalus membranaceus, Ophiopogon japonicus, Poria cocos and prepared licorice to deionized water, perform water extraction, filter, repeat water extraction, combine the two filtrates, concentrate to obtain concentrated solution; S2: Add ethanol solution to the concentrate from step S1, stir, let stand at room temperature, collect the precipitate, dissolve it with deionized water, and obtain polysaccharide phase concentrate. S3: Take a portion of the polysaccharide phase concentrate from step S2 as the fermentation fraction, adjust the soluble solids content, then inoculate with compound fermentation bacteria, perform static fermentation, heat sterilization, and then cool to obtain the fermentation broth. S4: Salvia miltiorrhiza, Scutellaria baicalensis, Morus alba root bark and Citrus reticulata peel were refluxed with ethanol, the reflux treatment was repeated, the two filtrates were combined, concentrated under reduced pressure, and treated with alcohol removal to obtain a concentrated phenolic acid flavonoid phase solution. S5: Sophora flavescens and Fritillaria cirrhosa were refluxed with ethanol, the filtrate was collected, citric acid was slowly added to adjust the pH, the mixture was allowed to stand, and the solution was concentrated under reduced pressure to obtain the alkaloid phase concentrate. S6: Mix the fermentation broth from step S3 with the unfermented polysaccharide concentrate retained from step S2. Then, mix the concentrated phenolic acid flavonoid phase solution from step S4 and the concentrated alkaloid phase solution from step S5 evenly, adjust the pH with a buffer, and add maltodextrin, β-cyclodextrin and glycerol in sequence. Stir at room temperature to obtain the compound solution. S7: Vacuum degas the compound solution from step S6; then adjust the solid content, spray dry, collect the powder, screen, and obtain the pharmaceutical composition.

6. A method for preparing a pharmaceutical composition for treating pulmonary fibrosis according to claim 5, characterized in that, In step S4, the liquid-to-solid ratio of the ethanol reflux treatment is 8-10 mL / g; the reflux temperature is 78-82°C.

7. A method for preparing a pharmaceutical composition for treating pulmonary fibrosis according to claim 6, characterized in that, In step S5, the liquid-to-solid ratio of the ethanol reflux treatment is 6–10 mL / g; the reflux temperature is 75–80°C.

8. A method for preparing a pharmaceutical composition for treating pulmonary fibrosis according to claim 7, characterized in that, In step S7, the spray drying parameters are: feed temperature 35-45℃, inlet air temperature 145-158℃, outlet air temperature 72-80℃, and atomization pressure 0.2-0.35MPa.

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