Application of Chinese honeylocust fruit powder in preparation of products for preventing and improving pulmonary fibrosis

Abstract

The invention discloses application of Chinese honeylocust fruit rice flour in preparation of products for preventing and improving pulmonary fibrosis, and a preparation method of the Chinese honeylocust fruit rice flour comprises the following steps: performing freezing pretreatment on Chinese honeylocust fruit rice, crushing, sieving, drying, and performing dose division to obtain the Chinese honeylocust fruit rice flour. And adding the prepared Chinese honeylocust fruit rice flour into boiling water (1: 70-1: 400 while stirring), soaking for 5-20 minutes, or boiling for 1-10 minutes with soft fire, thereby obtaining the Chinese honeylocust fruit rice flour. The preparation process is high in efficiency, the prepared Chinese honeylocust fruit rice flour can be used as a convenient edible product, and a reference basis can be provided for high-added-value terminal products in the Chinese honeylocust fruit rice industry. The prepared gleditsia sinensis powder has a remarkable effect of improving pulmonary fibrosis, and the mechanism of the gleditsia sinensis powder is related to regulation of intestinal flora structures, inhibition of excessive activation of TGF-beta1 / Smads signal channels, down-regulation of fibrosis-related protein expression and reduction of lung tissue damage. Scientific basis is provided for development and utilization of medicinal and edible products of the Chinese honeylocust fruit rice flour for improving pulmonary fibrosis, and reference can also be provided for development of healthy and nutritious Chinese honeylocust fruit rice foods.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of Gleditsia sinensis Lam. powder, in particular, the application of Gleditsia sinensis Lam. powder in the preparation of products for preventing and improving pulmonary fibrosis, belonging to the field of medical and food technology. BACKGROUND

[0002] Pulmonary fibrosis (PF) is a progressive lung disease characterized by abnormal deposition of extracellular matrix, infiltration of inflammatory cells and abnormal proliferation of fibroblasts. Its pathological features are the destruction of lung tissue structure and the loss of function. The early symptoms of the disease are occult, and with the progression of the disease, it can lead to dyspnea, persistent dry cough, and even respiratory failure and even endanger life in the later stage. Although the currently clinically used anti-fibrosis drugs (such as pirfenidone and nintedanib) have been approved by the US Food and Drug Administration, they can only delay the progression of the disease and have many adverse reactions. Lung transplantation is an effective treatment for end-stage, but it is limited by the scarcity of donors and high cost, which makes it difficult to popularize. Therefore, the development of new high-efficiency and low-toxicity pulmonary fibrosis improvement preparations has great clinical value.

[0003] Gleditsia sinensis Lam. (Gleditsia sinensis Lam.) is a legume plant. The dried mature seeds (Gleditsia sinensis Lam.) are processed and dried to remove the seed coat and endosperm of the embryo - Gleditsia sinensis Lam. powder, which is rich in dietary fiber, polysaccharides, proteins, amino acids and other nutrients. It is a low-fat, high-energy, high-value nutritional health food. Although Guizhou Bijie and other main producing areas have achieved large-scale planting, the industrial chain still remains at the primary agricultural product sales stage, resulting in low product added value.

[0004] There is no report on the use of Gleditsia sinensis Lam. powder for preventing and improving pulmonary fibrosis, and traditional consumption requires long soaking and cooking, lacking convenient product forms. Therefore, the present application proves through pharmacological tests that Gleditsia sinensis Lam. powder has the effects of preventing and improving pulmonary fibrosis and regulating intestinal flora, optimizes the preparation process, develops a convenient product form of Gleditsia sinensis Lam. powder, and improves its applicability as a functional food; explores its medicinal and health care functions of improving pulmonary fibrosis through regulating the TGF-β1 / Smads fibrosis signaling pathway and regulating intestinal flora, providing a new type of pharmaceutical and health product candidate for the treatment of pulmonary fibrosis. SUMMARY

[0005] To solve the above technical problems, the present application provides the application of Gleditsia sinensis Lam. powder in the preparation of products for preventing and improving pulmonary fibrosis, and proposes that Gleditsia sinensis Lam. powder can prevent and improve pulmonary fibrosis by regulating the TGF-β1 / Smads fibrosis signaling pathway and regulating intestinal flora. The problems of how to efficiently obtain Gleditsia sinensis Lam. powder and the inconvenience of traditional consumption are solved, and an effective drug and health product for improving pulmonary fibrosis is provided, providing a scientific basis for the development of pharmaceutical and food products.

[0006] The technical scheme of the present application: the application of the soapberry rice powder in the preparation of the product for preventing and improving pulmonary fibrosis.

[0007] The preparation method of the aforementioned soapberry rice powder is as follows: freeze pretreatment of soapberry rice, crushing, sieving, drying, and dosing, and the soapberry rice powder is obtained.

[0008] Specifically, the preparation method of the aforementioned soapberry rice powder is as follows: crushing the soapberry rice after freezing in liquid nitrogen for 1-60 min, sieving through a 5-9 mesh sieve, drying at 60-100℃, and dosing at 0.25-5 g per package, and the soapberry rice powder is obtained.

[0009] More specifically, the preparation method of the aforementioned soapberry rice powder is as follows: crushing the soapberry rice after freezing in liquid nitrogen for 1 min, sieving through a 6 mesh sieve, drying at 80℃, and dosing at 2.5 g per package, and the soapberry rice powder is obtained.

[0010] The aforementioned soapberry rice is the dried endosperm of the dried mature seeds of Gleditsia sinensis Lam. after removing the seed coat and cotyledon.

[0011] The aforementioned product for preventing and improving pulmonary fibrosis is a drug or a health food.

[0012] The aforementioned method for eating the health food is as follows: adding the soapberry rice powder into boiling water, the mass ratio of the soapberry rice powder to water being 1:70-400, stirring while adding, and soaking for 5-20 min or simmering for 1-10 min, and then eating.

[0013] Specifically, the aforementioned method for eating the health food is as follows: adding the soapberry rice powder into boiling water, the mass ratio of the soapberry rice powder to water being 1:72, stirring while adding, and soaking for 10 min or simmering for 5 min, and then eating.

[0014] The preparation method of the aforementioned drug is as follows: combining the soapberry rice powder with acceptable excipients in drugs, and processing according to the conventional method to prepare the corresponding drug.

[0015] The aforementioned drug is a tablet, a hard capsule, a granule, a pill, a paste, or an oral liquid.

[0016] The beneficial effects of the present application

[0017] 1.The application discloses application of soapberry rice powder in preparation of a medicine for improving pulmonary fibrosis.The experiment proves that the soapberry rice powder can inhibit TGF-β1 / Smads signal pathway activation,reduce the expression of fibrosis indexes Fibronectin,Collagen-I and alpha-SMA and the content of HYP,regulate intestinal flora structure,such as reducing the ratio of Firmicutes / Bacteroidetes,riching in beneficial bacteria genus Muribaculum,improving the weight loss of pulmonary fibrosis mice,reducing the lung coefficient,improving the pathological damage of lung tissue,reducing the lung tissue damage score and the percentage of fibrous tissue expression,so as to prevent and improve pulmonary fibrosis.

[0018] 2.The application breaks through the traditional eating limitation,has eating convenience of the prepared soapberry rice powder,and the preparation process of the soapberry rice powder is suitable for industrial production,provides a scientific basis for development and utilization of a medicine-food dual use product of the soapberry rice powder for improving pulmonary fibrosis through regulation of TGF-β1 / Smads fibrosis signal pathway and regulation of intestinal flora,and can also provide a reference for development of a healthy and nutritious soapberry rice food. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Soapberry rice powder preparation process and mechanism diagram;

[0020] Figure 2 : Grinding rate of soapberry rice powder prepared by ordinary grinding and grinding with different freezing time pretreatment (n=3);

[0021] Figure 3 : Time required for grinding rate reaching 80% of soapberry rice powder prepared by ordinary grinding and grinding with freezing pretreatment for 1 min and 10 min (n=3) (compared with ordinary grinding, **P<0.01);

[0022] Figure 4 : Infrared spectrum of soapberry rice powder prepared by different grinding methods;

[0023] Figure 5 : Change of polysaccharide content of soapberry rice powder prepared by different grinding methods (n=3);

[0024] Figure 6 : Appearance diagram of soapberry rice powder soaked in different volumes of boiling water (5 min) and 180 mL of boiling water (30 min);

[0025] Figure 7 : Appearance diagram of soapberry rice boiled in boiling water for 10 min, 20 min and 30 min;

[0026] Figure 8 : Appearance diagram of soapberry rice powder cooked on small fire for 1 min, 5 min and 10 min;

[0027] Figure 9 : Appearance diagram of soapberry rice cooked on small fire for 10 min, 30 min, 1 h and 2.5 h;

[0028] Figure 10 Body weight change of each group of mice (n = 8);

[0029] Figure 11 Lung coefficient change of each group of mice (n = 8) (compared with the blank group: ## indicates P < 0.01; compared with the model group: ** indicates P < 0.01);

[0030] Figure 12 HYP content change of lung tissue of each group of mice (n = 8) (compared with the blank group: ## indicates P < 0.01; compared with the model group: ** indicates P < 0.01);

[0031] Figure 13 HE staining of lung tissue of each group of mice (×400);

[0032] Figure 14 Lung tissue injury score of each group of mice (n = 5) (compared with the blank group: ## indicates P < 0.01; compared with the model group: ** indicates P < 0.01);

[0033] Figure 15 Masson staining of lung tissue of each group of mice (×400);

[0034] Figure 16 Percentage of fibrous tissue area in lung tissue of each group of mice (%) (n = 5) (compared with the blank group: ## indicates P < 0.01; compared with the model group: ** indicates P < 0.01);

[0035] Figure 17 TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I and α-SMA protein and internal reference protein banding diagram in lung tissue of each group of mice;

[0036] Figure 18 TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I and α-SMA protein expression in lung tissue of each group of mice (n = 6) (compared with the blank group: ## indicates P < 0.01; compared with the model group: * indicates P < 0.05, ** indicates P < 0.01);

[0037] Figure 19 OTU wein plot of intestinal microorganisms of each group of mice;

[0038] Figure 20 Quality assessment of intestinal microorganism sequencing of each group of mice;

[0039] Figure 21Alpha diversity analysis of intestinal flora of mice in each group (A: Chao1 index; B: Ace index; C: Simpson index; D: Shannon index) (n=6);

[0040] Figure 22 PCoA and NMDS results of intestinal flora of mice in each group (A: PCoA results of intestinal flora of mice in each group; B: NMDS results of intestinal flora of mice in each group) (n=6);

[0041] Figure 23 Species distribution column chart at the level of phylum (n=6);

[0042] Figure 24 Ratio of Firmicutes / Bacteroidetes (n=6) (compared with the blank group: # represents P<0.05);

[0043] Figure 25 Species distribution column chart at the level of genus (n=6);

[0044] Figure 26 Evolutionary branch diagram of relative abundance of flora among groups;

[0045] Figure 27 Species LDA value distribution column chart of significant abundance difference among groups;

[0046] Figure 28 Spearman correlation analysis of different genera of intestinal flora of mice in each group and pharmacodynamic indicators, TGF-β1 / Smads pathway. DETAILED DESCRIPTION

[0047] The application will be further described below in conjunction with examples, but it is not taken as the basis for limiting the application.

[0048] Example 1: Preparation of Ginkgo rice powder

[0049] Process: Ginkgo rice was frozen in liquid nitrogen for 1 min, then crushed, sieved through No. 6 sieve, dried at 80°C, and divided into 2.5 g per package for dosage, to obtain Ginkgo rice powder.

[0050] Example 2: Preparation of Ginkgo rice powder

[0051] Process: Ginkgo rice was frozen in liquid nitrogen for 1 min, then crushed, sieved through No. 5 sieve, dried at 60°C, and divided into 0.25 g per package for dosage, to obtain Ginkgo rice powder.

[0052] Example 3: Preparation of Ginkgo rice powder

[0053] Process: Ginkgo rice was frozen in liquid nitrogen for 60 min, then crushed, sieved through No. 9 sieve, dried at 100°C, and divided into 5 g per package for dosage, to obtain Ginkgo rice powder.

[0054] Example 4: Preparation of Ginkgo Rice Powder

[0055] Process: Ginkgo rice was frozen in liquid nitrogen for 40 min, then pulverized, passed through an 8-mesh sieve, dried at 70°C, and dosed at 0.3 g per package to obtain Ginkgo rice powder.

[0056] Example 5: Method of consumption

[0057] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:70) while stirring, and soaked for 20 min to be consumed.

[0058] Effect: Prevention and improvement of pulmonary fibrosis.

[0059] Example 6: Method of consumption

[0060] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:400) while stirring, and soaked for 5 min to be consumed.

[0061] Effect: Prevention and improvement of pulmonary fibrosis.

[0062] Example 7: Method of consumption

[0063] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:72) while stirring, and soaked for 10 min to be consumed.

[0064] Effect: Prevention and improvement of pulmonary fibrosis.

[0065] Example 8: Method of consumption

[0066] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:400) while stirring, and simmered for 1 min to be consumed.

[0067] Effect: Prevention and improvement of pulmonary fibrosis.

[0068] Example 9: Method of consumption

[0069] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:70) while stirring, and simmered for 10 min to be consumed.

[0070] Effect: Prevention and improvement of pulmonary fibrosis.

[0071] Example 10: Method of consumption

[0072] Ginkgo rice powder was added to boiling water (Ginkgo rice powder: boiling water = 1:72) while stirring, and simmered for 5 min to be consumed.

[0073] Effect: Prevention and improvement of pulmonary fibrosis.

[0074] Example 11: Preparation of hard capsules

[0075] Take 100 g of the soap kernel powder of Example 1, slowly add it to a water container, stir while adding, after boiling, turn to medium heat and cook for 30 min, heat and concentrate, after cooling, add a certain amount of sucrose powder and dextrin, dry granulation, sieve, capsule, 1000 capsules, hard capsule.

[0076] Effect: prevention and improvement of pulmonary fibrosis.

[0077] Usage and dosage: oral, 2 capsules at a time, twice a day.

[0078] Example 12: Preparation of granules

[0079] Take 100 g of the soap kernel powder of Example 1, slowly add it to a water container, stir while adding, after boiling, turn to medium heat and cook for 30 min, heat and concentrate, after cooling, add a certain amount of sucrose powder and dextrin, dry granulation, sieve, capsule, 1000 capsules, hard capsule.

[0080] Effect: prevention and improvement of pulmonary fibrosis.

[0081] Usage and dosage: oral, 2 capsules at a time, twice a day.

[0082] Example 13: Preparation of tablets

[0083] Take 100 g of the soap kernel powder of Example 1, slowly add it to a water container, stir while adding, after boiling, turn to medium heat and cook for 30 min, heat and concentrate, after cooling, add a certain amount of sucrose powder and dextrin, dry granulation, sieve, capsule, 1000 capsules, hard capsule.

[0084] Usage and dosage: oral, 2 capsules at a time, twice a day.

[0085] Treatment of disease: prevention and improvement of pulmonary fibrosis.

[0086] Example 14: Preparation of pills

[0087] Take 100 g of the soap kernel powder of Example 1, slowly add it to a water container, stir while adding, after boiling, turn to medium heat and cook for 30 min, heat and concentrate, after cooling, add a certain amount of sucrose powder and dextrin, dry granulation, sieve, capsule, 1000 capsules, hard capsule.

[0088] Usage and dosage: oral, 2 capsules at a time, twice a day.

[0089] Treatment of disease: prevention and improvement of pulmonary fibrosis.

[0090] The inventors have carried out a large number of experiments, and the following are the results of the preparation process of the soap kernel powder and the effect of improving pulmonary fibrosis described in the present application:

[0091] 1. Results of the research on the grinding process of soapberry rice flour:

[0092] 1.1 Properties and Appearance Uniformity of Soapberry Rice Flour Based on Different Grinding Methods

[0093] The properties and appearance uniformity of soapberry rice flour after ordinary grinding and grinding after different freezing time pretreatment are shown in Table 1.

[0094] Table 1. Properties and Appearance Uniformity of Soapberry Rice Flour

[0095]

[0096]

[0097] 1.2 Pulverization rate of soapberry rice flour using different pulverization methods

[0098] The grinding ratio of soapberry rice after ordinary grinding and pretreatment with different freezing times is as follows: Figure 2 As shown in the figure. The results indicate that freezing for 1 minute significantly improves the grinding efficiency. With increasing freezing time, the grinding rate continuously increases, reaching a peak at 20 minutes (5 times that of ordinary grinding). For 600g of saponin rice, when the grinding rate reaches 80%, ordinary grinding requires 49.10 minutes, freezing pretreatment for 1 minute requires 31.30 minutes, and freezing pretreatment for 10 minutes requires 38.18 minutes. Both freezing pretreatment for 1 minute and 10 minutes significantly reduce the grinding time. Figure 3 As shown.

[0099] 1.3 Infrared spectra of soapberry rice flour pulverized by different methods

[0100] Infrared spectroscopy is often used for qualitative analysis of material structure or chemical groups. By observing the positions of absorption peaks in an infrared spectrum, the chemical composition and structural characteristics of a substance can be inferred, thus aiding in the identification of compounds. For example... Figure 4 As shown, the saponin rice flour produced by ordinary grinding and grinding after different freezing pretreatment times has a thickness of 3432.2 cm⁻¹. -1 The surrounding area showed obvious absorption peaks, with little change in shape and width, but an increase in absorption intensity. Since this wavenumber range corresponds to the OH stretching vibration peak, a characteristic absorption peak of polysaccharides, it is speculated that the change in absorption peak intensity after pulverization may be related to the formation of more hydrogen bonds by hydroxyl groups during the pulverization process. (2800–3000 cm⁻¹) -1 The absorption peaks observed within this range can be attributed to the stretching vibrations of CH4 on the methyl and methylene groups in sugars, which are also characteristic absorption peaks of polysaccharides. Both normally ground and ground saponin rice flour with different freezing times showed an absorption peak at 2926 cm⁻¹. -1The strong absorption peak near 1640 cm -1 is the vibration peak caused by asymmetric contraction of sugar ring C=0; the peaks in the range of 1640-1375 cm -1 are C-H in-plane bending vibration peaks; the peaks in the range of 1250-1000 cm -1 are C-O stretching vibration peaks, which indicate that the polysaccharide contains pyranose ring; the peak at 871 cm -1 is methylene vibration, which indicates the presence of β-type glycosidic bond; the peak at 812 cm -1 is the characteristic absorption peak of mannose; the peak near 522 cm -1 is the carbonyl vibration deformation. The position and peak shape of the main absorption peaks in the spectrum did not change significantly, indicating that the common crushing and the crushing after different freezing time pretreatment of Ginkgo biloba L. seed powder were basically consistent in main components and structure. The presence of these characteristic absorption peaks indicates the stability of the chemical composition structure of Ginkgo biloba L. seed powder after freezing and crushing.

[0101] 1.4 Polysaccharide content of Ginkgo biloba L. seed powder crushed by different methods

[0102] The polysaccharide content of Ginkgo biloba L. seed powder crushed by common method and different freezing time pretreatment is shown in Table 1. Figure 5 The results showed that compared with common crushing, the crushing after different freezing time pretreatment did not significantly change the polysaccharide content, and the crushing after freezing pretreatment showed a non-linear fluctuation pattern among the treatment groups, with a total fluctuation range of 71.48-76.83%.

[0103] 1.5 Conclusion

[0104] There was no significant difference in the appearance and uniformity of Ginkgo biloba L. seed powder crushed by common method and different freezing time pretreatment; freezing pretreatment for 1 min could improve the crushing efficiency while maintaining the main structure and content of polysaccharide, the effective component of Ginkgo biloba L. seed powder. Based on the comprehensive consideration of crushing efficiency, industrial production, and component retention, freezing pretreatment for more than 1 min was determined as the preferred scheme for crushing Ginkgo biloba L. seed powder.

[0105] 2. Comparative study on Ginkgo biloba L. seed powder and Ginkgo biloba L. seed soaked in boiling water and cooked with small fire

[0106] 2.1 Comparison of Ginkgo biloba L. seed powder and Ginkgo biloba L. seed soaked in boiling water

[0107] The color, taste, solubility, and aroma of Ginkgo biloba L. seed powder soaked in different volumes of boiling water are shown in Table 2 and Table 3. Figure 6As shown in Table 2, when the amount of added water is 180 mL, the solubility is good, the taste is smooth, the overall feeling is comfortable, the plant fragrance is natural, and it will not be too thin; when the amount of added water is 200 mL, the solubility is good, the taste is smooth, but the plant fragrance begins to fade. Considering solubility, taste and aroma, the amount of added water of 180 mL is more appropriate, because it achieves a good balance in solubility and taste, while retaining the natural fragrance of the soapberry rice flour.

[0108] As shown in Table 3 and Table 4, the color, taste, solubility and aroma of the soapberry rice flour after being soaked in 180 mL of boiling water for 5, 10, 15, 20 and 30 minutes are as follows: Figure 6 As shown in Table 3 and Table 4, the color, taste, solubility and aroma of the soapberry rice flour after being soaked in 180 mL of boiling water for 5, 10, 15, 20 and 30 minutes are as follows:

[0109] As shown in Table 3 and Table 4, the color, taste, solubility and aroma of the soapberry rice flour after being soaked in 180 mL of boiling water for 5, 10, 15, 20 and 30 minutes are as follows: Figure 7 As shown in Table 3 and Table 4, the color, taste, solubility and aroma of the soapberry rice flour after being soaked in 180 mL of boiling water for 5, 10, 15, 20 and 30 minutes are as follows:

[0110] Table 2 Color, taste, solubility and aroma of soapberry rice flour after different volumes of boiling water soaking

[0111]

[0112]

[0113] Table 3 Color, taste, solubility and aroma of soapberry rice flour after different soaking times

[0114]

[0115] Table 4 Color, taste, solubility and aroma of soapberry rice after 10, 20 and 30 minutes of soaking

[0116]

[0117] 2.2 Comparison of soapberry rice flour and soapberry rice cooked with small fire

[0118] As shown in Table 5 and Table 6, the color, taste, solubility and aroma of the soapberry rice flour after being added to 180 mL of boiling water and continuing to cook with small fire for 1, 5 and 10 minutes are as follows: Figure 8As shown, with the extension of the boiling time, the solubility and mouthfeel of the Ginkgo rice powder gradually improved. There was still a granular feeling at 1 min, the granular feeling was weakened at 5 min, and the texture was more delicate at 10 min. Therefore, the Ginkgo rice powder was gradually delicate in texture, good in solubility, and comfortable in overall mouthfeel after being boiled for 5-10 min, which was a relatively ideal choice.

[0119] The color, taste, solubility and aroma of the Ginkgo rice after being boiled for 10 min, 30 min, 1 h and 2.5 h in 180 mL boiling water (supplemented with the evaporated water) were as shown in Table 6 and Table 7. Figure 9 As shown, with the extension of the boiling time, the color of the Ginkgo rice gradually changed, and the texture and mouthfeel also improved. At 2.5 h, the Ginkgo rice had a relatively uniform appearance and texture, was soft and glutinous in the middle, and still had a certain hardness and chewiness on the surface.

[0120] Table 5 Color, taste, solubility and aroma of Ginkgo rice powder after being boiled for 1 min, 5 min and 10 min

[0121]

[0122] Table 6 Color, taste, solubility and aroma of Ginkgo rice after being boiled for 10 min, 30 min, 1 h and 2.5 h

[0123]

[0124] 2.3 Conclusion

[0125] The Ginkgo rice powder can be eaten after being soaked in 180 mL boiling water for 10 min or being boiled for 5-10 min, while the traditional Ginkgo rice cannot be eaten even if the soaking time is extended, and its physical properties (such as hardness) do not change significantly. Moreover, the Ginkgo rice is not completely softened to a suitable state for eating even after a long time of cooking (2.5 h), and if the overall soft and glutinous texture is to be achieved, a longer time investment is needed to ensure that the texture and mouthfeel reach the ideal effect. The developed Ginkgo rice powder solves the problem of long cooking time of traditional Ginkgo rice, and significantly improves the convenience of eating.

[0126] 3. Effect of Ginkgo rice powder on improving pulmonary fibrosis

[0127] 3.1 Experimental materials

[0128] Gleditsia sinensis Lam. of the Leguminosae family, and was prepared by the following procedures: removing the pod, taking the seed, soaking, steaming, taking the endosperm, and drying. The Gleditsia sinensis Lam. powder was prepared by the Pharmaceutical Analysis Laboratory of Guizhou University of Chinese Medicine. Bleomycin hydrochloride and pirfenidone were purchased from Sellcek Company. Modified Masson's trichrome staining solution (potassium dichromate solution, wergert hematoxylin (b liquid + c liquid), acidic magenta fuchsin solution, phosphomolybdate solution, aniline blue solution) was purchased from Wuhan Seville Biological Technology Co., Ltd. Hematoxylin staining solution kit was purchased from Wuhan Google Biological Technology Co., Ltd. BCA protein concentration determination kit was purchased from Beijing Lanjiekeli Technology Co., Ltd. COL-I, SMAD2, SMAD3, α-SMA, TGF-β1, and FIB were purchased from Chengdu Zhengneng Biological Technology Co., Ltd.

[0129] 3.2 Experimental animals

[0130] SPF level C57BL / 6J male mice aged 6-8 weeks (body weight 22-24 g) were provided by Henan Skb Bioscience Co., Ltd. [License No.: SCXK (Yu) 2020-0005]. The mice were raised in the Animal Institute of Guizhou University of Chinese Medicine, and were adaptively fed for 5 days before the experiment. The feeding environment was SPF level, with room temperature of 23±2℃, humidity of 55±5%, and light control of 12h day-night cycle. The room was well ventilated, and the mice were fed in cages according to standard feed, with free access to water and food. All animal experiments were approved by the Animal Ethics Committee of Guizhou University of Chinese Medicine, with the ethical review number 20240818001.

[0131] 3.3 Experimental methods

[0132] 3.3.1 Preparation of drug solution and reagent

[0133] Preparation of Gleditsia sinensis Lam. powder solution: Gleditsia sinensis Lam. powder (Example 1) was accurately weighed and slowly added to a water container while stirring. After boiling, the medium-low heat was turned on for 30 minutes. The solution was concentrated to a certain volume, and the high, medium, and low dose Gleditsia sinensis Lam. powder solutions (doses of 325 mg / kg, 162.5 mg / kg, and 81.25 mg / kg) with concentrations of 16.25, 8.125, and 4.0625 mg / mL were prepared. The dosing volume was 0.2 mL / 10 g.

[0134] Preparation of bleomycin (BLM) solution: an appropriate amount of bleomycin was accurately weighed and dissolved in normal saline to prepare a bleomycin solution with a concentration of 0.75 mg / mL (dose of 1.5 mg / kg), which was prepared and used immediately. The dosing volume was 20 μL / 10 g.

[0135] Preparation of positive drug Pirfenidone (PFD) solution: accurately weigh a certain amount of pirfenidone, dissolve it in 0.5% sodium carboxymethyl cellulose to prepare a pirfenidone solution with a concentration of 5 mg / mL (dose 100 mg / kg), and use it immediately (complete gavage within 2 hours). The administration volume is 0.2 mL / 10 g.

[0136] Preparation of sodium pentobarbital solution: accurately weigh a certain amount of sodium pentobarbital, dissolve it in normal saline to prepare a sodium pentobarbital solution with a concentration of 6 mg / mL (dose 60 mg / kg), and use it immediately. The administration volume is 0.1 mL / 10 g.

[0137] 3.3.2 Animal grouping, modeling and administration

[0138] The mice were randomly divided into 6 groups: blank group (CON), model group (BLM), pirfenidone group (PFD), high-dose Gleditschia sinensis powder group (Gle-H), medium-dose Gleditschia sinensis powder group (Gle-M), and low-dose Gleditschia sinensis powder group (Gle-L).

[0139] Before modeling, all mice were weighed, and the required volume of sodium pentobarbital and bleomycin was calculated. The mice were intraperitoneally injected with sodium pentobarbital, and after the anesthesia took effect, the mice were fixed on the mouse plate in a supine position, the glottis was exposed (the glottis was observed to open and close under the larynx under the illumination of a cold light source using a small animal laryngoscope), and the trachea was cannulated with a retention needle. When the needle entered the glottis, the needle core was slowly withdrawn (verified by a water injection hose, if the water injection showed rhythmic fluctuation, it was confirmed that the cannula was in place). An appropriate amount of bleomycin solution was drawn into a fine needle syringe, and the bleomycin solution was injected into the trachea through the tracheal cannula. After injecting the drug, 30-50 μL of air was quickly injected, and then the cannula was pulled out. Then the mouse plate was quickly rotated left and right for about 1-2 min, and the chest was gently pressed to promote the uniform distribution of bleomycin in the lungs. The blank group mice were injected with the same volume of sterile normal saline in the same way. After injection, the mice were placed in a right lateral position and waited for them to wake up naturally to replicate the mouse model of pulmonary fibrosis.

[0140] Pirfenidone group: pirfenidone solution was given by gavage every day; Gleditschia sinensis powder group: the corresponding dose of Gleditschia sinensis powder liquid was given by gavage every day; blank group and model group: the same volume of distilled water was given by gavage every day; each administration was performed once in the morning and once in the evening at the same time every day, and the gavage was continued for 24 days.

[0141] 3.3.3 Sample collection

[0142] The mice were sacrificed by cervical dislocation on day 25 after modeling, and the lung tissue was taken out. After removing the excess tissue, the whole lung wet weight was accurately weighed, and the lung coefficient was calculated. The right lung was stored in a-80°C refrigerator for subsequent ELISA and Western blotting detection. The left lung was fixed in 4% paraformaldehyde solution for pathological detection. The mouse feces were placed in a sterile centrifuge tube and stored in a-80°C refrigerator for later use.

[0143] 3.3.4 Index detection

[0144] (1) General condition of mice

[0145] The body weight change and general state of mice were monitored daily after modeling, and the data were recorded.

[0146] (2) Calculation of lung coefficient of mice

[0147] The lung coefficient of mice was calculated according to the following formula: lung coefficient = lung mass (g) / mouse body mass (g) x 100%.

[0148] (3) Pathological observation of lung tissue

[0149] The fixed lung tissue was subjected to routine operations such as ethanol dehydration, xylene transparency, paraffin embedding, sectioning, HE staining, etc. to make lung tissue HE staining and Masson staining pathological sections. Image acquisition was performed on the sections using a microscopic imaging system to observe the morphological structure and lesion of the lung tissue. The fibrous tissue area in the collected images was measured using an Image-Pro Plus 6.0 image analysis system, and the fibrous tissue area percentage was calculated as fibrous tissue area / field area (pixel area).

[0150] (4) Western blotting detection of TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I and α-SMA protein expression in lung tissue

[0151] The lung tissue of each group of mice was ground with lysis buffer on ice, and the supernatant was taken after centrifugation for later use. The protein concentration was determined using a BCA protein quantification kit. Protein samples were added for electrophoresis, membrane transfer and blocking. The corresponding primary antibody was added, incubated at 4°C for a certain time, and then washed. The corresponding secondary antibody was added, incubated at room temperature, and then washed. The enhanced chemiluminescence reagent was used for color development, and the gray value analysis of the bands was performed using image analysis software. The results were expressed as the relative expression of the target protein.

[0152] (5) Analysis of changes in intestinal flora in mouse feces by 16s rRNA sequencing technology

[0153] Total genomic DNA was extracted from fecal samples of blank group (CON), model group (BLM) and Gleditsia sinensis powder high-dose group (Gle) mice using TGuide S96 Magnetic Soil / Stool DNA Kit kit instructions. The concentration of nucleic acids was detected using a microplate reader plus 1X dsDNA HS Working Solution, and the amplification was detected on a Bionics 1000 automatic according to the concentration and amplification region. After amplification, the PCR products were electrophoretically detected using 1.8% agarose, and their integrity was detected. Then library construction was performed, and the constructed library was used for double-end sequencing (2x250bp) of the amplicon library on Illumina novaseq 6000. USEARCH (version 10.0) was used to assign qualified sequences with a similarity threshold of more than 97% to an operational taxonomic unit (OTU). The SILVA database (version 138.1) was used to classify and annotate OTU / ASV based on the NaiveBayes classifier in QIIME2, with a confidence threshold of 70%. The online platform BMKCloud (https: / / www.biocloud.net) was used to analyze the sequencing data.

[0154] 3.3.5 Statistical processing

[0155] SPSS 27.0 statistical analysis software was used for data analysis. Measurement data was expressed as mean ± standard deviation ; for multiple measurement data between groups, if it was normally distributed and homoscedastic, one-way analysis of variance (ANOVA) was used, and the "LSD" test was used for comparison between groups. When the variance was not homogeneous, the "Tamhane's T2" method was used for analysis. For data that did not meet the one-way analysis of variance, non-parametric test was used. P<0.05 indicates a significant difference, and P<0.01 indicates a highly significant difference.

[0156] 3.4 Experimental results

[0157] 3.4.1 Effect of Gleditsia sinensis powder on body weight of mice

[0158] The body weight changes of mice in each group are shown in Figure 10 The body weight of mice in the blank group showed a stable and slow growth trend, while the body weight of mice in the other groups showed a sustained decline within one week after modeling. The body weight of mice in the Gleditsia sinensis powder groups began to increase gradually after one week, and the body weight growth rate or change was not significant after two weeks, while the body weight of mice in the model group continued to decline after two weeks.

[0159] 3.4.2 Effect of Gleditsia sinensis powder on lung coefficient of mice

[0160] Lung coefficient is an important indicator for evaluating the degree of pulmonary tissue edema, such as Figure 11 As shown in Figure 2, the lung coefficient of the model group mice was significantly higher than that of the blank group (P<0.01). Compared with the model group, the lung coefficient of the pirfenidone group mice was extremely significantly reduced (P<0.01); the lung coefficient of the each dose group of the Gromwell husk powder mice was reduced, but the difference was not statistically significant (P>0.05). The lung coefficient contents of the high dose group, the middle dose group and the low dose group of the Gromwell husk powder were only 1.42%, 1.41% and 1.50%, respectively.

[0161] 3.4.3 Effect of Gromwell husk powder on the content of HPY in the lung tissue of mice

[0162] In the process of pulmonary fibrosis, the content of hydroxyproline (HYP) is a key indicator for measuring the severity of fibrosis. The results of the content of HPY in the lung tissue of mice in each group are shown in Figure 3. Figure 12 As shown in Figure 3, compared with the blank group, the content of HPY in the lung tissue of the model group mice was extremely significantly increased (P<0.01). Compared with the model group, the content of HPY in the lung tissue of the each dose group of the Gromwell husk powder and the pirfenidone group mice was extremely significantly reduced (P<0.01), and the contents of hydroxyproline in the high dose group, the middle dose group and the low dose group of the Gromwell husk powder were only 0.34 ug / mL, 0.34 ug / mL and 0.35 ug / mL, respectively.

[0163] 3.4.4 Effect of Gromwell husk powder on the pathological morphology of lung tissue of mice

[0164] The lung tissue of mice in each group was observed by HE staining, and the lung tissue pathology score was conducted, and the results are shown in Figure 4. Figure 13 14 As shown in Figure 4, compared with the blank group, the model group showed obvious alveolar structure disorder and damage, obvious formation of pulmonary bulla, interstitial inflammatory cell infiltration and interstitial fibrosis, and the fibrosis was obvious, and the inflammatory cells were mainly lymphocytes and a small amount of neutrophils. Compared with the model group, the lung tissue of mice in each dose group of the Gromwell husk powder still had a certain degree of alveolar structure damage, inflammatory cell infiltration and interstitial fibrosis, but the severity of these pathological changes was obviously improved compared with the model group. The statistical results of the lung tissue pathology score showed that, compared with the blank group, the lung tissue score of the model group mice was extremely significantly increased (P<0.01), and compared with the model group, the lung tissue score of the pirfenidone group and the high dose group of the Gromwell husk powder was extremely significantly reduced (P<0.01), and the lung tissue score of the high dose group of the Gromwell husk powder was only 2.4, and the lung tissue scores of the middle dose group and the low dose group of the Gromwell husk powder were reduced, but the difference was not statistically significant (P>0.05).

[0165] The lung tissue of mice was observed by Masson staining, and the percentage of fibrous area in the lung tissue was calculated, and the results are shown in Figure 5. Figure 15 16 ​​The model group had almost no normal alveolar structure, and obvious collagen deposition was observed between alveoli. The blue-stained area was larger, and the percentage of fibrous tissue expression was significantly increased (P < 0.01) compared with the blank group. Compared with the model group, the lung tissues of the pirfenidone and each dose of the Ginkgo Corn Powder group still showed varying degrees of alveolar structure destruction and collagen deposition, but the blue-stained area was significantly reduced, and the percentage of fibrous tissue expression was significantly decreased (P < 0.01). The percentage of fibrous tissue expression in the high-dose Ginkgo Corn Powder group, the medium-dose Ginkgo Corn Powder group, and the low-dose Ginkgo Corn Powder group was only 9.16%, 9.55%, and 9.90%, respectively.

[0166] 3.4.5 Effect of Ginkgo Corn Powder on the expression of TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I, and α-SMA proteins in the lung tissues of mice

[0167] The protein expression of TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I, and α-SMA in the lung tissues of mice in each group is shown in Table 3.4.4. Figure 17 、 18 Compared with the blank group, the protein expression of TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I, and α-SMA in the lung tissues of the model group was significantly increased (P < 0.01). Compared with the model group, the protein expression of TGF-β1, Smad2, Smad3, Fibronectin, Collagen-I, and α-SMA in the lung tissues of the pirfenidone group was significantly decreased (P < 0.01). The protein expression of TGF-β1, Smad2, Smad3, Fibronectin, and α-SMA in the lung tissues of the high-dose Ginkgo Corn Powder group was significantly decreased (P < 0.01), and the protein expression of Collagen-I was significantly decreased (P < 0.05). The protein expression of Smad2, Smad3, Collagen-I, and α-SMA in the lung tissues of the medium-dose Ginkgo Corn Powder group was significantly decreased (P < 0.01), and the protein expression of TGF-β1 and Fibronectin was significantly decreased (P < 0.05). The protein expression of Smad2 in the lung tissues of the low-dose Ginkgo Corn Powder group was significantly decreased (P < 0.01), and there was no significant change in the protein levels of TGF-β1, Smad3, Fibronectin, Collagen-I, and α-SMA (P < 0.05).

[0168] 3.4.6 Effect of Ginkgo Corn Powder on the intestinal flora of mice

[0169] (1) Quality evaluation of intestinal microorganism sequencing

[0170] Based on the obtained OTU data, draw a Venn diagram. Figure 19 It was found that there were 317 shared OTUs among the three groups. The unique OTUs in the blank group, model group, and high-dose soapberry rice flour group were 1843, 1948, and 2766, respectively. The dilution curve can be used to determine whether the sequencing depth is sufficient: a sharp rise in the curve indicates insufficient sequencing depth, requiring an increase in the number of sequences; conversely, a flattening curve indicates sufficient sequencing depth for subsequent data analysis. The dilution curves for each group are shown below. Figure 20 It can be seen that the sequencing depth of the sample meets the analysis requirements.

[0171] (2) Alpha diversity analysis

[0172] Alpha diversity is used to measure the richness and diversity of species in a sample, and is mainly assessed using the following indicators: Chao1 and ACE indices are used to assess species richness, while Shannon and Simpson indices are used to measure species diversity. Compared with the control group, the model group showed an increase in Chao1 and ACE indices (P>0.05), and a decrease in Shannon and Simpson indices (P>0.05). Compared with the model group, the high-dose saponin rice powder group showed an increase in Chao1 and ACE indices, and a decrease in Shannon and Simpson indices, but the differences were not statistically significant (P>0.05), indicating that saponin rice powder intervention did not significantly change the richness and diversity of gut microbiota. Figure 21 As shown in AD.

[0173] (3) Beta diversity analysis

[0174] Principal Coordinate Analysis (PCoA) can extract key elements and structures from multidimensional datasets, serving as an effective dimensionality reduction technique to reveal differences in species diversity among samples. Non-metric Multidimensional Labelling (NMDS) is a method that simplifies research objects (whether samples or variables) in multidimensional space to a lower-dimensional space for location, analysis, and classification, while preserving the original relationships between objects. Differences between groups can be identified by observing the distribution of samples. When the stress value of NMDS analysis is below 0.2, it indicates that the analysis results have a certain degree of reliability. The results of PCoA and NMDS analyses based on weighted unifrace are shown below. Figure 22A, B, PCoA results showed that the sample points in each group were relatively clustered, indicating that the intestinal flora in each group tended to be consistent, and the contribution rates of pcoa1 and pcoa2 were 53.21% and 16.80%, respectively. The results observed in NMDS were consistent with the PCoA results, and the stress value of the NMDS results was 0.0708, which was less than 0.2, indicating that the results of the NMDS analysis were reliable.

[0175] (4) Effect of soapberry rice flour on the intestinal flora community structure of pulmonary fibrosis mice

[0176] The results of the relative abundance of species at the phylum level showed that compared with the blank group, the relative abundance of Firmicutes in the model group increased, and the relative abundance of Bacteroidota, Proteobacteria, Actinobacteriota, Desulfobacterota and Campylobacterota decreased. Compared with the model group, the relative abundance of Firmicutes in the high-dose soapberry rice flour group decreased, and the relative abundance of Bacteroidota, Proteobacteria, Actinobacteriota, Desulfobacterota and Campylobacterota increased. As shown in Figure 23 In addition, Firmicutes and Bacteroidota were the main groups in the intestinal flora, and the results of this study found that compared with the blank group, the F / B ratio in the model group increased significantly (P<0.05), and the F / B ratio decreased significantly after soapberry rice flour intervention. As shown in Figure 24 .

[0177] The relative abundance of species at the genus level was as shown in Figure 25The relative abundance of unclassified_Muribaculaceae, Enterorhabdus, Muribaculum and Desulfovibrio in the model group was lower than that in the blank group, and the relative abundance of Dubosiella and Ligilactobacillus was higher. Compared with the model group, the relative abundance of unclassified_Muribaculaceae, Lachnospiraceae_NK4A136_group, Enterorhabdus, Muribaculum and Desulfovibrio in the high-dose group of soap kernel rice powder increased, and the relative abundance of Dubosiella and Ligilactobacillus decreased.

[0178] (5) Analysis of intestinal differential flora of mice in different groups

[0179] The LEfSe analysis method, i.e. linear discriminant analysis (LDA), was used to further explore the classification differences between groups. At the level from phylum to species, 23 significantly different flora were found (LDA>4, P<0.05). Among them, the differential flora enriched in the blank group included 7, i.e. Coriobacteria, Coriobacteriales, Actinobacteriota, Muribaculum intestinale, Muribaculum, Alloprevotella and uncultured Bacteroidales bacterium. The differential flora enriched in the model group included 10, i.e. Firmicutes, unclassified Dubosiella, Staphylococcales, Staphylococcaceae, Jeotgalicoccus aerolatus, Jeotgalicoccus, Clostridium_sensu_stricto_1 and Mammaliicoccus. The differential flora in the high-dose group of soap kernel rice powder included 6, i.e. Turicibacter, Turicibacter_sp_LA61, Actinobacteria, Lachnospiraceae_NK4A136_group, etc. As shown in Figure 3, the relative abundance of unclassified_Muribaculaceae, Enterorhabdus, Muribaculum and Desulfovibrio in the model group was lower than that in the blank group, and the relative abundance of Dubosiella and Ligilactobacillus was higher. Compared with the model group, the relative abundance of unclassified_Muribaculaceae, Lachnospiraceae_NK4A136_group, Enterorhabdus, Muribaculum and Desulfovibrio in the high-dose group of soap kernel rice powder increased, and the relative abundance of Dubosiella and Ligilactobacillus decreased. Figure 26 、 27as shown.

[0180] (6) Correlation analysis of differential genera with pharmacodynamic indicators and TGF-β1 / Smads pathway

[0181] Spearman correlation analysis method was used to analyze the correlation between the differential genera at the genus level of intestinal microorganisms of each group of mice and the pharmacodynamic indicators of saponin rice powder for improving pulmonary fibrosis and the TGF-β1 / Smads pathway. As shown in Table 6, the differential genus Muribaculu was negatively correlated with Fibronectin, α-SMA, TGF-β1, Smad2, and Smad3; the differential genus Alloprevotella was negatively correlated with HYP, Fibronectin, Collagen-I, α-SMA, TGF-β1, Smad2, and Smad3; the differential genera Mammaliicoccus, Jeotgalicoccus, and Clostridium_sensu_stricto_1 were positively correlated with HYP, Fibronectin, Collagen-I, α-SMA, TGF-β1, Smad2, and Smad3; the differential genus Turicibacter was positively correlated with HYP, Smad2, and Smad3; and the differential genus Lachnospiraceae_NK4A136_group was positively correlated with Fibronectin, α-SMA, TGF-β1, and Smad2. Figure 28

[0182] 3.5 Conclusion

[0183] Saponin rice powder can promote the production of SCFAs and inhibit the activation of the TGF-β1 / Smads signaling pathway by regulating the intestinal flora structure, such as reducing the ratio of Firmicutes / Bacteroidetes and enriching beneficial genera such as Muribaculum, thereby reducing the expression of fibrosis indicators Fibronectin, Collagen-I, and α-SMA and the content of HYP, reducing the body weight loss and lung coefficient of pulmonary fibrosis mice, improving the pathological damage of lung tissue, reducing the lung tissue injury score and the percentage of fibrous tissue expression, and playing a role in preventing and improving pulmonary fibrosis.​

Claims

1. The use of Gleditsia sinensis powder in the preparation of a product for preventing and improving pulmonary fibrosis. 2.The use of the soap kernel rice flour according to claim 1 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The preparation method of the Gleditsia sinensis powder is: taking Gleditsia sinensis for cold pretreatment, crushing, sieving, drying, and dividing into doses to obtain the Gleditsia sinensis powder. 3.The use of the soap kernel rice flour according to claim 2 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The preparation method of the Gleditsia sinensis powder is: taking Gleditsia sinensis and freezing it in liquid nitrogen for 1-60 min, then crushing, sieving through a 5-9 mesh sieve, drying at 60-100 ℃, and dividing into doses of 0.25-5 g per package to obtain the Gleditsia sinensis powder.

4. The use of the soap kernel rice flour according to claim 3 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized by: The preparation method of the Gleditsia sinensis powder is: taking Gleditsia sinensis and freezing it in liquid nitrogen for 1 min, then crushing, sieving through a 6 mesh sieve, drying at 80 ℃, and dividing into doses of 2.5 g per package to obtain the Gleditsia sinensis powder.

5. The use of the soap kernel rice flour according to any one of claims 2-4 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The Gleditsia sinensis is the dried endosperm of the dried mature seeds of Gleditsia sinensis Lam. after removing the seed coat and cotyledons. 6.The use of the soap kernel rice flour according to claim 1 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The product for preventing and improving pulmonary fibrosis is a drug or a health food.

7. The use of the soap kernel rice flour according to claim 6 for the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The method for eating the health food is: adding Gleditsia sinensis powder to boiling water, stirring while adding, and soaking for 5-20 min or simmering for 1-10 min.

8. The use of the soap kernel rice flour according to claim 7 for the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The method for eating the health food is: adding Gleditsia sinensis powder to boiling water, stirring while adding, and soaking for 10 min or simmering for 5 min. 9.The use of the soap kernel rice flour according to claim 6 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The preparation method of the drug is: combining Gleditsia sinensis powder with acceptable excipients in drugs, and processing according to the conventional method to prepare the corresponding drug. 10.The use of the soap kernel rice flour according to claim 9 in the preparation of a product for preventing and improving pulmonary fibrosis, characterized in that: The drug is a tablet, a hard capsule, a granule, a pill, a decoction, or an oral liquid.

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