Application of epimedium extract in preventing and treating pulmonary / renal fibrosis and chronic kidney disease

Abstract

The invention discloses an application of an epimedium extract in preventing and treating pulmonary / renal fibrosis and chronic kidney disease, belonging to the field of medicine, and specifically relates to a new preparation method of an epimedium extract and its application in preventing and treating pulmonary fibrosis and chronic kidney disease drugs and renal fibrosis. The invention firstly finds that an extract of epimedium with phenolic acid, anthocyanin and condensed tannin as the core has a significant improvement effect on pulmonary fibrosis and chronic kidney disease drugs and renal fibrosis, and its activity is equivalent to that of pirfenidone. In addition, the preparation process of the extract is simple and convenient for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a method for preparing an epimedium extract and application of the extract in preparing a drug for preventing and / or treating pulmonary / renal fibrosis and chronic kidney disease. Background Art

[0002] Pulmonary fibrosis is a terminal change of a large class of lung diseases characterized by fibroblast proliferation and a large amount of extracellular matrix aggregation accompanied by inflammatory damage and tissue structure destruction. It is one of the main causes of death from lung diseases. It is mainly manifested by the progressive replacement of normal alveolar structure by dense connective tissue, thereby preventing normal gas exchange and ultimately affecting the normal physiological function of the lungs. Pulmonary fibrosis can be divided into idiopathic and secondary, of which the former is the main clinically. Idiopathic pulmonary fibrosis is a chronic, progressive and fibrotic interstitial pneumonia with a higher incidence in Europe and North America (2.8 to 18 cases per 100,000 people) and a lower incidence in Asia and South America (0.5 to 4.2 cases per 100,000 people). The mortality rate is higher than that of most tumors. It is called a "tumor-like disease". The average survival time of patients after diagnosis is less than 3 years, and the 5-year survival rate is less than 30%. At present, the only drugs approved internationally for the clinical treatment of idiopathic pulmonary fibrosis are pirfenidone and nintedanib, so anti-pulmonary fibrosis drug treatment methods are still in urgent need of development.

[0003] Chronic kidney disease (CKD) refers to chronic renal structural and functional disorders (history of renal damage > 3 months) caused by various reasons, including normal and abnormal pathological damage to glomerular filtration rate (GFR), abnormal blood or urine components and abnormal imaging examinations, or unexplained GFR decline (GFR < 60 mL / min) for more than 3 months. It is a series of clinical manifestations and metabolic disorder syndromes caused by various diseases, characterized by progressive deterioration or irreversible damage to renal structure and function. Diseases that cause CKD include various primary and secondary glomerulonephritis, tubular damage and renal vascular lesions. The course of the disease is irreversible and chronic, and eventually leads to end-stage renal disease (ESRD). ESRD patients need to receive lifelong renal replacement therapy (including maintenance hemodialysis or peritoneal dialysis or kidney transplantation), which not only seriously affects the quality of life of patients, but also brings heavy mental stress and economic burden to patients and their families, and consumes huge social medical resources. Renal fibrosis is a common pathological feature of CKD caused by various reasons progressing to ESRD, including glomerular sclerosis, tubulointerstitial fibrosis and vascular sclerosis. Therefore, inhibiting the development of renal fibrosis is crucial to delaying the progression of CKD. In recent years, the incidence of CKD in the population has increased year by year, and it has become a global public health problem that seriously endangers human health. Recent epidemiological studies have shown that the global prevalence of CKD was 9.1% in 2017. Since 1990, the mortality rate has increased by 41.5%, raising CKD from the 17th leading cause of death in the world to the 12th. At present, there is no specific drug for chronic kidney disease and renal fibrosis in clinical practice. Commonly used first-line drugs are antihypertensive angiotensin receptor blockers and angiotensin converting enzyme inhibitors, hypoglycemic sodium-glucose cotransporter 2 inhibitors, and non-steroidal mineralocorticoid receptor antagonists for the treatment of chronic kidney disease associated with type 2 diabetes. Although the above drugs have a certain effect in delaying the progression of CKD, they have certain side effects. Their use is likely to cause hyperkalemia in patients with mid-to-late stage CKD, and can also cause hypotension and hypoglycemia in CKD patients without hypertension or hyperglycemia. Therefore, it is urgent to find a safe, effective, and universal specific drug for chronic kidney disease and / or renal fibrosis.

[0004] Epimedium, also known as Xianlingpi, is a perennial herb of the genus Epimedium in the Berberidaceae family. It was first recorded in the "Shennong Bencao Jing". It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is used for kidney yang deficiency, impotence and spermatorrhea, tendon and bone weakness, rheumatic pain, numbness and cramps. It is included in the 2020 edition of the "Chinese Pharmacopoeia". Studies on the chemical composition of epimedium have shown that epimedium mainly contains flavonoids, polysaccharides, lignans and alkaloids, among which the highest content is flavonoids and polysaccharides. Modern pharmacological research also focuses on these two types of components. It is believed that the flavonoid components of epimedium have pharmacological activities such as treating impotence, anti-osteoporosis, anti-tumor, anti-fibrosis, anti-inflammatory, and antioxidant; polysaccharide components mainly have the effect of enhancing immunity. Studies have shown that icariin, the main isopentenyl flavonoid glycoside component in epimedium, can inhibit liver, kidney and lung fibrosis by exerting anti-oxidative stress and anti-inflammatory effects. However, the inventors discovered for the first time in previous studies an effective part of epimedium whose chemical composition is still unknown, and its anti-pulmonary fibrosis and renal fibrosis activity is significantly better than that of epimedium flavonoids and polysaccharides. Therefore, the anti-pulmonary fibrosis and renal fibrosis activity of the effective part was confirmed, and it is believed that it has the potential to be developed into an innovative Class I Chinese medicine for anti-pulmonary fibrosis and chronic kidney disease and / or renal fibrosis. Summary of the invention

[0005] The present invention aims to provide a novel preparation method of epimedium extract and use of the extract in preparing medicine for preventing and treating pulmonary fibrosis, CKD and / or renal fibrosis.

[0006] The method for preparing the epimedium extract of the present invention comprises the following steps:

[0007] Take the herb Epimedium, add 5 to 30 times of water or 1% to 100% ethanol (or methanol) by volume, extract 1 to 3 times, filter, combine the filtrate and concentrate under reduced pressure to obtain an extract, adsorb the extract on a macroporous resin, elute with water and 10% to 40% ethanol by volume in sequence, then concentrate the ethanol eluate under reduced pressure until there is no alcohol taste, and then freeze-dry it into powder to obtain the herb Epimedium extract.

[0008] The above preparation method, wherein:

[0009] Preferably, take the herb Epimedium, add 20 times the amount of water to extract three times, filter, combine the filtrate and concentrate it into an extract, adsorb it on a macroporous resin, elute it with water and 20% to 30% ethanol by volume, and concentrate the 20% to 30% ethanol eluate under reduced pressure until there is no alcohol taste, and then freeze-dry it into powder to obtain the herb Epimedium extract.

[0010] In the preparation method, polysaccharide components and flavonoid components in epimedium are removed by extraction, adsorption on macroporous resin and the like, so as to obtain an extract with phenolic acid, alkaloids, anthocyanin and condensed tannin as core components.

[0011] The present invention also provides the use of the epimedium extract in preparing a medicine for preventing and treating pulmonary fibrosis, CKD and / or renal fibrosis, wherein the content of the epimedium extract in the medicine is 0.5% to 50%.

[0012] The present invention also provides a pharmaceutical composition for preventing and treating pulmonary fibrosis, CKD and / or renal fibrosis, the pharmaceutical composition comprising the epimedium extract, the components of the epimedium extract are not based on polysaccharides and flavonoids, and the content of the epimedium extract in the pharmaceutical composition is 0.5% to 50%.

[0013] The pharmaceutical composition for preventing and treating pulmonary fibrosis, CKD and / or renal fibrosis also comprises pharmaceutically acceptable adjuvants and carriers.

[0014] The pharmaceutical composition for preventing and treating pulmonary fibrosis, CKD and / or renal fibrosis has a significant improvement effect on the progression of pulmonary fibrosis, chronic kidney disease and fibrosis thereof.

[0015] The epimedium extract of the present invention or the pharmaceutical composition for preventing and treating pulmonary fibrosis comprising the epimedium extract has a significant alleviating effect on pulmonary fibrosis, can significantly improve the hardness of A549 fibrotic cells, and reduce lung tissue inflammation and intercellular ECM deposition in bleomycin-induced pulmonary fibrosis mice.

[0016] The epimedium extract of the present invention or the pharmaceutical composition containing the epimedium extract for preventing and treating CKD and / or renal fibrosis has the effect of significantly delaying the progression of CKD, can significantly improve the renal function indicators of CKD rats, reduce renal tissue inflammation and intercellular ECM deposition, and reverse the expression of fibrosis-related proteins in the kidney.

[0017] Compared with the prior art, the advantages of the present invention are:

[0018] 1. Modern research on epimedium generally believes that flavonoids and polysaccharides are the main active ingredients in epimedium. Therefore, research and inventions on the anti-fibrosis and renal protection effects of epimedium are mainly focused on flavonoids. However, the HK-2 cell experiment induced by TGF-β1 in the present invention confirms that this extract in epimedium has more significant anti-fibrosis and renal protection activities than flavonoids.

[0019] 2. The TGF-β1-induced HK-2 cell experiment confirmed that the epimedium extract of the present invention has no statistical difference in improving the hardness of A549 fibrotic cells compared with pirfenidone, and the dosage used is much lower than pirfenidone, and has excellent anti-pulmonary fibrosis activity. At the same time, animal experiments have confirmed that the anti-renal fibrosis activity of the epimedium extract of the present invention is also comparable to that of pirfenidone, and has a significant improvement effect on CKD.

[0020] 3. The extract preparation process of the present invention is simple and convenient for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 H&E staining results of mouse lung tissue (400×).

[0022] Figure 2 Masson trichrome staining results of mouse lung tissue (400×).

[0023] Figure 3 Histopathological staining results of rat kidney tissue. A is H&E staining (200×), B is Masson trichrome staining (200×).

[0024] Figure 4 Immunohistochemical staining was used to detect the expression of EMT-related proteins α-SMA, E-cadherin and Fibronectin in rat kidney (200×).

[0025] Figure 5 EP-R-30 ultra-high performance liquid chromatography. DETAILED DESCRIPTION

[0026] Example 1

[0027] Preparation of Epimedium Extract

[0028] Take 1kg of Epimedium medicinal material, add 30, 15, and 15 times the amount of water in sequence, reflux and extract three times, filter, combine the filtrate and concentrate under reduced pressure to obtain an extract. The extract is adsorbed on the macroporous resin in a ratio of 1:1 between the mass of the medicinal material and the mass of the macroporous adsorption resin, and eluted with water and 30% ethanol by volume in sequence. The 30% ethanol eluate is concentrated under reduced pressure until there is no alcohol taste, and then freeze-dried into powder to obtain the Epimedium extract, which is named EP-R-30.

[0029] Example 2

[0030] Effect of EP-R-30 on the stiffness of A549 cell fibrosis induced by TGF-β1

[0031] 1. Experimental Methods

[0032] 1.1 MTT assay to detect the proliferation inhibitory activity of EP-R-30, total flavonoids of epimedium and icariin on A549 cells

[0033] Take A549 cells in the logarithmic growth phase, add trypsin to digest and prepare a cell suspension. Add 100 μL of the diluted cell suspension to each well of a 96-well plate, and control the seeding density to 5×10 3 The 96-well plate was placed in an incubator for culture, and the starvation culture was continued for 12 hours after the cells attached to the wall. Subsequently, the serum-free culture medium of the drug-treated group was replaced with a culture medium containing different concentrations of EP-R-30, total flavonoids of epimedium (EP-TF), icariin (ICA), positive control drug pirfenidone (PFD) and 2% serum. The control group and the model group were replaced with an equal volume of culture medium containing 2% serum. After continuing to culture in the incubator for 60 hours, the 96-well plate was taken out, 15 μL of MTT working solution was added to each well, and it was returned to the incubator for further incubation for 4 hours. After the incubation, the original culture medium in the culture plate was discarded, 150 μL of DMSO was added to each well, and it was placed on an oscillator for 10 minutes to fully dissolve the crystals, and the absorbance (OD) value of each well was detected at 492 nm using a multifunctional microplate reader. The cell proliferation inhibition rate was calculated according to the following formula, and the results were expressed as IC 20 Value representation.

[0034]

[0035] 1.2 Evaluation of the anti-pulmonary fibrosis activity of EP-R-30, EP-TF, ICA and PFD using AFM-based cell stiffness detection method

[0036] A549 cells in the logarithmic growth phase were inoculated into the culture dish at a certain ratio so that the cells would grow to 70%-80% of the whole dish during the hardness test. After the cells attached to the wall, the culture medium was discarded and replaced with serum-free culture medium for starvation culture for 12 hours to synchronize the cells. The culture medium was then discarded and the IC values ​​were calculated according to the IC values ​​obtained in "1.1". 20 Values, add culture medium containing appropriate concentrations of the corresponding test drugs and 2% serum to the EP-R-30, EP-TF, ICA and positive drug PFD groups, add culture medium containing 2% serum to the control group and model group, and continue to culture for 12 hours. Finally, except for the control group, all other groups were added with 10ng / mL TGF-β1 for continuous stimulation for 48 hours to create an in vitro pulmonary fibrosis model.

[0037] After 48h, the culture dish was removed from the incubator, the culture solution was discarded, and the cells were gently washed with PBS buffer to prevent them from falling off. Then the original buffer was discarded and 4mL of buffer was added again. The AFM workbench and console were turned on, and the culture dish was placed steadily on the workbench. First, the parameters were set in the force measurement mode so that the probe obtained the substrate Senstivity deflection value in the blank area of ​​the culture dish and recorded it to eliminate the error caused by the substrate difference of each culture dish. Subsequently, the probe was moved to the center of the cell to obtain the force curve, and the camera was taken after stabilization. The force curves of 20 cells were randomly collected for each culture dish, and ten were collected in parallel for each cell. The anti-pulmonary fibrosis activity of the extract was expressed by the cell Young's modulus (Kpa) and hardness inhibition rate (%). The larger the Young's modulus value, the more serious the pulmonary fibrosis; the higher the inhibition rate, the better the anti-pulmonary fibrosis activity of the extract.

[0038] The hardness suppression rate calculation formula is:

[0039]

[0040] SPSS 25.0 software was used to perform statistical analysis on the results, and the results were expressed as mean ± standard deviation.

[0041] 2. Experimental results

[0042] The results of the inhibitory effects of EP-R-30, EP-TF, ICA and PFD on the proliferation of A549 cells and the anti-TGF-β1-induced fibrosis of A549 cells are shown in Table 1. EP-R-30, EP-TF, ICA and PFD have no cytotoxicity to A549 cells; among the two effective parts of Epimedium, EP-R-30, EP-TF and the effective ingredient ICA, EP-R-30 has the best anti-fibrosis activity (Young's modulus value of 4.1Kpa, inhibition rate of 84%). Its Young's modulus value of 4.1Kpa is not statistically different from the positive control drug PFD 3.9Kpa, and the dose (14.7μg / mL) is much lower than PFD (500μg / mL).

[0043] Table 1 IC of EP-R-30, EP-TF, ICA and PFD on the proliferation inhibition of A549 cells 20 Value, Young's modulus value and inhibition rate of anti-fibrosis of A549 cells

[0044]

[0045] ### p<0.001, model group vs normal group; * p<0.05, ** p<0.01, ***p<0.001, model group vs each drug-treated group; △ p<0.05, PFD group vs each drug-treated group.

[0046] Example 3

[0047] Effect of EP-R-30 on BLM-induced pulmonary fibrosis in mice

[0048] 1. Experimental Methods

[0049] 1.1 Animals, grouping, modeling and drug administration

[0050] Twenty-four healthy SPF male C57BL / 6J mice (weight 16-20 g) were randomly divided into 4 groups: normal control group (Saline, 5 mg / kg), model group (BLM, 3 mg / kg), positive drug (BLM+PFD) group and Epimedium extract (BLM+EP-R-30) group, with 6 mice in each group. The dosages of PFD and EP-R-30 were 100 mg / kg and 200 mg / kg, respectively. After one week of adaptive breeding in the SPF laboratory, all mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (10 mL / kg). After the righting reflex and clamp finger web reflex of the mice disappeared, they were fixed on the mouse board, the neck was depilated and disinfected with iodine, and then the trachea was carefully exposed with ophthalmic surgical scissors and ophthalmic forceps, and the syringe needle was inserted through the tracheal cartilage toward the heart. The normal control group was given 0.9% saline, and the other mice were given 3 mg / kg of BLM. Immediately after the injection, the mice were upright and rotated left and right so that the drugs and saline could be smoothly and evenly distributed to the lungs. After the operation, the wound was sutured and disinfected with iodine. On the 7th day after modeling, PFD and EP-R-30 were given to the positive drug group and EP-R-30 group by gavage, and the normal control group and model group were given an equal amount of saline. The drugs were given for 21 consecutive days for treatment.

[0051] 1.2 Tissue embedding and preparation of paraffin sections

[0052] On the 28th day after modeling, all mice were dissected, and the chest cavity was opened for perfusion after anesthesia. After all the blood was drained and the effluent became clear, the perfusion was stopped and the lungs were removed. The left lung was immediately placed in the pre-prepared 4% paraformaldehyde, and the right lung was immediately placed in a cryotube and stored in a -80℃ refrigerator for later use. The left lung was fixed in 4% paraformaldehyde for 48 hours, then taken out and placed in an embedding box and rinsed with running water overnight. Then, the tissue was dehydrated in a gradient manner using 75% to 95% ethanol and anhydrous ethanol. After dehydration, the excess liquid was absorbed with filter paper, placed in xylene for permeabilization, and finally immersed in wax and embedded.

[0053] 1.3 Hematoxylin-Eosin (H&E) staining

[0054] H&E staining was used to examine the pathological changes in mouse lung tissue. 5 μm paraffin sections were dewaxed and gradiently immersed in water, and the sections were stained using a kit, naturally dried, and sealed with neutral gum for microscopic observation.

[0055] 1.4 Masson trichrome staining

[0056] The changes in collagen deposition in mouse lung tissue were detected using Masson trichrome staining. Paraffin sections were stained with Masson staining according to the kit instructions, and then the sections were dehydrated, transparent, and sealed. After sealing with neutral gum, microscopic observation can be performed, and the collagen quantification results are expressed as collagen volume fraction (CVF).

[0057] 2. Experimental results

[0058] H&E staining results Figure 1 As shown in the figure, the lung tissue sections of mice in the normal control group showed clear and neat alveolar morphology and structure, thin alveolar cavity walls and no fibers, and no obvious inflammatory cell infiltration around the trachea and blood vessels. In the lung tissue sections of mice in the BLM group, the alveolar morphology and structure were severely damaged, the alveolar cavity collapsed and disordered, and large continuous fibrotic lesions were visible in the field of view; the alveolar cavity of mice treated with PFD and EP-R-30 was less collapsed, the thickness of the alveolar septum increased, and nodular fibrosis was occasionally seen, and the pathological condition was significantly improved. The results of Masson trichrome staining are shown in the figure. Figure 2 As shown in the figure, only a very small amount of blue collagen fibers appeared in the alveolar septa in the lung tissue of the normal control group mice; obvious fibrosis lesions were seen in the lung tissue of the BLM group mice, and large collagen fibers were present around the trachea, which is a typical pathological manifestation of collagen fiber deposition; after treatment with EP-R-30 and PFD, although the thickness of the alveolar septa of the mice increased compared with the control group, there was only a small amount of collagen deposition in the alveolar wall. Semi-quantitative analysis of CVF in the EP-R-30 and PFD groups (Table 2) showed that EP-R-30 could significantly inhibit the deposition of collagen fibers in the lung tissue of BLM mice, and the effect was not statistically different from that of PFD.

[0059] Table 2 CVF analysis results of Masson trichrome stained sections of mouse lung tissue

[0060]

[0061] ### p<0.001, model group vs normal group; *** p<0.001, model group vs each drug-treated group

[0062] In summary, EP-R-30 has a significant inhibitory effect on the sclerosis of A549 cells after fibrosis induced by TGF-β1, and has a significant improvement effect on the inflammation and fibrotic lesions of lung tissue in the mouse pulmonary fibrosis model induced by bleomycin. Quantitative and semi-quantitative analysis of the above indicators found that there was no statistical difference in the effect of EP-R-30 compared with the positive control drug pirfenidone.

[0063] Example 4

[0064] Improvement of Epimedium extract on adenine-induced CKD and renal fibrosis in SD rats

[0065] 1. Experimental Methods

[0066] 1.1 Animal grouping, modeling and drug administration

[0067] 40 healthy male SD rats were randomly divided into 5 groups, 8 rats in each group, including normal control group (Control), model group (Adenine), low-dose group of epimedium extract (EP-R-30) Adenine + EP-R-30 (200 mg / kg), high-dose group of epimedium extract Adenine + EP-R-30 (400 mg / kg) and pirfenidone group (Adenine + PFD). Before daily gavage, all drugs were suspended with 0.5% sodium carboxymethylcellulose (CMC-Na) solution for use. After one week of adaptive feeding in the SPF laboratory, the body weight of the rats was weighed every two days. At 9 o'clock every day, except for the normal control group given the corresponding volume of 5% CMC-Na solution, the other groups were gavaged with adenine suspension at a dose of 200 mg / kg for 4 consecutive weeks to establish a rat chronic kidney disease model. During this period, except for the normal control group and the model group who were given corresponding volumes of 5% CMC-Na solution at 15:00 every day, the epimedium extract group was given corresponding doses of EP-R-30 suspension, and the pirfenidone group was given 160 mg / kg pirfenidone suspension by gavage.

[0068] 1.2 Biological sample collection and processing

[0069] After the last day of administration, the rats were immediately fasted but not watered and placed in metabolic cages, and 24-hour urine from all rats was collected on ice. After the collection was completed, the urine was centrifuged at 4200rpm for 20min, and the supernatant was divided into EP tubes and placed in a -80℃ refrigerator for use. The rats were anesthetized with 1% sodium pentobarbital at a dose of 40mg / kg. After blood was taken from the abdominal aorta, the rats' bilateral kidneys were quickly removed. After washing in ice saline, the left kidney was immediately placed in a -80℃ refrigerator for use, and the right kidney was fixed in 4% paraformaldehyde. The collected abdominal aorta whole blood was allowed to stand at room temperature for 1h and then centrifuged at 3000rpm for 10min. The serum was divided into EP tubes and placed in a -80℃ refrigerator for use.

[0070] 1.3 Detection of renal function biochemical indicators

[0071] Serum creatinine (Scr) and blood urea nitrogen (BUN) of rats were automatically detected using Chemray 240 automatic biochemical analyzer, and 24h urine protein (24h UP) was detected using ELISA kit.

[0072] 1.4 Tissue embedding and preparation of paraffin sections

[0073] The right kidney was fixed in 4% paraformaldehyde for 48 h, then taken out and placed in an embedding box and rinsed with running water overnight. Subsequently, the tissue was dehydrated in a gradient manner using 75% to 95% ethanol and anhydrous ethanol. After dehydration, the excess liquid was absorbed with filter paper, and the tissue was placed in xylene for permeabilization, and finally wax-immersion embedding was performed.

[0074] 1.5 H&E staining

[0075] H&E staining was used to examine the pathological changes of rat kidney tissue. 5μm paraffin sections were dewaxed and gradiently immersed in water, and the sections were stained using a kit, naturally dried, and sealed with neutral gum for microscopic observation.

[0076] 1.6 Masson trichrome staining

[0077] The changes of collagen deposition in rat kidney tissue were detected by Masson trichrome staining. Paraffin sections were stained with Masson staining according to the instructions of the kit, and then the sections were dehydrated, transparent, and sealed. Neutral gum was used to seal the sections for microscopic observation, and the collagen quantification results were expressed as collagen volume fraction (CVF).

[0078] 1.7 Immunohistochemistry (IHC) staining

[0079] After antigen repair and peroxidase blocking, rat kidney tissue sections were blocked with BSA for 30 minutes. After incubation with primary and secondary antibodies, DAB staining and DAPI cell nucleus counterstaining were performed, and finally dehydration, sealing, and microscopic examination were performed. The semi-quantitative analysis results were expressed as average optical density (AOD), and average optical density (AOD) = integrated optical density (integrated optical density) / tissue area (Area).

[0080] 2. Experimental results

[0081] 2.1 Effects of EP-R-30 on renal biochemical parameters in CKD rats

[0082] The results of the relevant indexes are shown in Table 3. The SCr, BUN and 24h UP of CKD rats were significantly higher than those of the normal group. After EP-R-30 intervention, the levels were significantly adjusted back to the normal group, and there was no statistical difference compared with the PFD group.

[0083] Table 3 Results of determination of renal function index levels in rats

[0084]

[0085] ### p<0.001, model group vs normal group; *** p<0.001, model group vs each drug-treated group

[0086] 2.2 EP-R-30 improves renal histopathology in CKD rats

[0087] like Figure 3 As shown in the figure, under the light microscope, it can be seen that the surface capsule of the renal tissue in the normal group is composed of dense connective tissue of uniform thickness; the renal parenchyma has a clear cortical-medullary boundary, the glomeruli are evenly distributed in the cortex, the number of cells and matrix in the glomeruli are uniform, the renal tubular epithelial cells are round and plump, the brush borders are arranged neatly and regularly, and there is no obvious abnormality in the medulla; there is no obvious hyperplasia in the renal interstitium and no obvious inflammatory changes. In the renal tissue of the adenine group, a large number of renal tubular dilatation can be seen, and necrotic cell fragments are often seen in the lumen. A large number of renal tubular atrophy is accompanied by connective tissue hyperplasia, lymphocyte infiltration and multinuclear giant cells. At the same time, a large number of urate crystals can be seen, often accompanied by granuloma formation, and compared with the normal group, a large number of collagen fibers are deposited in the renal interstitium of the adenine group. After intervention with EP-R-30, the renal tissue lesions were significantly alleviated, the fiber area was significantly reduced, and the tissue structure tended to be similar to that of the normal group. Meanwhile, the semi-quantitative analysis results of Masson's trichrome staining showed that there was no significant difference in CVF between the EP-R-30 group and the PFD group (Table 4).

[0088] Table 4 CVF analysis results of Masson trichrome stained sections of rat kidney

[0089]

[0090] ### p<0.001, model group vs normal group; *** p<0.001, model group vs each drug-treated group

[0091] 2.3EP-R-30 can inhibit epithelial-mesenchymal transition (EMT) and thus delay the progression of renal interstitial fibrosis

[0092] EMT is an important process of renal interstitial fibrosis, and detecting the levels of EMT-related factors is an important means to assess the degree of renal interstitial fibrosis. Figure 4As shown in the figure, the EMT-related factors, mesenchymal markers α-SMA and Fibronectin, were only expressed in very small amounts in the normal group, but were expressed in large amounts in the kidneys of CKD rats. E-cadherin is a protein that plays an important role in cell adhesion. It is highly expressed in healthy rats, but its expression level dropped sharply in the model group. After treatment with EP-R-30, the expression levels of the above EMT markers were significantly corrected, close to those of the control group, indicating that EP-R-30 can significantly reverse the changes in the levels of EMT factors and delay the progression of renal interstitial fibrosis. At the same time, the semi-quantitative analysis results showed that there was no significant difference in the AOD of the EP-R-30 group compared with the PFD group (Table 5).

[0093] Table 5 Analysis results of AOD detected by IHC staining of rat kidney

[0094]

[0095] ### p<0.001, model group vs normal group; *** p<0.001, model group vs each drug-treated group

[0096] In summary, EP-R-30 has a significant delaying effect on the course of adenine-induced CKD in rats, and there is no statistical difference compared with pirfenidone, indicating that the therapeutic effect of EP-R-30 on CKD, especially renal fibrosis, is equivalent to that of pirfenidone.

[0097] Example 5

[0098] EP-R-30 component analysis

[0099] 1. Chemical identification of ingredients

[0100] 1.1 Experimental methods

[0101] Weigh 10 mg of EP-R-30 into a 10 mL volumetric flask, dilute to the mark with water, and shake well to obtain the test sample.

[0102] Take 1 mL of EP-R-30 test sample in each stoppered glass test tube and react with different chemical reagents, observe and record the phenomena.

[0103] 1.2 Experimental Results

[0104] Chemical identification results show that EP-R-30 contains phenolic acid components, tannins, organic acids and alkaloids.

[0105] Table 6 Chemical identification results

[0106]

[0107]

[0108] 2. Determination of total ingredient content

[0109] 2.1 Determination of total polyphenol content

[0110] 2.1.1 Preparation of reagents

[0111] 7.5% Na2CO3 solution: Weigh 7.5g of anhydrous Na2CO3 into a 100mL volumetric flask, dilute to the mark with deionized water, and shake until dissolved.

[0112] Folin phenol reagent: Transfer 5 mL of Folin phenol reagent to a 25 mL volumetric flask, dilute to volume with water and shake well.

[0113] 2.1.2 Preparation of gallic acid standard stock solution

[0114] Accurately weigh 100.21 mg of gallic acid, place it in a 10 mL volumetric flask, dilute to the mark with deionized water, and shake well to obtain the gallic acid standard stock solution.

[0115] 2.1.3 Preparation of reference substance working solution

[0116] Pipette 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of gallic acid standard stock solution into 10 mL volumetric flasks, dilute to the mark with deionized water, shake well, and the concentrations are 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mg / mL, respectively.

[0117] 2.1.4 Preparation of test samples

[0118] Take EP-R-30 powder from different preparation batches, place it in a 10 mL volumetric flask, dilute to the mark with deionized water, shake well, and prepare the test sample with a concentration of 1 mg / mL.

[0119] 2.1.5 Drawing of standard curve

[0120] Pipette 0.1mL of the above working solution into a 10mL brown volumetric flask, add 1.5mL of Folin phenol reagent to each brown volumetric flask, shake well, react for 5min, add 0.7mL of 7.5% Na2CO3 solution, dilute to the scale with deionized water, shake well, and react for 1h in the dark. Use a 10mm cuvette, subtract blank solvent, and measure the absorbance (A) with a UV spectrophotometer at a wavelength of 765nm. Draw a standard curve with the concentration of the reference substance as the horizontal axis and the absorbance as the vertical axis.

[0121] 2.2 Determination of total proanthocyanidins content

[0122] 2.2.1 Preparation of test solution

[0123] Dilute hydrochloric acid: Measure 17mL of hydrochloric acid into a 100mL volumetric flask, add distilled water to the mark, mix well, and you have the product.

[0124] Preparation of 2% ammonium ferric sulfate: weigh 0.20 g of ammonium ferric sulfate into a 10 mL volumetric flask, add dilute hydrochloric acid to dissolve and dilute to the mark.

[0125] n-Butanol-hydrochloric acid reagent: Take an appropriate amount of n-butanol into a 100mL volumetric flask, accurately measure 5mL of hydrochloric acid, make up to volume with n-butanol, mix well, and set aside.

[0126] 2.2.2 Preparation of reference substances

[0127] Accurately weigh 4.71 mg of proanthocyanidin reference substance (PCB2), place in a 10 mL volumetric flask, add methanol to dissolve and dilute to the mark to obtain the proanthocyanidin reference substance stock solution. After dilution, 70.65, 94.2, 117.75, 141.3, 188.4, 211.95, and 235.5 μg / mL reference substance working solutions were obtained.

[0128] 2.2.3 Preparation of test samples

[0129] Accurately weigh 100.41 mg EP-R-30, place in a 10 mL volumetric flask, dilute to the mark with methanol, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 min, let it cool to room temperature, add methanol to the mark, shake well, centrifuge and take the supernatant as the test solution, and calculate the total proanthocyanidin content in the sample according to the standard curve.

[0130] 2.2.4 Preparation of standard curve

[0131] Accurately pipette 1 mL of proanthocyanidin working solution, accurately add 6 mL of hydrochloric acid-n-butanol solution and 0.2 mL of ammonium ferric sulfate solution, mix well, seal, heat in boiling water for 40 min, take out, and immediately put in an ice bath to room temperature. Determine the proanthocyanidin content by ultraviolet spectrophotometry, deduct the absorbance of the blank solvent, measure the absorbance at a wavelength of 546 nm, and measure 3 times in parallel. Take the average absorbance as the ordinate and the proanthocyanidin content as the abscissa to prepare a standard curve.

[0132] 2.2.5 Determination of proanthocyanidins

[0133] Accurately pipette 1 mL of the test sample, add 6 mL of hydrochloric acid-n-butanol solution and 0.2 mL of ammonium ferric sulfate solution, mix well, seal, heat in boiling water for 40 min, take out, immediately cool to room temperature in ice water, subtract the blank solvent, measure the absorbance at a wavelength of 546 nm, and measure three times in parallel.

[0134] 2.3 Results

[0135] 2.3.1 Total polyphenol content determination results

[0136] The linear relationship obtained in the range of 0.1 to 0.7 mg / mL is y = 1.1437x + 0.0234, R 2 =0.9995, and the total polyphenol content in the sample was calculated based on the standard curve. The experiment showed that the total polyphenol content in EP-R-30 was 27.53% to 30.91%.

[0137] Table 7 Gallic acid standard curve absorbance measurement data

[0138]

[0139] Table 8 Determination results of total polyphenol content of different batches of test samples

[0140]

[0141] 2.3.2 Results of proanthocyanidin content determination

[0142] The linear relationship of the proanthocyanidin standard curve obtained at 0.0942-0.2120 mg / mL is y=1.4239x+0.1344, R2=0.99, and the total proanthocyanidin content in the sample is calculated based on the standard curve. The experiment shows that the proanthocyanidin content in EP-R-30 is 0.98%.

[0143] Table 9 Proanthocyanidin standard curve absorbance determination data

[0144]

[0145] Table 10 Determination results of proanthocyanidin content in EP-R-30

[0146]

[0147] 3. Characterization of chemical composition of EP-R-30

[0148] 3.1 Liquid quality conditions

[0149] Liquid chromatograph (Agilent 1290uplc), mass spectrometer (Agilent Q-tof 6550); chromatographic column: WatersBEH C 18(2.1*100mm 1.7μm). Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile solution; Flow rate: 0.3mL / min; Injection volume: 5uL; Chromatographic elution program as shown in Table 11, Mass spectrometry scanning range: primary 50-1500m / z, sheath gastemp 350℃; sheath gas flow 12L / min, ESI+ mode: voltage 4000V, ESI- mode: voltage 3200V. Data analysis was performed using Agilent Masshunter.

[0150] Table 11 Chromatographic elution program

[0151]

[0152] 3.2 Experimental Results

[0153] UPLC-Q-TOF / MS analysis showed that EP-R-30 contained phenolic acid, alkaloids, anthocyanins and other types of compounds.

[0154] Table 12 Mass spectrometry information in negative ion mode

[0155]

[0156]

[0157] Table 13 Mass spectrum information of anthocyanins detected in positive ion mode

[0158]

[0159] 4.EP-R-30UPLC-DAD spectrum

[0160] 4.1 Preparation of test samples

[0161] Accurately weigh 5.92 mg of EP-R-30 and place it in a 2 mL volumetric flask. Add deionized water to the mark, shake well, and filter through a 0.22 μm aqueous filter membrane.

[0162] 4.2 Preparation of reference substances

[0163] Accurately weigh 1.24 mg of protocatechuic acid into a 1 mL volumetric flask, dilute to the mark with deionized water, and shake well to obtain the protocatechuic acid stock solution. Transfer 50 μL of the protocatechuic acid stock solution to a 1 mL volumetric flask, dilute to 1 mL with deionized water, and filter with a 0.22 μm water filter membrane to obtain the protocatechuic acid working solution.

[0164] Accurately weigh 1.42 mg of p-hydroxybenzoic acid into a 2 mL volumetric flask, dilute to the mark with chromatographic methanol, and shake well to obtain p-coumaric acid stock solution. Transfer 100 μL of p-hydroxybenzoic acid stock solution to a 2 mL volumetric flask, dilute to 1 mL with chromatographic methanol, and filter with a 0.22 μm water filter membrane to obtain p-hydroxybenzoic acid working solution.

[0165] Accurately weigh 0.26 mg of p-coumaric acid into a 1 mL volumetric flask, dilute to the mark with chromatographic methanol, and shake well to obtain the p-coumaric acid stock solution. Transfer 100 μL of the p-coumaric acid stock solution to a 1 mL volumetric flask, dilute to 1 mL with chromatographic methanol, and filter with a 0.22 μm water filter membrane to obtain the p-coumaric acid working solution.

[0166] Accurately weigh 0.81 mg of chlorogenic acid into a 1 mL volumetric flask, dilute to the mark with deionized water, and shake well to obtain the chlorogenic acid stock solution. Pipette 100 μL of the chlorogenic acid stock solution, dilute to 1 mL with deionized water, and filter with a 0.22 μm water filter to obtain the chlorogenic acid working solution.

[0167] Accurately weigh 1.16 mg of magnolia alkaloids into a 1 mL volumetric flask, dilute to the mark with deionized water, and shake well to obtain the magnolia alkaloids stock solution. Pipette 100 μL of the magnolia alkaloids stock solution, dilute to 1 mL with deionized water, and filter with a 0.22 μm water filter to obtain the magnolia alkaloids working solution.

[0168] 4.3 Chromatographic conditions

[0169] Shimadzu Nexera X2 LC-30A ultra-high performance liquid chromatograph; ACQUITY BEH C 18 Chromatographic column (2.1*100mm1.7μm), mobile phase is 0.1% FA / H2O (Pump A)-ACN (Pump B); column temperature is 30°C; flow rate is 0.30mL / min, injection volume is 2μL; detection wavelength is 280nm.

[0170] Table 14 Gradient elution time program

[0171]

[0172] 4.4 Results

[0173] See the attached chromatogram of EP-R-30 Figure 5, 14 characteristic peaks can be seen. Compared with the reference substance, peaks 3, 4, 7, 9, and 12 were identified as protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, p-coumaric acid, and magnolia alkaloids, respectively. The content of protocatechuic acid in this batch of EP-R-30 was determined by the external standard method to be 0.0099 mg / mg EP-R-30, 0.0021 mg / mg EP-R-30, 0.0180 mg / mg EP-R-30, 0.0061 mg / mg p-coumaric acid, and 0.0353 mg / mg EP-R-30.

[0174] Table 15 Chromatographic peak information table

[0175]

[0176] Table 16 Determination of chemical composition content of some EP-R-30 in this batch

[0177]

Claims

1. An application of an epimedium extract in the preparation of a drug for preventing and treating pulmonary fibrosis, chronic kidney disease and renal fibrosis. The preparation method of the epimedium extract comprises the following steps: taking an epimedium medicinal material, adding 5-30 times of water in turn, refluxing and extracting for 3 times, filtering, combining the filtrate and concentrating under reduced pressure to obtain an extract; adsorbing the extract on a macroporous resin in a ratio of 1:1 between the mass of the medicinal material and the mass of the macroporous adsorption resin, and eluting with water and ethanol with a volume concentration of 20%-40% in turn; concentrating the ethanol eluate under reduced pressure until there is no alcohol taste, and then freeze-drying it into powder to obtain the epimedium extract.

2. The use according to claim 1, characterized in that The epimedium extract components do not have polysaccharides and flavonoids as the core, and the content of the epimedium extract components in the medicine for preventing and treating pulmonary fibrosis, chronic kidney disease and renal fibrosis is 0.5% to 50%.

3. The use according to claim 2, characterized in that The components of the obtained epimedium extract are not centered on polysaccharides and flavonoids, but centered on phenolic acid, alkaloids, anthocyanins and condensed tannins.

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