Method for extracting pinoresinol from herba ephedrae and application of pinoresinol in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention relates to a method for extracting pinoresinol from herba ephedrae, which can effectively extract pinoresinol from herba ephedrae and realize the application in preparation of medicines for treating pulmonary arterial hypertension, and the preparation method comprises the following steps: taking dried roots and rhizomes of herba ephedrae, and carrying out water extraction and vacuum concentration to obtain an extract; and dispersing with water, eluting, dissolving with methanol, stirring with silica gel, carrying out gradient elution, detecting and identifying, merging the same fractions, loading the target components on column chromatography, eluting, separating and purifying, collecting the fractions with retention time, and drying to obtain the pinoresinol. The pinoresinol prepared by the invention has the effect of resisting abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), and realizes the application in preparation of medicines for treating pulmonary arterial hypertension. The preparation method is simple, the medicinal value of the pinoresinol and the medicinal value of the herba ephedrae are easily and industrially prepared, the application of the pinoresinol in preparation of the medicine for treating the pulmonary arterial hypertension is realized, and the medicine is a great innovation in preparation of the medicine for treating the pulmonary arterial hypertension and has practical popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and in particular to a method for extracting pinoresinin from Ephedra sinica and its application in the preparation of drugs for treating pulmonary hypertension. Background Technology

[0002] Pulmonary hypertension (PH) is a malignant and progressive pulmonary vascular disease caused by various etiologies and different pathogenesis mechanisms, leading to changes in the structure or function of pulmonary vessels, resulting in increased pulmonary artery pressure and pulmonary vascular resistance, which can then progress to right heart failure and even death, with a poor prognosis. Epidemiological studies show that it is estimated that at least 1% of the global population still suffers from PH, and the five-year mortality rate is as high as 50%. Understanding the pathogenesis and intervention methods of PH has always been a key focus and challenge in basic research and clinical treatment, posing a significant challenge to clinical diagnosis and treatment. Currently, there is no drug that can cure PH. Prostacyclin drugs and endothelin receptor antagonists used to treat PH have limited clinical application due to adverse reactions and high prices.

[0003] From the pathophysiological perspective of pulmonary hypertension (PH), it is mainly caused by changes in the structure and function of pulmonary blood vessels. This process primarily involves the proliferation and death of pulmonary artery smooth muscle cells (PASMCs) and the role of the extracellular matrix. Genetically controlled autophagy plays an indispensable role in cell development and the regulation of overall homeostasis. Mitophagy is a selective autophagy pathway similar to other autophagy pathways. Recent studies have shown that when cells are subjected to hypoxia, a series of stress changes occur. In the early stages of hypoxia, appropriate mitophagy can specifically clear damaged mitochondria and reduce cell damage. However, with prolonged hypoxia, mitophagy is promoted through mechanisms such as activation of the PINK1 / Parkin-mediated pathway, phosphorylation of the FUNDC1 receptor, and upregulation of HIF-1α and ROS, thereby damaging mitochondrial function, inhibiting apoptosis, and exacerbating cell proliferation. Since lung tissue is most sensitive to oxygen content, prolonged hypoxia can lead to abnormal pulmonary vasoconstriction and pathological remodeling of pulmonary arterioles, resulting in increased pulmonary circulatory resistance and ultimately evolving into hypoxic pulmonary hypertension. Mitophagy is closely related to this process.

[0004] In recent years, with the development of bioinformatics and artificial intelligence, and the continuous enrichment of medical big data, the application of informatics methods in the medical field has become increasingly frequent. Metabolomics and transcriptomics have made significant progress in the study of pulmonary hypertension (PH). Researchers have used blood, saliva, urine, and lung tissue samples from PH patients or animal models for metabolomics analysis, gradually revealing the composition and changes of metabolites during the disease process and discovering that metabolic abnormalities play a key role in the pathogenesis of PH. Transcriptomics can reveal the expression levels and patterns of genes under specific physiological or pathological states, thereby providing a deeper understanding of gene function and regulatory mechanisms in organisms. It can not only participate in revealing the mechanisms of PH occurrence and development but also provide new specific diagnostic biomarkers and potential therapeutic targets for its diagnosis and treatment, playing an important role in improving patients' quality of life and prognosis.

[0005] Ephedra root, also known as bitter toon vegetable, is the root of Ephedra sinica, a plant in the Ephedraceae family. Ephedra sinica Stapf) or Ephedra ( Ephedra intermedia The dried roots and rhizomes of *Ephedra sinica* (Schrenk et CAMey.) are a traditional Chinese medicine, listed in the *Pharmacopoeia of the People's Republic of China*. The modern pharmacological activities of *Ephedra sinica* root mainly include antihypertensive, antitumor, anti-inflammatory, and antioxidant effects. Pininsin is a lignan compound isolated from *Ephedra sinica* root. Previous experimental results showed that Pin has an anti-PASMCs abnormal proliferation effect, but its mechanism of action on pH is still unclear. Therefore, how to extract pininsin from *Ephedra sinica* and apply it in the preparation of drugs for treating pulmonary arterial hypertension has not yet been publicly reported. Summary of the Invention

[0006] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for extracting pinoresinin from ephedra sinica, which can effectively extract pinoresinin from ephedra sinica and realize its application in the preparation of drugs for treating pulmonary arterial hypertension.

[0007] The technical solution provided by this invention is: a method for extracting pinoresinin from Ephedra sinica, wherein the molecular structural formula of pinoresinin is:

[0008] Its preparation method is as follows: S1. Take the dried roots and rhizomes of Ephedra sinica and extract them twice with 15 times their weight volume of water using the decoction method, each time for 2 hours. After the extract is concentrated under reduced pressure, the total water extract is obtained. Weight volume refers to the solid being expressed in kg and the liquid in L (the same applies below). S2. The total extract was dispersed in water and then passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with 50% ethanol by volume to obtain the 50% ethanol fraction. S3 and the 70% ethanol fraction were dissolved in methanol, and then silica gel column chromatography was performed with silica gel mixed in a volume ratio of 1:1.2. The fractions were then eluted with a gradient of dichloromethane:methanol in volume ratios of 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1. The fractions were identified by TLC, and fractions with the same color development results were combined to obtain components Fr.1~Fr.4, Fr.5, and Fr.6. S4. Component Fr.4 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol (v / v), and purified by semi-preparative HPLC. The mobile phase was 25% acetonitrile, and the flow rate was 3 mL / min. The fraction with a retention time of 50-52 was collected, dried, and the resin was obtained.

[0009] The pinoresinin prepared by this invention has the effect of inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), enabling its application in the preparation of drugs for treating pulmonary arterial hypertension.

[0010] The preparation method of this invention is simple and easy to industrialize for the preparation of pinoresinin, thus expanding the medicinal value of ephedra and enabling its application in the preparation of drugs for the treatment of pulmonary hypertension. This is a major innovation in the treatment of pulmonary hypertension and has practical application value. Attached Figure Description

[0011] Figure 1 The molecular formula structure of the pinoresinin of this invention is shown in the diagram. Figure 2 The pinoresinin of the present invention 1 H-NMR spectrum (in 500 MHz, CD3OD); Figure 3 The pinoresinin of the present invention 13 C-NMR spectrum (in 125 MHz, CD3OD); Figure 4 The figure shows the effect of pinoresinin of the present invention on lung injury in mice with hypoxia-induced pulmonary hypertension. In the figure, (A) the effect of pin on pathological lung injury in hypoxia-induced PH mice (×400) (`x±s, n=6), (B) the effect of pin on pulmonary artery pressure in hypoxia-induced PH mice (`x±s, n=3), and (C) the effect of pin on lung function in hypoxia-induced PH mice (`x±s, n=3). Figure 5 The figure shows the effect of pinoresinin of the present invention on myocardial tissue damage in mice with hypoxia-induced pulmonary hypertension. (A) Effect of pin on right ventricular pathological damage in hypoxia-induced PH mice (×400) (`x±s, n=6), (B) Effect of pin on right ventricular function in hypoxia-induced PH mice (`x±s, n=3). Figure 6The figure shows the effect of pinoresinin of the present invention on the ROS and apoptosis levels of myocardial tissue in mice with hypoxia-induced pulmonary hypertension. (A) Effect of pin on the ROS level of myocardial tissue in hypoxia-induced PH mice (`x±s, n=3), and (B) Effect of pin on the apoptosis level of myocardial tissue in hypoxia-induced PH mice (`x±s, n=3). Figure 7 The figure shows the effect of pinoresinin of the present invention on mitochondrial function in hypoxia-induced pulmonary hypertension mice. In this figure, (A) Pin affects the calcium content of lung tissue in hypoxia-induced PH mice. 2+ (A) Effect of Pin on the level of mtROS in lung tissue of hypoxia-induced PH mice (x±s, n=3), (B) Effect of Pin on the level of mtROS in lung tissue of hypoxia-induced PH mice (x±s, n=3), (C) Effect of Pin on the level of MMP in lung tissue of hypoxia-induced PH mice (x±s, n=3). Figure 8 The effect of the pinoresinin of the present invention on ATP content in mice with hypoxia-induced pulmonary hypertension is shown in the figure (`x±s, n=3); Figure 9 The figure shows the effect of the pinoresinin of the present invention on mitochondrial autophagy in hypoxia-induced pulmonary hypertension mice. In the figure, (A) transmission electron microscopy (20000×) shows mitochondrial autophagy in lung tissue, (B) immunofluorescence detection shows the level of mitochondrial autophagy-related proteins in lung tissue (x±s, n=3), and (C) Western blotting shows the level of mitochondrial autophagy-related proteins in lung tissue (x±s, n=3). Yellow arrows indicate mitochondrial swelling, cristae disorder, and disappearance. Red arrows indicate mitochondrial autophagy. Figure 10 This is a graph showing the effect of the pinoresinin of the present invention on the serum metabolic profile of pulmonary hypertension mice, wherein (A) is the PCA score (ESI) graph of the NC group, M group, and Pin group. + , R 2 X =0.459, Q 2 =0.272; ESI - , R 2 X =0.494, Q 2 =0.341), (B) OPLS-DA, S-plot, and permutation test plots (ESI) of NC and M groups. + , R 2 X =0.714, R 2 Y =0.997, Q 2 =0.985; ESI- , R 2 X =0.574, R 2 Y =0.981, Q 2 =0.916), (C) OPLS-DA, S-plot, and permutation test plots (ESI) of M group and Pin group. + , R 2 X =0.596, R 2 Y =0.998, Q 2 =0.913; ESI - , R 2 X =0.568, R 2 Y =0.997, Q 2 =0.900), (D) Cluster heatmap analysis, metabolic pathway analysis and key differential metabolite network analysis diagram; Figure 11 The diagram shows the effect of the pinoresinin of the present invention on gene expression in mice with pulmonary hypertension, wherein (A) is a volcano plot of differentially expressed genes, (B) is a heatmap of differentially expressed gene clusters, and (C) is a GO and KEGG enrichment analysis of differentially expressed genes. Figure 12 The figure shows the effect of the pinoresinin of the present invention on cholesterol metabolism in mice with hypoxia-induced pulmonary hypertension. In the figure, (A) the expression of cholesterol metabolism-related proteins was detected by immunohistochemistry (`x±s, n=3), and (B) the expression of cholesterol metabolism-related proteins was detected by Western blotting (`x±s, n=3). Note: Compared with the NC group, ** P <0.01; compared with group M, # P <0.05, ## P <0.01. Detailed Implementation

[0012] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0013] The present invention is illustrated by the following embodiments: The technical solution provided by this invention is: a method for extracting pinoresinin from Ephedra sinica, wherein the molecular structural formula of pinoresinin is: ; Its preparation method is as follows: S1. Take 40 kg of dried roots and rhizomes of Ephedra sinica and extract them twice with 600 L of water for 2 hours each time by decoction. After the extract is concentrated under reduced pressure, the total water extract (4.07 kg) is obtained. S2. The total extract was dispersed in water and then eluted with 50% ethanol by volume through a Diaion HP-20 macroporous adsorption resin column to obtain the 50% ethanol fraction (97.80 g). S3, the 50% ethanol fraction (97.80 g) was dissolved in methanol, and then silica gel column chromatography was performed with silica gel mixing at a volume ratio of 1:1.2. Elution was then performed using a gradient of dichloromethane:methanol at volume ratios of 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1. TLC analysis was performed, and fractions with the same colorimetric results were combined to obtain components Fr.1~Fr.5 and Fr.6. S4. Component Fr.4 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol (v / v), and purified by semi-preparative HPLC. The mobile phase was 25% acetonitrile, and the flow rate was 3 mL / min. The fraction with a retention time of 50-52 was collected, dried, and the resinin (16.30 mg) was obtained.

[0014] The pinoresinin prepared by this invention has the effect of inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), enabling its application in the preparation of drugs for treating pulmonary arterial hypertension.

[0015] According to the method given in the above embodiments, any amount of pinoresinin can be prepared as needed. The given embodiments are only used to illustrate specific implementation methods of the present invention, and are not intended to limit the scope of protection of the present invention. The core technology protected by the present invention is the preparation method of pinoresinin and its application.

[0016] The preparation method of this invention is simple and easy to operate, and can effectively extract pinoresinin from Ephedra sinica. Pinoresinin has an anti-proliferation effect on pulmonary artery smooth muscle cells (PASMCs), enabling its application in the preparation of drugs for treating pulmonary hypertension. Experiments have shown very good and beneficial technical results, as detailed below: I. Physical Morphology and Structural Identification The compound prepared by the method of this invention is a colorless oil. The structure of the compound was determined using 1H and 1C spectrophotometer spectroscopy, as shown in the following figures. Figure 2 , 3 As shown, ESI-MS m / z 359 [M+H]+ .exist 1 H-NMR (500 MHz, DMSO- d 6) In the spectrum, the aromatic region δ H 6.89 (2H, d, J = 1.8 Hz, H-2, 2'), 6.73 (2H, d, J = 8.1 Hz, H-5,5'), 6.75 (2H, dd, J = 8.1, 1.8 Hz, H-6, 6') represents the hydrogen proton signal on the benzene ring of the ABX coupling system. δ H 4.60 (2H, d, J = 4.0 Hz, H-7, 7') is the hydrogen proton signal on the hydroxymethyl group. δ H 3.03 (2H, m, H-8, 8') represents the hydrogen proton signal of the methine group. δ H 4.12 (2H, m, H-9a, 9'a) and 3.72 (2H, m, H-9b, 9'b) are hydrogen proton signals on the oxymethylene group, suggesting that the compound is a completely symmetrical bicyclic oxylignan, and the above signals are hydrogen signals on the bis-tetrahydrofuran ring. δ H 3.76 (6H, s, 3, 3'-OCH3) represents the hydrogen proton signal of the methoxy group. 13 C-NMR (125 MHz, DMSO- d 6) In the spectrum, except δ C Apart from the methoxy carbon signal at 55.6 (3, 3'-OCH3), the remaining signals are carbon signals from C6-C3 structural units. δ C 132.2 (C-1, 1'), 110.4 (C-2, 2'), 146.0 (C-3, 3'), 147.6 (C-4, 4'), 115.2 (C-5, 5'), and 118.7 (C-6, 6') are carbon signals on the benzene ring. δ C85.2 (C-7, 7'), 53.6 (C-8, 8'), and 70.9 (C-9, 9') are characteristic carbon signals at positions 7, 7', 8, 8', 9, and 9' on the bis-tetrahydrofuran ring of diepoxylignans. Based on the above NMR data analysis combined with the study of the chemical constituents of the whole herb *Serissa japonica* [J]. Chinese Medicinal Herbs, 2016, 39(01):94-7, the compound can be identified as pinoresinin, and the carbon and hydrogen data are shown in Table 1.

[0017]

[0018] The structural formula of the compound is: .

[0019] II. Activity Test 2.1 Establishment, grouping, and administration of animal models Male C57BL / 6N mice were acclimatized for one week and then randomly divided into four groups: normal control group (NC), model group (M), positive control drug sildenafil group (Y), total extract of ephedra root group (MHG), low-dose Pin group (Pin-L), and high-dose Pin group (Pin-H). The mice were placed in a hypoxic chamber for 12 hours daily for 35 consecutive days, and drug administration began by gavage on days 28-35. The dosages for each group were: Y group (20 mg / kg), MHG group (3 g / kg), Pin-L group (10 mg / kg), and Pin-H group (20 mg / kg). The NC and M groups received an equal volume of distilled water by gavage.

[0020] 2.2 Detection of pulmonary artery pressure and right ventricular function Pulmonary artery pressure (PAT) and right ventricular function parameters in mice were measured using a small animal ultrasound diagnostic instrument. Mice were anesthetized with isoflurane. A high-frequency matrix probe with a frequency of 400 Hz was used to locate the parasternal long-axis right ventricular and pulmonary artery sections. After adjusting the B-mode ultrasound image to a suitable interface, the values ​​were measured on the mid-section of the echocardiogram. The data were collected and the results were analyzed to obtain PAT / PET, right ventricular free wall thickness, and right ventricular diameter.

[0021] 2.3 HE and Masson staining After modeling and drug administration, the heart and lung tissues of mice were dissected, fixed in 4% formaldehyde, embedded in paraffin, sectioned, and stained with HE and Masson staining. The pathological damage of the heart and lung tissues was observed under a microscope.

[0022] 2.4. Mouse lung function test The changes in lung function indicators of mice within 5 minutes were recorded using a small animal pulmonary function testing (WBP) system to obtain the respiratory rate (F), tidal volume (TVb), and minute ventilation (MVb).

[0023] 2.5. Detection of calcium in mouse lung tissue 2+ Mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP) levels After modeling and drug administration, fresh lung tissue from mice was collected, added to PBS, minced, centrifuged, and the supernatant was discarded. Trypsin without EDTA was added for 5 min, followed by centrifugation and discarding of the supernatant. Red blood cell lysis buffer was added for 5 min, followed by centrifugation and discarding of the supernatant. Cells were resuspended in PBS buffer and filtered through a 0.75 µm filter. The Ca2+ levels in the tissue cells were detected using a Fluo-3AM fluorescent probe. 2+ The levels of mtROS in tissue cells were detected using the MitoSOX Red fluorescent probe, and the levels of MMP in tissue cells were measured using the JC-1 fluorescent probe.

[0024] 2.6. Detection of reactive oxygen species (ROS) and apoptosis levels in mouse myocardial tissue Fresh mouse myocardial tissue was collected, minced, and centrifuged in pre-cooled PBS buffer, with the supernatant discarded. EDTA-free trypsin was added for 5 min, followed by centrifugation and supernatant discarding. Red blood cell lysis buffer was added for 5 min, followed by centrifugation and supernatant discarding. Cells were resuspended in PBS buffer and filtered through a 0.75 µm filter. ROS levels in the tissue cells were detected using the DCFH-DA fluorescent probe, and apoptosis levels were detected using a PE-conjugated Annexin-V apoptosis assay kit.

[0025] 2.7 Determination of ATP content ATP extraction from serum: The serum volume to extraction liquid volume was 1:5-10, thoroughly shaken, and centrifuged at 10000g, 4℃ for 10 min. The supernatant was transferred to another EP tube, 500 μL of chloroform was added, and the mixture was thoroughly shaken and centrifuged at 10000g, 4℃ for 3 min. The supernatant was collected and placed on ice for analysis. The ATP content was determined according to the ATP assay kit instructions.

[0026] 2.8 Transmission Electron Microscopy Observe the lung tissue structure of mice in each group, and take 1 mm of lung tissue from each mouse. 3 The samples were fixed in 2.5% glutaraldehyde solution, dehydrated, embedded, polymerized into resin blocks, and then sectioned and stained to make copper mesh sections, which were then observed and photographed under a transmission electron microscope.

[0027] 2.9 Immunofluorescence staining detection Immunofluorescence staining was used to detect the levels of VDAC1, HSP60, and LAMP1 in lung tissues of each group. Lung tissue sections were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 30 min. They were blocked with BSA blocking solution for 30 min, the blocking solution was discarded, and then incubated overnight with primary antibody (1:500). The next day, after washing, they were incubated with fluorescent secondary antibody (1:500) for 2 h. Then, after washing, the cell nuclei were stained with 1 μg / mL DAPI. After washing, the slides were sealed and images were taken under a fluorescence microscope.

[0028] 2.10 Metabolomics Analysis 60 μL of serum was collected, 5 times the volume of acetonitrile was added, the mixture was vortexed for 1 min, sonicated for 10 min, and incubated at -20 ℃ for 4 h. After centrifugation at 12000 rpm for 15 min, the supernatant was collected, 2 times the volume of acetonitrile was added, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was then transferred to a vial for later use. Chromatographic separation was performed using an Acclaim™ RSLC 120 C18 column (2.2 μm, 2.1 mm × 100 mm). The mobile phase consisted of solvent A (0.1% formic acid aqueous solution) and solvent B (acetonitrile). The elution program was gradient elution (0–6 min, 10–70% B; 6–14 min, 70–80% B; 14–16 min, 80–90% B); the column oven was set to 40 ℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. Mass spectrometry analysis was performed using quadrupole time-of-flight mass spectrometry, scanning in both positive and negative ion modes. Mass scan range: m / z 50~1500, scan rate: 1.0 Hz, nebulizer gas pressure: 2.0 bar, desolvation temperature: 230℃, dry gas (N2) flow rate: 8 L / min, capillary voltage: 3500 V and 3200 V (positive and negative ion modes), ion source energy: 3.0 eV, real-time calibration solution (sodium formate) flow rate: 50 μL / h. The raw data acquired by UPLC-Q / TOF-MS were imported into Profile Analysis software for preprocessing such as peak matching, noise reduction, normalization, and correction of missing data. Then, the data were imported into SIMCA-P14.1 software for unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA) to obtain the corresponding score plots. Differences between the model group and the drug-treated group were compared using the score plots to screen for differentially expressed metabolites (Variable importance in the projection, VIP) > 3. P<0.05) Potential biomarkers were identified and screened using the mass spectrometry software DataAnalysis and online databases including HMDB (http: / / hmdb.ca), KEGG (http: / / www.genome.jp / kegg / ), MassBank (http: / / massbank.eu), and MetaboAnalyst (http: / / www.metaboanalyst.ca). Semi-quantitative analysis of potential biomarkers was performed using Compass Quant Analysis software (version 4.8.0), and heatmap clustering analysis was conducted using Mev software (version 4.9.0). Finally, metabolic pathway enrichment and the construction of metabolic pathway networks were performed using MetaboAnalyst and the KEGG database.

[0029] 2.11 Transcriptomics Analysis Lung tissues were collected from mice in each group. The RNA from the lung tissues was isolated and purified by Zhejiang Hangzhou Lianchuan Biotechnology Co., Ltd. The quantity and purity of total RNA were quality controlled, and RNA integrity was tested. The purified mRNA was fragmented and reverse transcribed to construct the final cDNA library, which was then sequenced. The final clean data were analyzed using Omic Studio (https: / / www.omicstudio.cn / index) and the Cluster Profiler R package to generate volcano plots, heatmaps, and KEGG analyses of differentially expressed genes. The screening criteria for differentially expressed genes were |log2FC|≥1 and... P <0.05. Functional enrichment analysis, including GO functional annotation and KEGG pathway enrichment analysis of differentially expressed genes.

[0030] 2.12 Western Blot (WB) of Proteins Mouse lung tissue was collected, and total protein was extracted by adding RIPA lysis buffer containing PMSF and PPI according to the protein extraction kit instructions. Protein concentration was determined using the BCA method, and the loading amount was determined based on the protein concentration. Proteins were separated by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% BSA for 1 h, the membrane was incubated with primary antibody overnight at 4 °C, washed five times with 0.2% PBST for 5 min each time, and then incubated with secondary antibody for 1 h in the dark. The membrane was then washed three times with 0.2% PBST and once with PBS for 5 min each time. Using β-actin as an internal control, the target protein was semi-quantitatively analyzed using an Odyssey dual-color imaging system combined with Image Studio software.

[0031] 2.13 Immunohistochemical detection Immunofluorescence staining was used to detect the levels of ABCA1 in lung tissues of each group. Lung tissue sections were fixed with 4% paraformaldehyde, embedded in paraffin, and then dewaxed. The tissue sections were boiled in citrate buffer for 10 min and rinsed with distilled water. 3% hydrogen peroxide solution was added and incubated for 10 min, followed by washing with water again. Primary and secondary antibodies were added, followed by DAB staining, hematoxylin re-staining, dehydration and clearing, mounting, microscopic examination, and imaging under a fluorescence microscope.

[0032] 2.14 Statistical Analysis Data statistical analysis was performed using SPSS 20.0 software. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare differences between groups. P <0.05 indicates a significant difference. P A value <0.01 is considered highly significant.

[0033] III. Experimental Results 3.1 Effects of Pin on Lung Injury in Hypoxia-Induced Pulmonary Hypertension Mice HE staining of lung tissue showed that in group M, the pulmonary vessel walls were thickened, the lumen was narrowed, and inflammatory cells were accumulated around the vessels; after drug administration, these conditions were alleviated to varying degrees. Masson staining of lung tissue showed that there was a large amount of collagen fiber deposition around the pulmonary vessels in group M; after drug administration, collagen fiber deposition decreased. This suggests that Pin can improve the pathological damage of lung tissue in PH mice to varying degrees, such as… Figure 4 A. Compared with the NC group, the PAT / PET ratio of mice in the M group was lower. Figure 4 B) Tidal volume (TVb), lung ventilation (MVb) Figure 4 C) Significantly reduced ( P <0.01), respiratory rate (F) ( Figure 4 C) significantly increased ( P <0.01); After administration, the above indicators improved ( P <0.05 or P <0.01). PAT / PET is an important indicator of pulmonary artery pressure; the lower the PAT / PET ratio, the higher the pulmonary artery pressure. This indicates that Pin can reduce pulmonary artery pressure, improve lung function, and alleviate lung tissue damage in mice.

[0034] 3.2 Effects of Pin on Myocardial Tissue Injury in Mice with Hypoxia-Induced Pulmonary Hypertension HE staining of myocardial tissue showed that the myocardial tissue in group M was disordered and disorganized, with breaks and extensive inflammatory cell infiltration; after drug administration, the myocardial tissue became more orderly. Masson staining of myocardial tissue showed that there was extensive collagen fiber deposition in the myocardial tissue of group M; after drug administration, collagen fiber deposition decreased. This suggests that Pin can alleviate pathological damage to myocardial tissue in mice with pulmonary hypertension, such as... Figure 5 A. M mice showed significantly increased RVHI, RVFW, and RVID ( P <0.01); After administration, RVHI, RVFW, and RVID in mice were significantly reduced ( P <0.05 or P <0.01), such as Figure 5 B. This suggests that elevated pulmonary artery pressure leads to myocardial tissue damage in mice, resulting in right ventricular hypertrophy and right ventricular dysfunction. Pin, however, can improve myocardial damage, right ventricular hypertrophy, and right ventricular function in mice with pulmonary hypertension.

[0035] 3.3 Effects of Pin on ROS and Apoptosis Levels in Myocardial Tissue of Hypoxia-Induced Pulmonary Hypertension Mice Flow cytometry results showed that, compared with the NC group, the ROS (reactive oxygen species) in the myocardial tissue of the M group mice was significantly lower. Figure 6 A) and apoptosis Figure 6 B) Level significantly increased ( P <0.01); After administration, ROS and apoptosis levels were significantly reduced ( P <0.01). This suggests that myocardial tissue cell damage exists in PH mice, and Pin can improve myocardial tissue cell damage.

[0036] 2.4. The effect of Pin on Ca in lung tissue of mice with hypoxia-induced pulmonary hypertension 2+ The influence of mtROS and MMP levels Flow cytometry results showed that, compared with the NC group, the lung tissue calcium levels in the M group mice were significantly lower. 2+ ( Figure 7 A) and mtROS ( Figure 7 B) Level significantly increased ( P <0.01), MMP( Figure 7 C) levels significantly decreased ( P <0.01); after administration, all of the above indicators were reversed. P <0.05 or P <0.01). This suggests that PH mice have mitochondrial dysfunction, and that Pin can improve mitochondrial function in PH mice.

[0037] 2.5 Effect on ATP content in mice with hypoxia-induced pulmonary hypertension The experimental results showed that, compared with the NC group, the ATP content of mice in the M group was significantly reduced.P <0.01); After administration, ATP levels increased significantly, such as Figure 8 As shown ( P <0.05 or P <0.01). This suggests that PH mice have mitochondrial dysfunction, and that Pin can improve mitochondrial function in PH mice.

[0038] 2.6 Effects of Pin on Mitochondrial Autophagy in Hypoxia-Induced Pulmonary Hypertension Mice Transmission electron microscopy examination of pulmonary artery mitochondrial autophagy revealed that, for example Figure 9 Compared with the NC group, mice in the M group exhibited mitochondrial swelling, cristae disorganization, and mitophagy. Pin intervention improved these symptoms. Immunofluorescence assays of VDAC1, HSP60, and LAMP1 expression levels showed that, compared with the NC group, the M group mice exhibited significantly elevated levels of these three expression levels. P <0.01), and achieved high co-localization. After Pin intervention, the expression levels of the three proteins were significantly reduced ( P <0.05 or P <0.01). Western blotting was used to detect the expression levels of mitophagy-related proteins. The results showed that, compared with the NC group, the expression levels of Parkin, PINK1, Beclin1, and LC3Ⅱ / Ⅰ were significantly increased in the M group mice. P <0.01), the expression level of P62 was significantly reduced ( P <0.01), Pin intervention can reverse protein expression ( P <0.05 or P <0.01), the above results indicate that Pin intervention can improve mitochondrial autophagy in PH mice.

[0039] 2.7 Effects of Pin on Intestinal Tissue Injury and Intestinal Function in Hypoxia-Induced Pulmonary Hypertension Mice To investigate the metabolic pathways significantly affected by Pin, serum metabolomics studies were conducted in PH mice. In the PCA scoring diagram, the NC and M groups were separated, with the drug-treated groups moving closer to the NC group. Figure 10 A. OPLS-DA analysis see Figure 10 B and 10C were used to screen for 24 shared biomarkers, with 16 positive and 8 negative patterns. Cluster heatmap analysis and metabolic pathway analysis of these differentially expressed metabolites are as follows: Figure 10 As shown in D, the metabolic pathways they significantly affect mainly include primary bile acid biosynthesis, taurine and hypoturine metabolism. This suggests that Pin may improve pulmonary hypertension in mice by influencing cholesterol metabolism.

[0040] 2.8 Transcriptomic analysis of hypoxia-induced pulmonary hypertension mice Transcriptomic results showed that compared with the NC group, the M group had 223 significantly different genes, 35 significantly upregulated genes, and 188 significantly downregulated genes. Compared with the M group, the Pin group had 905 significantly different genes, 178 significantly upregulated genes, and 727 significantly downregulated genes. Figure 11 A indicates that drug administration significantly altered the gene expression profile in the lung tissue of PH mice, inhibiting the expression of most genes. The differentially expressed gene clustering heatmap showed obvious clustering between the NC and Pin groups, such as... Figure 11 B indicates that Pin has a callback effect on most genes. For example... Figure 11 Figure C shows the GO functional annotation and KEGG enrichment analysis results of differentially expressed genes. Biological processes include phospholipid efflux, cellular components include high-density lipoprotein particles and very low-density lipoprotein particles, and molecular functions include lipase inhibitory activity, phospholipid binding, lipid binding, apolipoprotein receptor binding, and phosphatidylcholinesterol O-acyltransferase activity. The KEGG pathway is enriched in pathways such as cholesterol metabolism.

[0041] 2.9 Effects of Pin on Cholesterol Metabolism in Hypoxia-Induced Pulmonary Hypertension Mice Immunohistochemical and Western blot analysis of cholesterol metabolism pathway-related proteins revealed that the expression levels of cholesterol metabolism-related proteins ABCA1, APOA1, and SOAT2 were significantly decreased in group M. P <0.05 or P <0.01), after Pin intervention, the protein expression level increased significantly ( P <0.05 or P <0.01). This indicates that Pin can improve pulmonary hypertension by improving abnormal cholesterol metabolism, see [reference needed]. Figure 12 .

[0042] IV. Conclusion Pinoresinin has a good anti-tumor effect on human cells derived from breast cancer. It can inhibit the proliferation of mouse myoblasts through the Akt / mTOR signaling pathway. In addition, it can also inhibit the growth of ovarian cancer cells in vitro and in vivo by inducing autophagy, inhibiting cell invasion, mitochondrial membrane potential loss, and inhibiting the Ras / MEK / ERK signaling pathway.

[0043] Pulmonary hypertension (PH) is a malignant progressive disease with insidious onset and high mortality. Its pathogenesis involves multiple factors, including cell proliferation, immunity, and inflammation. Pulmonary artery constriction and pulmonary vascular remodeling are the main pathophysiological basis of PH. Pulmonary vessels are composed of cells such as pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). Under the influence of intrinsic factors such as immunity and inflammation, or extrinsic factors such as hypoxia and drugs, PASMCs undergo abnormal proliferation and participate in pulmonary vascular remodeling and vasoconstriction. The abnormal proliferation of PASMCs is very similar to the malignant proliferation of tumor cells, but the role of pinoresinin in PH is still unclear. Therefore, an experiment was designed to investigate this.

[0044] A mouse model of pulmonary hypertension (PH) was established using hypoxia-induced induction. Results showed that Pin significantly reduced pulmonary artery pressure, improved lung tissue damage in PH mice, enhanced lung function, and reduced pulmonary vascular thickening. Furthermore, Pin improved myocardial tissue damage in PH mice, reduced right ventricular load, decreased collagen fiber deposition, and improved right ventricular remodeling; Pin's effects were superior to those of total extract from Ephedra root. Hypoxia often leads to mitochondrial damage and dysfunction. Mitophagy, the selective clearance of damaged mitochondria by cells, plays a crucial role in the abnormal proliferation of PASMCs.

[0045] This experiment detected MMP and Ca. 2+The levels of mtROS and ATP were measured, indicating that PH mice exhibited mitochondrial dysfunction, and Pin could improve mitochondrial functional damage caused by hypoxia. Electron microscopy revealed increased mitophagy in PASMCs of PH mouse lung tissue, which significantly decreased after Pin intervention. Immunofluorescence analysis of the co-localization of mitochondrial membrane protein (VDAC1), mitochondrial matrix protein (HSP60), and lysosome-associated membrane protein 1 (LAMP1) showed high co-localization of these three proteins in PH mice, which was significantly improved after Pin intervention. PTEN-induced kinase 1 (PINK1) is a key component of mitophagy. When mitochondrial membrane potential is impaired, the pathway for PINK1 to enter the inner mitochondrial membrane is blocked, leading to stable aggregation of PINK1 on the cytoplasmic surface of the outer mitochondrial membrane. Simultaneously, this recruits and activates E3 ubiquitin ligase (Parkin), whose spatial conformation changes to become activated E3 ubiquitin ligase, which then ubiquitinates proteins on the mitochondria. PINK1 and Parkin interact to jointly regulate mitophagy to maintain mitochondrial quality. Microtubule-associated protein light chain 3 (LC3) is an important protein involved in autophagy, existing in two forms: LC3Ⅰ and LC3Ⅱ. The LC3Ⅱ / Ⅰ ratio reflects the level of autophagy. During autophagy, LC3Ⅰ binds to phosphatidylethanolamine to form LC3Ⅱ. Selective autophagy receptor chelate 1 (p62) specifically binds to LC3Ⅱ, recruiting ubiquitinated protein aggregates or other cellular components to autophagosomes, where they are then degraded within autolysosomes. Autophagy effector protein (Beclin-1) is an essential molecule in autophagosome formation, mediating the localization of other autophagy proteins to phagosomes, thereby regulating autophagosome formation and maturation. Western blotting analysis of autophagy-related protein expression showed that Pin can improve mitophagy.

[0046] To further explore the mechanism by which Pin improves pulmonary hypertension, metabolomics and transcriptomics techniques were employed. Metabolomics analysis of serum from PH mice revealed significant changes in the metabolic profile. Pin intervention regulated differentially expressed metabolites in PH mice, significantly affecting pathways including primary bile acid biosynthesis, taurine and hypotaurine metabolism. Cholesterol can be converted into bile acids, which can then bind with taurine to form conjugated bile acids; therefore, these pathways are closely related to cholesterol metabolism. Transcriptomics analysis showed that Pin altered the gene expression profile in the lung tissue of PH mice, exhibiting a reversal effect on the expression of most genes. GO and KEGG enrichment analyses of differentially expressed genes revealed biological processes including phospholipid efflux, cellular components including high-density lipoprotein particles and very low-density lipoprotein particles, and molecular functions including lipase inhibition activity, phospholipid binding, lipid binding, apolipoprotein receptor binding, and phosphatidylcholinesterol O-acyltransferase activity. KEGG pathway enrichment included cholesterol metabolism and other pathways. Similar to the results of metabolomics, pinoresinin can improve pulmonary hypertension in mice by affecting cholesterol metabolism. Therefore, differentially expressed genes in the cholesterol metabolism pathway were detected using immunohistochemistry and Western blotting to examine the expression of cholesterol reverse transporter ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (APOA1), and cholesterol acyltransferase 2 (SOAT2), an enzyme regulating cholesterol esterification. The results showed that pin significantly increased the expression of ABCA1 and APOA1 proteins and significantly decreased the expression of SOAT2 protein.

[0047] In summary, the preparation method of this invention is simple and easy to operate, and can effectively extract the compound Pin from Ephedra sinica. This compound can improve pulmonary hypertension in mice by affecting cholesterol metabolism, and can improve mitochondrial dysfunction and reduce mitophagy by regulating abnormal cholesterol metabolism, thereby exerting a therapeutic effect on pulmonary hypertension. It can be used in the preparation of drugs for the treatment of pulmonary hypertension and has practical clinical application value.

Claims

1. A method for extracting pinoresinin from Ephedra sinica, characterized in that, The molecular structure of the pinoresinin is as follows: ； Its preparation method is as follows: S1. Take dried roots and rhizomes of Ephedra sinica and extract them twice with 15 times their weight volume of water using the decoction method, each time for 2 hours. The extract is concentrated under reduced pressure to obtain an aqueous extract. Weight volume refers to kg for solids and L for liquids. S2. The extract was dispersed in water and then passed through a Diaion HP-20 macroporous adsorption resin column, and eluted with 50% ethanol by volume to obtain the 50% ethanol fraction. S3 and the 70% ethanol fraction were dissolved in methanol, and then silica gel column chromatography was performed with silica gel mixed in a volume ratio of 1:1.

2. The fractions were then eluted with a gradient of dichloromethane:methanol in volume ratios of 50:1, 30:1, 20:1, 10:1, 5:1, and 1:

1. The fractions were identified by TLC, and fractions with the same color development results were combined to obtain components Fr.1~Fr.4, Fr.5, and Fr.

6. S4. Component Fr.4 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol (v / v), and purified by semi-preparative HPLC. The mobile phase was 25% acetonitrile, and the flow rate was 3 mL / min. The fraction with a retention time of 50-52 was collected, dried, and the resin was obtained.

2. The method for extracting pinoresinin from Ephedra sinica according to claim 1, characterized in that, The preparation method is as follows: S1. Take 40 kg of dried roots and rhizomes of Ephedra sinica and extract them twice with 600 L of water for 2 h each time by decoction. After concentration under reduced pressure, 4.07 kg of water extract was obtained. S2. The extract was dispersed in water and then passed through a Diaion HP-20 macroporous adsorption resin column. It was eluted with 50% ethanol by volume to obtain 97.80g of the 50% ethanol fraction. S3, 97.80 g of the 50% ethanol fraction was dissolved in methanol, and then silica gel column chromatography was performed with silica gel mixing at a volume ratio of 1:1.

2. Elution was then performed using a gradient of dichloromethane:methanol at volume ratios of 50:1, 30:1, 20:1, 10:1, 5:1, and 1:

1. TLC analysis was performed, and fractions with the same colorimetric results were combined to obtain components Fr.1~Fr.5 and Fr.

6. S4. Component Fr.4 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol (v / v), and purified by semi-preparative HPLC. The mobile phase was 25% acetonitrile, and the flow rate was 3 mL / min. The fraction with a retention time of 50-52 was collected, dried, and 16.30 mg of pinoresinin was obtained.

3. The use of the pinoresinin prepared by any one of claims 1-2 in the preparation of drugs for treating pulmonary hypertension.

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