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

Abstract

The invention relates to a method for extracting ephedrine E from herba ephedrae, which can effectively extract ephedrine E from herba ephedrae and realize application in preparation of medicines for treating pulmonary arterial hypertension. The preparation method comprises the following steps: taking dried roots and rhizomes of ephedra sinica stapf, extracting by adopting a water decoction method, concentrating under reduced pressure to obtain total extract, dispersing with water, eluting, dissolving with methanol, stirring with silica gel, carrying out gradient elution, identifying, merging the same fractions, loading a target component on column chromatography, eluting, purifying, collecting the fractions within retention time, and drying to obtain ephedrine E; the preparation method is simple, the ephedrine E is easy to industrially prepare, the medicinal value of ephedra is expanded, the application in preparation of the medicine for treating the pulmonary arterial hypertension is realized, and the ephedrine E is a great innovation in preparation of the medicine for treating the pulmonary arterial hypertension and has practical popularization and application value.

Description

Technical Field

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

[0002] Pulmonary hypertension (PH) is a clinical and pathophysiological syndrome caused by peripulmonary inflammation and pulmonary artery remodeling due to various etiologies and different pathogenesis mechanisms, leading to increased pulmonary vascular resistance and pulmonary artery pressure. It can progress to right heart failure and even death, and is characterized by high morbidity and mortality. Generally, pulmonary hypertension can be diagnosed when pulmonary artery systolic pressure (RVSP) >30 mmHg or hemodynamic monitoring shows mean pulmonary artery pressure (mPAP) ≥25 mmHg. PH does not have specific clinical symptoms, but it typically presents with exertional dyspnea, fatigue, chest pain, syncope, and symptoms and signs of progressive right heart failure.

[0003] The pathogenesis of pulmonary vasculitis (PH) is complex, involving multiple biological pathways and molecular mechanisms. Metabolism, as a fundamental characteristic of life, is closely related to pathophysiological changes in pulmonary vessels, and is crucial for maintaining vascular structure and function. Metabolic dysregulation is a key factor in the pathogenesis of PH; PH patients often exhibit energy metabolism disorders, lipid metabolism abnormalities, and significant alterations in amino acid metabolism. These metabolic changes are closely related to pathological processes such as pulmonary vascular remodeling, inflammatory responses, and oxidative stress. Inflammation is a key factor in the occurrence and development of PH. The gut microbiota can regulate the host's immune environment, and changes in gut microbiota composition and activation of inflammation participate in the progression of PH.

[0004] 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*. Related studies have shown that the chemical components of *Ephedra sinica* roots are mainly alkaloids, flavonoids, phenolic acids, and polysaccharides. Ephedrain E (MaE) is a flavonoid compound isolated from *Ephedra sinica* roots. Experiments have shown that MaE has a good effect on the abnormal proliferation of PASMCs, but its mechanism of action on pH is still unclear. Therefore, how to extract ephedrain E 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

[0005] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for extracting ephedrine E from ephedra sinica, which can effectively extract ephedrine E from ephedra sinica and realize its application in the preparation of drugs for treating pulmonary arterial hypertension.

[0006] The technical solution provided by this invention is: a method for extracting ephedrine E from ephedra sinica, wherein the molecular structural formula of ephedrine E is: ; 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 eluted with 70% ethanol by volume through a Diaion HP-20 macroporous adsorption resin column to obtain the 70% 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.5 and Fr.6. S4. Component Fr.5 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol, and purified by semi-preparative HPLC. The flow rate of the mobile phase was 3 mL / min, and the fraction with a retention time of 30-31 was collected, dried, and ephedrine E (MaE) was obtained. The mobile phase was a solution prepared by adding 0.3% trifluoroacetic acid to 54% methanol by volume.

[0007] The ephedrine E prepared by this invention has the effect of inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), thus enabling its application in the preparation of drugs for treating pulmonary arterial hypertension.

[0008] The preparation method of this invention is simple and easy to prepare Ephedra E industrially, which expands the medicinal value of Ephedra sinica and realizes its application in the preparation of drugs for treating pulmonary hypertension. It is a major innovation in the treatment of pulmonary hypertension and has practical application value. Attached Figure Description

[0009] Figure 1 This is a molecular formula diagram of Ephedrine E of the present invention; Figure 2 The ephedrine E of the present invention 1 H-NMR spectrum (in 500 MHz, CD3OD); Figure 3 The ephedrine E of the present invention 13 C-NMR spectrum (in 125 MHz, CD3OD); Figure 4The figure shows the effect of Ephedrine E (MaE) on lung injury in mice with hypoxia-induced pulmonary hypertension. In the figure, (A) the effect of MaE on pathological lung damage in hypoxia-induced PH mice (×400) (`x±s, n=6), (B) the effect of MaE on pulmonary artery pressure in hypoxia-induced PH mice (`x±s, n=3), and (C) the effect of MaE on lung function in hypoxia-induced PH mice (`x±s, n=3). Figure 5 The figure shows the effect of Ephedrine E (MaE) on myocardial tissue damage in mice with hypoxia-induced pulmonary hypertension. (A) Effect of MaE on right ventricular pathological damage in hypoxia-induced PH mice (×400) (`x±s, n=6), (B) Effect of MaE on right ventricular function in hypoxia-induced PH mice (`x±s, n=3). Figure 6 The figure shows the effect of Ephedrine E (MaE) on mitochondrial function in hypoxia-induced pulmonary hypertension mice. (A) The effect of MaE on lung tissue calcium in hypoxia-induced PH mice. 2+ (A) Effect of MaE on the level of mtROS in lung tissue of hypoxia-induced PH mice (x±s, n=3), (B) Effect of MaE on the level of mtROS in lung tissue of hypoxia-induced PH mice (x±s, n=3), (C) Effect of MaE on the level of MMP in lung tissue of hypoxia-induced PH mice (x±s, n=3). Figure 7 The figure shows the effect of MaE on the ROS and apoptosis levels of myocardial tissue in hypoxia-induced pulmonary hypertension mice. (A) Effect of MaE on the ROS level of myocardial tissue in hypoxia-induced PH mice (`x±s, n=3), and (B) Effect of MaE on the apoptosis level of myocardial tissue in hypoxia-induced PH mice (`x±s, n=3). Figure 8 This is a graph showing the effect of ephedrine E on the serum metabolic profile of mice with pulmonary hypertension, where (A) is the PCA score (ESI) graph of the NC group, M group, and MaE group. + , R 2 X =0.458, Q 2 =0.29; ESI - , R 2 X =0.531, Q 2 =0.383), (B) OPLS-DA, S-plot, and permutation test plots (ESI) of NC and M groups. + , R 2 X =0.712, R 2Y =0.997, Q 2 =0.982; ESI - , R 2 X =0.593, R 2 Y =0.983, Q 2 =0.915), (C) OPLS-DA, S-plot, and permutation test plots (ESI) of M group and MaE group + , R 2 X =0.634, R 2 Y =0.991, Q 2 =0.895; ESI - , R 2 X =0.529, R 2 Y =0.973, Q 2 =0.864), (D) Cluster heatmap analysis, metabolic pathway analysis and key differential metabolite network analysis diagram; Figure 9 The figure shows the effect of Ephedrine E (MaE) on the intestinal flora of mice with hypoxia-induced pulmonary hypertension. (A) Effect of MaE on α diversity in hypoxia-induced PH mice, (B) Effect of MaE on β diversity in hypoxia-induced PH mice, and (C) Effect of MaE on differential flora in hypoxia-induced PH mice. Figure 10 The figure shows the effect of Ephedrine E (MaE) on intestinal tissue damage in hypoxia-induced pulmonary hypertension mice. In the figure, (A) the effect of MaE on small intestinal tissue damage in hypoxia-induced PH mice (×200) (`x±s, n=6), (B) the effect of MaE on colonic tissue damage in hypoxia-induced PH mice (×100) (`x±s, n=6), and (C) the effect of MaE on intestinal function in hypoxia-induced PH mice (`x±s, n=3). Figure 11 The figure shows the effect of Ephedrine E on SIgA in the lung and intestinal tissues of mice with hypoxia-induced pulmonary hypertension (PH). (A) Effect of Ephedrine E on SIgA in the lung tissue of PH mice with hypoxia-induced PH (`x±s, n=6), and (B) Effect of Ephedrine E on SIgA in the intestinal tissue of PH mice with hypoxia-induced PH (`x±s, n=6). Figure 12 The figure shows the effects of ephedrine E (MaE) on the inflammatory pathway and arachidonic acid metabolism in hypoxia-induced pulmonary hypertension (PH) mice. (A) Effects of MaE on hypoxia-induced PH inflammation and arachidonic acid metabolism-related proteins (`x±s, n=3), (B) Effects of MaE on pulmonary inflammatory factors in hypoxia-induced PH mice (`x±s, n=3), and (C) Effects of MaE on pulmonary inflammation in hypoxia-induced PH mice (`x±s, n=3).

[0010] Note: Compared with the NC group, ** P <0.01; compared with group M, # P <0.05, ## P <0.01. Detailed Implementation

[0011] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0012] The present invention is illustrated by the following embodiments: Example

[0013] The technical solution provided by this invention is: a method for extracting ephedrine E from ephedra sinica, wherein the molecular structural formula of ephedrine E 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 70% ethanol by volume through a Diaion HP-20 macroporous adsorption resin column to obtain the 70% ethanol fraction (53.40 g). S3, the 70% ethanol fraction (53.40 g) was dissolved in methanol, and then silica gel column chromatography was performed with silica gel mixed in a volume ratio of 1:1.2. Elution was then performed using a gradient of dichloromethane:methanol in 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.5 was subjected to Toyopearl HW-40 column chromatography, eluted isocratically with 50% methanol, and purified by semi-preparative HPLC. The flow rate of the mobile phase was 3 mL / min, and the fraction with a retention time of 30-31 was collected, dried, and ephedrine E (6.70 mg) was obtained.

[0014] The ephedrine E prepared by this invention has the effect of inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), thus 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 ephedrine E can be prepared as needed. The given embodiments are only used to illustrate the specific implementation 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 ephedrine E and its application.

[0016] The preparation method of this invention is simple and easy to operate, and can effectively extract ephedrine E from ephedra sinica. Ephedrine E 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. Relevant information is as follows: I. Physical Morphology and Structural Identification The compound prepared by the method of this invention is an orange-red amorphous powder. The structure of the compound was determined using 1H and 1C spectrophotometer spectroscopy. The 1H and 1C spectrophotometer spectroscopy results are as follows: Figure 2 , 3 As shown, ESI-MS m / z :541 [M+H] + .exist 1 In the H-NMR (500 MHz, CD3OD) spectrum, the aromatic region δ H 7.47 (2H, d, J = 8.3 Hz, H-2', 6'), 6.82 (2H, overlap, H-3', 5'), 7.49 (2H, d, J = 8.3 Hz, H-2''', 6''') and 6.83 (2H, overlap, H-3''', 5''') are the hydrogen proton signals on the para-substituted benzene rings. δ H 5.97 (1H, d, J = 2.3 Hz, H-6), 5.99 (1H, d, J = 2.3 Hz, H-8) is the meta hydrogen proton signal on the benzene ring, which is the characteristic hydrogen signal at positions 6 and 8 of ring A in flavonoids. δ H 6.15 (1H, s, H-6'') represents the signal of an isolated hydrogen proton on the benzene ring. δ H 2.12 (2H, m, H-3) is a signal of two hydrogen protons on a methylene group. δ H4.44 (1H, t, J = 3.3 Hz, H-4) is the hydrogen proton signal on the methine group. δ H 4.97 (1H, s, H-2'') and 4.22 (1H, m, H-3'') are the hydrogen proton signals on the two oxymethine groups. δ H 2.96 (1H, dd, J = 17.2, 4.9 Hz, H-4''a), 2.76 (1H, dd, J = 17.2, 2.6 Hz, H-4''b) are the two hydrogen proton signals of the methylene group. The above four hydrogen signals are the characteristic hydrogen signals at positions 2, 3, and 4 on the C ring of flavan-3-ol compounds. 13 The C-NMR (125 MHz, CD3OD) spectrum yielded a total of 30 carbon signals. δ C 99.7 (C-2), 35.4 (C-3), and 21.7 (C-4) are characteristic carbon signals at positions 2, 3, and 4 of the C ring of flavan compounds. The presence of oxygen-bonded substitution at position 2 places its carbon signal in a low field. δ C 81.8 (C-2''), 67.1 (C-3''), and 30.0 (C-4'') are characteristic carbon signals at positions 2, 3, and 4 of the C ring in flavan-3-ol compounds. The remaining 24 carbon signals: δ C 154.7 (C-5), 96.6 (C-6), 155.5 (C-7), 97.0 (C-8), 158.1 (C-9), 107.5 (C-10), 134.4 (C-1'), 128.0 (C-2', 6'), 115.9 (C-3', 5'), 158.8 (C-4'), 156.3 (C-5''), 97.9 (C-6''), 152.8 (C-7''), 107.7 (C-8''), 152.2 (C-9''), 102.2 (C-10''), 130.6 (C-1'''), 130.1 (C-2''', 6'''), 115.8 (C-3''', 5'''), 158.4 (C-4''') represents the carbon signals on the four benzene rings, where δ C96.6 (C-6), 97.0 (C-8), and 97.9 (C-6'') are carbon signals at positions 6 and 8 of ring A and position 6'' of ring D. Based on the analysis of the above NMR data and the study of the chemical composition of the traditional Chinese medicine Ephedra root [J]. Chinese Patent Medicine, 2010, 32(10): 1758-60, the compound was identified as Mahuangning E (MaE), 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), sildenafil positive control group (Y), total extract of ephedra root group (MHG), low-dose MaE group (MaE-L), and high-dose MaE group (MaE-H). The mice were placed in a hypoxic chamber for 12 hours daily for 35 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), MaE-L group (10 mg / kg), and MaE-H group (20 mg / kg). The NC and M groups were administered 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, lung and intestinal 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, lung and intestinal 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 tissue2+ 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 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 correction 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.

[0026] 2.8 Intestinal flora detection Fresh feces were collected from each group of mice, and PCR amplification of the V3-V4 region of the 16S rDNA gene fragment of the gut microbiota was performed by Zhejiang Hangzhou Lianchuan Biotechnology Co., Ltd. Cluster analysis was performed on the final clean data, with sequences having a similarity >97% considered as an operational taxonomic unit (OTU). After singleton filtering, the final OUT abundance and representative sequences were further analyzed using α-diversity analysis, β-diversity analysis, and LDA effect size difference analysis (LEfSe) to assess changes in the gut microbiota of each group of mice.

[0027] 2.9 Enzyme-linked immunosorbent assay (ELISA) Mouse serum samples were collected and centrifuged at 3000 rpm for 10 min at 4 ℃. The supernatant was collected, and the levels of MLT and GT were measured according to the instructions of the ELISA kits. Mouse lung and intestinal tissues were thawed, homogenized at low temperature, centrifuged at 3000 r / min for 20 min at 4 ℃, and the supernatant was collected. The levels of secretory immunoglobulin A (SIgA) in the lung and intestinal tissues were measured according to the instructions of the ELISA kits.

[0028] 2.10 Detection of D-xylose excretion rate After modeling, mice were deprived of water for 12 h, and then administered 5% D-xylose solution (1 mL / 100g) by gavage. They were placed in metabolic cages for 5 h, and urine was collected and the volume recorded. The urine samples were obtained by centrifugation at 3000 rpm for 30 min at 4 ℃, and the supernatant was collected. The changes in D-xylose excretion rate in each group of mice were then detected according to the instructions of the D-xylose kit.

[0029] 2.11 Western Blot (WB) of Proteins Mouse lung tissue was collected, and total protein, cytoplasmic protein, and nuclear protein were extracted using RIPA lysis buffer containing PMSF and PPI, following the instructions of the protein extraction kit. 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 and Histone H3 as internal controls, semi-quantitative analysis of the target protein was performed using an Odyssey dual-color imaging system combined with Image Studio software.

[0030] 2.12 Real-time quantitative polymerase chain reaction (RT-PCR) Mouse lung tissue samples were collected and placed on ice. After being cut into small pieces, they were placed in enzyme-free EP tubes, and total RNA was extracted from the lung tissue using an RNA extraction kit. The RNA concentration was measured using an RNA analyzer. A mixture was then prepared in the enzyme-free EP tubes and placed in a PCR instrument for reverse transcription to obtain cDNA. Quantitative real-time PCR was then performed. According to the instructions, primer mixtures and cDNA mixtures were prepared and loaded into enzyme-free eight-tube strips. After loading, the tubes were capped, centrifuged at 3000 rpm for 2 min, and then loaded into the PCR instrument. The program was set to 95 ℃ for 5 min, 95 ℃ for 5 s, and 60 ℃ for 10 s, for 40 cycles. Data were saved. 2^ -△△Ct Methods were used to analyze the data; primer sequences are shown in Table 2.

[0031]

[0032] 2.13 Near-infrared in vivo imaging Mice in each group were injected with the IRDyes 800CW 2-DG optical probe via the tail vein at a dose of 10 nmol / 25 g. After 72 h, the mice were anesthetized with isoflurane and near-infrared imaging was performed. The results were analyzed using Image Studio software.

[0033] 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.

[0034] III. Experimental Results 3.1 Effects of MaE 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 MaE 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 1 C) Significantly reduced ( P <0.01), respiratory rate (F) ( Figure 4C) significantly increased ( P <0.01); After administration, the above indicators improved ( 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 MaE can reduce pulmonary artery pressure, improve lung function, and alleviate lung tissue damage in mice.

[0035] Note: Compared with the NC group, ** P <0.01; compared with group M, # P <0.05, ## P <0.01 3.2 Effects of MaE 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 MaE 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. MaE, however, can improve myocardial damage, right ventricular hypertrophy, and right ventricular function in mice with pulmonary hypertension.

[0036] Note: Compared with the NC group, ** P <0.01; compared with group M, # P <0.05, ## P <0.01 3.3. Effects of MaE 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 6 A) and mtROS ( Figure 6 B) Level significantly increased ( P <0.01), MMP( Figure 6C) Level significantly decreased ( P <0.01); after administration, all of the above indicators were reversed. P <0.01). This suggests that mitochondrial dysfunction exists in PH mice, and MaE can improve mitochondrial function in PH mice.

[0037] 2.4 Effects of MaE 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 7 A) and apoptosis Figure 7 B) Level significantly increased ( P <0.01); After administration, ROS and apoptosis levels were significantly reduced ( P <0.05 or P <0.01). This suggests that myocardial tissue cell damage exists in PH mice, and MaE can improve myocardial tissue cell damage.

[0038] 2.5 Effects of MaE on serum metabolic profile in mice with hypoxia-induced pulmonary hypertension To investigate the metabolic pathways significantly affected by MaE, serum metabolomics studies were conducted in PH mice. In the PCA scoring plot, the NC and M groups were separated, with the drug-treated groups moving closer to the NC group. Figure 8 A. OPLS-DA analysis see Figure 8 B and 5C identified 27 shared biomarkers, with 20 positive and 7 negative patterns. Cluster heatmap analysis and metabolic pathway analysis of these differentially expressed metabolites are as follows: Figure 8 As shown in D, the metabolic pathways they significantly affect mainly include arachidonic acid metabolism, taurine and hypoturine metabolism, and primary bile acid biosynthesis. This suggests that MaE may improve pulmonary hypertension in mice by influencing inflammatory responses and lipid metabolism.

[0039] 2.6 Effects of MaE on gut microbiota in mice with hypoxia-induced pulmonary hypertension The results of α-diversity studies showed that, compared with the NC group, the Chao1 index of mice in the M group was significantly reduced ( P <0.01), compared with group M, the Chao1 index of group MaE was significantly higher ( P <0.05), such as Figure 9 A indicates that pulmonary hypertension causes a decrease in gut microbiota diversity, and MaE can improve the decrease in gut microbiota diversity caused by pulmonary hypertension. The β-diversity results showed a clear separation trend between the NC and M groups, and significant clustering between the NC and MaE groups, such as... Figure 9B indicates that pulmonary hypertension can cause intestinal flora imbalance, and MaE can improve the intestinal flora imbalance caused by pulmonary hypertension. Differential flora were analyzed using LEfSe with LDA ≥ 3 as the criterion, and the results are as follows: Figure 9 As shown in C, at the genus level, the bacterial communities that are significantly different between the NC group and the M group, and between the M group and the MaE group, are: g_ Alistipes, g_Rikenella, g_Clostridia_vadinBB60_group and g_Odoribacter In group M, the abundance of these four bacterial communities showed a decreasing trend, while in group MaE, it showed an increasing trend. Literature review revealed... g_Alistipes It has anti-inflammatory properties and a protective effect against colitis. g_Rikenella Closely related to inflammation and regulation of intestinal barrier function, g_Clostridia_ vadinBB60_group Associated with intestinal mucosal damage, g_Odoribacter It can suppress inflammation and is associated with the improvement of colitis. The above results indicate that pulmonary hypertension causes intestinal flora dysbiosis, and MaE can improve intestinal flora dysbiosis caused by pulmonary hypertension.

[0040] 2.7 Effects of MaE on intestinal tissue damage and intestinal function in hypoxia-induced pulmonary hypertension mice Pathological damage was examined in the small intestine and colon. HE staining results showed that in group M, the villi of the small intestine were shortened and detached, and their arrangement was disordered. The villi of the colon were also shortened, with surrounding inflammatory cell infiltration. After MaE intervention, these symptoms improved. The right figure shows the pathological damage score, indicating that MaE can effectively improve intestinal tissue pathological damage. P <0.01), detect intestinal function-related indicators, such as Figure 10 C. The results showed that the excretion rate of D-xylose and the content of MTL were significantly reduced in group M. P <0.01), GT content increased significantly ( P <0.01), after MaE intervention, D-xylose excretion rate and MTL content significantly increased ( P <0.01), GT content decreased significantly ( P <0.05 or P <0.01). The above results indicate that MaE can significantly improve intestinal tissue damage and intestinal function in PH mice.

[0041] 2.8 Effects of MaE on SIgA in Lung and Intestinal Tissue of Hypoxia-Induced Pulmonary Hypertension Mice The results showed that, compared with the NC group, the expression level of SIgA in the lung and intestinal tissues of mice in the M group was significantly reduced. P <0.01), compared with the M group, the expression level of SIgA in the lung and intestinal tissues of mice in the MaE group was significantly increased ( P <0.01). This suggests that the lungs and intestines influence each other, and MaE can simultaneously reduce the immune inflammatory response in the lungs and intestines.

[0042] 2.9 Effects of MaE on inflammation and arachidonic acid metabolism in hypoxia-induced pulmonary hypertension mice Western blot analysis was performed on mouse lung tissue to detect the expression levels of proteins related to inflammation and arachidonic acid metabolism. The results showed that the expression levels of TLR4, p-NF-κB, ALOX12, SEH, and COX2 proteins were significantly increased in the M group. P <0.05 or P <0.01), MaE can significantly reduce the expression levels of the above proteins ( P <0.05 or P <0.01), such as Figure 12 As shown in Figure A; RT-PCR was used to detect the expression levels of inflammatory factors, and the results showed that the expression levels of inflammatory factors IL6, IL-1β, and TNF-α were significantly increased in group M. P <0.05 or P <0.01), MaE can significantly reduce the expression level of inflammatory factors ( P <0.05 or P <0.01), such as Figure 12 As shown in B; near-infrared imaging results show that the fluorescence intensity of group M is significantly increased ( P <0.01), MaE can significantly reduce fluorescence intensity ( P <0.01), such as Figure 12 As shown in Figure C, MaE can improve the inflammatory response in mice with pulmonary hypertension.

[0043] IV. Conclusion In the pathological progression of pulmonary embolism (PH), the pulmonary artery lumen progressively narrows and becomes occluded, leading to pulmonary vascular damage, thickening, and fibrosis. This continuously increases pulmonary vascular resistance, causing right ventricular overload and ultimately right ventricular failure, even death. Studies show that the global prevalence of PH is 28.5 per million, with a 3-year survival rate of only 55%–65%. In the treatment of PH, Western medicine is expensive, its clinical efficacy is uncertain, and its combined use often results in serious adverse reactions. Currently, research on the intervention of traditional Chinese medicine in PH is gradually deepening, and increasing evidence suggests that traditional Chinese medicine and its active ingredients have great potential for the treatment of PH.

[0044] Ephedra extract (MaE) has the effect of inhibiting the malignant proliferation of tumor cells. PASMCs are one of the main cells that make up the pulmonary vascular wall. Under hypoxia-induced PH pathological conditions, PASMCs will exhibit abnormal proliferation, which is very similar to the malignant proliferation of tumor cells. Previous experimental results have also shown that MaE has an anti-proliferative effect, but the effect of MaE on PH is still unclear.

[0045] To clarify the intervention effect of MaE on pulmonary hypertension (PH), this invention established a pulmonary hypertension model using hypoxia-induced methods. Experimental results showed that MaE could alleviate lung tissue damage, reduce pulmonary artery pressure, improve lung function, reduce pulmonary vascular thickening, improve mitochondrial dysfunction, and reduce inflammatory response in PH mice. Furthermore, MaE could alleviate myocardial tissue damage, reduce right ventricular load, decrease collagen fiber deposition, and improve right ventricular remodeling in PH mice, and the effect of MaE was superior to that of total extract of Ephedra root.

[0046] To further explore the mechanism of action of MaE in improving pulmonary hypertension, metabolomics was used to analyze the serum of PH mice. The results showed that the metabolic profile of PH mice was significantly altered, and MaE intervention could reverse differential metabolites in PH mice. The metabolic pathways significantly affected by these differential metabolites mainly included arachidonic acid metabolism, primary bile acid biosynthesis, and taurine and hypotaurine metabolism. These metabolic pathways are closely related to inflammation and lipid metabolism. Furthermore, 16S rDNA technology was used to detect the gut microbiota in the feces of PH mice. The results showed that PH caused gut microbiota dysbiosis, and MaE could improve this dysbiosis. At the genus level, four bacterial groups showed significant differences between the NC group and the M group, and between the M group and the MaE group. These four groups were all closely related to inflammation, suggesting that the gut microbiota may be one of the material bases connecting the lung and gut, participating in the regulatory pathway of MaE's lung-gut combined therapy, and possibly closely related to anti-inflammatory pathways. The lungs and large intestine are internally and externally related. Pathological examination of intestinal tissue, specifically HE staining, revealed shortened and dislocated small intestinal villi in group M, along with shortened colonic villi and surrounding inflammatory cell infiltration. MaE intervention improved these symptoms. Further analysis of intestinal function indicators showed that MaE effectively improved intestinal function in PH mice, indicating intestinal damage in PH mice and demonstrating that MaE can treat both the lungs and intestines simultaneously. SIgA, secreted by both the lung and intestinal mucosa, is a crucial molecular biological basis for the "internal and external relationship between the lungs and large intestine." Analysis of lung and intestinal SIgA levels showed a significant decrease in group M and a significant increase in group MaE, further indicating the interaction between the lungs and intestines. SIgA is mediated by the TLR4 / NFκB pathway, and the arachidonic acid metabolites 11,12-epoxyeicosatetrienoic acid (11,12-DiHETrE), 12-hydroxyeicosatetraenoic acid (12-HETE), and thromboxane are regulated by key arachidonic acid metabolism enzymes, epoxidase (SHE), arachidonic acid 12-lipoxygenase (ALOX12), and cyclooxygenase 2 (COX2). Therefore, this study investigated the expression of inflammatory pathways and enzymes regulating arachidonic acid metabolism, as well as the levels of inflammatory factors. The results showed that MaE can inhibit the expression of arachidonic acid metabolism-related enzymes and suppress the expression of inflammatory factors.

[0047] In summary, the preparation method of this invention is simple and easy to operate, and can effectively extract compound MaE from Ephedra sinica. This compound can reduce the inflammatory response in PH mice by regulating arachidonic acid metabolism, thereby improving pulmonary hypertension. At the same time, MaE can improve intestinal damage and abnormal intestinal function in PH mice and has an anti-proliferation effect on pulmonary artery smooth muscle cells (PASMCs), thus realizing its application in the preparation of drugs for the treatment of pulmonary hypertension and having practical clinical application value.

Claims

1. A method for extracting ephedrine E from Ephedra sinica, characterized in that, The molecular structural formula of the ephedrine NING E is: ； The preparation method is: S1, taking dry roots and rhizomes of Ephedra sinica, using water decoction method, extracting 2 times with 15 times weight volume of water, each time for 2 hours, and then concentrating the extract liquid under reduced pressure to obtain water extract infusion; S2, dispersing the infusion with water, and then eluting the Diaion HP-20 macroporous adsorption resin column with 70% ethanol by volume to obtain the 70% ethanol part; S3, dissolving the 70% ethanol part with methanol, then mixing silica gel, and then eluting the silica gel mixture with dichloromethane:methanol=50:1, 30:1, 20:1, 10:1, 5:1, 1:1 by volume ratio in gradient, identifying by TLC, and then combining the same flow parts according to the color results to obtain components Fr.1~Fr.5 and Fr.6; S4, eluting the component Fr.5 with Toyopearl HW-40 column chromatography with 50% methanol by volume, and then separating and purifying by semi-preparative HPLC, with the flow rate of the mobile phase being 3 mL / min, collecting the flow part with retention time of 30-31, and drying to obtain ephedrine NING E; the mobile phase is a solution prepared by adding 0.3% trifluoroacetic acid in methanol with a volume concentration of 54%.

2. The method of claim 1, wherein the extraction of ephedrine E from Ephedra sinica is characterized by, The preparation method is: S1, taking 40 kg of dry roots and rhizomes of Ephedra sinica, using water decoction method, extracting 2 times with 600 L of water, each time for 2 hours, and then concentrating the extract liquid under reduced pressure to obtain 4.07 kg of water extract infusion; S2, dispersing the infusion with water, and then eluting the Diaion HP-20 macroporous adsorption resin column with 70% ethanol by volume to obtain 53.40 g of 70% ethanol part; S3, dissolving the 70% ethanol part with methanol, then mixing silica gel, and then eluting the silica gel mixture with dichloromethane:methanol=50:1, 30:1, 20:1, 10:1, 5:1, 1:1 by volume ratio in gradient, identifying by TLC, and then combining the same flow parts according to the color results to obtain components Fr.1~Fr.5 and Fr.6; S4, eluting the component Fr.5 with Toyopearl HW-40 column chromatography with 50% methanol by volume, and then separating and purifying by semi-preparative HPLC, with the flow rate of the mobile phase being 3 mL / min, collecting the flow part with retention time of 30-31, and drying to obtain ephedrine NING E 6.70 mg.

3. The use of ephedrine NING E prepared by the method of any one of claims 1-2 in the preparation of a drug for treating pulmonary arterial hypertension.

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