Application of Ephedra Root Ethyl Acetate in Preventing or Treating Pulmonary Hypertension

Abstract

This invention discloses the use of the ethyl acetate fraction (ERE) of Ephedrae root for the prevention or treatment of pulmonary hypertension. This invention is the first to use ERE as a drug for the treatment of pulmonary hypertension. Efficacy experiments have demonstrated that the resulting drug can treat pulmonary hypertension in rats induced by both hypoxia alone and SU5416 combined with hypoxia.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine application, and in particular to application of ethyl acetate fraction of ephedra root in preventing or treating pulmonary hypertension. Background Art

[0002] Pulmonary arterial hypertension (PH) is a pulmonary vascular disease characterized by a progressive increase in pulmonary artery pressure, caused by a variety of etiologies. It is characterized by excessive pulmonary vasoconstriction and occlusive vascular remodeling. Various lung diseases, such as COPD and interstitial lung disease, can cause persistent pulmonary hypoxia, leading to pulmonary vasoconstriction and remodeling, ultimately leading to hypoxic PH. Clinically, PH is often treated with oxygen inhalation and vasodilators. Severe and terminally ill patients require lung transplantation or combined heart-lung transplantation. However, some commonly used vasodilators, such as endothelin receptor antagonists and phosphodiesterase-5 inhibitors, are not only ineffective in controlling pulmonary vascular remodeling, but long-term use can also cause gas exchange impairment, further exacerbating hypoxia and increasing mortality. Therefore, research and development of novel therapeutic agents for hypoxic PH is of great theoretical and practical significance.

[0003] Ephedra root is a traditional Chinese medicine derived from the dried roots and rhizomes of Ephedra sinica Stapf or Ephedra intermedia Schrenk et C. Amey., both members of the Ephedraceae family. Ephedra root has the properties of strengthening the exterior and stopping perspiration, and is primarily used for conditions such as spontaneous sweating and night sweats. Modern pharmacological studies have revealed that Ephedra root contains macrocyclic spermine alkaloids and various flavonoids, which exhibit diverse biological activities, including antihypertensive, anti-inflammatory, and anti-tumor properties. However, a small number of compounds, such as tyrosine betaine, also possess pressor activity. The pathogenesis of hypoxic pulmonary hypertension remains unclear, but oxidative stress and inflammation have been shown to contribute to the progression of the disease. Previous studies have shown that the ethylacetate extract (ERE) of Ephedra root exhibits significant antioxidant and anti-inflammatory activities. Therefore, research on its pharmacological activity has primarily focused on inflammatory diseases, and further exploration of its therapeutic effects and mechanisms remains to be explored.

[0004] So far, there has been no report on the use of this drug in the prevention and treatment of hypoxic pulmonary hypertension. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of the ethyl acetate fraction of Ephedra root (ERE) in preventing or treating pulmonary hypertension, which is a new use of the drug.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides the use of the ethyl acetate fraction of Ephedra root in the preparation of a drug for preventing or treating pulmonary hypertension.

[0007] The pulmonary hypertension is induced by hypoxia alone or by SU5416 combined with hypoxia.

[0008] The ethyl acetate fraction of the root of ephedra treats pulmonary hypertension by inhibiting RVSP, RVHI and lung index.

[0009] The ethyl acetate fraction of the root of ephedra can also treat pulmonary hypertension by inhibiting the production of MDA and promoting the increase of SOD.

[0010] The ethyl acetate fraction of the ephedra root improves pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension rats to treat pulmonary hypertension.

[0011] The ethyl acetate fraction of the root of ephedra treats pulmonary hypertension by inhibiting the increase of RVSP, the increase of RVHI, the increase of lung oxidative stress level and the pulmonary vascular remodeling induced by simple hypoxia or SU5416 combined with hypoxia.

[0012] Wherein, the medicine includes a single component or a compound preparation.

[0013] The dosage form of the drug includes one of tablets, capsules, oral liquid, syrup, pills, injection dosage form or freeze-dried powder injection dosage form.

[0014] Wherein, in the application, the concentration of the ethyl acetate fraction of the ephedra root is 300-600 mg / kg animal.

[0015] Beneficial effects: Compared with the existing technology, the present invention has the following advantages: The present invention uses ERE as a drug for the treatment of pulmonary hypertension for the first time. Animal efficacy experiments have verified that ERE can significantly inhibit the increase in RVSP, RVHI, increased lung oxidative stress level and pulmonary vascular remodeling caused by simple hypoxia and SU5416 combined with hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of ERE on RVSP, RVHI, lung index and oxidative stress level in rats with pulmonary hypertension induced by simple hypoxia (Mean±SD, n=6)

[0017] Figure 2 Effects of ERE on pulmonary arteriolar vascular remodeling in rats with pulmonary hypertension induced by simple hypoxia;

[0018] Figure 3 Effects of ERE on RVSP, RVHI and various ultrasound indices in SuHx rats (Mean±SD, n=6);

[0019] Figure 4Effects of ERE on pulmonary arteriolar vascular remodeling in SuHx rats;

[0020] Figure 5 Effects of ERE on lung oxidative stress levels in SuHx rats. DETAILED DESCRIPTION

[0021] Experimental animals of the present invention:

[0022] Wistar rats (SPF male, 6–8 weeks old, 180–200 g) were purchased from the Center for Comparative Medicine of Yangzhou University. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Yangzhou University, and all procedures were performed in accordance with the Regulations of Laboratory Management of China (2017) under project license (SYXK(SU)2017-0044). Rats were housed in a room maintained at 22–25°C and 45%–55% humidity, with a 12-h day / night cycle. Rats had free access to water and food during the experiment.

[0023] Sprague-Dawley (SD) rats (SPF, male, 6-8 weeks old, 180-200 g) were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (SCXK (Zhejiang) 2019-0001). Animal quality certificate number: 20230712Aazz0619000863. All rats were housed in a room with a room temperature of 22-25°C and an air humidity of 45%-55%, under a 12-hour daylight cycle. During the experiment, rats had free access to water and food. Rats were acclimated to feeding for one week before the start of the experiment, and their body weights were recorded daily.

[0024] The drugs, reagents and equipment of the present invention:

[0025] SU 5416: TCI (Shanghai) Chemical Industry Development Co., Ltd., batch number: 4LSOH-SR; Ephedra root: Xuzhou Guandong Changjiu Chinese Medicinal Planting Co., Ltd.; α-SMA antibody: CST (USA), catalog number: 19245; Goat Anti-Rabbit IgG (H+L)-HRP: Formase Biotechnology Co., Ltd., catalog number: FMS-Rb01; Chloral hydrate: Aladdin Reagent Co., Ltd., catalog number: C2026060; Small animal hypoxia chamber: Changsha Changjin Technology Co., Ltd., model number: CJ-DO2245; Biological blood pressure sensor: Chengdu Taimeng Software Co., Ltd., model number: PT-103. Malondialdehyde (MDA) test kit: Nanjing Jiancheng Bioengineering Institute, catalog number: A003-1; Superoxide dismutase (SOD) assay kit: Nanjing Jiancheng Bioengineering Institute, catalog number: A001-3; BCA protein concentration assay kit: Shanghai Beyotime Biotechnology Co., Ltd. The ethyl acetate fraction of Ephedra root used in the embodiments of the present invention is the ethyl acetate fraction of Ephedra root (ERE) obtained by the preparation method of Example 1 in Chinese patent application CN115463164A, the preparation method of the ethyl acetate fraction of Ephedra root and its medicine and use, and the ERE solutions of different concentrations prepared in Example 1.

[0026] 1. Establishment of a rat model of hypoxia-induced pulmonary hypertension

[0027] Eighteen male Wistar rats were acclimated to normoxia for one week and then randomly divided according to body weight into three groups of six rats each: a normal control group (Control), a model group (Model), and a group treated with ethyl acetate extract of Ephedra root (350 mg / kg) (ERE). The normal control group was maintained in a normoxia environment, while the model and drug-treated groups were placed in the same hypoxic chamber at an oxygen concentration of 9% for four weeks. Calcium chloride and sodium hydroxide solutions were placed in the hypoxic chamber to absorb water vapor and carbon dioxide, respectively.

[0028] 2. Establishment of a pulmonary hypertension rat model induced by SU 5416 combined with hypoxia (SuHx)

[0029] Based on body weight recorded on the last day of the acclimation period, SD rats were randomly divided into five groups, each consisting of six rats: a normal control group (Control), a model group (SuHx), a low-dose Ephedra root ethyl acetate extract-treated group (ERE-LD), a high-dose Ephedra root ethyl acetate extract-treated group (ERE-HD), and a positive drug group (SD). On the first day of the experiment, rats in both the model and drug groups received a single subcutaneous injection of 20 mg / kg of Sugen (SU5416) and were then placed in the same hypoxic chamber with an oxygen concentration of 10% for three weeks. Calcium chloride and sodium hydroxide solutions were placed in the hypoxic chamber to absorb water vapor and carbon dioxide, respectively. Rats in the normal control group received a subcutaneous injection of the same volume (1 mL / 200 g rat) of vehicle solution (5% DMSO, 5% Tween-80, 40% PEG300, 50% Saline) and were then placed in a normoxic environment. The rats in the low-dose drug group and the high-dose drug group were orally administered with 300 mg / kg and 600 mg / kg of ethyl acetate extract of Ephedra root, respectively, every day. The rats in the positive drug group were orally administered with 25 mg / kg of sildenafil citrate every day.

[0030] 3. Color Doppler ultrasound measurement

[0031] After 3 weeks of hypoxia induction, SD rats were anesthetized with 4% isoflurane, and then the isoflurane concentration was adjusted to 2% to maintain anesthesia, and the heart rate was kept at 300-350 beats / minute. The rat's chest hair was shaved, and the rat was placed in a supine position on the testing table. A 37°C constant temperature heating pad was turned on to maintain the rat's body temperature, and the rat's limbs were fixed to the electrode sheet. A coupling agent was applied to the rat's chest, and an ultrasound probe was used for detection. During the detection, the position of the probe and the angle of the platform were continuously adjusted according to the ultrasound viewing angle. Different sections were selected to measure the corresponding indicators, and no less than 3 cardiac cycles were recorded and the average value was taken.

[0032] 4. Right ventricular systolic pressure measurement

[0033] On the last day of the experiment, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg / kg) before blood pressure measurement. Subsequently, the rats were placed on a thermostatic operating table in the supine position. A skin incision of about 3 cm in length was made along the right midclavicular line on the right clavicle, and the right jugular vein was bluntly separated. After the mesentery on the vein was peeled off cleanly, two surgical sutures were inserted vertically under the vein, the surgical suture at the distal end was ligated, and the surgical suture at the proximal end was tied with a slipknot. A V-shaped incision was made in the venous segment between the two surgical sutures using ophthalmic scissors, and a polyethylene (PE 50) catheter filled with heparin solution was inserted into the right jugular vein and introduced into the right ventricle. The other end of the catheter was connected to a blood pressure recorder for measuring right ventricular contraction (RVSP).

[0034] 5. Determination of right ventricular hypertrophy index

[0035] After RVSP measurement, the rats were sacrificed, the thoracotomy was performed, and the hearts were removed and rinsed with ice-cold saline. The left and right atrial tissue and remaining major vessels were removed. The right ventricle was excised along the edge of the ventricular septum, and the blood clot was rinsed. After blotting with filter paper, the right ventricle (RV) and left ventricle plus ventricular septum (LV+S) of each group were weighed. The RV / LV+S ratio was calculated to assess the degree of right ventricular hypertrophy.

[0036] 6. Lung Histopathological Analysis

[0037] Fixed lung tissue specimens were dehydrated with ethanol, permeabilized with xylene, and then embedded in melted paraffin. The paraffin blocks were sliced ​​into 5-μm sections on a microtome. After staining with hematoxylin and eosin and mounting with neutral gum, pathological changes in the pulmonary vasculature were observed and photographed under a microscope. Two experts, unaware of the experimental protocol and group assignments, selected peripheral arteries with a diameter ranging from 50 to 150 μm and measured the wall thickness percentage (WT%) and wall area percentage (WA%) using ImageJ software according to formulas (1-1) and (1-2), respectively.

[0038]

[0039]

[0040] WT% represents the ratio of pulmonary arteriolar wall thickness to vessel diameter, while WA% represents the ratio of pulmonary arteriolar wall area to total vessel area. WT represents vessel wall thickness, ED represents vessel external diameter, TA represents total vessel area, and VA represents lumen area.

[0041] 7. Immunohistochemical staining analysis

[0042] Lung tissue sections were dewaxed, rehydrated, inactivated endogenous peroxidase, permeabilized, and microwave-heat-repaired. Subsequently, 5% bovine serum albumin (BSA) was used for blocking at room temperature for 30 minutes. Incubate with α-SMA antibody overnight at 4°C, and then incubate with secondary antibody (Goat Anti-Rabbit IgG (H+L)-HRP) at 37°C for 20 minutes. Use DAB colorimetric agent to develop the sections, observe the state of the sections in real time, determine the final color development time, and place them in distilled water to stop the color development. Use hematoxylin for nuclear staining, followed by dehydration and transparent treatment. After the sections are blocked with neutral glue, they are observed and photographed under a microscope. The integrated optical density (IOD) of α-SMA is calculated using ImageJ software. IOD is determined as the ratio of total optical density to total area.

[0043] 8. SOD and MDA determination

[0044] The rat lung tissue homogenate was taken and the assay was performed according to the requirements of the kit.

[0045] 9. Statistical processing

[0046] Data are presented as mean ± standard deviation (X ± SD). Differences between groups were analyzed using one-way analysis of variance. When data did not follow a normal distribution, the Kruskal-Wallis rank sum test was used. Additionally, when data followed a normal distribution but with unequal variances, the Dunnett's T3 multiple comparison test was used. P < 0.05 indicated statistical significance.

[0047] 10. Results Analysis

[0048] (1) ERE significantly reduced RVSP and lung oxidative stress levels in rats with hypoxia-induced pulmonary hypertension

[0049] After four weeks of simple hypoxia induction, the pulmonary hemodynamics and lung oxidative stress levels of rats in each group were detected. RVSP of rats in each group was measured by right cardiac catheterization. Figure 1 As shown in B, the right ventricular systolic pressure in the model group was significantly higher than that in the control group, and ERE could significantly inhibit the increase of RVSP (P<0.01). Figure 1 C is the right ventricular hypertrophy index (RVHI) of rats in each group. Hypoxia treatment significantly increased RVHI (P < 0.001), while ERE treatment significantly inhibited the increase of RVHI (P < 0.05). Figure 1 D is the ratio of right ventricle to body weight of rats in each group. Lung index of rats in each group is shown in Figure 1 E, After four weeks of hypoxia treatment, the lung index of the model group rats increased significantly, and ERE significantly inhibited the increase of lung index (P < 0.05). In addition, by detecting the SOD ( Figure 1 F) and MDA levels ( Figure 1 G) to reflect the level of oxidative stress in lung tissue. Compared with the control group, the MDA level in the lung tissue of rats in the model group was significantly increased (P < 0.001), and the SOD level was significantly decreased (P < 0.001). The MDA level in the lung tissue of rats in the ERE group was significantly lower than that in the model group (P < 0.01), and the SOD level was significantly higher than that in the model group (P < 0.01). The above results indicate that ERE can inhibit the increase of RVSP, RVHI and lung index induced by hypoxia in rats, inhibit the production of MDA, promote the increase of SOD, regulate the oxidative stress response, and thus treat hypoxia-induced pulmonary hypertension in rats.

[0050] (2) ERE significantly improved pulmonary vascular remodeling in rats with hypoxia-induced pulmonary hypertension

[0051] The results of H&E staining and immunohistochemistry of rat lung tissue are shown in Figure 2 .according to Figure 2 A, In the control group, the walls of the pulmonary arterioles were thin and regular, with no intimal damage. After hypoxia induction, the walls of the pulmonary arterioles were significantly thickened, with uneven thickness, and were infiltrated with numerous inflammatory cells, intimal necrosis, significant smooth muscle cell proliferation, and reduced lumen size. These pathological changes were significantly improved in the ERE group. Figure 2 B and Figure 2 C are WT% and WA%, respectively. After hypoxia treatment, the WT% and WA% of the model group increased significantly (P<0.001), and the ERE group showed significant improvement. α-SMA antibody was used to label the proliferating pulmonary artery smooth muscle cells. Figure 2 D is the integrated optical density of α-SMA. The IOD value of the model group was significantly higher than that of the control group. ERE treatment significantly inhibited the proliferation of pulmonary artery smooth muscle cells (P < 0.001). These results indicate that ERE can inhibit pulmonary vascular remodeling in rats with pulmonary hypertension induced by simple hypoxia.

[0052] (3) ERE significantly reduced RVSP and RVFW in rats with pulmonary hypertension induced by SU5416 combined with hypoxia (SuHx)

[0053] Three weeks after the injection of SU5416 combined with hypoxia induction, the pulmonary hemodynamics of the rats in each group were detected. The RVSP of the rats in each group was measured by right cardiac catheterization. Figure 3 As shown in A, the right ventricular systolic pressure in the model group was significantly higher than that in the control group, and high-dose ERE drugs could significantly inhibit the increase of RVSP (P < 0.01). Figure 3 E is a representative picture of the pressure waveform of rats in each group. The right ventricular hypertrophy index (RVHI) of rats in each group is shown in Figure 3 B. The RVHI of the rats in the drug group was significantly lower than that in the model group (P < 0.05). After three weeks of hypoxia treatment, the rats in the model group showed obvious symptoms such as tachypnea, cyanosis of the mouth and paws, lack of energy, messy and dull hair, fatigue and drowsiness. The ERE-LD group showed some improvement compared with the model group. The above symptoms were significantly delayed and alleviated in the ERE-HD and SD groups. Figure 3 C, Ultrasound results showed that the pulmonary artery acceleration time (PAT) was significantly shortened after modeling and improved after drug administration, and the ERE-HD group and SD group had extremely significant differences compared with the model group (P < 0.001). Figure 3 The results showed that compared with the normal group (Control), the right ventricular free wall thickness (RVFW) of the model group (SuHx) rats was significantly increased, and high-dose ERE and positive drugs could significantly inhibit the increase in wall thickness (P < 0.01). Figure 3 F is a representative picture of the ultrasound results of rats in each group.

[0054] (4) ERE significantly inhibited pulmonary vascular remodeling in rats with pulmonary hypertension induced by SU5416 combined with hypoxia

[0055] The results of H&E staining and immunohistochemistry of rat lung tissue are shown in Figure 4 .according to Figure 4 A, The walls of the pulmonary arterioles in the control group were thin and regular; in the model group, the walls of the pulmonary arterioles were significantly thickened, and the wall thickness was uneven. Intima necrosis and desheathing, and thickening of the middle layer were observed, causing the lumen to become smaller. However, after treatment with ERE, the above pathological changes were significantly inhibited. Compared with the model group, the WT% in the ERE-HD group was significantly reduced (P < 0.001). α-SMA antibody was used to mark the proliferating pulmonary artery smooth muscle cells, and the immunohistochemical results ( Figure 4 B) shows significant proliferation of pulmonary artery smooth muscle cells in the model group, which was significantly improved after ERE treatment. These results indicate that ERE can inhibit pulmonary vascular remodeling in rats with pulmonary hypertension induced by SU5416 combined with hypoxia.

[0056] (5) ERE significantly reduced the oxidative stress level in lung tissue of rats with pulmonary hypertension induced by SU5416 combined with hypoxia

[0057] The expression levels of MDA (malondialdehyde) and SOD (superoxide dismutase) in rat lung tissue were measured. Figure 5 Compared with the control group, the MDA level in the lung tissue of rats in the model group was significantly increased (P < 0.001), and the SOD level was significantly decreased (P < 0.001). The MDA level in the lung tissue of rats in all ERE dose groups was significantly lower than that in the model group (P < 0.05), and the SOD level was significantly higher than that in the model group (P < 0.01). These results indicate that ERE can inhibit the production of MDA and promote the increase of SOD, thereby regulating the oxidative stress response.

Claims

1. The use of the ethyl acetate fraction of Ephedra root in the preparation of a medicament for preventing or treating pulmonary hypertension, characterized in that: The preparation method of the ethyl acetate fraction of Ephedra root comprises the following steps: 1) 220 g of Ephedra root was crushed into granules with a particle size of less than 2 mm. The granules were soaked in 95% ethanol at a solid-to-liquid ratio of 1:10 at room temperature for 12 h. The granules were then ultrasonically extracted twice at 60°C with reflux for 2 h each time. The extract was collected, filtered, and the solvent was recovered under reduced pressure. The extract was then dried at 60°C to obtain 15 g of extract. 2) Add 150 mL of distilled water to 15 g of the extract, soak for 12 hours, extract with 150 mL of petroleum ether five times and ethyl acetate nine times, each extraction time 0.5 h. Combine the extracted ethyl acetate fractions and dry them to obtain 5 g of the ethyl acetate fraction of Ephedra root.

2. The use according to claim 1, characterized in that The pulmonary hypertension is induced by hypoxia alone or by SU5416 combined with hypoxia.

3. The use according to claim 1 or 2, characterized in that The ethyl acetate fraction of the root of ephedra treats pulmonary hypertension by inhibiting RVSP, RVHI or lung index.

4. The use according to claim 1 or 2, characterized in that The ethyl acetate fraction of the root of ephedra can also treat pulmonary hypertension by inhibiting the production of MDA and promoting the increase of SOD.

5. The use according to claim 1 or 2, characterized in that: The ethyl acetate fraction of the root of ephedra treats pulmonary hypertension by improving pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia.

6. The use according to claim 1 or 2, characterized in that The ethyl acetate fraction of the root of ephedra treats pulmonary hypertension by inhibiting the increase of RVSP, the increase of RVHI, the increase of lung oxidative stress level and the pulmonary vascular remodeling induced by simple hypoxia or SU5416 combined with hypoxia.

7. The use according to claim 1 or 2, characterized in that The medicine includes a single component or a compound preparation.

8. The use according to claim 1 or 2, characterized in that The dosage form of the drug includes one of tablets, capsules, oral liquid, syrup, dripping pills, injection dosage form or freeze-dried powder injection dosage form.

9. The use according to claim 1 or 2, characterized in that: The concentration of the ethyl acetate fraction of the ephedra root is 300-600 mg / kg.

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