Application of geum aleppicum total triterpenes in treating hypertension and preparation method of geum aleppicum total triterpenes

Abstract

The invention discloses application of geum aleppicum total triterpenes in treatment of hypertension and a preparation method of the geum aleppicum total triterpenes, the geum aleppicum total triterpenes are obtained by step-by-step extraction and purification of geum aleppicum, and the geum aleppicum total triterpenes comprise bitter raspberry sweet F1, 2-hydroxyl oleanolic acid, 2-hydroxyl ursolic acid, 20beta, 20beta, 20beta, 20beta, 20beta, 20beta, 20beta, 20beta, 20beta The traditional Chinese medicine composition is prepared from the following raw materials in percentage by weight: 28-epoxy-28-hydroxytaxasan-3 beta-ol (taraxane triterpenes), ursolic acid, maslinic acid, rosa multiflora acid and tormentic acid. According to the present invention, the geum aleppicum total triterpenes are extracted and purified from the geum aleppicum step by step, and the geum aleppicum total triterpenes can significantly increase the total cross-sectional area of the vascular bed of the ischemic tissue or organ, such that the low blood pressure can meet the blood perfusion for maintaining the life activity so as to achieve the blood pressure lowering purpose; the compound can be widely applied to the field of hypertension prevention and treatment medicines.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of total triterpenes from Myrica rubra in the treatment of hypertension. Background Technology

[0002] Essential hypertension (EH) is a common cardiovascular disease and is recognized worldwide as one of the top ten diseases. Hypertension refers to a condition where the resistance to blood flow in blood vessels exceeds the normal range. If left untreated, it can lead to serious complications such as heart disease, stroke, and kidney damage. Its core characteristic is a persistently elevated arterial blood pressure. Currently, the antihypertensive drugs used clinically are mainly Western medicines. Their main characteristics are targeted treatment, rapid onset of action, and small daily doses with fewer frequency of administration, but relatively significant side effects. Traditional Chinese medicine (TCM) treatment of hypertension focuses on regulating bodily functions and addressing both the symptoms and root cause. Its effects are gentler and relatively fewer side effects, but the daily dose is larger and more frequent, and its onset of action is slower. my country has abundant TCM resources, and the use of TCM components for lowering blood pressure has great potential for application and development. In Traditional Chinese Medicine (TCM), *Myrica rubra* (hairy water berry) is believed to tonify the kidneys and liver, promote blood circulation, and reduce swelling. It is used to treat dizziness, infantile convulsions, impotence, seminal emission, consumptive cough, rheumatic pain, irregular menstruation, sores and swelling, and injuries from falls and blows. There are also reports on the use of *Myrica rubra* extract to treat various diseases. For example, Chinese patent application number 2015107668297 discloses a drug for preventing and treating thrombosis, and its preparation method. This involves extracting the active component of a compound from *Myrica rubra* and administering it to the human body. This active component primarily works by inhibiting thrombus formation and dissolving existing thrombi, thereby preventing and treating different disease states caused by thrombosis in different tissues or organs. However, there are currently no reports in the literature on the use of total triterpenes from *Myrica rubra* to treat hypertension. Summary of the Invention

[0003] In view of the existing technology, the purpose of this invention is to provide a total triterpenoid of Myrica rubra. It has been verified that the total triterpenoid of Myrica rubra can significantly increase the total cross-sectional area of ​​the vascular bed in ischemic tissues or organs, so that lower blood pressure can meet the blood flow perfusion required to maintain life activities, thereby achieving the purpose of lowering blood pressure. This invention first provides a total triterpenoid from Myrica rubra var. rubra, obtained by stepwise extraction and chromatographic purification, including rutin F1, 2-hydroxyoleanolic acid, 2-hydroxyursolic acid, 20β,28-epoxy-28-hydroxytaraxasteran-3β-ol (taraxasteran-type triterpenoid), ursolic acid, hawthorn acid, rosolic acid, and cinnamyl acid. In another aspect, the present invention provides the use of total triterpenes from Myrica rubra in the preparation of a medicament for treating hypertension. In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of total triterpenes from Myrica rubra. Preferably, the drug is an oral preparation. In another aspect, the present invention provides a method for preparing total triterpenes from Myrica rubra var. rubra, comprising the following steps: 1) Dry the fuzzy bayberries and cut them into pieces; 2) Soak the fragments in a 50% ethanol solution and extract at room temperature. Repeat the extraction 3-6 times, collect and combine the ethanol extracts, and spray dry them into solid powder to obtain the ethanol extract. 3) The ethanol extract was dissolved in water and then extracted successively with C6 alkyl, KOAc and alcohols selected from C1-C4 alcohols at 45-50℃ under negative pressure to obtain a fractional extract. 4) After each extraction step is completed, the extract is dried before proceeding to the next extraction step. Finally, the extract is dried by an alcohol selected from C1-C4 alcohols to obtain total triterpenoid powder of Myrica rubra. To further purify the total triterpenes of Myrica rubra, a purification step 5) is set after step 4): the total triterpenes-rich powder of Myrica rubra obtained in step 4) is added to the chromatographic column and eluted with an aqueous solution containing gradually increasing concentrations of alcohols selected from C1-C4 alcohols. The beneficial effects of the present invention are as follows: The present invention extracts and purifies the total triterpenes of Myrica rubra from Myrica rubra in steps by step and by chromatography. The total triterpenes of Myrica rubra can significantly increase the total cross-sectional area of ​​the vascular bed in ischemic tissues and organs, so that lower blood pressure can meet the blood flow perfusion required to maintain life activities, thereby achieving the purpose of lowering blood pressure. The present invention will now be described in more detail with reference to the accompanying drawings and embodiments. Attached Figure Description Figure 1 This is a diagram showing the formation of capillary-like cavities in cell experiments during the present invention. Figure 2 This is a bar chart showing the total length of capillary-like cavities under different concentrations of total triterpenoids from Myrica rubra in this invention. Figure 3 This diagram shows the total number of blood vessels in the ischemic area after coronary artery ligation surgery of the left anterior descending artery of the heart according to the present invention. Figure 4 This diagram shows the total number of blood vessels in the ischemic area after bilateral common carotid artery ligation surgery according to the present invention. Figure 5 a is a graph showing the blood pressure changes in SHR rats after administering saline solution. Figure 5b is a graph showing the blood pressure changes in SHR rats after administration of total triterpenes from Myrica rubra. Detailed Implementation Example 1: A total triterpenoid of Myrica rubra var. ... The total triterpenes of this *Myrica rubra* can be used to prepare drugs for treating hypertension. As a component of a pharmaceutical composition, this component can significantly increase the total cross-sectional area of ​​the vascular bed in ischemic tissues and vital organs, thereby achieving the purpose of lowering blood pressure. The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention can be in an oral dosage form. Example 2: A method for preparing total triterpenes from Myrica rubra var. rubra, comprising the following steps: 1) Wash, dry, and cut the fuzzy bayberries into pieces; 2) The fragments are soaked in a 50% ethanol solution at 25-28℃ for extraction. The extraction is repeated 3-6 times, and the ethanol extracts are collected and combined and spray-dried into solid powder to obtain the ethanol extract. It is preferable to repeat the extraction 5 times. Generally, the weight of the ethanol solution added is 10 times the weight of the dried hairy water berries to be extracted. 3) The ethanol extract is suspended in water and extracted sequentially with a C6 alkane, KOAc, and an alcohol selected from C1-C4 alcohols to obtain an extract. The C6 alkane includes cyclic and acyclic alkanes having 6 carbon atoms, such as cyclohexane, n-hexane, and neohexane. C1-C4 alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. The weight of the organic solvent used (C6 alkane, KOAc, or an alcohol selected from C1-C4 alcohols) is typically 1-10 times the weight of the powder to be further extracted. 4) After each extraction step, the drying step is completed at 45-50℃ and under negative pressure (-0.1~-1.0 kPa) or by electrospray drying. Then, the next extraction step is carried out to finally obtain the total triterpenoid powder of Myrica rubra. That is, in the first step, the ethanol extract is suspended in water and extracted with C6 alkyl, and then dried to obtain C6 alkyl extract powder; then the C6 alkyl extract powder is dissolved in water again and extracted with KOAc, and then dried to obtain KOAc extract powder; then the KOAc extract powder is dissolved in water and extracted with an alcohol selected from C1-C4 alcohols, and then dried to finally obtain the total triterpenoid powder of Myrica rubra. The C6 alkyl extraction removes chlorophyll impurities from the total triterpenes of Myrica rubra, and the KOAc extraction removes triterpenoid aglycones impurities from the total triterpenes of Myrica rubra. Finally, extraction with an alcohol selected from C1-C4 alcohols removes polysaccharides and total polyphenols from the total triterpenes of Myrica rubra, thereby improving the purity of the total triterpenes of Myrica rubra. 5) Purification: The total triterpenoid powder of Myrica rubra obtained in step 4) was added to a chromatographic column and eluted with an aqueous solution containing gradually increasing concentrations of alcohols selected from C1-C4 alcohols to remove impurities, such as flavonoids. For example, molecular sieve column chromatography or reversed-phase column chromatography can be used. The alcohol used can be any of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. NMR analysis revealed that the total triterpenoids mainly included maltose F1, 2-hydroxyoleanolic acid, 2-hydroxyursolic acid, 20β,28-epoxy-28-hydroxytaraxasteran-3β-ol (taraxasteran-type triterpenoid), ursolic acid, crataegolic acid, rosmarinic acid, and potassium oleate. Example 3: Cell Experiment Ham's F-12 three-dimensional vascular endothelial cell culture medium was used to promote the formation of vascular-like lumens in three-dimensional cell culture. The preparation of the medium was briefly described as follows: a fibrinogen matrix was prepared by polymerizing fibrinogen solution (5 mg fibrinogen per mL of serum-free medium) with thrombin (2.5 units per mL). After polymerization, the gel was immersed in Ham's F-12 medium containing 10% fetal bovine serum and incubated at 37ºC for 3 hours to inactivate α-thrombin. Purified total triterpenes from Myrica rubra were added to three-dimensional vascular endothelial cell culture medium to promote the formation of vascular-like lumen structures by human coronary artery endothelial cells (HCAECs). Specifically, HCAECs were seeded on the surface of three-dimensional culture medium in 96-well plates, and the target cultured cells were treated with different concentrations (10-60 µg / ml) of total triterpenes from Myrica rubra. The control group received the same amount of culture medium without the drug. The culture plates were then incubated at 37ºC in a 5% CO2 balanced incubator for 2 hours. Subsequently, the cultured cells were photographed under an inverted microscope. Figure 1As shown in Figure 2, the control group also formed a small number of tubular networks after 2 hours of culture. However, the number of capillary-like lumen networks formed by cells treated with total triterpenes of Myrica rubra (50 μg / ml) for 2 hours was significantly higher in a dose-dependent manner than that of cells treated with culture medium (p<0.001). The effect of total triterpenes of Myrica rubra on promoting vascular lumen formation was quantitatively assessed by counting the total number of lumens formed at each concentration. The results showed that the number of capillary-like lumen networks formed by cells treated with total triterpenes of Myrica rubra was significantly higher in a dose-dependent manner than that of cells treated with culture medium, as shown in Figure 2. Within the low concentration range (20-50 μg / ml), the total length of the formed capillary-like lumens significantly increased with the gradual increase of the concentration of total triterpenes of Myrica rubra. Among all tested concentrations, the total triterpenoid concentration that reached the maximum lumen length was 50 μg / ml (p<0.001), while when the total triterpenoid concentration exceeded 50 μg / ml (50-60 μg / ml), the total length of the formed capillary-like lumen no longer increased. Experiments have shown that total triterpenes of Myrica rubra var. rubra upregulate the expression of angiogenic factors such as VEGF, FGF, EGF, and bFGF, promote the regeneration of collateral circulation in ischemic areas of tissues and organs, thereby increasing the cross-sectional area of ​​the effective blood supply vessels to these ischemic tissues and organs, and reducing the vascular resistance of these tissues and organs. Therefore, it has a significant therapeutic effect on hypertension, while also improving blood supply to ischemic areas of tissues and organs and reshaping the microenvironment for cell survival. The total triterpenoid combination significantly enhances the expression levels of vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2), thereby promoting angiogenesis. VEGF can promote vascular endothelial cell proliferation, migration, and lumen formation. Furthermore, total triterpenoids can reduce inflammatory responses and inhibit the production of inflammatory factors, mitigating oxidative stress and inflammation, thus creating a favorable microenvironment for angiogenesis. This aspect of research is based on existing techniques, as discussed in the paper "Antiinflammatory activity of natural triterpenes—An overview from 2006 to 2021" published by Phytotherapy Research on February 28, 2022. Simultaneously, existing experiments have shown that VEGF expression is promoted. VEGF plays a crucial role in activating vascular endothelial cells, maintaining vascular integrity, angiogenesis, and wound healing, as discussed in the paper "Enhanced myoblast-mediated gene transfer into myocytes for the production of therapeutic proteins" published in the journal Medical & Biological Engineering & Computing in 2005. Example 4: Animal experiments on the use of total triterpenes from Myrica rubra of the present invention for the treatment of hypertension. Experiment 1: SHR Model Experiment Experimental Methods: Ten healthy SHR rats with an average blood pressure of 160 mmHg were selected as the SHR model treatment group, and another 10 healthy SHR rats with an average blood pressure of 160 mmHg were selected as the SHR model control group. SHR rats are spontaneously hypertensive rats (Wuhan Shulaibao). SHR rats exhibit characteristics such as age-related increases in blood pressure and cardiovascular lesions, which are similar to the pathophysiological process of human hypertension, and can simulate human essential hypertension. The SHR model treatment group was administered the total triterpenes of Myrica rubra var. pubescens from Example 1 via gavage at a dose of 50 mg / kg once daily for 8 weeks, and changes in blood pressure were recorded and observed. Figure 5 As shown in b. The SHR model control group was fed the same volume of physiological saline, also administered via gavage, once daily for 8 weeks, and blood pressure changes were recorded and observed. Figure 5 As shown in a. Efficacy evaluation indicator: Systolic blood pressure (SBP) measurement. The systolic blood pressure (SBP) of the experimental animals was measured weekly, and the changes in blood pressure among the groups were compared. Results: Eight weeks after treatment with total triterpenes, the systolic blood pressure in the SHR model treatment group was approximately 130 mmHg, while the systolic blood pressure in the SHR model control group remained at approximately 150-160 mmHg (P<0.01). The systolic blood pressure in the SHR model treatment group was significantly lower than that in the SHR model control group after treatment, indicating that the administration of total triterpenes from Myrica rubra can significantly reduce blood pressure and has a good therapeutic effect on hypertension. Experiment 2: Rats with Ligation of the Anterior Descending Coronary Artery Experimental methods: Forty SD rats with a body weight of 200±20g were selected, half male and half female (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After one week of acclimatization feeding, the blood pressure of all SD rats was measured using a non-invasive rat tail artery blood pressure measurement system. Twenty rats (weight 200±10g, blood pressure 120±10 mmHg) were selected for the experiment. Ten rats were randomly selected as the experimental group and ten rats were selected as the control group. The experimental group consisted of rats whose anterior descending coronary artery was completely ligated. Blood pressure was measured after ligation using a non-invasive rat tail artery blood pressure measurement system. After one week of ligation and once the blood pressure stabilized, the rats were given the total triterpenes of Myrica rubra from Example 1 via gavage at a dose of 50 mg / kg once a day for 8 weeks. Changes in blood pressure were recorded and observed. The control group consisted of rats that underwent complete ligation of the left anterior descending coronary artery. Blood pressure was measured after ligation using a non-invasive rat tail artery blood pressure measurement system. After one week of ligation and once the blood pressure stabilized, the rats were given an equal volume of water (such as saline) by gavage once a day for eight consecutive weeks. Changes in blood pressure and the total number of vessels per high-power field were recorded and observed. Recordings showed that one week after ligation of the left anterior descending coronary artery, the systolic blood pressure of all SD rats increased by approximately 15% (140 mmHg); after 8 weeks of treatment, the blood pressure of the experimental group significantly decreased, essentially returning to pre-ligation blood pressure (approximately 120 mmHg). Histological examination, using a histological microscope at 200x magnification, revealed a significant increase in the total number of vessels in the ischemic area of ​​the heart within each high-power field, such as... Figure 3 As shown by the black circle in the right-hand image, the increased total number of blood vessels increases the cross-sectional area supplied to the heart. In contrast, the same area of ​​the heart in the control group SD rats showed a smaller total number of blood vessels under the same microscope magnification. Figure 3(Left side), so the cross-sectional area for blood supply to the heart is smaller. Therefore, although the blood pressure in the control group also decreased slightly, by about 4%, the blood pressure value was still significantly higher, remaining at around 135 mmHg. Conclusion: After 8 weeks of total triterpenoid treatment, the blood pressure in the experimental group basically returned to the normal pre-LAD ligation blood pressure, that is, a decrease of about 15%. The blood pressure in the control group remained at the level one week after ligation, that is, an increase of about 11%. Experiment 3: Bilateral common carotid artery ligation in mice Experimental methods: Forty SD rats with a body weight of 200±20g were selected, half male and half female (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After one week of acclimatization feeding, the blood pressure of all SD rats was measured using a non-invasive rat tail artery blood pressure measurement system. (40) rats (body weight 200±10g, blood pressure 120±10 mmHg) were selected for the experiment, of which (20) rats were randomly selected as the experimental group and (20) rats were selected as the control group. The experimental group: After bilateral carotid artery ligation surgery, there is a certain mortality rate. The surviving rats with bilateral carotid artery ligation (10 rats) were used as the experimental group. The blood pressure value after ligation was measured by a non-invasive rat tail artery blood pressure measurement system. After one week of ligation and when the blood pressure value stabilized, the rats were given the total triterpenes of Myrica rubra in Example 1 by gavage at a dose of 50 mg / kg once a day for 8 consecutive weeks. The changes in blood pressure were recorded and observed. The control group: There is a certain mortality rate after bilateral carotid artery ligation surgery. Ten surviving rats with bilateral carotid artery ligation were obtained as the control group. The blood pressure value after ligation was measured by a non-invasive rat tail artery blood pressure measurement system. After one week of ligation and when the blood pressure value stabilized, the rats were given an equal amount of water (such as physiological saline) by gavage once a day for 8 consecutive weeks. The changes in blood pressure were recorded and observed. Records showed that one week after bilateral common carotid artery ligation, the systolic blood pressure of all SD rats increased by approximately 25% (around 150 mmHg). After 8 weeks of treatment, the experimental group showed a significant decrease in blood pressure, reducing it by approximately 20% (around 125 mmHg). Histological examination, using a histological microscope at 100x magnification, revealed a significant increase in the total number of blood vessels in the corresponding frontal cortex region of the rat brain within each high-power field of view. Figure 4 As shown by the black circle in the right-hand image, the increased total number of blood vessels increases the cross-sectional area of ​​blood supply to the brain. In contrast, the control group rats showed fewer blood vessels in the frontal cortex under the same microscope magnification. Figure 4As shown by the black circle in the left-hand image, the cross-sectional area for blood supply to the brain is smaller. Therefore, although the blood pressure in the control group also decreased slightly, by about 4%, the blood pressure value was still significantly higher (around 145 mmHg). After 8 weeks of total triterpenoid treatment, the blood pressure in the experimental group basically returned to the normal pre-ligation blood pressure (a decrease of about 20%), while the blood pressure in the control group remained at the level one week after ligation (an increase of about 11%). The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the inventive concept and technical solution; or the direct application of the inventive concept and technical solution to other situations without modification, are all within the protection scope of the present invention.

Claims

1. A total triterpenoid of Myrica rubra var. rubra, characterized in that: It was obtained by stepwise extraction and purification of the soft-haired water berry.

2. The total triterpenes of Myrica rubra according to claim 1, characterized in that: It includes bitterberry F1, 2-hydroxyoleanolic acid, 2-hydroxyursolic acid, taraxasterane-type triterpenoids, ursolic acid, hawthorn acid, rosolic acid and cinnamyl acid.

3. Use of the total triterpenes of Myrica rubra according to claim 1 or 2 in the preparation of a medicament for treating hypertension.

4. A pharmaceutical composition comprising an effective amount of the total triterpenes of Myrica rubra as described in claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition is an oral preparation.

6. The method for preparing total triterpenes from Myrica rubra as described in claim 1, characterized in that, Includes the following steps: 1) Dry the fuzzy bayberries and cut them into pieces; 2) Soak the fragments in a 50% ethanol solution for extraction. Repeat the extraction 3-6 times and collect and combine the ethanol extracts. Then spray dry them into solid powder to obtain the ethanol extract. 3) The ethanol extract was dissolved in water and then sequentially extracted with C6 alkylene, KOAc, and an alcohol selected from C1-C4 alcohols to obtain the extract: 4) After each extraction step is completed, the extract is dried before proceeding to the next extraction step. Finally, the extract is dried by an alcohol selected from C1-C4 alcohols to obtain total triterpenoid powder of Myrica rubra.

7. The method for preparing total triterpenes from Myrica rubra according to claim 6, characterized in that: Step 4) is followed by step 5) purification: the total triterpenoid powder of Myrica rubra obtained in step 4) is added to the chromatographic column and eluted with an aqueous solution containing an increasing concentration of alcohols selected from C1-C4 alcohols.

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