Application of melastoma dodecandrum in preparation of antihypertensive drugs and drugs for treating hypertension complications

Abstract

The invention discloses application of melastoma dodecandrum lour in preparation of antihypertensive drugs and drugs for treating hypertension complications. It is found for the first time that by means of melastoma dodecandrum extract treatment, AKT1 and EGFR protein expression related to hypertension can be lowered, blood pressure (Plt, 0.05) and cardiac fibrosis degree (Plt, 0.01) of spontaneously hypertensive rats (SHR) can be remarkably lowered, inflammatory response (Plt, 0.05) can be inhibited, and the effect of treating the melastoma dodecandrum extract can be achieved. The melastoma dodecandrum not only has the effect of reducing blood pressure, but also has the functions of improving myocardial fibrosis and inhibiting inflammatory response; moreover, the melastoma dodecandrum lour has no toxic or side effect on the body, is safe and reliable to use, shows that the melastoma dodecandrum lour has good research and development prospects of reducing blood pressure, improving myocardial fibrosis and resisting ventricular remodeling, and provides a new effective way for hypertension and corresponding complications.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of ground rhizome in preparing blood pressure lowering drugs and drugs for treating complications of hypertension. Background Art

[0002] Hypertension, a global chronic disease characterized by high morbidity and a high incidence of serious complications, has become a major public health issue as the most common and leading risk factor for cardiovascular and cerebrovascular disease. Myocardial fibrosis is a prominent pathological feature of hypertension. It is a series of structural and functional abnormalities in myocardial cells caused by changes in the internal and external environment of hypertension. It is often present in various cardiovascular diseases and is characterized by an imbalance in the proportion and disorder of collagen.

[0003] During the development and progression of hypertension, activation of the RAAS, increased levels of angiotensin II and aldosterone, and elevated levels of norepinephrine can further lead to myocardial damage and fibrosis. Endothelial dysfunction, the release of inflammatory factors, the massive infiltration of cardiomyocytes, and accelerated oxidative stress can induce myocardial fibrosis, affecting cardiac structure and function. These abnormalities are major risk factors for increased morbidity and mortality associated with structural and functional abnormalities in various organs, including heart failure, stroke, coronary heart disease, renal failure, and aortic aneurysms, severely jeopardizing human health and quality of life.

[0004] At present, drugs for treating hypertension are usually chemically synthesized drugs, such as diuretics, angiotensin receptor antagonists, calcium antagonists, beta-blockers, angiotensin-converting enzyme inhibitors, etc.; however, these first-line clinical drugs all have significant side effects. For example, the use of diuretics can cause adverse reactions such as hypokalemia, hyperuricemia, hypercholesterolemia, and impaired glucose tolerance. There is an urgent need to study safer and more reliable hypertension treatment drugs and clinical treatment plans.

[0005] Ground mulberry is a special medicinal material commonly used by the She ethnic group for both medicinal and edible purposes. Its main chemical components include flavonoids, alkaloids, polyphenols, tannins, organic acids, polysaccharides, volatile oils, etc. It has the effects of clearing heat and detoxifying, astringent and hemostatic, promoting blood circulation and regulating menstruation, diuretic and detumescent, antibacterial, anti-tumor, anti-inflammatory, and antioxidant. Currently, there are no reports on the use of ground mulberry in lowering blood pressure and preventing hypertensive myocardial fibrosis. Summary of the Invention

[0006] The invention aims to provide the use of ground rhizome in the preparation of antihypertensive drugs and drugs for treating hypertension complications, thereby providing a new and effective approach for treating hypertension and its corresponding complications.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: Application of Diren in the preparation of antihypertensive drugs; and, use of Diren in preparing drugs for treating complications of hypertension; The present invention found for the first time that the treatment with the extract of Radix Dirunculatae could not only downregulate the expression of AKT1 and EGFR proteins related to hypertension, but also significantly reduce the blood pressure of spontaneously hypertensive rats (SHR). P <0.05) and cardiac fibrosis degree ( P <0.01), and can also inhibit inflammatory response ( P <0.05); indicating that Di Ren has the effect of lowering blood pressure, but also has the function of improving myocardial fibrosis and inhibiting inflammatory response; moreover, Di Ren has no toxicity or side effects on the body and is safe and reliable to use, indicating that Di Ren has good research and development prospects for lowering blood pressure, improving myocardial fibrosis, and resisting ventricular remodeling.

[0008] Preferably, the drugs for treating hypertension complications include at least drugs for treating hypertensive myocardial fibrosis and drugs for treating hypertensive inflammation.

[0009] Preferably, the blood pressure-lowering drug or the drug for treating complications of hypertension is an oral preparation, an intravenous injection preparation or an external preparation.

[0010] Preferably, the application comprises: obtaining a water extract or an alcohol extract of the rhizome of ground rhizome, and using the water extract or the alcohol extract of the rhizome of ground rhizome to prepare a blood pressure lowering drug or a drug for treating complications of hypertension.

[0011] As further preferred, the method for obtaining the water extract or alcohol extract of Radix Glehniae comprises the following steps: S1: crushing the ground rhizome and extracting it with water or alcohol by reflux to obtain an extract; Wherein, the alcohol includes at least methanol and ethanol; the number of reflux extractions is preferably 1-3 times; S2: After concentrating the extract, the water extract or alcohol extract of the Radix Glehniae is obtained.

[0012] Compared with the prior art, the beneficial effects of the present invention are embodied in: The present invention found for the first time that the treatment with the extract of Radix Dirunculatae could not only downregulate the expression of AKT1 and EGFR proteins related to hypertension, but also significantly reduce the blood pressure of spontaneously hypertensive rats (SHR). P <0.05) and cardiac fibrosis degree ( P <0.01), and can also inhibit inflammatory response ( P <0.05); indicating that Di Ren has the effect of lowering blood pressure, but also has the function of improving myocardial fibrosis and inhibiting inflammatory response; moreover, Di Ren has no toxicity or side effects on the body and is safe and reliable to use, indicating that Di Ren has good research and development prospects for lowering blood pressure, improving myocardial fibrosis, and resisting ventricular remodeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effects of different drug treatments on rat body weight; Wherein, Weight (g) indicates body weight (grams), Control indicates blank control group, Model indicates negative control group, MDL+low indicates low-dose dill treatment group (administered with dill water extract 2.1 g / kg / d), MDL+High indicates high-dose dill treatment group (administered with dill water extract 8.4 g / kg / d), Telmisartan indicates positive control group (administered with telmisartan tablets 8.4 mg / kg / d) (n=8); the same below; Figure 2 The effects of different drug treatments on systolic blood pressure in rats; Among them, SBP (mmHg) represents systolic blood pressure (mmHg); * represents the difference between the control group and the model group. P <0.05, ** indicates comparison with the model group P <0.01, # indicates comparison with the positive drug group P <0.05, the same below; Figure 3 The effects of different drug treatments on diastolic blood pressure in rats; Wherein, DBP (mmHg) represents diastolic blood pressure (millimeters of mercury); Figure 4 The effects of different drug treatments on the mean blood pressure of rats; Wherein, MAP (mmHg) represents mean pressure (millimeters of mercury); Figure 5 This is the electrophoresis diagram of AKT1 and EGFR protein detected by Western blot; Figure 6 The relative expression level of AKT1 protein was analyzed by Western blot. Figure 7 The relative expression level of EGFR protein was analyzed by Western blot. Figure 8 The results of staining of myocardial tissue sections of rats treated with different drugs are shown; Among them, Masson indicates Masson staining, Sirius-red indicates Sirius-red staining; Figure 9 Effects of different drug treatments on the collagen fiber content in rat heart tissue (Masson staining); Figure 10 Effects of different drug treatments on the collagen fiber content in rat heart tissue (Sirius-red staining); Figure 9 and Figure 10 In the figure, Area of ​​fibrosis / Sirus-red Positive area (%) indicates the percentage of cardiac tissue collagen area to the total heart area; Figure 11 The results of IL-1β analysis in rats treated with different drugs; Among them, IL-1β indicates interleukin-1β; Figure 12 The results of TGF-β analysis in rats treated with different drugs; TGF-β indicates transforming growth factor-β; Figure 13 The results of TNF-α analysis in rats treated with different drugs; Here, TNF-α represents tumor necrosis factor-α. DETAILED DESCRIPTION

[0014] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0015] The medicinal materials, experimental animals, reagents, and instruments used in the embodiments of the present invention are as follows: (1) Medicinal materials: The Radix Direnzi used in the experiment was sourced from the Chinese medicine warehouse of the unit and was inspected and accepted by the unit’s Chinese medicine preparation acceptance team. It was found to be in compliance with the relevant provisions of the 2015 edition of the Chinese Pharmacopoeia.

[0016] (2) Experimental animals: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0017] (3) Reagents: Anhydrous ethanol (item number: SJ003614) was purchased from Hangzhou Hongda Chemical Instrument Co., Ltd.; xylene (item number: 10023418) was purchased from Sinopharm Chemical Reagent Co., Ltd.; hematoxylin staining solution (item number: HK2053) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.; hydrochloric acid alcohol differentiation solution (item number: HK2054) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.; eosin (item number: HK2051) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.; ammonia bluing solution (item number: HK2055) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.; neutral gum (item number: 10004160) was purchased from Sinopharm Chemical Reagent Co., Ltd.; n-butanol (item number: 71-36-3) was purchased from MacLean Chemical Reagent Co., Ltd.; Masson staining kit (item number: HK2020) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.; picrosirius red staining solution (item number: HK1038) was purchased from Hangzhou Haoke Biotechnology Co., Ltd.

[0018] (4) Instruments: Dehydrator (JT-12S) was purchased from Wuhan Junjie Electronics Co., Ltd.; Biological tissue automatic embedding machine (JB-P5) was purchased from Wuhan Junjie Electronics Co., Ltd.; Paraffin embedding machine (cold stage) (JB-L5) was purchased from Wuhan Junjie Electronics Co., Ltd.; Paraffin embedding machine (cold stage) (JB-P5) was purchased from Hubei Beinuo Medical Technology Co., Ltd.; Tissue spreader (JK-5) was purchased from Wuhan Junjie Electronics Co., Ltd.; KD-P tissue spreader (51A011) was purchased from Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.; Rotary microtome (HistoCoreBI OCUT was purchased from Leica Microsystems Shanghai Co., Ltd.; oven (GFL125) was purchased from Tianjin Labterui Instrument Equipment Co., Ltd.; oven (HGPF-50) was purchased from Shanghai Yuejin Medical Instrument Co., Ltd.; cover glass (710510) was purchased from Jiangsu Huida Medical Instrument Co., Ltd.; slide (188109) was purchased from Haimen Shenying Experimental Instrument Factory; Dakoway coverslipper (CS500) was purchased from Shenzhen Dakoway Medical Technology Co., Ltd.; Dakoway stainer (DP260s) was purchased from Shenzhen Dakoway Medical Technology Co., Ltd.; microscope (ECLIPSE E100) was purchased from NIKON; Jiangfeng scanner (KF-PRO-120) was purchased from Ningbo Jiangfeng Bioinformatics Co., Ltd.

[0019] The present invention uses GraPhPad Prism 8.3.0 software for data processing and plotting. All data are expressed as mean ± standard deviation (x ± s), and one-way ANOVA is used for comparative analysis of multiple groups of data.

[0020] Example 1 Application of Radix Dirunculatae in the preparation of antihypertensive drugs

[0021] 1. Preparation of water extract of Diren Specifically, the ground rhizome is crushed and reflux-extracted three times with water as an extraction solution, filtered, and all filtrates are combined; the filtrate is concentrated to obtain a concentrated extract, which is the ground rhizome water extract.

[0022] 2. The blood pressure lowering function of the water extract of Diren To investigate the antihypertensive effect of Radix Dioscoreae, 8-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto rats were selected as experimental subjects, with a body weight of 200±220 g. After 3 days of adaptive feeding, the Wistar rats were used as the blank control group, and the SHR rats were divided into a negative control group, a low-dose Radix Dioscoreae treatment group (administered 2.1 g / kg / d of Radix Dioscoreae aqueous extract), a high-dose Radix Dioscoreae treatment group (administered 8.4 g / kg / d of Radix Dioscoreae aqueous extract), and a positive control group (administered 8.4 mg / kg / d of telmisartan tablets). Physical signs of rats in each group (n=5) were measured after 2 months of continuous gavage treatment.

[0023] Specifically, each rat was weighed using a scale, and the tail artery systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) of each group of rats were measured using a CODA Monitor mouse and rat blood pressure non-invasive blood pressure meter (Kent Scientific, USA). After the measurements, blood was collected from the abdominal aorta under 2% isoflurane anesthesia. After blood collection, the rat heart was quickly removed, and residual blood stains and connective tissue on the organ were cleaned with cold saline. The heart was then divided into two halves. One heart tissue was fixed in 4% paraformaldehyde for tissue section staining (as described in Example 2); the other heart tissue was immediately snap-frozen in liquid nitrogen for subsequent detection of tissue protein levels.

[0024] (1) Weight and blood pressure measurement Before the experiment and after 2 months of treatment, the body weight of each rat was measured 3 times and the average value was taken. Figure 1 The tail artery systolic pressure, diastolic pressure and mean arterial pressure of each group of rats were measured three times and the average value was taken. Figure 2 、 Figure 3 and Figure 4 shown.

[0025] Depend on Figure 1 It can be seen that after 2 months of different drug treatments, there was no significant difference in the body weight of the rats in each group, indicating that the extract of Radix Diruncatulae had no damage to the rats' bodies and had no toxicity or side effects.

[0026] Depend on Figure 2 、 Figure 3 and Figure 4 It can be seen that compared with the negative control group, the systolic blood pressure of the low-dose and high-dose Diren treatment groups ( P <0.05), diastolic blood pressure (low dose P <0.05, high dose P <0.01) and mean depressed dose ( P <0.05, high dose P <0.01) were significantly reduced, while the diastolic and mean blood pressures in the positive control group were significantly decreased ( P <0.05), but there was no significant change in systolic blood pressure.

[0027] Compared with the positive control group, the diastolic blood pressure of rats in the high-dose Diren treatment group ( P <0.05) and mean pressure ( P <0.05) were significantly lower, indicating that the antihypertensive efficacy of the water extract of Direngren was better than that of the positive drugs.

[0028] (2) Analysis of the expression of hypertension-related proteins AKT1 and EGFR Immunoblotting (Western blot) was used to detect the expression of hypertension-related proteins AKT1 and EGFR. Specifically, myocardial tissue proteins were extracted using a lysis buffer containing a protease inhibitor and a phosphatase inhibitor mixture, and quantified using a BCA protein analysis kit. The proteins were separated by electrophoresis and then transferred to a PVDF membrane. After blocking for 2 hours, the membrane was incubated with primary antibodies against AKT1 (Proteintech 1:1200 rabbit source), EGFR (abcam 1:1200 rabbit source) and GAPDH (weiao 1:2000 mouse source) at 4°C overnight. After washing, goat anti-rabbit (1:2000) was added and incubated at room temperature for 1 hour. The supersensitive ECL chemiluminescence ready-to-use substrate was used for color development, and the grayscale of the bands was analyzed using ImageJ software (GAPDH was used as an internal reference). The analysis results are shown in Figure 2. Figure 5 、 Figure 6 and Figure 7 shown.

[0029] Depend on Figure 5 、 Figure 6 and Figure 7 It can be seen that compared with the blank group, the expression levels of hypertension-related proteins AKT1 and EGFR in the model group were significantly decreased (P<0.05); and compared with the model group, the expression levels of AKT1 and EGFR in the low-dose group and the high-dose group were significantly decreased ( P <0.01), the above results showed that the water extract of Radix Diranthis Bidentatae could inhibit the expression of related proteins involved in the formation of hypertension.

[0030] Example 2 Application of Radix Dirunculatae in the preparation of drugs for treating complications of hypertension

[0031] 1. Analysis of the efficacy of water extract of Diren on myocardial fibrosis in hypertension In order to study the efficacy of the aqueous extract of Radix Dirunculatae on hypertensive myocardial fibrosis, after anesthetizing the rats, the heart tissues of the rats in each group were quickly isolated and fixed in 4% polyformic acid. After being embedded in paraffin, the sections were sliced ​​and stained with Masson and Sirius-red respectively, and the percentage of collagen content was measured to study the morphology of the myocardial tissue.

[0032] The specific steps of Masson staining are as follows: (1) Dewaxing of paraffin sections: Place the sections in xylene I for 12 minutes, xylene II for 12 minutes, anhydrous ethanol I for 6 minutes, 95% alcohol for 6 minutes, and 85% alcohol for 6 minutes, followed by washing with distilled water.

[0033] (2) Potassium dichromate mordant overnight: After the sections are dewaxed and hydrated, they are placed in potassium dichromate standard solution at room temperature overnight (about 17 hours).

[0034] (3) Ponceau staining: Stain with Ponceau acid fuchsin solution in the Masson staining kit for 5-10 minutes, and rinse quickly with distilled water.

[0035] (4) Phosphomolybdic acid treatment: Treat with the phosphomolybdic acid aqueous solution in the Masson staining kit for about 1-2 minutes.

[0036] (5) Aniline blue staining: No need to wash with water, directly use the aniline blue solution in the Masson staining kit to counterstain for 5 seconds.

[0037] (6) Differentiation: Treat with three cylinders of 1% glacial acetic acid for 5-10 seconds.

[0038] (7) Dehydration and sealing: Three cylinders of anhydrous ethanol and two cylinders of n-butanol are used for rapid dehydration for 15-30 seconds respectively, xylene is used for transparency, and neutral resin glue is used for sealing.

[0039] (8) Microscopic examination: image acquisition and analysis.

[0040] Results: Collagen fibers, mucus, and cartilage appear blue; muscle fibers, cellulose, and red blood cells appear red.

[0041] The specific steps of Sirius-red staining are as follows: (1) Dewaxing of paraffin sections: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and then wash with distilled water.

[0042] (2) Staining with picric acid picrosirius red staining solution: Stain the sections in saturated picric acid picrosirius red staining solution for 8 minutes.

[0043] (3) Rinse: Rinse the slices in anhydrous alcohol for several minutes and observe under a microscope until you are satisfied.

[0044] (4) Sealing: Bake the slices in a 60° oven, then transparentize them in xylene for 5 minutes and seal them with neutral gum.

[0045] (5) Microscopic examination, image acquisition and analysis.

[0046] Staining results: Collagen fibers are red and the background is yellow under an optical microscope.

[0047] Staining and analysis results Figure 8 、 Figure 9 and Figure 10 shown.

[0048] It can be seen that compared with the model group, the myocardial collagen fiber content of rats in the low-dose and high-dose Diran treatment groups was significantly reduced ( P <0.01); and, compared with the positive control group, the myocardial collagen fiber content of rats in the low-dose and high-dose Diran treatment groups was significantly lower ( P <0.05), indicating that the extract of Radix Diranthis Bidentatae has the function of anti-myocardial fibrosis and its efficacy is better than that of positive drugs.

[0049] 2. Analysis of the efficacy of the water extract of Diren on hypertension and inflammation The serum obtained after centrifugation of the whole blood was tested for IL-1β, TGF-β, and TNF-α levels according to the kit instructions. The test results are shown in Figure 11 、 Figure 12 and Figure 13 shown.

[0050] Depend on Figure 11 、 Figure 12 and Figure 13 It can be seen that compared with the normal group, the levels of IL-1β, TGF-β, and TNF-α in the model group rats were significantly increased ( P <0.01), indicating that hypertension caused an inflammatory response and increased the levels of inflammatory factors. Compared with the model group, the levels of IL-1β, TGF-β, and TNF-α in the rats in the low- and high-dose groups of Diren were significantly reduced ( P <0.01, P <0.05). This indicates that Diren can inhibit a series of inflammatory reactions caused by hypertension.

Claims

1. Application of Diren in the preparation of antihypertensive drugs.

2. The use according to claim 1, characterized in that include: The water extract or alcohol extract of the rhizome of ground dwarf is obtained, and the water extract or alcohol extract of the rhizome of ground dwarf is used to prepare a blood pressure lowering drug.

3. The use according to claim 2, characterized in that The method for obtaining the water extract or alcohol extract of Radix Glehniae comprises the following steps: S1: crushing the ground rhizome and extracting it with water or alcohol by reflux to obtain an extract; S2: After concentrating the extract, the water extract or alcohol extract of the Radix Glehniae is obtained.

4. The use according to claim 3, characterized in that The alcohol includes at least methanol and ethanol.

5. The use according to claim 1, characterized in that The blood pressure lowering drug is an oral preparation, an intravenous injection preparation or an external preparation.

6. Application of Di Ren in the preparation of drugs for the treatment of complications of hypertension.

7. The use according to claim 6, characterized in that The drugs for treating hypertension complications at least include drugs for treating hypertensive myocardial fibrosis and drugs for treating hypertension inflammation.

8. The use according to claim 6, characterized in that include: The water extract or alcohol extract of the rhizome of ground rhizome is obtained, and the water extract or alcohol extract of the rhizome of ground rhizome is used to prepare a drug for treating complications of hypertension.

9. The use according to claim 6, characterized in that The method for obtaining the water extract or alcohol extract of Radix Glehniae comprises the following steps: S1: crushing the ground rhizome and extracting it with water or alcohol by reflux to obtain an extract; S2: After concentrating the extract, the water extract or alcohol extract of the Radix Glehniae is obtained.

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