Application of asiatic toddalin in preparation of medicine for treating hypertension and / or Parkinson's disease

Abstract

The invention provides application of asiatic toddalin in preparation of a medicine for treating hypertension and / or Parkinson's disease, and belongs to the technical field of biological medicine. According to the application, the asiatic toddalin can be used for remarkably inhibiting the activation of CaV1.2 voltage-gated ion channels of different species. The CaV1.2 is inhibited as a receptor closely related to animal peripheral blood pressure perception, and can passivate the blood pressure induction of animals within a certain period of time. Experiments on hypertension model mice show that the endurance capacity of the mice to acute blood pressure induced by L-NAME can be reduced through treatment of the asiatic toddalin, and the blood pressure value of the mice is remarkably reduced. In conclusion, the asiatic toddalin reduces the blood pressure of a hypertension model mouse, can be developed into a potential antihypertensive molecule as a resource, and is applied to blood pressure reduction of human beings and other animals. Secondly, the asiatic toddalin acts on a receptor CaV1.2 in a targeting manner, and can be applied to research on related ion channel diseases and used as a potential therapeutic drug.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular relates to the application of pyrocaulin in the preparation of drugs for treating hypertension and / or Parkinson's disease. Background Art

[0002] Toddaculin is a coumarin compound derived from the plant Toddalia asiatica of the Rutaceae family. Currently, there is no report on the effect of toddaculin on the perception of blood pressure in humans and animals. Summary of the invention

[0003] The purpose of the present application is to provide the use of pyralidin in the preparation of drugs for treating hypertension and / or Parkinson's disease.

[0004] The present application provides a method for preparing a method for preventing and / or treating Ca V 1.2 Application in drugs for ion channel-related diseases; the chemical structural formula of the palmatum is shown in Formula I:

[0005]

[0006] Preferably, the Ca V 1.2 Ion channel-related diseases include one or more of hypertension, Parkinson's disease, arrhythmia, epilepsy and Alzheimer's disease.

[0007] Preferably, when the Ca V 1.2 When the ion channel-related disease is hypertension, the drug is used for at least one of the following aspects: 1) lowering blood pressure; 2) assisting in lowering blood pressure; 3) improving the resistance and / or perception of humans or animals to increased blood pressure.

[0008] Preferably, the hypertension includes acute hypertension or chronic hypertension.

[0009] Preferably, the hypertension includes hypertension caused by nitroso-L-arginine methyl ester.

[0010] Preferably, the drug is directed to humans or mammals.

[0011] Preferably, the dosage form of the drug includes an oral dosage form or an injection dosage form.

[0012] Preferably, the physalis hematin is the only active ingredient of the drug.

[0013] The present application also provides a method for preparing Ca V 1.2 Application in ion channel inhibitors; The chemical structural formula of the palmatum is shown in Formula I:

[0014]

[0015] Preferably, the palm blood element is the Ca V 1.2 The only active ingredient of ion channel inhibitor.

[0016] The present application provides a method for preparing a method for preventing and / or treating Ca V 1.2 Application in drugs for ion channel-related diseases. In this application, phyllanthin can significantly inhibit the Ca2+ expression of different species. V 1.2 Activation of voltage-gated ion channels. Electrophysiological experiments showed that pyrocalyx acts on Ca V 1.2 receptor, which inhibits Ca V 1.2 Activation of receptors. V 1.2 receptors are closely related to the perception of peripheral blood pressure in animals. When inhibited, they will blunt the animal's perception of blood pressure for a certain period of time. Experiments in hypertensive model mice have shown that feroxamine treatment can reduce the tolerance of mice to acute blood pressure induced by L-NAME, and the blood pressure values ​​of mice are significantly reduced. In summary, feroxamine reduces the blood pressure of hypertensive model mice and can be used as a resource to develop into a potential antihypertensive molecule for use in humans and other animals to reduce blood pressure. Secondly, feroxamine targets the receptor Ca V 1.2, can be applied to the study of related ion channel diseases and as potential therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 Ca V 1.2 Effect result diagram, where 1 is the control, 2 is 80mmol potassium chloride, 3 is 80mmol potassium chloride + 25μmol feroxanthin, and 4 is after elution;

[0019] Figure 2 The inhibition of Ca by phytosporin V 1.2 current and concentration effect curve, where A is the inhibition of Ca by phytosporin V 1.2 Current effect curve, B is the inhibition of Ca by phytomycin V 1.2 Concentration effect curve;

[0020] Figure 3 This is the curve of contraction of rat thoracic aorta rings vasodilated by cypermethrin;

[0021] Figure 4 This is the statistical result of the relaxation of rat thoracic aorta ring tension induced by high potassium by cypermethrin;

[0022] Figure 5 This is the time-blood pressure relationship diagram of the blood pressure lowering effect of cypermethrin;

[0023] Figure 6 This is a graph showing the changes in average blood pressure of mice after 4 weeks of administration of Phellodendron chinense blood. DETAILED DESCRIPTION

[0024] The present application provides a method for preparing a method for preventing and / or treating Ca V 1.2 Application in drugs for ion channel-related diseases; the chemical structural formula of the palmatum is shown in Formula I:

[0025]

[0026] In the present application, the toddalia hemoglobin is purchased from MedChemExpress or is obtained by collecting and extracting Toddalia asiatica of the Rutaceae family and the genus Toddalia, and after functional screening, separating and purifying the toddalia hemoglobin using techniques such as FCC, TLC, and Sephadex LH20.

[0027] In the specific implementation process of this application, the Ca V 1.2 Ion channel-related diseases include one or more of hypertension, Parkinson's disease, cognitive decline and memory impairment.

[0028] In the specific implementation process of this application, when the Ca V 1.2 When the ion channel-related disease is hypertension, the drug is used for at least one of the following aspects: 1) lowering blood pressure; 2) assisting in lowering blood pressure; 3) improving the resistance and / or perception of humans or animals to increased blood pressure.

[0029] In the specific implementation of the present application, the hypertension includes acute hypertension or chronic hypertension.

[0030] In the specific implementation of the present application, the hypertension includes hypertension caused by nitroso-L-arginine methyl ester.

[0031] In the specific implementation process of this application, the drug targets humans or mammals. In this application, the mammals include mice.

[0032] The present application does not specifically limit the dosage form of the antihypertensive molecule, and conventional dosage forms in the art can be used. The present application preferably applies the antihypertensive molecule to humans and animals by oral administration or injection to reduce blood pressure, overcome acute and chronic blood pressure in a short period of time (taking effect half an hour after administration), etc. In the specific implementation process of the present application, the dosage form of the drug includes an oral dosage form or an injection dosage form.

[0033] In the specific implementation of the present application, the phytomycin is the only active ingredient of the drug.

[0034] The present application also provides a method for preparing Ca V 1.2 Application in ion channel inhibitors; The chemical structural formula of the palmatum is shown in Formula I:

[0035]

[0036] Ca V 1.2 is a type of blood pressure-related voltage-gated calcium channel, which is abundantly expressed in peripheral nerve cells, especially in vascular smooth muscle. V The special distribution of 1.2, Ca V 1.2 was found to be closely related to various physiological and pathological functions of blood pressure. In the present application, the palm blood can specifically target the receptor Ca V 1.2, thereby inhibiting Ca V 1.2 Voltage activation of calcium ion channels can lower the blood pressure of animals within a certain period of time (taking effect half an hour after administration). Therefore, pyrocatecholamine can be used as a resource to develop into a potential antihypertensive drug for use in humans and other animals to lower blood pressure. V 1.2, can be applied to the research of related ion channel diseases, such as anti-hypertension, cognitive decline, memory impairment and Parkinson's disease, and as a potential therapeutic drug. In this application, the application of phyllophaga hemoglobin on different species is universal.

[0037] In the specific implementation process of this application, the dragon palm blood element is the Ca V 1.2 The only active ingredient of ion channel inhibitor.

[0038] In order to further illustrate the present application, the application of the palm blood provided by the present application in the preparation of drugs for treating hypertension and / or Parkinson's disease is described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0039] Example 1

[0040] Pterocarpin and mCa V 1.2 Electrophysiological experiments targeting blood pressure-related receptors

[0041] mCa V 1.2 Plasmids were transiently transfected and overexpressed in HEK293T cell lines. All HEK293T cell lines were cultured with DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0042] Under an inverted microscope, cells with smooth cell membranes and uniform cytoplasm were selected, and patch clamp experiments were performed at room temperature of 20-25°C. WPI 0.86mm thin-walled borosilicate glass capillaries were selected as glass electrode materials. The glass electrodes were drawn on a drawing instrument (P-97, Shutter) in 5 steps. The diameter of the tip of the glass electrode was 1.5-3.0μm after thermal polishing. After drawing, the intracellular fluid was filled into the glass electrode. The initial resistance of the glass electrode was 1.5-2.5MΩ. After a high-impedance GΩ seal was formed between the electrode and the cell membrane, the fast capacitance of the electrode was compensated. Then a short and strong negative pressure was applied to quickly break the cell membrane clamped in the electrode, and then compensate the slow capacitance of the cell. After the whole-cell recording mode was formed, the cell was clamped to 0mV, and the cell was stabilized for 10min before the current was recorded using an appropriate pulse voltage (80 / -80mV). The drugs were perfused using BiolabRS200, and the switching speed between drugs was 50ms. The series resistance (Rs) was kept constant within the range of 5-8MΩ during the experiment, and the system series resistance compensation was generally between 30% and 60%. The experimental data were analyzed using PatchMaster software, and the data were further analyzed using Igor software. All results are expressed in the form of mean ± standard error (SEM), and n represents the number of experimental data.

[0043] The cells are slowly lifted by the electric micromanipulator system until they are flush with the bottom edge of the drug-adding port of the rapid drug-adding switching system (RSC-200). The drug-adding switching system is directly connected to the amplifier, and all experimental data are recorded in the computer software PatchMaster through the amplifier. Cells are stimulated and treated with drugs through PatchMaster and the switching drug-adding system, and the changes in channel current are recorded to detect the activity of different drugs on the channel. To ensure the accuracy of the experimental data, the sealing resistance and series resistance must be kept stable throughout the recording process.

[0044] Ca V1.2 Channel external solution: 2.5mM CsCl, 140mM TEA-Cl, 0.6mM MgCl2, 5mM CaCl2, 10mMglucose and 10mM HEPES, adjusted to pH 7.4 with NaOH. The drugs used in the experiment need to be dissolved and prepared with the above electrode external solution.

[0045] Ca V 1.2 Channel solution: 140 mM CsCl, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPESand 5 mM Mg-ATP, adjusted to pH 7.3 with CsOH.

[0046] The results are as follows Figure 1 As shown, pyrenophytin inhibits Ca V 1.2 channel activation, and showed concentration dependence.

[0047] Example 2

[0048] Ca V 1.2 Channel inhibitory activity

[0049] Electrophysiological experiments were performed at room temperature using an EPC-10 amplifier controlled by Patch-Master software (HEKA Elektronik, Lambrecht, Germany). For whole-cell recordings, 80% series resistance compensation was used to reduce all possible voltage errors. Capacitive artifacts were eliminated using the computer control circuit of the patch-clamp amplifier. Current sampling was performed at 10 kHz and filtered at 2.9 kHz. The electrode tubes were made of very clean borosilicate glass and then polished with heating resistor wire to achieve a resistance of 3 to 5 MΩ as the optimal resistance. Conductance-voltage experiments were solved by evaluating the current with voltage steps starting from deeply hyperpolarizing voltages. Dose-response relationships were determined by perfusing different doses of the test sample using a solution exchanger (RSC-200, Bioscience Instruments) with the tubes connected. The specific tube number sent by the solution exchanger was fed to the analog input of the EPC-10 patch-clamp amplifier, and the stable current amplitudes of different doses before and after perfusion were recorded simultaneously. Recording of Ca V 1.2 channels, the electrode internal solution consisted of (mM): 110CsCl, 5MgSO4, 10EGTA, 25HEPES and 5Mg-ATP (pH7.2), and the electrode external solution consisted of (mM): 125TEA-Cl, 10BaCl2, 5D-glucose and 5HEPES (pH 7.4).

[0050] By whole-cell electrophysiological recording, it was found that 100 μM pyroheparin almost completely inhibited the CaV 1.2 channel current, while 75 μM cypermethrin inhibited 72.90% of the channel current. Cypermethrin inhibited Ca V 1.2 channels, the half inhibition concentration is 12.56μM, the results are shown in Figure 2 .

[0051] Example 3

[0052] Vasodilatory activity of pyralidin in thoracic aorta

[0053] Male Sprague Dawley rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) and subsequently killed by cervical dislocation. The rat thoracic aorta was removed and placed in cold (4°C) Kleinstein solution, the solution composition of which is as follows (mM): 117NaCl, 25NaHCO3, 1.2NaH2PO4, 4.7KCl, 1.2MgCl2, 2.5CaCl2 and 11.5glucose, and oxygenated by a mixture of 95% O2 and 5% CO2 to maintain a pH of 7.4. The aorta was stripped of attached connective tissue and cut into 4 mm long rings. The aortic segment was placed on two stainless steel wires passed through the heart tube. The isometric tension of the aortic ring was recorded using a device connected to a recording device (Taimeng). Results can be found in Figure 3 and Figure 4 It can be seen that Toddaculin relaxes the constriction of rat arterial rings induced by high potassium in a concentration-dependent manner.

[0054] Example 4

[0055] Antihypertensive activity of physalis

[0056] Construction and administration of mouse hypertension model: Female C57BL / 6 mice aged 6 to 8 weeks (19 to 26 g) were housed under specific pathogen-free conditions, with controlled temperature (23 ± 2 °C) and humidity (55 ± 5%) and a 12-h light-dark cycle. Animals were housed according to standard regulations, 4 per cage, with free access to water and standard food. All mice were randomly assigned to different experimental groups and numbered. Data collection and testing for all experiments were performed double-blind.

[0057] The experiment was conducted under constant temperature (23±2℃) and humidity (55±5%) control and 12h light-dark cycle. 300mg / L nitroso-L-arginine methyl ester (L-NAME) was used to establish a hypertension model. The drug was dissolved in drinking water and mice were free to drink water. No drug was added to the drinking water of mice in the blank control group. The drug treatment was carried out according to the following dosing schedule:

[0058] Group 1: drinking water + solvent;

[0059] Group 2: 300 mg / LL-NAME (in drinking water) + solvent;

[0060] Group 3: 300mg / LL-NAME+30mg / kg chloranthine.

[0061] The solvent was 2% DMSO + olive oil. Each group was set up with 8 replicates. The drug was given by gavage once a day at 3 pm for 30 consecutive days.

[0062] The purified cannabinoids are administered according to the following dosing regimen (taking Cannabis sativa L. as an example):

[0063] Group 4: drinking water + solvent;

[0064] Group 5: 300 mg / LL-NAME (in drinking water) + solvent;

[0065] Group 6: 300 mg / LL-NAME + 5 mg / kg Nifedipine (positive control, nifedipine);

[0066] Group 7: 300mg / LL-NAME+5mg / kg phytosphingolipids;

[0067] Group 8: 300mg / LL-NAME+10mg / kg chloranthine.

[0068] The solvent was 2% DMSO + olive oil. Each group was set up with 8 replicates, and the administration method and time were the same as above.

[0069] Gavage is completed in a negative pressure workbench. Use a 1ml sterile syringe to draw the experimental drug solution, replace the syringe needle with a round-headed gavage needle, and set it aside after expelling the bubbles. Take out the mouse to be gavaged and place it in a mouse cage. Pull the mouse's tail. When it is in a state of crawling forward, take the opportunity to grab the fur on the back of the neck from the tail forward. The tightness of the fixation should be moderate to prevent the mouse's esophagus and trachea from being blocked. Use your other hand to gently pinch the syringe, insert the gavage needle head into the mouse's mouth from the corner of its mouth, slide along the upper palate wall into the esophagus, and reach the end of the esophagus. If the mouse has a strong resistance reaction during the insertion of the needle, it may be that the needle has entered the trachea. The gavage needle should be withdrawn in time and reinserted. See results for details. Figure 5 and 6 .

[0070] The above-mentioned mouse-related behavioral experiments showed that intraperitoneal injection of pyrocatecholamine can help mice lower their blood pressure. Pyrocatecholamine reduces the blood pressure of L-NAME-induced hypertensive mice in a dose-dependent manner and enhances the resistance of mice to blood pressure. It can be used as a resource to develop into a potential antihypertensive molecule for use in humans and other animals to lower blood pressure. Secondly, pyrocatecholamine targets the receptor CaV 1.2, can be applied to the study of related ion channel diseases and as potential therapeutic drugs.

[0071] Although the above-mentioned embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. Application of Pterocarpin in the Preparation of Drugs for the Prevention and / or Treatment of Ca V 1.2 Application in drugs for ion channel-related diseases; the chemical structural formula of the palmatum is shown in Formula I:

2. The use according to claim 1, characterized in that: The Ca V 1.2 Ion channel-related diseases include one or more of hypertension, Parkinson's disease, arrhythmia, epilepsy and Alzheimer's disease.

3. The use according to claim 2, characterized in that: When the Ca V 1.2 When the ion channel-related disease is hypertension, the drug is used for at least one of the following aspects: 1) Lowering blood pressure; 2) Assist in lowering blood pressure; 3) Improve the resistance and / or perception of increased blood pressure in humans or animals.

4. The use according to claim 2 or 3, characterized in that: The hypertension includes acute hypertension or chronic hypertension.

5. The use according to claim 4, characterized in that: The hypertension includes hypertension caused by nitroso-L-arginine methyl ester.

6. The use according to claim 1, characterized in that: The subjects of the drug include humans or mammals.

7. The use according to claim 1, characterized in that: The dosage form of the drug includes an oral dosage form or an injection dosage form.

8. The use according to claim 1, characterized in that: The palm blood is the only active ingredient of the drug.

9. Preparation of Ca V 1.2 Application in ion channel inhibitors; The chemical structural formula of the palmatum is shown in Formula I:

10. The use according to claim 9, characterized in that: The palm blood element is the Ca V 1.2 The only active ingredient of ion channel inhibitor.

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