WO2021208985A1 - USE OF QUERCETIN-3'-O-β-D-GLUCOSIDE AS CALCIUM CHANNEL INHIBITOR - Google Patents

Abstract

Provided is the use of quercetin-3'-O-β-D-glucoside as a calcium channel inhibitor, which belongs to the medical field. Also provided is the use of the quercetin-3'-O-β-D-glucoside in the preparation of a drug for treating cardiovascular diseases, coronary heart disease, atherosclerosis, advanced renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases. The present invention demonstrates that the quercetin-3'-O-β-D-glucoside can inhibit a calcium channel, especially the TRPC ion channel, and expands the use of the quercetin-3'-O-β-D-glucoside.

Description

Application of quercetin-3'-O-β-D-glucoside as an inhibitor of calcium ion channels Technical field

The technology of the present invention belongs to the field of medicine, and specifically relates to the application of quercetin-3'-O-β-D-glucoside.

Background technique

Cardiovascular disease is the most harmful disease to human life and health in modern society. According to the World Health Organization, approximately 17.9 million people died of cardiovascular disease globally in 2016, accounting for 31% of the total global deaths. It can be seen that the medical needs of new drugs for the prevention and treatment of cardiovascular diseases are very urgent. Relevant studies have proved that TRPC channels are important pharmacological targets for the development of new drugs for cardiovascular diseases such as cardiomyopathy, heart failure, hypertension, and cerebrovascular diseases. Patent application WO2006/074802A1 discloses the use of TRPC channels in the treatment of cardiovascular and cerebrovascular diseases. Its research shows that the use of genetic technology in rabbit atherosclerosis models can significantly improve by inhibiting the activity of vascular endothelial cells TRPC3\TRPC6 and TRPC7 Vascular function and vascular pathological changes of atherosclerosis.

The TRPC channel is a ^{non-selective ion channel permeated by Ca 2+} , which is widely present in mammalian tissues. According to structural homology and functional orientation, the TRPC family can be divided into four subgroups: TRPC1 and TRPC2 each constitute a subgroup; there is about 65% amino acid homology between TRPC4 and TRPC5, so they are classified as The same subgroup; TRPC3, TRPC6 and TRPC7 have 70-80% amino acid homology, and the three are classified into the same subgroup. TRPC3, TRPC6, and TRPC7 channels share a common activation mechanism. The endogenous ligands are currently known as diacylglycerol (DAG) and 4-ethyl-(3-(4-fluorophenyl)-7-hydroxy-2 -Methylpyrazole[1,5-a]-pyrimidin-5-yl)piperidine-1-carboxylate (M085). Currently known organic inhibitors of TRPC include 2-aminoethoxydiphenylboronic acid (2-APB), SKF96365, YM-58483 (BTP2), and inorganic blockers (such as Gd ³⁺ and La ³⁺ ), etc. However, they lack sufficient effectiveness and specificity. At present, the natural composition, activation mechanism, physiological function and role of TRPC in pathophysiology and disease are still unresolved issues. Because of their extensive and partially overlapping distribution, potential heteromultimerization, similar electrophysiological properties, and lack of compound tools to clearly track these channels, it is difficult to identify natural TRPC channels in situ.

The research of Dietrich et al. proved that through the study of transgenic mouse models, some possible physiological functions of TRPC may be unlocked, and the potential of TRPC3, 6, and 7 subfamily in vitro and in vivo heteroterminalization was summarized, and they provided the down-regulation of channel activity. Preliminary data on physiological function analysis in isolated tissue and gene defect mouse models. However, due to the lack of specific channel blockers, it is susceptible to the compensation effect of the channels closely related to the TRPC channel. Therefore, it is impossible to determine the physiological relevance of the TRPC homotype or heteroisomer in the complex organ function of the entire body. This must be overcome. Defects require targeted gene inactivation in embryonic stem cells and subsequent production of gene-deficient mouse models for each channel and channel subfamily. The generation and analysis of these model systems is time-consuming and cost-intensive, and there are certain limitations.

The hollyhock flower is the dried flower of Abelmoschus Manihot (L.) Medic, a plant belonging to the okra family of the Malvaceae. The hollyhock flower was first recorded in "Jiayou Materia Medica". It is widely distributed and rich in resources. Cold, slippery and non-toxic. It mainly treats urinary drenching and promotes birth, treats all malignant sores and puss that have not succumbed for a long time.

The hollyhock flower contains a variety of chemical components, including: gallic acid, 5-hydroxymethyl-2-furanoic acid, protocatechuic acid-3-O-β-D-glucoside, protocatechuic acid, acortatarin A, cotton Cortin-3-O-β-D-glucose-8-O-β-D-glucuronide, quercetin-3-O-[β-D-xylosyl(1→2)-α- L-rhamnosyl 1→6)]-β-D-galactoside, myricetin-3-O-β-D-galactoside, myricetin-3-O-β-D-glucoside, quercetin Cortin-3-O-β-D-xylosyl-(1→2)-β-D-galactoside, quercetin-3-O-lochoside, rutin, hyperoside, isoquerque Cortex, myricetin-3′-O-β-D-glucoside, gospelin-3′-O-β-D-glucoside, gospelin-8-O-β-D-glucuronide , Myricetin, quercetin-3'-O-β-D-glucoside, quercetin, etc.

At present, there is no research showing the inhibitory effect of quercetin-3'-O-β-D-glucoside on TRPC channels, and the preparation and treatment of TRPC channel-related cardiovascular diseases, coronary heart disease, atherosclerosis, and advanced renal disease. Use in medicines for failure, neurological diseases, chronic pain, acute pain or inflammatory diseases.

In view of this, the present invention provides a hollyhock flower extract as an inhibitor of TRPC ion channels and preparation for treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease The use of the drug proves that the quercetin-3′-O-β-D-glucoside in its extract is a new pharmacological tool that can selectively inhibit TRPC ion channels and can neutralize TRPC subfamily There are internal distinctions, which can clarify the role of different channels under physiological and pathophysiological conditions, open up ideas for cardiovascular and cerebrovascular diseases, and expand the use of hollyhock flower extracts.

Summary of the invention

The technical scheme of the present invention is as follows:

the term:

1. The terms "TRPC channel", "TRPC ion channel" or "TRPC" in the context of the present invention refer to a non-selective cation channel ^{permeable to Ca 2+.} It refers to any one of the following list of typical ion channels for transient receptor potentials: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7. Particularly preferred are TRPC3, TRPC6 and TRPC7.

Such TRPC ion channels can be derived from any vertebrate, and particularly mammals (such as dogs, horses, cows, mice, rats, canines, rabbits, chickens, apes, humans, or others). TRPC can be isolated from the tissue detection agent of this vertebrate organism, or can be manufactured by a method of recombinant biological material capable of expressing TRPC protein.

This term can refer to natural polypeptides, polymorphic variants, mutants, and interspecies homologs.

2. The term "pharmacological tool" in the context of the present invention refers to compounds and compound combinations whose functional properties can be used to study how drugs interact with living organisms to produce changes in the intended function, so as to be able to study new pharmaceutical compositions, and Nature, interaction, toxicology, treatment, medical treatment and disease resistance. Moreover, the term refers to compounds that can be used to characterize potential targets in the development of new drugs, such as characterizing their natural components, activation mechanisms, physiological functions, and their role in pathophysiology and disease.

3. The term "TRPC ion channel modulator" in the context of the present invention refers to a TRPC channel modulator molecule, especially an inhibitory or activating molecule ("inhibitor" or "activator"), especially a molecule according to the present invention The method can identify inhibitors of TRPC channels. The inhibitor is usually a compound, as described in detail above, for example, binding, partially or totally blocking activity, reducing, preventing, delaying activation, inactivating, desensitizing or down-regulating the activity or expression of at least one TRPC channel . The activator is usually a compound, as described in detail above and preferably, for example, increasing, opening, activating, promoting, enhancing activation, sensitizing, agonizing or up-regulating the activity or expression of at least one TRPC channel. Such modulators include genetically modified versions of TRPC channels, preferably inactivated mutants of TRPC channels, and naturally-occurring or synthetic ligands, antagonists, agonists, peptides, cyclic peptides, nucleic acids, antibodies, antisense molecules, Ribozymes, small organic molecules, etc. Examples of TRPC activators are diacylglycerols, especially 1-oleoyl-2 acetyl-sn-glycerol (OAG); Gq-coupled receptor agonists, such as phenylephrine, especially trypsin; stimulate receptors An agonist of tyrosine kinase such as epidermal growth factor (EGF); or a diacylglycerol generating enzyme such as phospholipase or its activator. An example of the measurement of the regulation of TRPC ion channel activity in the presence of a test compound is as follows: Generally, cells expressing the TRPC channel are provided. Such cells can be produced using genetic methods known to those skilled in the art. After the induction of expression of the TRPC channel, the cells are usually placed in, for example, a microplate and grown. Usually the cells grow and fix on the bottom of the multiwell plate. Then, wash these cells routinely and add a dye in a suitable loading buffer, preferably a fluorescent dye such as fluo4am. After removing the loading buffer, the cells are incubated with test compounds or modulators (especially the above-mentioned biochemical or chemical test compounds, for example in the form of a chemical compound library). The Ca ²⁺ measurement can be read by using, for example, fluorescence imaging. In order to stimulate the Ca2+ influx through the TRPC channel, channel activators such as OAG and 4-ethyl-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyridine are usually used. Azole[1,5-a]-pyrimidin-5-yl)piperidine-1-carboxylate (M085). The expected effect of the inhibitor is, for example, a decrease in the increase in fluorescence. The activator causes, for example, the activator to induce fluorescence Further increase, or induce, for example, an activator-independent fluorescence enhancement. After that, suitable modulators, especially inhibitors, can be analyzed and/or separated. Preferably, chemical compounds are analyzed using high-throughput analysis known to the skilled person or commercially available Screening of the library.

4. The term "TRPC-expressing cell" in the context of the present invention refers to a cell or recombinant cell that endogenously expresses the ion channel of interest. The cells are usually mammalian cells, such as human cells, mouse cells, rat cells, Chinese hamster cells, and the like. Cells that have been found to be convenient to use include MDCK, HEK 293, HEK 293T, BHK, COS, NIH3T3, Swiss3T3 and CHO cells, with HEK293 cells being preferred.

5. The term "tissue" in the context of the present invention refers to any type of tissue product, or part of a tissue or organ (such as brain, liver, spleen, kidney, heart, blood vessel, muscle, skin, etc.), and also refers to any type of tissue Body fluids such as blood, saliva, lymph fluid, synovial fluid, etc.) are preferably derived from vertebrates, and more preferably derived from mammals such as humans. Tissue samples can be obtained by well-known techniques, such as blood sampling, tissue puncture, or surgical techniques.

6. The term "drug" in the context of the present invention refers to a therapeutic agent containing a therapeutically effective amount of quercetin-3'-O-β-D-glucoside, or a plant extract containing the compound. The drug can be administered systemically or locally in any traditional way. This can be done, for example, by oral dosage forms such as tablets, granules or capsules, by mucosal methods such as the nasal cavity or oral cavity, depot preparations implanted under the skin, by injections, infusions or gels containing the medicament according to the invention. Methods. If appropriate, in order to treat the above-mentioned specific diseases, it can also be administered topically and locally in the form of liposome complexes. The drug can also be administered in the form of injection or infusion. If it is only a relatively small amount of solution or suspension, for example, about 1 to 20 mL, injection is generally used to administer the body.

In one aspect, the present invention provides the use of quercetin-3'-O-β-D-glucoside in the preparation of drugs for inhibiting calcium ion channels.

As an exemplary or preferred example, in the application, quercetin-3'-O-β-D-glucoside is added in the form of the main active ingredient.

As an example, the present invention provides the application of quercetin-3'-O-β-D-glucoside in the preparation of drugs for inhibiting calcium ion channels.

As an exemplary or preferred example, the above-mentioned calcium ion channel is a TRPC channel (or referred to as a TRPC ion channel).

As an exemplary or preferred example, the above-mentioned TRPC channel TRPC3, TRPC6 or TRPC7 channel.

As an exemplary or preferred example, the aforementioned calcium ion channel refers to a calcium ion channel that quercetin-3'-O-β-D-glucoside is used to inhibit in vitro and in vivo.

On the other hand, the present invention provides quercetin-3′-O-β-D-glucoside in the preparation, diagnosis, treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, Use in medicine for chronic pain, acute pain or inflammatory diseases.

As an example, the present invention provides quercetin-3'-O-β-D-glucoside in the preparation of diagnosis, treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic Use in medicine for pain, acute pain or inflammatory diseases.

As an exemplary or preferred example, the medicament further contains one or more pharmaceutically acceptable carriers or adjuvants. Pharmaceutically acceptable carriers or auxiliary agents are, for example, physiological buffer solutions such as sodium chloride solution, demineralized water, stabilizers such as protease or nuclease inhibitors, or chelating agents such as EDTA.

On the other hand, the present invention provides a plant extract which contains more than 0.5% by weight of quercetin-3'-O-β-D-glucoside; further said The plant extract contains 0.5-2.0% by weight of quercetin-3′-O-β-D-glucoside; further, the plant extract contains 0.8-1.6% by weight of quercetin Cortin-3'-O-β-D-glucoside; preferably, the plant is a Hibiscus plant, a Malvaceae plant, and more preferably a yellow sunflower, a hollyhock, a sunflower, a grape leaf hibiscus, a goat carob, and a millet One or more of.

On the other hand, the present invention provides the application of the above-mentioned plant extracts in the preparation of medicines for treating diseases mediated by calcium ion channels.

As an exemplary or preferred example, the plant extract is Abelmoschus manihot flower extract. The hollyhock flower extract is an ethanol extract, preferably an extract with 50-95% ethanol reflux, and more preferably an extract with 80-95% ethanol reflux.

The hollyhock flower extract of the present invention can be prepared by the following method: taking hollyhock flower medicinal materials, extracting with ethanol under heating and refluxing, filtering, concentrating the filtrate, and drying. Further, it is preferably prepared by the following method: the hollyhock flower is extracted with 85%-95% ethanol at reflux for 1 to 3 times, each for 1 to 2 hours, filtered, the filtrate is combined to recover the ethanol, and the filtrate is concentrated to a specific gravity of 1.20 to 1.35. The concentrated solution is allowed to stand at 0°C to 4°C for 24 to 48 hours to remove the oil layer of the cold storage solution, adjust the pH to 6.0 to 7.0, concentrate, and dry the thin layer quickly or vacuum dry to obtain the hollyhock flower extract.

The preparation method of the hollyhock flower extract of the present invention can be: the hollyhock flower is extracted twice with 95% ethanol, refluxed for 1 hour each time, filtered, the filtrate is combined to recover the ethanol, the filtrate is concentrated to a specific gravity of 1.20, and the concentrated solution is at 0 Let stand for 24 to 48 hours at ℃～4℃, remove the oil layer of the refrigerating liquid, adjust the pH value to 6.0, slowly add the refrigerating liquid into the thin-layer quick-drying drum tank, so that the liquid level of the refrigerated liquid in the drum tank just touches the surface of the drum body , Preheat the surface temperature of the roller body to 140℃～150℃, the air pressure is 0.4～0.5Mpa, turn on the roller rolling start button, the rotation speed of the cylinder body is 3～3.5 minutes/revolution, and apply the rolled extract liquid on the poly four The temperature of the vinyl fluoride board is lowered, the materials to be dried are cooled and become brittle, crushed, and put into a clean double-layer plastic bag to obtain the hollyhock flower extract.

The conditions for the fast drying operation of the thin layer are as follows: preheat the surface temperature of the thin layer fast drying drum to 135℃～160℃, the air pressure is 0.3～0.6Mpa, the rotation speed of the drum is 2～4.5 minutes/revolution, and the coated plate is a plastic plate. Or stainless steel plate, plastic plate is selected from polyethylene plate, PVC plastic plate, PP plastic plate, PE plastic plate, polytetrafluoroethylene plate, preferably polytetrafluoroethylene plate.

The preferred preparation method of the hollyhock flower extract of the present invention is: the hollyhock flower is extracted twice with 95% ethanol, refluxed for 1 hour each time, filtered, the filtrate is combined to recover the ethanol, the filtrate is concentrated to a specific gravity of 1.20, and the concentrated solution is at 0 Let stand for 24 to 48 hours at ℃～4℃, remove the oil layer of the refrigerated liquid, adjust the pH value to 6.0, and slowly add to the vacuum belt dryer for vacuum belt drying after concentration.

As an example, the preparation method of the above-mentioned hollyhock flower extract is: take 4000g of the medicinal hollyhock flower, use 15 times (mass/volume ratio) of 95% ethanol, reflux for 2 times, 1 hour each time, filter, combine the filtrate to recover the ethanol , Concentrate the filtrate to a specific gravity of 1.20, let the concentrate stand for 24 hours at 0℃～4℃, remove the oil layer of the refrigerated liquid, adjust the pH to 6.0, slowly add to the dryer after concentration, dry, crush, and put it into a clean double-layer plastic bag In the middle, the hollyhock flower extract is obtained.

On the other hand, the present invention provides a hollyhock flower extract which contains more than 0.5% by weight of quercetin-3'-O-β-D-glucoside Further, the hollyhock flower extract contains 0.5-2.0% by weight of quercetin-3'-O-β-D-glucoside; further, the hollyhock flower extract contains by weight It counts as 0.8-1.6% of quercetin-3'-O-β-D-glucoside.

On the other hand, the present invention provides the application of the above-mentioned Abelmoschus manihot flower extract in the preparation of medicines for the treatment of diseases mediated by calcium ion channels.

On the other hand, the present invention provides the use of the above-mentioned hollyhock flower extract for the diagnosis, treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease. Use in medicine.

In another aspect, the present invention provides a medicine for the treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease, said medicine comprising Quercetin-3'-O-β-D-glucoside.

As an example, the present invention provides a medicine for the treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease, and the medicine comprises quercetin Element-3'-O-β-D-glucoside.

On the other hand, the present invention provides a new pharmacological tool that can distinguish between and within TRPC subfamilies. This can clarify the role of different channels under physiological and pathophysiological conditions. That is, the present invention provides a pharmacological tool for characterizing channels belonging to different TRPC subfamily, and the pharmacological tool includes quercetin-3'-O-β-D-glucoside.

According to the present invention, this is achieved by inhibiting TRPC3, TRPC6 and TRPC7 with quercetin-3'-O-β-D-glucoside. Therefore, quercetin-3'-O-β-D-glucoside can pharmacologically distinguish channels belonging to different TRPC subfamily. In addition, quercetin-3'-O-β-D-glucoside does not interfere with the common G protein-coupled receptor, Gq, and phospholipase Cβ pathways that mediate the activation of TRPC channels in many cells. These characteristics make quercetin-3'-O-β-D-glucoside a preferred tool for identifying and regulating TRPC3, TRPC6 and TRPC7.

As an inhibitor of TRPC3, TRPC6 and TRPC7, Quercetin-3′-O-β-D-glucoside can be used as a pharmacological tool to characterize channels belonging to different TRPC subfamily and distinguish TRPC3/6 /7 subfamily members and other ion channel family members (Figure 1-6).

As such an inhibitor, quercetin-3'-O-β-D-glucoside can be further used as a tool compound for developing and validating assays to measure the activity of related ion channels. An example of such an assay is shown in Figure 1-3.

On the other hand, the present invention provides: under physiological and pathophysiological conditions, the use of quercetin-3'-O-β-D-glucoside for differential analysis of channel function of TRPC3/6/7 subfamily members . This can be done as described in the examples. The analysis can be performed in cells, tissues or animals. The animal may be a rodent, preferably a mouse or a rat.

According to a preferred embodiment, the regulation of natural TRPC by quercetin-3'-O-β-D-glucoside can be studied using the HEK293 cell line, which is used to study endogenously expressed TRPC ion channels Validation model system. Further details of this preferred measurement system are given in the Examples and Figures 1-6.

On the other hand, the present invention provides a method for determining the effect of quercetin-3'-O-β-D-glucoside on the activity of TRPC channels. Preferably, the TRPC ion channels are TRPC3, TRPC6 and TRPC7.

Generally speaking, cells expressing TRPC ion channels are in contact with quercetin-3′-O-β-D-glucoside, and the effect of quercetin-3′-O-β-D-glucoside on TRPC is measured or detected. The effect of ion channel activity.

On the other hand, the present invention provides a method for identifying modulators of TRPC ion channels, preferably TRPC ion channels are TRPC3, TRPC6 and TRPC7.

Generally, cells expressing TRPC ion channels are contacted with a test compound, and the effect of the test compound on the activity of the TRPC ion channel is measured or detected.

In an embodiment, the cells used in the above methods are fluorescent cells.

The preferred cells according to the present invention are MDCK, HEK293, HEK293T, BHK, COS, NIH3T3, Swiss3T3 or CHO cells, especially HEK293 cells.

The activity of TRPC channels can be measured or detected by, for example, patch clamp techniques, whole cell currents, radiolabeled ion currents, or especially fluorescence (for example, using voltage-sensitive dyes or ion-sensitive dyes) to measure or detect changes in ion currents, especially Ca ²⁺ ion currents. To perform measurement or detection.

An example of a TRPC channel activity assay is an assay that includes the following steps:

(1) Contact quercetin-3'-O-β-D-glucoside with fluorescent cells expressing TRPC ion channels, and use a channel activator to stimulate Ca ^{2+ influx} before, at the same time or after the contact;

(2) Detect changes in the activity of TRPC ion channels.

In another aspect, the present invention provides: a method for describing the selectivity of quercetin-3'-O-β-D-glucoside to TRPC channels, comprising evaluating quercetin-3'-O-β-D- The ability of glucoside to inhibit the activity of TRPC channels.

The present invention has the following beneficial effects:

(1) The present invention provides a pioneering use of one or more compositions of quercetin-3'-O-β-D-glucoside, and its application in the preparation of drugs for inhibiting calcium ion channels And its related uses.

(2) In order to prepare drugs for the treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, advanced renal failure, neurological diseases, chronic pain, acute pain and inflammatory diseases, especially TRPC channel-related drugs, and to develop the selectivity of TRPC ion channels Inhibitors provide new ideas;

(3) Expanded the use of the extract of Abelmoschus manihot.

Description of the drawings

Figure 1 is a graph showing the changes ^{of Ca 2+} fluorescence intensity in TRPC3HEK293 cells caused by different doses of quercetin-3'-O-β-D-glucoside over time;

Figure 2 is a graph showing the changes in the fluorescence intensity ^{of Ca 2+} in TRPC6HEK293 cells caused by different doses of quercetin-3'-O-β-D-glucoside over time;

Fig. 3 is a graph showing the changes ^{of Ca 2+} fluorescence intensity in TRPC7HEK293 cells caused by different doses of quercetin-3'-O-β-D-glucoside over time.

Figure 4 is a graph showing changes in the fluorescence intensity ^{of Ca 2+} in TRPC3HEK293 cells caused by extracts of Abelmoschus manihot over time;

Figure 5 is a graph showing changes in the fluorescence intensity ^{of Ca 2+} in TRPC6HEK293 cells caused by the extract of Abelmoschus manihot over time;

Fig. 6 is a graph showing changes in the fluorescence intensity ^{of Ca 2+} in TRPC7HEK293 cells caused by the extract of Abelmoschus manihot over time.

Detailed ways

In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the following examples are combined to further illustrate the present invention. However, the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the implementation manners, other examples obtained by those skilled in the art without creative work shall fall within the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The structure of the quercetin-3'-O-β-D-glucoside compound involved in the embodiment of the present invention is:

Preparation Example: Preparation of Abelmoschus manihot flower extract

Take 3000g of hollyhock flower, and extract hollyhock flower with 19 times 95% ethanol, reflux for 2 times, 1 hour each time, filter, combine the filtrate to recover ethanol, concentrate the filtrate to a specific gravity of 1.20, and let the concentrate stand at 0℃～4℃ for 48 After hours, remove the oil layer of the refrigerated liquid, adjust the pH value to 6.0, slowly add to the vacuum belt dryer after concentration, dry at 100°C, pulverize, and put into a clean double-layer plastic bag to obtain the hollyhock flower extract. Among them, it contains quercetin-3-O-robin glycoside 5.6 (mg/g), isoquercitrin 12.2 (mg/g), quercetin-3′-O-β-D-glucoside 11.2 (mg/g) g).

Example 1-6

(1) Transfect the cDNA plasmid vector containing TRPC3, TRPC6 or TRPC7 ion channels into the HERK293 cell line, and then select the corresponding antibiotic incubation cells according to the resistance of the plasmid to selectively screen the successfully transfected cells. Perform functional tests on surviving cells to confirm the expression and function of channel proteins, and then clone and purify them through a "limiting dilution" process to obtain stable cell lines stably expressing specific channels.

(2) Add 150 μL of cell suspension to each well of the stable cells obtained in step (1) at 10-13×104 cells/mL and add them to a 96-well plate with a black wall bottom. After 24h in the incubator, it can be used for follow-up experiments.

(3) Observe the cells, and after confirming that the cells are in good condition, load the dye (fluo-4) for 60 minutes.

(4) Prepare channel activator and inhibitor solutions as follows:

1. Preparation of TRPC6 agonist (M085): Weigh an appropriate amount of M085 and dissolve it in dimethyl sulfoxide (DMSO) to obtain a mother liquor of M085 with a concentration of 10 mM. In the FLIPR experiment, Locke’s buffer and other reagents were added according to the experimental procedure to make the final concentration of M085 1μM.

2. Pharmaceutical preparation: Alibaba hollyhock flower extract solution: weigh an appropriate amount of the hollyhock flower extract in the preparation example, and dissolve it in DMSO to obtain a mother liquor of the hollyhock flower extract with a concentration of 300 mg/mL. In the FLIPR experiment, Locke’s buffer and other experimental reagents were added according to the experimental procedure to make the final concentration of the hollyhock flower extract 50μg/mL.

Quercetin-3'-O-β-D-glucoside monomer compound solution: Weigh an appropriate amount of monomer compound and dissolve it in DMSO to obtain a monomer compound mother solution with a concentration of 10 mM. In the FLIPR experiment, Locke's buffer and other reagents were added according to the experimental procedure to make the final concentration of the monomer compound 0.1, 0.3, 3, 10, and 30 μM, respectively.

(5) Dosing: The activator used is M085, and the dosage is 1 μM; the inhibitor used is quercetin-3′-O-β-D-glucoside, the hollyhock flower extract of the preparation example (see Table 1 for details), Five gradient dosages of 0.1 μM, 0.3 μM, 3 μM, 10 μM, and 30 μM were set for the inhibitor of each example, and a control group without medicine (Veh) was set at the same time.

(6) After the drug addition is completed, use FLIPR (Molecular Devices, Sunnyvale, CA, USA) to measure the intracellular calcium ion concentration.

The inhibitors and ion channel types used in Examples 1-6 are shown in Table 1:

Table 1.

Example	group name	Inhibitor	Ion channel type
Example 1	HK-15-TRPC3	Quercetin-3′-O-β-D-glucoside	TRPC3
Example 2	HK-15-TRPC6	Quercetin-3′-O-β-D-glucoside	TRPC6
Example 3	HK-15-TRPC7	Quercetin-3′-O-β-D-glucoside	TRPC7
Example 4	HK-D-total-extract	Abelmoschus manihot flower extract of preparation example	TRPC3
Example 5	HK-D-total-extract	Abelmoschus manihot flower extract of preparation example	TRPC6
Example 6	HK-D-total-extract	Abelmoschus manihot flower extract of preparation example	TRPC7

The detection results of the above embodiments are shown in Figures 1-6. The abscissa in the figure is time and the unit is (S). Except for the figure in Example 4, the group with the highest peak shape around 400S in all other examples is the M085 group. In Figures 1-3, the peak shape around 400S is the M085 group and 0.1μM from high to low. Dosage, 0.3μM dosage, 3μM dosage, 10μM dosage, 30μM dosage, and no drug control group (Veh). In Figure 5-6, in the peak shape around 400S, from high to low, they are M085 group, HK-D-total-extract group, and no drug control group (Veh). In Figure 4, in the peak shape around 400S, from high to low, the HK-D-total-extract group, the M085 group, and the no-drug control group (Veh) are in order.

The detection results of Examples 1-3 are shown in Figures 1-3, respectively. Quercetin-3'-O-β-D-glucoside in different dose groups caused ^{different changes in intracellular Ca 2+} fluorescence intensity over time. Different doses of quercetin-3'-O-β-D-glucoside can reduce the degree of increase in intracellular Ca ²⁺ fluorescence intensity caused by M085, and present a dose-dependent manner. In each group in Figure 1 and Figure 3, the intracellular Ca ²⁺ fluorescence intensity reached a peak at about 400 s, and the peak time was basically the same. After 600 s, the intracellular Ca ²⁺ fluorescence intensity stabilized. Figure 2 In each group, the intracellular Ca ²⁺ fluorescence intensity reached a peak after 400s, and the intracellular calcium ion fluorescence intensity stabilized after 600s. It shows that quercetin-3'-O-β-D-glucoside can inhibit the calcium influx caused by the opening of TRPC3/6/7. In general, the IC50 of quercetin-3'-O-β-D-glucoside to TRPC6=7.518μM.

The detection results of Examples 4-6 are shown in Figures 4-6, respectively. A 50 μg/mL hollyhock flower extract can inhibit the calcium ion influx in TRPC6-HEK293 cells and TRPC7-HEK293 cells caused by M085.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. Within the scope of protection.

Claims (12)

1. The application of quercetin-3'-O-β-D-glucoside in the preparation of drugs for inhibiting calcium ion channels.
2. The application according to claim 1, wherein in the application, quercetin-3'-O-β-D-glucoside is added in the form of the main active ingredient.
3. The application according to claim 1, wherein the calcium ion channel is a TRPC channel.
4. The application according to claim 3, wherein the TRPC channel is a TRPC3, TRPC6 or TRPC7 channel.
5. Quercetin-3′-O-β-D-glucoside is used in the preparation of diagnosis, treatment or adjuvant treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory Use in medicine for diseases.
6. An application of a plant extract in the preparation of a medicine for the treatment of calcium ion channel-mediated diseases, characterized in that the plant extract contains more than 0.5% by weight of quercetin-3'-O-β -D-glucoside; further the plant extract contains 0.5-2.0% by weight of quercetin-3'-O-β-D-glucoside; further, the plant extract Contains 0.8-1.6% by weight of quercetin-3'-O-β-D-glucoside; preferably the plant is a Hibiscus plant, a Malvaceae plant, and more preferably a yellow sunflower, a hollyhock, or golden flower One or more of sunflower, grape-leaf hibiscus, croissant bean, and mopan grass.
7. An application of hollyhock flower extract in the preparation of a medicine for the treatment of calcium ion channel-mediated diseases, characterized in that the hollyhock flower extract contains more than 0.5% by weight of quercetin-3'-O -β-D-glucoside; further, the hollyhock flower extract contains 0.5-2.0% by weight of quercetin-3'-O-β-D-glucoside; further, the Abelmoschus manihot flower extract contains 0.8-1.6% by weight of quercetin-3'-O-β-D-glucoside.
8. The use of claim 6 or claim 7, wherein the calcium channel-mediated diseases are cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain Or inflammatory disease.
9. The application of claim 6 or claim 7, wherein the calcium ion channel is a TRPC channel, and further, is a TRPC3, TRPC6, or TRPC7 channel.
10. The application according to claim 7, characterized in that the hollyhock flower extract is prepared by the following method: taking the hollyhock flower medicinal material, extracting by heating with ethanol, filtering, concentrating the filtrate, and drying to obtain the hollyhock flower extract.
11. A medicine for the treatment or auxiliary treatment of cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease, characterized in that the medicine contains quercetin- 3'-O-β-D-glucoside.
12. A pharmacological tool for characterizing channels belonging to different TRPC subfamily is characterized in that the pharmacological tool comprises quercetin-3'-O-β-D-glucoside.

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