WO2021208984A1 - Application of abelmoschi corolla extract as trpc ion channel inhibitor - Google Patents

Abstract

An application of an Abelmoschi Corolla extract in preparation of a drug for inhibiting a calcium channel and treating a calcium channel-mediated disease. The disease is a cardiovascular disease, coronary heart disease, atherosclerosis, end-stage renal failure, a neurological disorder, chronic pain, acute pain, or an inflammatory disease. The Abelmoschi Corolla extract contains, by weight, at least one of 0.2% or more of quercetin-3-O-robinoside, 0.5% or more of isoquercitrin, and 0.5% or more of quercetin-3'-O-β-D-glucoside.

Description

Use of Abelmoschus manihot flower extract as TRPC ion channel inhibitor Technical field

The technology of the present invention belongs to the field of medicine, and specifically relates to the application of an extract of Abelmoschus manihot.

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 vascular function by inhibiting the activity of vascular endothelial cells TRPC3\TRPC6 and TRPC7 And atherosclerotic blood vessel pathological changes.

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 protocatechuic acid, quercetin-3-O-robinosides, isoquercitrin and quercetin-3′-O-β-D-glucoside on TRPC channels, and Use in the preparation of a medicine for treating cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease related to TRPC channel.

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 proved that the extracts of protocatechuic acid, quercetin-3-O-robin glycoside, isoquercitrin and quercetin-3′-O-β-D-glucoside as a new Pharmacological tools can selectively inhibit TRPC ion channels, distinguish between and within TRPC subfamilies, so as to clarify the role of different channels under physiological and pathophysiological conditions, and open up ideas for cardiovascular and cerebrovascular diseases. At the same time, the use of hollyhock flower extract is expanded.

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 therapeutically effective amount of quercetin-3-O-robin glycoside, isoquercitrin and quercetin-3'-O-β-D-glucoside One or more of the therapeutic agents, or plant extracts containing these compounds. 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 an application of hollyhock flower extract in the preparation of a medicine for inhibiting calcium ion channels, wherein the hollyhock flower extract contains more than 0.2% by weight of quercetin-3-O- At least one of locust glycoside, isoquercitrin 0.5% or more, and quercetin-3′-O-β-D-glucoside 0.5% or more, further, the hollyhock flower extract contains by weight Calculated as 0.2-1.2% of quercetin-3-O-robin glycoside, 0.5-2.0% of isoquercitrin and 0.5-2.0% of quercetin-3′-O-β-D-glucoside At least one; further, the hollyhock flower extract contains 0.4-0.8% by weight of quercetin-3-O-robin glycoside, 0.8-1.6% of isoquercitrin and 0.8-1.6% At least one of quercetin-3'-O-β-D-glucoside.

The application of the hollyhock flower extract provided by the present invention in the preparation of a medicine for inhibiting calcium ion channels, said calcium ion channel said calcium ion channel is TRPC channel; preferably TRPC3, TRPC6 or TRPC7 channel.

On the other hand, the present invention also provides an application of a plant extract in the preparation of a medicine for calcium ion channel-mediated diseases, characterized in that the plant extract contains more than 0.2% by weight of quercetin At least one of -3-O-locust glycoside, 0.5% or more isoquercitrin and 0.5% or more quercetin-3'-O-β-D-glucoside; further, the plant extract The substance contains 0.2-1.2% quercetin-3-O-robin glycoside, 0.5-2.0% isoquercitrin and 0.5-2.0% quercetin-3′-O-β-D- by weight At least one of glucoside; further, the plant extract contains 0.4-0.8% by weight of quercetin-3-O-locust glycoside, 0.8-1.6% of isoquercitrin and 0.8% -1.6% of at least one of quercetin-3'-O-β-D-glucoside. The calcium ion channel The calcium ion channel is a TRPC channel; preferably a TRPC3, TRPC6 or TRPC7 channel.

The calcium channel-mediated diseases are cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.

The plant of the present invention is a plant of the genus Hibiscus and Malvaceae, and is further preferably one or more of yellow sunflower, hollyhock, hollyhock, grape leaf hibiscus, croissant, and mulberry.

As an exemplary or preferred example, the plant extract is an extract of hollyhock flowers, further, the plant extract is an ethanol extract of hollyhock flowers, preferably an extract with 50-95% ethanol reflux, further Preferably, it is an extract extracted by refluxing with 80-95% ethanol.

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 under 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 refrigerated solution, adjust the pH to 6.0 to 7.0, concentrate and dry, preferably by thin-layer rapid drying or vacuum drying, 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.

As an example, the present invention provides a drug 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 drug contains protozoa One or more of tea acid, quercetin-3-O-locust glycoside, isoquercitrin and quercetin-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 contains quercetin-3-O-loci glycoside, isoquercitrin and quercetin-3' -One or more plant extracts of O-β-D-glucoside.

According to the present invention, this is inhibited by using a composition containing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3'-O-β-D-glucoside Realized by TRPC3, TRPC6 and TRPC7. Therefore, a composition containing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside can pharmacologically distinguish between different TRPCs Channel of subfamily. In addition, a composition containing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside will not interfere in many cells Common G protein-coupled receptor, Gq, and phospholipase Cβ pathways that mediate the activation of TRPC channels. These characteristics make a composition containing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside to identify and regulate TRPC3 and TRPC6 And the preferred tool of TRPC7.

As an inhibitor of TRPC3, TRPC6 and TRPC7, containing one or more of quercetin-3-O-robinosides, isoquercitrin, and quercetin-3′-O-β-D-glucoside The composition can be used as a pharmacological tool to characterize channels belonging to different TRPC subfamily and distinguish TRPC3/6/7 subfamily members from other ion channel family members.

As such an inhibitor, Abelmoschus manihot flower extract can be further used as a tool compound to develop and validate 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, containing quercetin-3-O-robin glycoside, isoquercitrin, quercetin-3'-O-β-D-glucoside Use of one or more compositions of TRPC3/6/7 subfamily members for differential analysis of channel function. 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, a composition containing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside is effective against natural TRPC The regulation of TRPC can be studied using HEK293 cell line, which is a validated model system for studying endogenously expressed TRPC ion channels. Further details of this preferred measurement system are given in the Examples and Figures 1-3.

In another aspect, the present invention provides: determination of one or more compositions containing quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside As a method for affecting the activity of TRPC channels, the preferred TRPC ion channels are TRPC3, TRPC6 and TRPC7.

Generally speaking, cells expressing TRPC ion channels are combined with one or more of quercetin-3-O-locobinoside, isoquercitrin, and quercetin-3′-O-β-D-glucoside. The composition is contacted, and the effect of the composition on the activity of the TRPC ion channel is measured or detected.

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) Make a composition containing one or more of quercetin-3-O-robinoid glycoside, isoquercitrin or quercetin-3′-O-β-D-glucoside and express TRPC ion channel Contact with the fluorescent cells, 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.

On the other hand, the present invention provides: for describing one or more of quercetin-3-O-robin glycoside, isoquercitrin, and quercetin-3'-O-β-D-glucoside A method for the selectivity of the composition to the TRPC channel, comprising evaluating one of quercetin-3-O-robinosides, isoquercitrin, quercetin-3′-O-β-D-glucoside or The ability of various compositions to inhibit the activity of TRPC channels.

The present invention has the following beneficial effects:

(1) The present invention provides a pioneering preparation containing one or more of quercetin-3-O-robin glycoside, isoquercitrin and quercetin-3′-O-β-D-glucoside The new use of the composition, the application in the preparation of medicines for inhibiting calcium ion channels and the 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 in the fluorescence intensity ^{of Ca 2+} in TRPC3HEK293 cells caused by the extract of Abelmoschus manihot over time;

Figure 2 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. 3 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 compound involved in the embodiment of the present invention is:

Quercetin-3-O-Robin Glucoside:

Isoquercitrin:

Quercetin-3′-O-β-D-glucoside:

Preparation Example 1: 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).

Preparation Example 2: 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-robinoid glycoside 5.6 (mg/g), isoquercitrin 10.1 (mg/g), quercetin-3′-O-β-D-glucoside 13.8 (mg/g) g).

Example 1-3

(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. Medicine preparation: Alibaba hollyhock flower extract solution: weigh an appropriate amount of the hollyhock flower extract in Preparation Example 1, 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.

(5) Dosing: The activator used was M085, and the dosage was 1 μM; the inhibitor used was the hollyhock flower extract of Preparation Example 1 (see Table 1 for details), and the inhibitor of each example was set to 0.1 μM, 0.3 μM, Five gradient dosages of 3μM, 10μM, and 30μM, and a non-medicated control group (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 1-3 are shown in Table 1:

Table 1.

Example	group name	Inhibitor	Ion channel type
Example 1	HK-D-total-extract	The hollyhock flower extract of Preparation Example 1	TRPC3
Example 2	HK-D-total-extract	The hollyhock flower extract of Preparation Example 1	TRPC6
Example 3	HK-D-total-extract	The hollyhock flower extract of Preparation Example 1	TRPC7

The detection results of the above embodiments are shown in Figures 1-3. The abscissa in the figure is time and the unit is (S). Except for the figure of Example 1, the group with the highest peak shape around 400S in all other examples is the M085 group. In Figure 2-3, the peak shape around 400S is the M085 group and HK- D-total-extract group, no drug control group (Veh). In Figure 1, in the peak shape around 400S, from high to low, they are the HK-D-total-extract group, the M085 group, and the no-drug control group (Veh).

The detection results of Examples 1-3 are shown in Figures 1-3, respectively. A 50 μg/mL hollyhock flower extract can inhibit 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. An application of hollyhock flower extract in the preparation of a medicine for inhibiting calcium ion channels, characterized in that the hollyhock flower extract contains more than 0.2% by weight of quercetin-3-O-robin glycoside, 0.5% % Or more of isoquercitrin and 0.5% or more of quercetin-3'-O-β-D-glucoside.
2. The application according to claim 1, characterized in that the hollyhock flower extract contains 0.2-1.2% by weight of quercetin-3-O-robin glycoside, 0.5-2.0% of isoquercitrin, 0.5-2.0% of at least one of quercetin-3'-O-β-D-glucoside.
3. The application according to claim 1, characterized in that the hollyhock flower extract contains 0.4-0.8% by weight of quercetin-3-O-robin glycoside, 0.8-1.6% of isoquercitrin, 0.8-1.6% of at least one of quercetin-3'-O-β-D-glucoside.
4. The application of claim 1, wherein the calcium ion channel of the calcium ion channel is a TRPC channel; preferably a TRPC3, TRPC6 or TRPC7 channel.
5. An application of a plant extract in the preparation of a medicine for calcium ion channel-mediated diseases, characterized in that the plant extract contains more than 0.2% by weight of quercetin-3-O-robin glycoside, At least one of 0.5% or more isoquercitrin and 0.5% or more quercetin-3′-O-β-D-glucoside; further, the plant extract contains 0.2% by weight At least one of 1.2% quercetin-3-O-robinoid glycoside, 0.5-2.0% isoquercitrin, 0.5-2.0% quercetin-3′-O-β-D-glucoside; Furthermore, the plant extract contains 0.4-0.8% by weight of quercetin-3-O-robin glycoside, 0.8-1.6% of isoquercitrin, and 0.8-1.6% of quercetin- At least one of 3'-O-β-D-glucoside.
6. The application of claim 5, wherein the calcium ion channel of the calcium ion channel is a TRPC channel; preferably a TRPC3, TRPC6 or TRPC7 channel.
7. The application of claim 5, wherein the calcium ion channel-mediated disease is cardiovascular disease, coronary heart disease, atherosclerosis, advanced renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.
8. The application according to claim 5, characterized in that the plant is a Hibiscus plant, a Malvaceae plant, and is further preferably one of yellow sunflower, hollyhock, golden flower sunflower, grape leaf hibiscus, goat carob, and millet grass. Kind or more.
9. The application of claim 5, wherein the plant extract is an extract of hollyhock flowers, and further, the plant extract is an ethanol extract of hollyhock flowers, preferably 50-95% ethanol refluxed The extract is further preferably an extract extracted by refluxing with 80-95% ethanol.
10. The application according to claim 9, characterized in that, the hollyhock flower extract is prepared by the following method: taking hollyhock flower medicinal materials, extracting with 50-95% ethanol under heating and refluxing, filtering, and concentrating the filtrate to obtain the hollyhock flower extract Things.
11. The application according to claim 10, characterized in that, the hollyhock flower extract is prepared by the following method: the hollyhock flower is extracted with 85%-95% ethanol, refluxed for 1 to 3 times, each time for 1 to 2 hours, and filtered , Combine the filtrate to recover ethanol, concentrate the filtrate to a specific gravity of 1.20 to 1.35, and let the concentrated solution stand at 0°C to 4°C for 24 to 48 hours, remove the oil layer of the refrigerated liquid, adjust the pH to 6.0 to 7.0, concentrate and dry to obtain the hollyhock Flower extract.
12. The application of claim 11, wherein the hollyhock flower extract is prepared by the following method: the hollyhock flower is extracted twice with 95% ethanol, refluxed for 1 hour each time, filtered, and the filtrate is combined to recover the ethanol and concentrated The filtrate has a specific gravity of 1.20, and 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 value to 6.0, concentrate and dry to obtain the hollyhock flower extract.

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