Use of protocatechuic acid as an inhibitor of calcium ion channels

Abstract

The application relates to application of protocatechuic acid as an inhibitor of a calcium ion channel, belongs to the field of medicine, and also provides use of protocatechuic acid in preparation of drugs for treating cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, nervous diseases, chronic pain, acute pain or inflammatory diseases, proves that the protocatechuic acid can inhibit the calcium ion channel, especially a TRPC ion channel, and expands the use of the protocatechuic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to the application of protocatechuic acid. BACKGROUND

[0002] Cardiovascular disease is the most harmful disease to human life and health in modern society. According to the report of the World Health Organization, about 17.9 million people died of cardiovascular disease in the world in 2016, accounting for 31% of the total number of deaths in the world. Therefore, the medical demand for new drugs for preventing and treating cardiovascular disease is very urgent. Relevant studies have proved that TRPC channels are important pharmacological targets for the development of new drugs for some cardiovascular diseases such as myocardial disease, heart failure, hypertension and cerebrovascular disease. Patent application WO2006 / 074802A1 discloses the use of TRPC channels for the treatment of cardiovascular and cerebrovascular diseases. Studies have shown that by using gene technology, in a rabbit atherosclerosis model, by inhibiting the activity of TRPC3, TRPC6 and TRPC7 of vascular endothelial cells, the vascular function and the vascular pathological changes of atherosclerosis can be significantly improved.

[0003] TRPC channels are a kind of Ca 2+ Permeable non-selective ion channels, which exist widely in mammalian tissues. According to the structural homology and functional tendency, the TRPC family can be divided into four subgroups: TRPC1 and TRPC2 each constitute a subgroup; TRPC4 and TRPC5 have about 65% amino acid homology, so they are classified into 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 have a common activation mechanism, and the currently known endogenous ligands are diacylglycerol (DAG) and 4-ethyl-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazole[1,5-a]-pyrimidin-5-yl)piperidine-1-carboxylate (M085). The currently known TRPC organic inhibitors are 2-aminoethoxydiphenyl boronic acid (2-APB), SKF96365, YM-58483 (BTP2) and inorganic blockers (such as Gd 3+ and La 3+ ) etc., but all lack sufficient effectiveness and specificity. At present, the natural composition, activation mechanism, physiological function and role in pathophysiology and diseases of TRPC are still pending issues. Because of their wide and partially overlapping distribution, potential heteromultimerization, similar electrophysiological properties and lack of clear tracking of these channel compounds, it is difficult to achieve in situ identification of natural TRPC channels.

[0004] Dietrich et al. demonstrated that the study of transgenic mouse models can unravel some of the possible physiological functions of certain TRPCs, summarized the heteroextremal potential of TRPC3, 6, 7 subfamily in vitro and in vivo, and provided preliminary data of their physiological functions in isolated tissues and in genetically deficient mouse models under down-regulation of channel activity. However, due to the lack of specific channel blockers, it is easy to be affected by the compensation effect of channels closely related to TRPC channels, so it is difficult to determine the physiological relevance of TRPC homo- or hetero-isoforms in the complex organ function of the whole body. To overcome this defect, it is necessary to target gene inactivation in embryonic stem cells and subsequently produce genetically deficient mouse models for each channel and channel subfamily. The generation and analysis of these model systems are very time-consuming and costly, and there are certain limitations.

[0005] The flowers of Abelmoschus Manihot (L.) Medic are the dried flowers of the mallow family of Abelmoschus Manihot (L.) Medic. The flowers of Abelmoschus Manihot (L.) Medic were first recorded in "Jiayou Bencao". It is widely distributed and rich in resources. "Bencao Gangmu" records that the flowers of Abelmoschus Manihot (L.) Medic have sweet, cold, and slippery odors, and are non-toxic. They are used to treat stranguria and induce labor, and are used to treat various bad sores and pus water that do not heal for a long time. They are also used to treat scalds and burns.

[0006] The flowers of Abelmoschus Manihot (L.) Medic contain a variety of chemical components, including gallic acid, 5-hydroxymethyl-2-furancarboxylic acid, protocatechuic acid-3-O-β-D-glucoside, protocatechuic acid, acortatarin A, gossypetin-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-3-O-β-D-xylosyl-(1→2)-β-D-galactoside, quercetin-3-O-robinoside, rutin, hyperoside, isoquercitrin, myricetin-3'-O-β-D-glucoside, gossypetin-3'-O-β-D-glucoside, gossypetin-8-O-β-D-glucuronide, myricetin, quercetin-3'-O-β-D-glucoside, quercetin, and the like.

[0007] Currently, there is no research indicating that protocatechuic acid has an inhibitory effect on TRPC channels and its use in the preparation of drugs for treating TRPC channel-related cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain, or inflammatory diseases.

[0008] In view of this, the present application provides the use of Abelmoschus flower extract as an inhibitor of TRPC ion channels and the preparation of a drug for treating cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, proving that the protocatechuic acid in its extract is a new pharmacological tool that can selectively inhibit TRPC ion channels, can distinguish between and within the TRPC subfamily, thus elucidating the role of different channels under physiological and pathophysiological conditions, opening up new ideas for cardiovascular and cerebrovascular diseases, and expanding the use of Abelmoschus flower extract. SUMMARY

[0009] The technical solution of the present application is as follows:

[0010] Terms:

[0011] 1. The term "TRPC channel", "TRPC ion channel" or "TRPC" in the summary means a non-selective cation channel permeable to Ca 2+ It refers to any one of the following list of transient receptor potential canonical ion channels: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7. Particularly preferred are TRPC3, TRPC6 and TRPC7.

[0012] Such TRPC ion channels can be derived from any vertebrate, and particularly a mammalian species (e.g., dog, horse, cow, mouse, rat, canine, rabbit, chicken, simian, human or other). TRPCs can be isolated from tissue extracts of such vertebrate organisms, or produced by recombinant biological materials capable of expressing TRPC proteins.

[0013] This term can refer to native polypeptides, polymorphic variants, mutants, and interspecies homologs.

[0014] 2. The term "pharmacological tool" in the summary means a compound and combination of compounds whose functional properties can be studied to investigate how drugs interact with living organisms to produce changes in the desired function, thus allowing the study of new drug compositions, as well as properties, interactions, toxicology, therapeutic, medical 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 role in pathophysiology and disease.

[0015] 3. The term "TRPC ion channel modulator" in the context of the present application means a modulating molecule of a TRPC channel, in particular an inhibiting or activating molecule ("inhibitor" or "activator"), in particular an inhibitor of a TRPC channel which can be identified according to the methods of the present application. An inhibitor is typically a compound which, as preferably described in detail above, e.g. binds to, partially or totally blocks the activity, reduces, prevents, delays activation, inactivates, desensitizes or down-regulates the activity or expression of at least one TRPC channel. An activator is typically a compound which, as preferably described in detail above, e.g. increases, opens, activates, facilitates, enhances activation, sensitizes, agonizes or up-regulates the activity or expression of at least one TRPC channel. Such modulators include genetically engineered versions of TRPC channels, preferably inactivating mutants of TRPC channels, as well as naturally occurring or synthetic ligands, antagonists, agonists, peptides, cyclic peptides, nucleic acids, antibodies, antisense molecules, ribozymes, small organic molecules and the like. Examples of TRPC activators are diacylglycerol, in particular 1-oleoyl-2 acetyl-sn-glycerol (OAG); Gq-coupled receptor agonists, such as phenylephrine, in particular trypsin; agonists stimulating receptor tyrosine kinases such as epidermal growth factor (EGF); or diacylglycerol generating enzymes such as phospholipases or activators thereof. Examples of measurements of the modulation of TRPC ion channel activity in the presence of a test compound are as follows: In general, cells expressing a TRPC channel are provided. Such cells can be generated using genetic methods known to the skilled person. After induction of expression of the TRPC channel, the cells are typically put into e.g. a microtiter plate and grown. Usually the cells are grown on the bottom of the multiwell plate and immobilized. Then, the cells are routinely washed and a dye, preferably a fluorescent dye such as fluo 4 am, is added in an appropriate loading buffer. After removal of the loading buffer, the cells are incubated with the test compound or modulator, in particular a biochemical or chemical test compound as described above, e.g. in the form of a chemical compound library, Ca2+and a suitable indicator for Ca2+fluxes, e.g. a fluorescent indicator such as fluo 4 am. The cells are then incubated for a suitable time, e.g. 1 to 60 minutes, and the fluorescence is measured. The fluorescence is typically measured by fluorescence imaging using a suitable instrument such as a FLIPR. The expected effect of an inhibitor is e.g. a reduction of the fluorescence increase. The expected effect of an activator is e.g. a further increase of the activator-induced fluorescence, or an induction of a fluorescence increase independent of the activator. Thereafter, suitable modulators, in particular inhibitors, can be analyzed and / or isolated. Screening of chemical compound libraries is preferably performed using high-throughput assays known to the skilled person or commercially available. 2+ Measurements can be performed by using e.g. a fluorescence imaging plate reader (FLIPR). To stimulate Ca2+influx through the TRPC channel, a channel activator such as OAG and 4-ethyl-(3-(4-fluorophenyl)-7-hydroxy-2-methylpyrazolo[1,5-a]-pyrimidin-5-yl)piperidine-1-carboxylate (M085) is typically applied. The expected effect of an inhibitor is e.g. a reduction of the fluorescence increase. The activator is expected to cause e.g. a further increase of the activator-induced fluorescence, or an induction of a fluorescence increase independent of the activator. Thereafter, suitable modulators, in particular inhibitors, can be analyzed and / or isolated. Screening of chemical compound libraries is preferably performed using high-throughput assays known to the skilled person or commercially available.

[0016] 4. The term "cell expressing TRPC" in the context of the present application means a cell which endogenously expresses the ion channel of interest or a recombinant cell. The cell is typically a mammalian cell, e.g. a human cell, a mouse cell, a rat cell, a Chinese hamster cell, etc. Conveniently used cells include MDCK, HEK 293, HEK 293T, BHK, COS, NIH 3T3, Swiss 3T3 and CHO cells, preferably HEK 293 cells.

[0017] 5. The term "tissue" in the context of the present application means any type of tissue preparation or a part of a tissue or organ (e.g. brain, liver, spleen, kidney, heart, blood vessels, muscle, skin, etc. and also any type of body fluid such as blood, saliva, lymph, synovial fluid, etc.), preferably if derived from a vertebrate, more preferably from a mammal such as a human. Tissue samples can be obtained by well-known techniques such as blood taking, tissue puncture or surgical techniques.

[0018] 6. The term "medicament" in the context of the present application means a therapeutic agent comprising a therapeutically effective amount of protocatechuic acid or a plant extract comprising the compound. The medicament can be administered systemically or locally in any conventional manner. This can be, for example, by oral administration such as tablets, granules or capsules, by way of a mucous membrane such as the nasal or oral cavity, depot preparations implanted under the skin, by injection, infusion or gel comprising the medicament according to the application. If appropriate, the medicament can also be administered topically and locally in the form of liposome complexes for the treatment of the particular disease mentioned above. The medicament can also be administered in the form of an injection or infusion, if only relatively small amounts of solution or suspension are used, for example about 1 to 20 mL, which are administered to the body in general using injection solutions.

[0019] In one aspect, the present application provides the use of protocatechuic acid or a composition comprising the same for the manufacture of a medicament for inhibiting a calcium ion channel.

[0020] As an example, the present application provides the use of protocatechuic acid for the manufacture of a medicament for inhibiting a calcium ion channel.

[0021] As an exemplary or preferred example, the calcium ion channel is a TRPC channel (or TRPC ion channel).

[0022] As an exemplary or preferred example, the TRPC channel is a TRPC3, TRPC6 or TRPC7 channel.

[0023] As an exemplary or preferred example, the calcium ion channel is a calcium ion channel which is selectively inhibited by protocatechuic acid in vitro and in vivo.

[0024] In another aspect, the present application provides the use of protocatechuic acid or a composition comprising the same in the manufacture of a medicament for the diagnosis, treatment or adjunctive treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end-stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.

[0025] As an example, the present application provides the use of protocatechuic acid in the manufacture of a medicament for the diagnosis, treatment or adjunctive treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end-stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.

[0026] As an exemplary or preferred example, the medicament further comprises one or more pharmaceutically acceptable carriers or auxiliaries. Pharmaceutically acceptable carriers or auxiliaries 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.

[0027] In another aspect, the present application provides a plant extract comprising 0.1% or more protocatechuic acid by weight, further comprising 0.1-2.5% protocatechuic acid by weight, and more further comprising 0.4-1.2% protocatechuic acid by weight. Preferably, the plant is one or more of a plant of the genus Hibiscus, a plant of the family Malvaceae, and more preferably one or more of Abelmoschus moschatus, Abelmoschus manihot, Abelmoschus crinitus, Hibiscus laevis, Dolichos lablab, and Malvastrum tricuspidatum.

[0028] As an exemplary or preferred example, the plant extract is an extract of Abelmoschus manihot flower. The extract of Abelmoschus manihot flower is an ethanol extract, preferably an extract by refluxing with 50-95% ethanol, and more preferably an extract by refluxing with 80-95% ethanol.

[0029] The extract of Abelmoschus manihot flower can be prepared by the following method: taking the flower of Abelmoschus manihot, refluxing with ethanol, filtering, concentrating the filtrate, and drying. Further, preferably, the extract is prepared by the following method: refluxing the flower of Abelmoschus manihot with 85-95% ethanol for 1-3 times, each time for 1-2 hours, filtering, recovering ethanol from the combined filtrate, concentrating the filtrate to a specific gravity of 1.20-1.35, standing the concentrated solution at 0-4°C for 24-48 hours, removing the oil layer of the refrigerated solution, adjusting the pH to 6.0-7.0, concentrating, and thin-layer quick-drying or vacuum-drying to obtain the extract of Abelmoschus manihot flower.

[0030] The preparation method of the Ipomoea nil flower extract can be as follows: the Ipomoea nil flower is extracted with 95% ethanol by reflux extraction for 2 times, 1 hour each time, filtration, recovery of ethanol from the combined filtrate, concentration of the filtrate to a specific gravity of 1.20, standing of the concentrated solution at 0-4°C for 24-48 hours, removal of the oil layer of the refrigerated solution, adjustment of the pH value to 6.0, slow addition of the refrigerated solution into a thin-layer rapid drying drum slot, just making the refrigerated solution liquid surface of the drum slot contact the drum body surface, preheating the drum body surface temperature to 140-150°C, air pressure of 0.4-0.5 MPa, opening of the drum rolling start button, drum body rotating speed of 3-3.5 minutes / revolution, coating of the rolled extract liquid on a polytetrafluoroethylene plate for cooling, knocking and crushing of the dried material after cooling and becoming brittle, and packing of the crushed material in clean double-layer plastic bags to obtain the Ipomoea nil flower extract.

[0031] The conditions of the above thin-layer rapid drying operation are as follows: preheating of the thin-layer rapid drying drum body surface temperature to 135-160°C, air pressure of 0.3-0.6 MPa, drum rotating speed of 2-4.5 minutes / revolution, and the coating plate being a plastic plate or a stainless steel plate, the plastic plate being selected from polyethylene plate, PVC plastic plate, PP plastic plate, PE plastic plate and polytetrafluoroethylene plate, and the polytetrafluoroethylene plate being preferred.

[0032] The preferred preparation method of the Ipomoea nil flower extract is as follows: the Ipomoea nil flower is extracted with 95% ethanol by reflux extraction for 2 times, 1 hour each time, filtration, recovery of ethanol from the combined filtrate, concentration of the filtrate to a specific gravity of 1.20, standing of the concentrated solution at 0-4°C for 24-48 hours, removal of the oil layer of the refrigerated solution, adjustment of the pH value to 6.0, and slow addition of the concentrated solution into a vacuum belt dryer for vacuum belt drying.

[0033] As an example, the preparation method of the above Ipomoea nil flower extract is as follows: 4000 g of the medicinal material Ipomoea nil is extracted with 15 times (mass / volume ratio) of 95% ethanol by reflux extraction for 2 times, 1 hour each time, filtration, recovery of ethanol from the combined filtrate, concentration of the filtrate to a specific gravity of 1.20, standing of the concentrated solution at 0-4°C for 24 hours, removal of the oil layer of the refrigerated solution, adjustment of the pH value to 6.0, slow addition of the concentrated solution into a dryer for drying, crushing, and packing in clean double-layer plastic bags to obtain the Ipomoea nil flower extract.

[0034] In another aspect, the present application provides an Ipomoea nil flower extract containing 0.1% or more of protocatechuic acid by weight, further containing 0.1-2.5% of protocatechuic acid by weight, and more further containing 0.4-1.2% of protocatechuic acid by weight.

[0035] In another aspect, the present application provides the use of the above Ipomoea nil flower extract in the preparation of a drug for treating a calcium ion channel-mediated disease.

[0036] In another aspect, the present application provides the use of the above-mentioned extract of Abelmoschus flowers in the manufacture of a medicament for the diagnosis, treatment or adjuvant treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases.

[0037] In another aspect, the present application provides a medicament for the diagnosis, treatment or adjuvant treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, said medicament comprising protocatechuic acid.

[0038] As an example, the present application provides a medicament for the diagnosis, treatment or adjuvant treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, said medicament comprising protocatechuic acid.

[0039] In another aspect, the present application provides a new pharmacological tool that is able to discriminate between and within the TRPC subfamily. Thus, it is possible to elucidate the role of different channels under physiological and pathophysiological conditions. Namely, the present application provides a pharmacological tool that characterizes channels belonging to different TRPC subfamilies, said pharmacological tool comprising protocatechuic acid.

[0040] According to the present application, this is achieved by inhibiting TRPC3, TRPC6 and TRPC7 with protocatechuic acid. Thus, protocatechuic acid is able to pharmacologically discriminate between channels belonging to different TRPC subfamilies. Moreover, protocatechuic acid does not interfere with the common G protein-coupled receptor, Gq, phospholipase C beta pathway that mediates TRPC channel activation in many cells. These properties make protocatechuic acid a preferred tool for the identification and modulation of TRPC3, TRPC6 and TRPC7.

[0041] As an inhibitor of TRPC3, TRPC6 and TRPC7, protocatechuic acid can be used as a pharmacological tool that is able to characterize channels belonging to different TRPC subfamilies, discriminate between TRPC3 / 6 / 7 subfamily members and other ion channel subfamily members (Figures 1-6).

[0042] As such an inhibitor, protocatechuic acid can further be used as a tool compound for the development and validation of assays to measure activity related to ion channels. An example of such an assay is shown in Figure 1-3

[0043] ​In another aspect, the present application provides the use of protocatechuic acid for differential analysis of the channel function of members of the TRPC3 / 6 / 7 subfamily under physiological and pathophysiological conditions. This can be done as described in the examples. The analysis can be performed in cells, tissues or animals. The animals can be rodents, preferably mice or rats.

[0044] According to a preferred embodiment, the modulation of native TRPCs by protocatechuic acid can be studied using a HEK293 cell line, wherein the HEK293 cell line is a validated model system for studying endogenously expressed TRPC ion channels. Further details of such a preferred assay system are given in the examples and Figure 1-3

[0045] In another aspect, the present application provides a method for determining the influence of protocatechuic acid on TRPC channel activity, preferably the TRPC ion channel is TRPC3, TRPC6 and TRPC7.

[0046] Generally, cells expressing the TRPC ion channel are contacted with protocatechuic acid and the influence of protocatechuic acid on the TRPC ion channel activity is measured or detected.

[0047] In another aspect, the present application provides a method for identifying a modulator of a TRPC ion channel, preferably the TRPC ion channel is TRPC3, TRPC6 and TRPC7.

[0048] Generally, cells expressing the TRPC ion channel are contacted with a test compound and the influence of the test compound on the TRPC ion channel activity is measured or detected.

[0049] In embodiments, the cells used in the above methods are fluorescent cells.

[0050] According to a preferred embodiment, the cells are MDCK, HEK 293, HEK 293T, BHK, COS, NIH3T3, Swiss 3T3 or CHO cells, in particular HEK 293 cells.

[0051] The activity of the TRPC channel can be measured or detected by patch clamp technique, whole cell currents, radioactive ion flux, or in particular fluorescence (e.g. using voltage sensitive dyes or ion sensitive dyes) measuring or detecting ion flux, in particular Ca 2+ changes in ion flux.

[0052] One example of a TRPC channel activity assay is an assay comprising the following steps:

[0053] ​(1) Contact protocatechuic acid with fluorescent cells expressing TRPC ion channels, and stimulate Ca2+ expression with a channel activator before, during, or after contact. 2+ internal flow;

[0054] (2) Detect changes in TRPC ion channel activity.

[0055] On the other hand, the present invention provides a method for describing the selectivity of protocatechuic acid for TRPC channels, comprising evaluating the ability of protocatechuic acid to inhibit TRPC channel activity.

[0056] The present invention has the following beneficial effects:

[0057] (1) This invention provides a novel use of protocatechuic acid, including its application in the preparation of drugs that inhibit calcium ion channels and its related uses.

[0058] (2) It provides new ideas for the preparation of drugs for the treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain and inflammatory diseases, especially TRPC channel-related diseases, as well as for the development of selective inhibitors of TRPC ion channels;

[0059] (3) It expands the applications of okra flower extract. Attached Figure Description

[0060] Figure 1 Different doses of protocatechuic acid induced intracellular calcium deficiency in TRPC3HEK293 cells. 2+ Graph showing the change in fluorescence intensity over time;

[0061] Figure 2 Different doses of protocatechuic acid induced intracellular calcium deficiency in TRPC6HEK293 cells. 2+ Graph showing the change in fluorescence intensity over time;

[0062] Figure 3 Different doses of protocatechuic acid induced intracellular calcium deficiency in TRPC7HEK293 cells. 2+ Graph showing the change in fluorescence intensity over time;

[0063] Figure 4 The extract of Hibiscus syriacus flower induced intracellular calcium in TRPC3HEK293 cells 2+ Graph showing the change in fluorescence intensity over time;

[0064] Figure 5 The extract of Hibiscus syriacus flower induced intracellular calcium in TRPC6HEK293 cells 2+ Graph showing the change in fluorescence intensity over time;

[0065] Figure 6Ca2+ influx induced by the extract of Abelmoschus flower in TRPC7 HEK293 cells 2+ Fluorescence intensity versus time. DETAILED DESCRIPTION

[0066] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific embodiments are further described, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0067] The experimental methods in the following examples are all conventional methods, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0068] The structure of the protocatechuic acid compound involved in the embodiments of the present application is as follows:

[0069]

[0070] Preparation example: preparation of Abelmoschus flower extract

[0071] Take 3000g of medicinal material Abelmoschus flower, and reflux extract the Abelmoschus flower with 19 times 95% ethanol for 2 times, 1 hour each time, filter, recover ethanol from the combined filtrate, concentrate the filtrate to a specific gravity of 1.20, and let the concentrated solution stand at 0°C-4°C for 48 hours. Remove the oil layer of the refrigerated liquid, adjust the pH value to 6.0, slowly add it into a vacuum belt dryer after concentration, dry at 100°C, crush, and pack into clean double plastic bags to obtain the Abelmoschus flower extract. Among them, protocatechuic acid is contained.

[0072] Examples 1-6

[0073] (1) Transfect the cDNA plasmid vector containing TRPC3, TRPC6 or TRPC7 ion channel into HERK293 cell line, then incubate the cells according to the resistance of the plasmid to select the corresponding antibiotics, so as to selectively screen the successfully transfected cells. Perform functional test on the survived cells to confirm the expression and function of the channel protein, and then clone and purify by "limiting dilution" process to obtain a stable cell line stably expressing a specific channel.

[0074] (2) The stable cells obtained in step (1) are added at 10-13×104 / mL, 150μL of cell suspension is added to each well of a black wall bottom transparent 96-well plate. After 24h of incubation in an incubator, it can be used for subsequent experiments.

[0075] (3) Observe the cells, and after confirming that the cells are in good condition, load the dye (fluo-4) for 60min.

[0076] (4) Preparation of channel activator and inhibitor solutions, as follows:

[0077] 1. Preparation of TRPC6 agonist (M085): An appropriate amount of M085 was weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain a M085 stock solution with a concentration of 10 mM. In the FLIPR experiment, Locke's buffer and other experimental reagents were added according to the experimental procedure to make the final concentration of M085 1 μM.

[0078] 2. Preparation of drug: Sphaeranthus indicus extract solution: An appropriate amount of Sphaeranthus indicus extract was weighed and dissolved in DMSO to obtain a Sphaeranthus indicus extract stock solution 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 Sphaeranthus indicus extract 50 μg / mL.

[0079] Monomeric compound solution of protocatechuic acid: An appropriate amount of monomeric compound was weighed and dissolved in DMSO to obtain a monomeric compound stock solution with a concentration of 10 mM. In the FLIPR experiment, Locke's buffer and other experimental reagents were added according to the experimental procedure to make the final concentration of monomeric compound 0.1, 0.3, 3, 10 and 30 μM, respectively.

[0080] (5) Drug addition: The activator used was M085 at a dosage of 1 μM; the inhibitors used were protocatechuic acid, Sphaeranthus indicus extract of the preparation example (see Table 1 for details), and each example of the inhibitor was set at 0.1 μM, 0.3 μM, 3 μM, 10 μM, 30 μM, respectively, with a no-drug control group (Veh) also set.

[0081] (6) After the addition of drugs was completed, intracellular calcium ion concentration was measured using FLIPR (Molecular Devices, Sunnyvale, CA, USA).

[0082] The inhibitors and ion channel types used in Examples 1-6 are shown in Table 1:

[0083] Table 1.

[0084] Examples Group Name Inhibitor Ion Channel Type Example 1 HK-12-TRPC3 Protocatechuic Acid TRPC3 Example 2 HK-12-TRPC6 Protocatechuic Acid TRPC6 Example 3 HK-12-TRPC7 Protocatechuic Acid TRPC7 Example 4 HK-D-total-extract Example 1's Okra Flower Extract TRPC3 Example 5 HK-D-total-extract Example 1's Okra Flower Extract TRPC6 Example 6 HK-D-total-extract Example 1's Okra Flower Extract TRPC7

[0085] The detection results of the above examples are shown in Figure 1-6 The horizontal axis of the graph is time, in seconds. Except for the graph of Example 4, the highest peak of all the graphs is the M085 group at about 400 s, Figure 1-3In Example 1, from high to low in the peak shape around 400s were M085 group, 0.1 μΜ dosage, 0.3 μΜ dosage, 3 μΜ dosage, 10 μΜ dosage, 30 μΜ dosage, and no drug control group (Veh). In Example 2, from high to low in the peak shape around 400s were M085 group, HK-D-total-extract group, and no drug control group (Veh). Figure 4-6 In Example 1, from high to low in the peak shape around 400s were M085 group, 0.1 μΜ dosage, 0.3 μΜ dosage, 3 μΜ dosage, 10 μΜ dosage, 30 μΜ dosage, and no drug control group (Veh). In Example 2, from high to low in the peak shape around 400s were M085 group, HK-D-total-extract group, and no drug control group (Veh). Figure 4 In Example 1, from high to low in the peak shape around 400s were M085 group, 0.1 μΜ dosage, 0.3 μΜ dosage, 3 μΜ dosage, 10 μΜ dosage, 30 μΜ dosage, and no drug control group (Veh). In Example 2, from high to low in the peak shape around 400s were M085 group, HK-D-total-extract group, and no drug control group (Veh).

[0086] The detection results of Example 1-3 are shown in Figures 1-3, respectively. In the HEK293 cells of HK-12-TRPC3, HK-12-TRPC6, and HK-12-TRPC7 groups, different dosages of protocatechuic acid caused intracellular Ca Figure 1-3 fluorescence intensity to change over time. It can be seen that M085 significantly increased the intracellular Ca 2+ fluorescence intensity, and the addition of protocatechuic acid reduced the increase in intracellular Ca 2+ fluorescence intensity caused by M085, and showed a dose-dependent effect. In Example 1 and Example 3, the intracellular Ca 2+ fluorescence intensity of each group reached a peak around 400s, and the peak time was basically the same. After 600s, the intracellular Ca 2+ fluorescence intensity tended to be stable. In Example 2, the intracellular Ca 2 fluorescence intensity of each group reached a peak after 400s, and after 600s, the intracellular Ca + fluorescence intensity tended to be stable. In Example 2, the intracellular Ca 2+ fluorescence intensity of each group reached a peak after 400s, and after 600s, the intracellular Ca 2+ fluorescence intensity tended to be stable. Different dosages of protocatechuic acid could reduce the increase in intracellular Ca 2+ fluorescence intensity caused by M085 to different degrees, indicating that the protocatechuic acid had an inhibitory effect on the Ca 2+ influx caused by the opening of TRPC3 / 6 / 7 ion channels. In general, the IC50 of protocatechuic acid for TRPC6 was 7.958 μΜ, and the IC50 for TRPC7 was 1.574 μΜ.

[0087] The detection results of Example 4-6 are shown in Figures 4-6, respectively. 50 μg / mL of the extract of the flower of the Ipomoea purpurea could inhibit the calcium ion influx in TRPC6-HEK293 cells and TRPC7-HEK293 cells caused by M085.

[0088] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of protocatechuic acid as the sole active ingredient for the preparation of a pharmacological tool for the in vitro differentiation of channels belonging to different TRPC subfamily members, said differentiation of channels belonging to different TRPC subfamily members being differentiated by different characterizations of the decrease of intracellular calcium concentration, said TRPC subfamily members channels being selected from the group consisting of TRPC3, TRPC6 or TRPC7.

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