CN111991382A - Use of protocatechuic acid as inhibitors of calcium ion channels - Google Patents
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Abstract
The invention relates to application of protocatechuic acid as an inhibitor of a calcium ion channel, belongs to the field of medicines, and also provides application of protocatechuic acid in preparing medicines for treating cardiovascular diseases, coronary heart diseases, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of protocatechuic acid.
Background
Cardiovascular disease is the disease most harmful to the human life health in modern society, and about 1790 million people die of cardiovascular disease in 2016, accounting for 31% of the total death in the world, according to the report of the world health organization. It follows that the medical need for new drugs for the prevention and treatment of cardiovascular diseases is very urgent. Related researches prove that the TRPC channel is an important pharmacological target for developing new drugs for cardiovascular diseases such as cardiomyopathy, heart failure, hypertension, cerebrovascular diseases and the like. The patent application WO2006/074802A1 discloses that TRPC channels are used for treating cardiovascular and cerebrovascular diseases, and research shows that by utilizing gene technology, in a rabbit atherosclerosis model, the vascular function and vascular pathological changes of atherosclerosis can be obviously improved by inhibiting the activities of vascular endothelial cells TRPC3\ TRPC6 and TRPC 7.
The TRPC channel is a Ca2+The permeated non-selective ion channel is widely present in mammalian tissues. The TRPC family can be divided into four subgroups based on structural homology and functional predilection: TRPC1 and TRPC2 each constituted a subset; TRPC4 and TRPC5 have approximately 65% amino acid homology between them, thus dividing them into the same subpopulation; TRPC3, TRPC6 and TRPC7 have 70-80% amino acid homology and are classified into the same subgroup. TRPC3, TRPC6 and TRPC7 channels share a common activation mechanism, and the endogenous ligands thereof are known to be Diacylglycerol (DAG) and 4-ethyl- (3- (4-fluorophenyl) -7-hydroxy-2-methylpyrazole [1,5-a ]]-pyrimidin-5-yl) piperidine-1-carboxylic acid salt (M085). Organic inhibitors of TRPC known to date are 2-aminoethoxydiphenylboronic acid (2-APB), SKF96365, YM-58483(BTP2) and inorganic blockers (e.g. Gd)3+And La3+) Etc., but all lack sufficient effectiveness and specificity. There are still pending issues with respect to the natural composition, activation mechanism, physiological function of TRPC and its role in pathophysiology and disease etc. Achieving in situ identification of native TRPC channels presents certain difficulties because of their widespread and partially overlapping distribution, potential heteropolymerization, similar electrophysiological properties, and the lack of compound tools to specifically track these channels.
Studies by Dietrich et al demonstrated that it is possible to unravel some of the TRPC's possible physiological functions by studying transgenic mouse models, summarizing the hetero-ultimate potential of TRPC3, 6, 7 subfamilies in vitro and in vivo, and provide preliminary data for their analysis of physiological functions in isolated tissue and gene-deficient mouse models with down-regulated channel activity. However, due to the lack of specific channel blockers, which are susceptible to compensatory effects from channels closely related to the TRPC channel, it is impossible to determine the physiological relevance of TRPC homo-or heteroisomers in the function of complex organs throughout the body, and to overcome this drawback, targeted gene inactivation in embryonic stem cells and subsequent production of gene-deficient mouse models for each channel and channel subfamily are required, and the generation and analysis of these model systems is very time-consuming and costly, and has certain limitations.
Abelmoschus Manihot is a dried flower of Abelmoschus Manihot (L.) Medic of Abelmoschus of Malvaceae, is recorded in Jiayouben, is widely distributed and has abundant resources, and is recorded in Ben Cao compendium: the flowers have sweet, cold, smooth and nontoxic smell, are mainly used for treating urinary stranguria and hastening the growth of the patients, are used for treating patients with malignant sores and pus for a long time and are healed after being applied for powder, are the main medicines for sores, eliminate gangrene swelling, soak oil and spread soup for fire injury and the like.
The flower of abelmoschus manihot contains various chemical components, including: gallic acid, 5-hydroxymethyl-2-furancarboxylic acid, protocatechuic acid-3-O-beta-D-glucoside, protocatechuic acid, acortarin A, gossypetin-3-O-beta-D-glucose-8-O-beta-D-glucuronide, quercetin-3-O- [ beta-D-xylosyl (1 → 2) -alpha-L-rhamnosyl 1 → 6) ] -beta-D-galactoside, myricetin-3-O-beta-D-glucoside, quercetin-3-O-beta-D-xylosyl- (1 → 2) -beta-D-galactoside, quercetin-3-O-locust glucoside, rutin, hyperoside, isoquercitrin, myricetin-3 '-O-beta-D-glucoside, gossypetin-8-O-beta-D-glucuronide, myricetin, quercetin-3' -O-beta-D-glucoside, quercetin and the like.
At present, no research has shown the inhibitory effect of protocatechuic acid on TRPC channel and its use in preparing medicine for treating TRPC channel related cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases.
In view of the above, the invention provides the use of the abelmoschus manihot extract as an inhibitor of TRPC ion channels and the preparation of drugs for treating cardiovascular diseases, coronary heart diseases, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, and proves that protocatechuic acid in the extract is used as a new pharmacological tool, can selectively inhibit TRPC ion channels, and can be distinguished between TRPC subfamilies and inside TRPC subfamilies, so that the effects of different channels under physiological and pathophysiological conditions can be clarified, ideas are developed for cardiovascular and cerebrovascular diseases and the like, and the use of the abelmoschus manihot extract is expanded.
Disclosure of Invention
The technical scheme of the invention is as follows:
the terms:
1. the terms "TRPC channel", "TRPC ion channel" or "TRPC" in the context of the present invention refer to Ca permeable2+Non-selective cation channels of (a). It refers to any one of the following list of transient receptor potentials typical ion channels: TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC 7. Particularly preferred are TRPC3, TRPC6 and TRPC 7.
Such TRPC ion channels may be derived from any vertebrate, and in particular mammalian species (e.g., dog, horse, cow, mouse, rat, dog, rabbit, chicken, ape, human, or others). TRPC can be isolated from tissue probes of such vertebrate organisms or produced by methods of recombinant biomaterials capable of expressing TRPC proteins.
The term may refer to native polypeptides, polymorphic variants, mutants, and interspecies homologs.
2. The term "pharmacological tool" in the context of the present invention refers to compounds and combinations of compounds whose functional properties enable the study of how drugs interact with living organisms to produce changes in the function of interest, thereby enabling the study of new pharmaceutical compositions, as well as the properties, interactions, toxicology, therapy, therapeutic utility and disease resistance. Moreover, the term refers to compounds that can be used to characterize potential targets in new drug development, e.g., to characterize their natural components, activation mechanisms, physiological functions, and roles in pathophysiology and disease.
3. The term "TRPC ion channel modulator" in the context of the present invention refers to a regulatory molecule of the TRPC channel, in particular an inhibitory or activating molecule ("inhibitor" or "activator"), in particular an inhibitor of the TRPC channel identifiable according to the method of the present invention. The inhibitor is typically a compound, as described in detail preferably above, for example, that binds, partially or totally blocks 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, as described in detail preferably above, for example, that increases, opens, activates, promotes, enhances activation, sensitizes, agonizes or upregulates the activity or expression of at least one TRPC channel. Such modulators include genetically engineered versions of the TRPC channel, preferably inactivating mutants of the TRPC channel, 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, particularly trypsin; agonists that stimulate receptor tyrosine kinases such as Epidermal Growth Factor (EGF); or a diacylglycerol-producing enzyme such as a phospholipase or an activator thereof. Examples of measurements of modulation of TRPC ion channel activity in the presence of test compounds are as follows: in general, cells expressing a TRPC channel are provided. Such cells can be produced using genetic methods known to those skilled in the art. After performing the induced expression of the TRPC channel, the cells are typically placed in, for example, a microplate and grown. Typically cells are grown and fixed at the bottom of a multi-well plate. The cells are then washed routinely and a dye, preferably a fluorescent dye such as fluo4am, is added in a suitable loading buffer. After removal of the loading buffer, the cells are contacted with the test compound or modulator (particularly as described above)Biochemical or chemical test compounds, e.g. Ca incubated with chemical compound libraries2+The measurement can be performed using, for example, a fluorescence imaging plate reader (FLIPR). To stimulate Ca2+ influx through the TRPC channel, channel activators such as OAG and 4-ethyl- (3- (4-fluorophenyl) -7-hydroxy-2-methylpyrazole [1,5-a ] are generally used]-pyrimidin-5-yl) piperidine-1-carboxylic acid salt (M085). The expected effect of the inhibitor is, for example, a decrease in the increase in fluorescence. An activator will result in, for example, a further increase in fluorescence induced by the activator, or induce, for example, an increase in fluorescence 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 artisan or commercially available.
4. The term "TRPC expressing cell" in the context of the present invention refers to a cell or recombinant cell endogenously expressing an ion channel of interest. The cell is typically a mammalian cell, such as a human cell, a mouse cell, a rat cell, a chinese hamster cell, and the like. Cells which have been found to be convenient include MDCK, HEK293, HEK 293T, BHK, COS, NIH3T3, Swiss3T3 and CHO cells, preferably HEK293 cells.
5. The term "tissue" in the context of the present invention refers to 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., but also to any type of bodily fluid such as blood, saliva, lymph, synovial fluid, etc.), preferably if derived from a vertebrate, more preferably a mammal such as a human. Tissue samples can be obtained by well-known techniques, such as blood sampling, tissue penetration, or surgical techniques.
6. The term "drug" in the context of the present invention refers to a therapeutic agent comprising a therapeutically effective amount of protocatechuic acid, or a plant extract comprising the compound. The drug can be administered systemically or locally in any conventional manner. This can be done, for example, by means of oral dosage forms such as tablets, granules or capsules, by means of mucous membranes such as the nasal or oral cavity, depot preparations for subcutaneous implantation, by means of injections, infusions or gels containing the medicaments according to the invention. If appropriate, for the treatment of a particular disease of the above-mentioned type, it is also possible to administer the drug locally (topocally and locally) in the form of a liposome complex. The drug may also be administered in the form of an injection or infusion solution, and if only a relatively small amount of solution or suspension, for example about 1 to 20mL, is administered to the body, an injection solution is generally used.
In one aspect, the invention provides the use of protocatechuic acid or a composition containing the same in the preparation of a medicament for inhibiting calcium ion channels.
By way of example, the present invention provides the use of protocatechuic acid in the manufacture of a medicament for inhibiting calcium ion channels.
As an illustrative or preferred example, the calcium channel is a TRPC channel (or referred to as a TRPC ion channel).
As an illustrative or preferred example, the TRPC channel TRPC3, TRPC6 or TRPC7 channel described above.
As an illustrative or preferred example, the calcium channel described above refers to a calcium channel for which protocatechuic acid is used for selective inhibition in vitro and in vivo.
In another aspect, the invention provides the use of protocatechuic acid or a composition containing it in the manufacture of a medicament for the diagnosis, treatment or adjunct treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.
By way of example, the present invention provides the use of protocatechuic acid in the manufacture of a medicament for the diagnosis, treatment or adjunct treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end-stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.
As an illustrative or preferred example, the medicament may be formulated with one or more pharmaceutically acceptable carriers or adjuvants. Pharmaceutically acceptable carriers or adjuvants are, for example, physiological buffer solutions such as sodium chloride solutions, demineralised water, stabilisers such as protease or nuclease inhibitors, or chelating agents such as EDTA.
In another aspect, the present invention provides a plant extract comprising 0.1% by weight or more of protocatechuic acid, and further comprising 0.1 to 2.5% by weight of protocatechuic acid; more particularly, the plant extract contains 0.4-1.2% by weight of protocatechuic acid; preferably, the plant is selected from Hibiscus plant and Malvaceae plant, and more preferably one or more of Abelmoschus manihot, Hibiscus viniferus, Hibiscus esculentus and Phyllanthus niruri.
As an illustrative or preferred example, the plant extract is an extract of abelmoschus manihot. The flos Abelmoschi Manihot extract is ethanol extract, preferably 50-95% ethanol reflux extract, and more preferably 80-95% ethanol reflux extract.
The abelmoschus manihot flower extract can be prepared by the following method: heating and refluxing flos Abelmoschi Manihot with ethanol, filtering, concentrating the filtrate, and drying. Further, it is preferably prepared by the following method: extracting the flower of abelmoschus manihot with 85-95% ethanol under reflux for 1-3 times, each time for 1-2 hours, filtering, combining the filtrates, recovering ethanol, concentrating the filtrate until the specific gravity is 1.20-1.35, standing the concentrated solution at 0-4 ℃ for 24-48 hours, removing an oil layer of a refrigerating solution, adjusting the pH value to 6.0-7.0, concentrating, and performing thin-layer quick drying or vacuum drying to obtain the flower of abelmoschus manihot extract.
The preparation method of the abelmoschus manihot flower extract can comprise the following steps: extracting flos Abelmoschi Manihot with 95% ethanol under reflux for 2 times, each for 1 hr, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.20, standing the concentrated solution at 0-4 deg.C for 24-48 hr, removing oil layer of the cold storage solution, adjusting pH to 6.0, slowly adding the cold storage solution into thin layer rapid drying roller tank until the liquid level of the cold storage solution in the roller tank just contacts with the surface of the roller, preheating the surface temperature of the roller to 140-150 deg.C, and controlling air pressure to 0.4-0.5 Mpa, turning on the roller rolling start button, controlling the rotation speed of the roller to 3-3.5 min/r, coating the rolled extract liquid on a polytetrafluoroethylene plate to cool, cooling to become brittle, breaking, and packaging into clean double-layer plastic bags to obtain flos Abelmoschi Manihot extract.
The conditions of the thin layer rapid drying operation are as follows: the surface temperature of the preheating thin layer quick drying roller body is 135-160 ℃, the air pressure is 0.3-0.6 Mpa, the rotating speed of the roller is 2-4.5 minutes/revolution, the coating plate is a plastic plate or a stainless steel plate, the plastic plate is selected from a polyethylene plate, a PVC plastic plate, a PP plastic plate, a PE plastic plate and a polytetrafluoroethylene plate, and the polytetrafluoroethylene plate is preferred.
The preferable preparation method of the abelmoschus manihot flower extract comprises the following steps: extracting flos Abelmoschi Manihot with 95% ethanol under reflux for 2 times, each for 1 hr, filtering, mixing filtrates, recovering ethanol, concentrating the filtrate to specific gravity of 1.20, standing the concentrated solution at 0-4 deg.C for 24-48 hr, removing oil layer of the cold storage solution, adjusting pH to 6.0, concentrating, and slowly adding into vacuum belt drier for vacuum belt drying.
As an example, the preparation method of the abelmoschus manihot extract comprises the following steps: taking 4000g of abelmoschus manihot, performing reflux extraction for 2 times by using 15 times (mass/volume ratio) of 95% ethanol, performing 1 hour each time, filtering, combining filtrates, recovering the ethanol, concentrating the filtrate to the specific gravity of 1.20, standing the concentrated solution for 24 hours at the temperature of 0-4 ℃, removing an oil layer of a refrigerating solution, adjusting the pH value to 6.0, slowly adding the concentrated solution into a dryer after concentration, drying, crushing, and filling into a clean double-layer plastic bag to obtain the abelmoschus manihot extract.
In another aspect, the present invention provides a sunflower flower extract comprising more than 0.1% by weight of protocatechuic acid, further comprising 0.1-2.5% by weight of protocatechuic acid; further, the sunset abelmoschus flower extract contains 0.4-1.2% by weight of protocatechuic acid.
In another aspect, the invention provides the use of the above sunset abelmoschus flower extract in the preparation of a medicament for treating a calcium channel mediated disease.
In another aspect, the present invention provides the use of the above abelmoschus manihot extract in the preparation 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.
In another aspect, the invention provides a medicament for the treatment or co-treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease, said medicament comprising protocatechuic acid.
As an example, the invention provides a medicament for the treatment or co-treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, late renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, said medicament comprising protocatechuic acid.
In another aspect, the present invention provides a new pharmacological tool that is able to distinguish between and within TRPC subfamilies. Thereby enabling elucidation of the role of the different channels under physiological and pathophysiological conditions. Namely, the present invention provides a pharmacological tool for characterizing channels belonging to different TRPC subfamilies, said pharmacological tool comprising protocatechuic acid.
According to the present invention, this is achieved by inhibiting TRPC3, TRPC6 and TRPC7 with protocatechuic acid. Thus, protocatechuic acids are able to pharmacologically distinguish channels belonging to different TRPC subfamilies. In addition, protocatechuic acid does not interfere with the common G protein-coupled receptor, Gq, phospholipase C β pathway that mediates TRPC channel activation in many cells. These properties make protocatechuic acid a preferred tool for identifying and modulating TRPC3, TRPC6 and TRPC 7.
As an inhibitor of TRPC3, TRPC6 and TRPC7, protocatechuic acid may be used as a pharmacological tool to characterize channels belonging to different TRPC subfamilies, distinguishing TRPC3/6/7 subfamily members from other ion channel subfamily members (fig. 1-6).
As such an inhibitor, protocatechuic acid can further be used as a tool compound for developing and validating assays to measure the activity of the ion channel of interest. An example of such an assay is shown in FIGS. 1-3.
In another aspect, the present invention provides: the use of protocatechuic acid for differential analysis of channel function of TRPC3/6/7 subfamily members under physiological and pathophysiological conditions. This can be done as described in the examples. The assay may 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 modulation of native TRPC by protocatechuic acid can be studied using the 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 in FIGS. 1-3.
In another aspect, the present invention provides: a method for determining the effect of protocatechuic acid on TRPC channel activity, preferably TRPC ion channels TRPC3, TRPC6 and TRPC 7.
Generally, a cell expressing a TRPC ion channel is contacted with protocatechuic acid, and the effect of protocatechuic acid on the TRPC ion channel activity is measured or detected.
In another aspect, the present invention provides: preferred TRPC ion channels for the method of identifying a TRPC ion channel modulator are TRPC3, TRPC6 and TRPC 7.
Generally, a cell expressing a TRPC ion channel is contacted with a test compound, and the effect of the test compound on the TRPC ion channel activity is measured or detected.
In embodiments, the cells used in the above methods are fluorescent cells.
Preferred cells according to the invention are MDCK, HEK293, HEK 293T, BHK, COS, NIH3T3, Swiss3T3 or CHO cells, in particular HEK293 cells.
The activity of a TRPC channel can be measured or detected by e.g. patch clamp techniques, whole cell currents, radiolabeled ion fluxes, or especially fluorescence (e.g. using a voltage or ion sensitive dye), especially Ca2+The change in ion current is measured or detected.
An example of an assay for TRPC channel activity is an assay comprising the following steps:
(1) contacting protocatechuic acid with a fluorescent cell expressing a TRPC ion channel and stimulating Ca using a channel activator before, simultaneously with or after the contacting2+Internal flow;
(2) detecting a change in TRPC ion channel activity.
In another aspect, the present invention provides: directed to methods for describing the selectivity of protocatechuic acid for TRPC channels comprising assessing the ability of protocatechuic acid to inhibit TRPC channel activity.
The invention has the following beneficial effects:
(1) the invention provides a new application of protocatechuic acid, an application in preparing a medicament for inhibiting a calcium ion channel and a related application thereof.
(2) Provides a new idea for preparing medicines for treating cardiovascular diseases, coronary heart diseases, atherosclerosis, advanced renal failure, neurological diseases, chronic pain, acute pain and inflammatory diseases particularly related to TRPC channels and developing selective inhibitors of TRPC ion channels;
(3) the application of the abelmoschus manihot extract is expanded.
Drawings
FIG. 1 shows that different doses of protocatechuic acid cause TRPC3HEK293 intracellular Ca2+A graph of fluorescence intensity versus time;
FIG. 2 shows that different doses of protocatechuic acid cause TRPC6HEK293 intracellular Ca2+A graph of fluorescence intensity versus time;
FIG. 3 shows that different doses of protocatechuic acid cause TRPC7HEK293 intracellular Ca2+A graph of fluorescence intensity versus time;
FIG. 4 shows TRPC3HEK293 intracellular Ca induced by Abelmoschus manihot extract2+A graph of fluorescence intensity versus time;
FIG. 5 shows TRPC6HEK293 intracellular Ca caused by Abelmoschus manihot extract2+A graph of fluorescence intensity versus time;
FIG. 6 shows TRPC7HEK293 intracellular Ca induced by Abelmoschus manihot extract2+Graph of fluorescence intensity as a function of time.
Detailed Description
In order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easily understood, the present invention will be further explained with reference to specific embodiments, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The protocatechuic acid compound related in the embodiment of the invention has the structure as follows:
preparation example: preparation method of flos Abelmoschi Manihot extract
Taking 3000g of abelmoschus manihot as a medicinal material, carrying out reflux extraction on the abelmoschus manihot with 19 times of 95% ethanol for 2 times, carrying out 1 hour each time, filtering, combining the filtrates, recovering the ethanol, concentrating the filtrate to a specific gravity of 1.20, standing the concentrated solution at 0-4 ℃ for 48 hours, removing an oil layer of a refrigerating solution, adjusting the pH value to 6.0, slowly adding the concentrated solution into a vacuum belt type drying machine after concentration, drying at 100 ℃, crushing, and filling into a clean double-layer plastic bag to obtain the abelmoschus manihot extract. Wherein the extract contains protocatechuic acid.
Examples 1 to 6
(1) cDNA plasmid vectors containing TRPC3, TRPC6 or TRPC7 ion channels were transfected into HERK293 cell lines, and then cells were incubated with corresponding antibiotics selected for plasmid resistance to selectively screen for successfully transfected cells. The surviving cells are subjected to a functional test to confirm the expression and the function of the channel protein, and then are cloned and purified through a limiting dilution process, so that a stable cell line which stably expresses a specific channel is obtained.
(2) The stable cells obtained in step (1) were added to a black-walled bottom-penetrating 96-well plate at a rate of 10-13X 104 cells/mL per well of 150. mu.L of cell suspension. After 24 hours of culture in the incubator, the culture medium can be used for carrying out subsequent experiments.
(3) Cells were observed and, after confirming that the cells were in good condition, dye (fluo-4) was loaded for 60 min.
(4) Channel activator and inhibitor solutions were formulated as follows:
TRPC6 agonist (M085) was prepared by weighing an appropriate amount of M085 in dimethyl sulfoxide (DMSO) to obtain a 10mM M085 stock solution. In the FLIPR experiment, Locke's buffer and other experimental reagents were added according to the experimental procedure to give a final concentration of M085 of 1. mu.M.
2. The preparation of the medicine comprises the following steps: weighing a proper amount of the sunset abelmoschus flower extract, and dissolving the sunset abelmoschus flower extract in DMSO to obtain a sunset abelmoschus flower extract mother solution with the 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 Abelmoschus manihot extract 50 μ g/mL.
Protocatechuic acid monomer compound solution: an appropriate amount of monomer compound was weighed and dissolved in DMSO to obtain a monomer compound mother liquor having a concentration of 10 mM. In the FLIPR experiment, Locke's buffer and other experimental reagents were added according to the experimental procedure to give final concentrations of monomeric compounds of 0.1, 0.3, 3, 10 and 30. mu.M, respectively.
(5) Adding medicine: the activating agent is M085, and the dosage is 1 mu M; the inhibitor is protocatechuic acid, flos Abelmoschi Manihot extract of preparation example (see Table 1), and the inhibitor of each example is set at five gradient dosages of 0.1 μ M, 0.3 μ M, 3 μ M, 10 μ M, and 30 μ M, respectively, and is set at the same time as non-medicated control group (Veh).
(6) After dosing was complete, intracellular calcium ion concentration measurements were performed using FLIPR (Molecular Devices, Sunnyvale, Calif., USA).
The inhibitors and ion channel types used in examples 1-6 are shown in table 1:
table 1.
| Examples | Group name | Inhibitors | Type of ion channel |
| 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 | Preparation example Abelmoschus manihot extract | TRPC3 |
| Example 5 | HK-D-total-extract | Preparation example Abelmoschus manihot extract | TRPC6 |
| Example 6 | HK-D-total-extract | Preparation example Abelmoschus manihot extract | TRPC7 |
The results of the tests of the above examples are shown in FIGS. 1 to 6. The abscissa in the figure is time in(s). In all the examples except the graph of example 4, the group with the highest peak shape in about 400s is M085 group, and in FIGS. 1 to 3, the group with the highest peak shape in about 400s is M085 group, 0.1. mu.M dose, 0.3. mu.M dose, 3. mu.M dose, 10. mu.M dose, 30. mu.M dose, and non-medicated control group (Veh) in the order from high to low. In FIGS. 4 to 6, the M085 group, the HK-D-total-extract group, and the non-medicated control group (Veh) were arranged in the order of the peak shape from high to low at around 400 s. In FIG. 4, the HK-D-total-extract group, the M085 group, and the non-medicated control group (Veh) were arranged in the order of the height from the top to the bottom in the peak pattern of about 400 s.
The results of the assays of examples 1-3 are shown in FIGS. 1-3, respectively, and different doses of protocatechuic acid in HEK293 cells of groups HK-12-TRPC3, HK-12-TRPC6, and HK-12-TRPC7 caused intracellular Ca2+The change in fluorescence intensity with time is significantly different. It can be seen that M085 significantly increased Ca in cells2+Fluorescence intensity, addition of protocatechuic acid reduced M085-induced intracellular Ca2+The degree of increase in fluorescence intensity and the dose dependence exhibited. Intracellular Ca at around 400s for each of the groups in examples 1 and 32+The fluorescence intensity reaches a peak and the time of the peak is almost similar, and the intracellular Ca is generated after 600s2 +The fluorescence intensity tends to be stable. Example 2 groups intracellular Ca after 400s2+The fluorescence intensity peaks and the intracellular Ca is reached after 600s2+The fluorescence intensity tends to be stable. The different amounts of protocatechuic acid can reduce intracellular Ca caused by M085 to different degrees2+The increase of fluorescence intensity indicates that the protocatechuic acid opens Ca caused by TRPC3/6/7 ion channel2+The internal flow showed inhibitory effect. In general, protocatechuic acid had an IC50 ═ 7.958 μ Μ for TRPC6 and an IC50 ═ 1.574 μ Μ for TRPC 7.
The detection results of examples 4-6 show that 50. mu.g/mL sunset abelmoschus flower extract can inhibit M085-induced calcium ion influx in TRPC6-HEK293 cells and TRPC7-HEK293 cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. Application of protocatechuic acid in preparing medicine for inhibiting calcium ion channel is provided.
2. The use according to claim 1, wherein the calcium channel is the TRPC channel.
3. The use as claimed in claim 2, wherein the TRPC channel is the TRPC3, TRPC6 or TRPC7 channel.
4. Use of protocatechuic acid for the preparation of a medicament for the diagnosis, treatment or co-treatment of cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, in particular diseases related to TRPC channels.
5. The application of a plant extract in preparing a medicament for treating calcium ion channel mediated diseases is characterized in that the plant extract contains more than 0.1 percent of protocatechuic acid by weight, and further contains 0.1 to 2.5 percent of protocatechuic acid by weight; further, the plant extract contains 0.4-1.2% by weight of protocatechuic acid; preferably, the plant is selected from Hibiscus plant and Malvaceae plant, and more preferably one or more of Abelmoschus manihot, Hibiscus viniferus, Hibiscus esculentus and Phyllanthus niruri.
6. The application of a flower extract of abelmoschus manihot in preparing a medicament for treating a calcium ion channel mediated disease is characterized in that the flower extract of abelmoschus manihot contains more than 0.1% by weight of protocatechuic acid, and further the flower extract of abelmoschus manihot contains 0.1-2.5% by weight of protocatechuic acid; further, the sunset abelmoschus flower extract contains 0.4-1.2% by weight of protocatechuic acid.
7. The use according to claim 5 or claim 6, wherein the disease mediated by calcium ion channels is cardiovascular disease, coronary heart disease, atherosclerosis, end stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.
8. The use as claimed in claim 5 or claim 6, wherein the calcium channel is a TRPC channel, further a TRPC3, TRPC6 or TRPC7 channel.
9. A medicament for the treatment or co-treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease, wherein the medicament comprises protocatechuic acid.
10. A pharmacological tool for characterizing channels belonging to different TRPC subfamilies, characterized in that said pharmacological tool comprises protocatechuic acid.
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