Background
Cardiovascular diseases are the most dangerous diseases for human life and health in modern society, and according to the report of the world health organization, about 1790 million people die of cardiovascular diseases in 2016 years all over the world, accounting for 31 percent of the total death all over the world. Thus, 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. Patent application WO2006/074802A1 discloses that TRPC channel can be used for treating cardiovascular and cerebrovascular diseases, and the research shows that by using gene technology, in rabbit atherosclerosis model, the vascular function and vascular pathological changes of atherosclerosis can be obviously improved by inhibiting activity of vascular endothelial cells TRPC3\ TRPC6 and TRPC7.
The TRPC channel is a Ca 2+ 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 constitute a subpopulation; there is about 65% amino acid homology between TRPC4 and TRPC5, thus classifying them as the same subgroup; 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 at present, diacylglycerol (DAG) and 4-ethyl- (3- (4-fluorophenyl) -7-hydroxy-2-methylpyrazole [1,5-a ] are known as endogenous ligands]-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 (BTP 2) and inorganic blockers (e.g. Gd) 3+ And La 3 + ) Etc., but 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. Because of their widespread and partially overlapping distribution, potential heteropolymerization, similar electrophysiological properties, and the lack of compounds that specifically track these channelsTools, there are certain difficulties in achieving in situ identification of native TRPC 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 potential for heteroterminal of the 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 isoforms or isoforms 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 Jiayou Ben (a general plant of Jiayou) for the earliest time, is widely distributed and is abundant in resources, and is recorded in Ben gang mu: the flower smell is sweet, cold, slippery and nontoxic, and is mainly used for treating urinary stranguria and hastening the growth, and for treating malignant sores and pus which are not healed for a long time, the medicament is used as a main medicament for sores, can eliminate gangrene swelling, is soaked in oil and is applied to soup and injured by fire, and the like.
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, quercetin-3-beta-D-glucoside, quercetin, etc.
At present, no research shows that the inhibition effect of quercetin-3-O-robinin on TRPC channel and the application thereof in preparing the medicine for treating cardiovascular diseases, coronary heart disease, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases related to the TRPC channel are provided.
In view of this, the invention provides the application of the abelmoschus manihot extract as an inhibitor of a TRPC ion channel and the preparation of a medicament for treating cardiovascular diseases, coronary heart diseases, atherosclerosis, end-stage renal failure, neurological diseases, chronic pain, acute pain or inflammatory diseases, and proves that quercetin-3-O-robioside in the extract is used as a new pharmacological tool, can selectively inhibit the TRPC ion channel, 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 elucidated, ideas are developed for cardiovascular and cerebrovascular diseases and the like, and the application 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 permeable 2+ 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 TRPC7. TRPC3, TRPC6 and TRPC7 are particularly preferred.
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, medically and antiviral abilities. 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 to, 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 generally 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: generally, a cell expressing a TRPC channel is 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 cell is usually placed in, for example, a microplate and grown. Typically cells are grown and fixed at the bottom of a multi-well plate. These cells are then washed routinely and a dye, preferably a fluorescent dye such as fluo4am, is added in a suitable loading buffer. At the time of removingAfter the buffer has been applied, the cells are incubated with test compounds or modulators (in particular the biochemical or chemical test compounds mentioned above, for example in the form of chemical compound libraries) Ca 2+ The measurement can be performed by using, for example, a fluorescence imaging plate reader (FLIPR). To stimulate Ca2+ influx through TRPC channels, 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 may 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 cells" in the context of the present invention refers to cells or recombinant cells that endogenously express the 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 from a mammal such as a human. Tissue samples can be obtained by well-known techniques, such as blood sampling, tissue lancing, or surgical techniques.
6. The term "medicament" in the context of the present invention refers to a therapeutic agent comprising a therapeutically effective amount of quercetin-3-O-robinoside, or a plant extract comprising these compounds. The medicament 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 buccal 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, usually using an injection solution.
In one aspect, the invention provides the use of quercetin-3-O-robinin in the preparation of a medicament for the treatment of a calcium channel mediated disease.
As an example, the invention provides application of quercetin-3-O-robioside in preparation of a medicine for inhibiting a calcium ion channel.
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 channels TRPC3, TRPC6 or TRPC7 channels described above.
As an illustrative or preferred example, the above calcium channel refers to a calcium channel used by quercetin-3-O-robioside for inhibition in vitro and in vivo.
In another aspect, the present invention provides the use of quercetin-3-O-robinoside in the preparation 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.
By way of example, the present invention provides the use of quercetin-3-O-robinin 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 more than 0.2% by weight of quercetin-3-O-robioside; further, the plant extract contains 0.2-1.2% by weight of quercetin-3-O-robioside; further, the plant extract contains 0.4-0.8% by weight of quercetin-3-O-robioside; 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 flower of abelmoschus manihot. The flos Abelmoschi Manihot flower extract is ethanol extract, preferably 50-95% ethanol reflux extract, and more preferably 80-95% ethanol reflux extract.
The abelmoschus manihot 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 the 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 extract of the flower of abelmoschus manihot.
The preparation method of the sunset abelmoschus flower extract comprises the following steps: extracting the flower of abelmoschus manihot with 95% ethanol under reflux for 2 times, each time for 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating the filtrate until the specific gravity is 1.20, standing the concentrated solution at 0-4 ℃ for 24-48 hours, removing an oil layer of the refrigerating solution, adjusting the pH value to 6.0, slowly adding the refrigerating solution into a thin-layer quick-drying roller tank, enabling the liquid level of the refrigerating solution in the roller tank to be just in contact with the surface of a roller body, preheating the surface temperature of the roller body to 140-150 ℃, controlling the air pressure to 0.4-0.5 Mpa, opening a roller rolling start button, controlling the rotating speed of the roller body to be 3-3.5 minutes/revolution, coating the rolled extract liquid on a polytetrafluoroethylene plate for cooling, cooling until the dried material becomes brittle after cooling, breaking, and filling the dried material into a clean double-layer plastic bag to obtain the flower of abelmoschus 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, and 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, preferably the polytetrafluoroethylene plate.
The preferable preparation method of the sunset abelmoschus 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 as a medicinal material, 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 liquid, adjusting the pH value to 6.0, slowly adding the concentrated solution into a drier 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 0.2% by weight quercetin-3-O-robioside; further, the sunset abelmoschus flower extract contains 0.2-1.2% by weight of quercetin-3-O-robioside; further, the sunset abelmoschus flower extract contains 0.4-0.8% by weight of quercetin-3-O-robioside.
On the other hand, the invention provides the application of the abelmoschus manihot extract in preparing a medicament for inhibiting a calcium ion channel.
In another aspect, the invention provides the use of the above abelmoschus manihot extract in the manufacture of a medicament for the diagnosis, treatment or co-treatment of cardiovascular disease, coronary heart disease, atherosclerosis, end-stage renal failure, neurological disease, chronic pain, acute pain or inflammatory disease.
In another aspect, the present 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 quercetin-3-O-robioside.
As an example, the present 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 quercetin-3-O-robioside.
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. That is, the present invention provides a pharmacological tool for characterizing channels belonging to different TRPC subfamilies, said pharmacological tool comprising quercetin-3-O-robioside.
According to the present invention, this is achieved by inhibiting TRPC3, TRPC6 and TRPC7 with quercetin-3-O-robioside. Thus, quercetin-3-O-robioside enables pharmacological differentiation of channels belonging to different TRPC subfamilies. In addition, quercetin-3-O-robioside 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 quercetin-3-O-robioside a preferred tool for identifying and modulating TRPC3, TRPC6 and TRPC7.
As an inhibitor of TRPC3, TRPC6 and TRPC7, quercetin-3-O-robioside may be used as a pharmacological tool, capable of characterizing channels belonging to different TRPC subfamilies, distinguishing TRPC3/6/7 subfamily members from other ion channel family members (fig. 1-6).
As such an inhibitor, quercetin-3-O-robioside may further be used as a tool compound for developing and validating assays to measure 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 quercetin-3-O-robioside for differential analysis of channel function of TRPC3/6/7 subfamily members under physiological and pathological 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 quercetin-3-O-robioside 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 measuring the influence of quercetin-3-O-robioside on TRPC channel activity, preferably TRPC ion channels TRPC3, TRPC6 and TRPC7.
Generally, a cell expressing a TRPC ion channel is contacted with quercetin-3-O-robioside, and the effect of quercetin-3-O-robioside on the TRPC ion channel activity is measured or detected.
In another aspect, the present invention provides: for the method of identifying a TRPC ion channel modulator, preferred TRPC ion channels are TRPC3, TRPC6 and TRPC7.
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) 2+ 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 quercetin-3-O-robioside with fluorescent cells expressing TRPC ion channels and stimulating Ca with a channel activator before, simultaneously with or after the contacting 2+ Internal flow;
(2) Detecting a change in TRPC ion channel activity.
In another aspect, the present invention provides: directed to methods of describing the selectivity of quercetin-3-O-robioside for the TRPC channel, comprising assessing the ability of quercetin-3-O-robioside to inhibit the TRPC channel activity.
The invention has the following beneficial effects:
(1) The invention provides a new application of quercetin-3-O-robinin, 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, end-stage 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.
Detailed Description
The present invention will be further described with reference to specific examples, which are intended to illustrate various embodiments of the present invention. Other embodiments obtained by persons skilled in the art without making creative efforts based on the embodiments in the implementation belong to the protection scope of the 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 structure of the quercetin-3-O-robioside compound related in the embodiment of the invention is 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 the specific gravity of 1.20, standing the concentrated solution at the temperature of 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 the temperature of 100 ℃, crushing, and filling into a clean double-layer plastic bag to obtain the abelmoschus manihot extract. Wherein the composition contains quercetin-3-O-robioside 5.6 (mg/g), isoquercitrin 12.2 (mg/g), and quercetin-3' -O-beta-D-glucoside 11.2 (mg/g).
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 the corresponding antibiotics selected for plasmid resistance to selectively screen for cells that were successfully transfected. 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 for stably expressing 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 10-13X 104 cells/mL, 150. Mu.L of cell suspension per well. 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 60min.
(4) Channel activator and inhibitor solutions were formulated as follows:
TRPC6 agonist (M085) preparation weighing the appropriate amount of M085 in dimethyl sulfoxide (DMSO) to obtain 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 give a final concentration of M085 of 1. Mu.M.
2. The preparation of the medicine comprises the following steps: weighing an appropriate amount of the flower extract of abelmoschus manihot in the preparation example, and dissolving the flower extract of abelmoschus manihot in DMSO to obtain a flower extract mother liquor with a concentration of 300 mg/mL. In the FLIPR experiment, locke's buffer and other reagents were added according to the experimental procedure to make the final concentration of Abelmoschus manihot extract 50 μ g/mL.
Quercetin-3-O-robinin monomeric compound solution: the appropriate amount of the four monomeric compounds was weighed and dissolved in DMSO to obtain a monomeric compound stock solution with a concentration of 10 mM. In the FLIPR experiments, 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 quercetin-3-O-robioside, flos Abelmoschi Manihot extract (see Table 1), and the inhibitor of each example is set at five gradient doses of 0.1 μ M, 0.3 μ M, 3 μ M, 10 μ M, and 30 μ M, and is set at Veh-free control group.
(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.
The results 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 graphs of examples 2 and 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. 5 to 6, the M085 group, the HK-D-total-extract group, and the non-medicated control group (Veh) were arranged in the order from the top to the bottom in the peak shape of about 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 tests of examples 1-3 are shown in FIGS. 4-6, respectively, and different doses of quercetin-3-O-robioside induced intracellular Ca in HEK293 cells of the HK-12-TRPC3, HK-12-TRPC6, HK-12-TRPC7 groups 2+ The change in fluorescence intensity with time is significantly different. Quercetin-3-O-robioside with different dosages each dosage group can reduce intracellular Ca caused by M085 2+ The increase in fluorescence intensity was substantial and dose-dependent. Intracellular Ca at 400s for each of the groups in FIGS. 4 and 6 2+ The fluorescence intensity reaches the peak, the peak reaching time is almost the same, and the intracellular calcium ion fluorescence intensity tends to be stable after 600 s. Among them, quercetin-3-O-robioside at a dose of 30. Mu.M had the best effect of inhibiting the fluorescence intensity of intracellular calcium ions. Each group of quercetin-3-O-robinin can reduce intracellular Ca caused by M085 2+ The increase of fluorescence intensity indicates that Ca caused by the opening of the quercetin-3-O-robinin on TRPC3/6/7 2+ The inflow showed inhibitory effect. Overall, IC50=6.746 μ Μ for TRPC6 and IC50= for TRPC7 for quercetin-3-O-robininoside9.007μM。
The test results of examples 4-6 are shown in fig. 4-6, respectively, and 50 μ g/mL of 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.