CN102552372A - Novel purpose of eucommia chemical components as blood vessel protective agent - Google Patents
Novel purpose of eucommia chemical components as blood vessel protective agent Download PDFInfo
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- CN102552372A CN102552372A CN2011103028359A CN201110302835A CN102552372A CN 102552372 A CN102552372 A CN 102552372A CN 2011103028359 A CN2011103028359 A CN 2011103028359A CN 201110302835 A CN201110302835 A CN 201110302835A CN 102552372 A CN102552372 A CN 102552372A
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Abstract
The invention relates to a novel purpose of eucommia chemical components as blood vessel protective agents. Specifically, the invention relates to a purpose of eucommia or eucommia extracts in preparing medicines used as blood vessel protective agents and/or antihypertensive agents, or in preparing medicines used for treating and/or preventing vascular proliferation diseases. The eucommia extracts comprise at least one component selected from the following: geniposide, wogonin, Qianye extract A, alpha-oxygen-beta-D-glucopyranoside-4,2', 4'-trihydroxyl dihydrochalcone, and betulic acid. According to the invention, the 5 chemical components or the eucommia extracts comprising the 5 chemical components can be used as effective blood vessel protective agents and/or antihypertensive agents, and can be used for treating and/or preventing vascular proliferation diseases.
Description
Technical field
The present invention relates to the new purposes of the some chemical constituents in the Chinese medicine material Cortex Eucommiae; Particularly the Chiba element A in the Cortex Eucommiae, wogonin, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide be as the pharmaceutical applications of blood vessel protective agent.
Background technology
The human blood circulatory system has the homergy equilibrium function of himself, but it is unusual vascular function to occur, can cause the infringement to vascular system.As vascular smooth muscle cell proliferation be atheromatous plaque form with coronary angioplasty after the pathologic basis of generation restenosis; The calcium ion concentration rising can cause vasoconstriction in the smooth muscle cell, and vasomotoricity can cause hypertensive generation unusually.Hypotensive effect is brought into play in the rising of calcium ion concentration and the vasoconstriction that the antagonism norepinephrine causes in the VSMC that some blood vessel protective agents can cause through the high potassium of antagonism; Form through suppressing vascular smooth muscle cell proliferation control atheromatous plaque; Thereby keep the normal physiological function of blood vessel, in preventing and treating hypertension, atherosclerosis, postangioplasty restenosis, coronary heart disease, cerebrovascular, have important effect.
Well-known; Some have been applied to clinical blood vessel protective agent and have had many useful physiological roles; For example can reduce capillary permeability and fragility, prevent because of vascular permeability raise the edema cause, blood vessel injury, protection vascular endothelial cell that the gentle kassinin kinin of medmain is caused, promote neovascularization to promote side Zhi Xunhuan, anticoagulant, prevent thrombosis, the acute ischemic brain injury is had remarkable protective effect; They also have effects such as anti-lonizing radiation operation, antiinflammatory, antiallergic, antiulcer, syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before the hemiplegia that causes applicable to obliterated cerebral vascular disease, aphasia, the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis etc.Blood vessel protective agent refluxes, reduce edema, blood viscosity lowering, correction albumins/globulins ratio, reduce hematoblastic high aggregation and prevent thrombosis, improve the erythrocyte pliability thereby can be used for reducing the wall of micrangium permeability, build up one's resistance to disease, improve lymph fluid.Blood vessel protective agent also can be used for suppressing the high penetration effect that vaso-active substance (for example histamine, 5-hydroxy tryptamine, Kallidin I, hyaluronidase, prostaglandin etc.) causes blood capillary, reduces the tunica intima damage, improves the biosynthesis of basement membrane collagen.Clinically applicable to diabetic machine nethike embrane pathological changes, various microangiopathys, varicosis etc.
The Cortex Eucommiae is the bark of Eucommiaceae plant Cortex Eucommiae Eucommia ulmoides Oliv., has another name called to think celestial (" herbal classic "), and wood is continuous, think secondary (" not Lu "), Shi Sixian (" Amplification on Materia Medica addendum ").The little suffering of sweet in the mouth is warm in naturely gone into liver, kidney channel.Has invigorating the liver and kidney, bone and muscle strengthening, antiabortive effect.Be used to treat the spinal column ache, foot and knee flaccidity, dribbling urination, uncontrolled urination is itched, and vaginal bleeding during pregnancy is desired to fall, frequent fetal movement.
In view of the good effect of the Cortex Eucommiae, the medicine scholar has carried out extensive studies to its chemical constituent.Isolating bioactive ingredients mainly contains three types from the Cortex Eucommiae at present, lignin, chemical constituents such as iridoids and flavonoid.Modern pharmacological research shows that the hypotensive effect of the Cortex Eucommiae is relevant with pinoresinol diglucoside, alkaloid, chlorogenic acid, aucubin and saccharide.Antitumor action and lignin, phenylpropyl alcohol element are relevant with iridoid.Its decocting liquid and alcohol extract all have the kidney invigorating, enhance immunity effect.Cortex Eucommiae medical material also has antioxidation, defying age, aging, antibiotic, the antiviral effect of anti-muscle skeleton.
CN101347418 (one Chinese patent application number: 200710044099.5; Open day: on January 21st, 2009) studied the purposes of 8-O-4 ' type lignin in preparation ACA medicine; This invention it is said that separation and Extraction obtains chemical compound 8-O-4 ' type lignanoid from the Chinese medicine Cortex Eucommiae; Confirm that through experiment in vitro the classics in complement system are activated the cell haemolysis that is caused with alternative pathway has inhibition, the classics and the alternative pathway activation of complement system had obvious inhibitory action; Pharmacological tests proves, have significant anticomplementary action, and valid density is low.
CN101260131 (one Chinese patent application number: 200810031122.1, open day: on JIUYUE 10th, 2008) disclose from the Cortex Eucommiae, extract obtain iridoid active site and monomer geniposidic acid (geniposidic acid, GPA), geniposide (geniposide; GP) and aucubin (aucubin; AU) method, this method is a raw material with the Cortex Eucommiae (skin, leaf or seed), adopts conventional extraction, ultrasonic extraction or microwave extraction method that raw material is extracted; With methods such as solvent extraction, extraordinary adsorbents adsorb separation extracting solution is carried out preliminary purification; Obtaining iridoid active site (the iridoid total amount is not less than 50%), adopt the preparative high-performance liquid chromatographic technology again, is mobile phase with the alcohol-water solution; GPA, GP and AU are separated; Cryoconcentration and lyophilization obtain above three kinds of monomers, and its content all is not less than 90%.
Still need the new blood vessel protective agent and/or the method for blood pressure lowering in the clinical treatment.
Summary of the invention
The purpose of this invention is to provide effective blood vessel protective agent and hypotensive agent, be the approach that provides new that treats and/or prevents of relevant disease clinically.The phytochemistry that the inventor carries out system to the Cortex Eucommiae separates, finds unexpectedly, and the Chinese crude drug Cortex Eucommiae, particularly its extract, some monomer chemical constituent of perhaps from the Cortex Eucommiae, extracting has the effect of effective blood vessel protective agent and/or blood pressure lowering etc.The present invention is based on above-mentioned discovery and be accomplished.
For this reason, first aspect present invention provides the Cortex Eucommiae to be used for the purposes as the medicine of blood vessel protective agent and/or hypotensive agent in preparation, perhaps is used for treating and/or preventing the purposes of the medicine of vascular proliferative disease in preparation.
According to each purposes of first aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular.
According to each purposes of first aspect present invention, wherein said vascular proliferative disease is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.
According to each purposes of first aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
According to each purposes of first aspect present invention, wherein said medicine gives mammal, and its dosage is that said mammal every kg body weight every day is equivalent to Cortex Eucommiae medical material 1-40g, preferred 2-20g, more preferably 5-10g.
Second aspect present invention provides Cortex Eucommiae extract to be used for the purposes as the medicine of blood vessel protective agent and/or hypotensive agent in preparation, perhaps is used for treating and/or preventing the purposes of the medicine of vascular proliferative disease in preparation.
According to each purposes of second aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.
According to each purposes of second aspect present invention, wherein said vascular proliferative disease is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.
According to each purposes of second aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
According to each purposes of second aspect present invention; Comprise at least a following composition that is selected from the wherein said Cortex Eucommiae extract: geniposide (can be described as EUB30-1), wogonin (can be described as EUC-4), Chiba element A (can be described as EUC-6), α-oxygen-β-D-glucopyanosyl base-4 at this paper at this paper at this paper; 2 ', 4 '-trihydroxy dihydrochalcone (can be described as EUE-3) and betulic acid (can be described as EUC-2) at this paper at this paper.
According to each purposes of second aspect present invention; Comprise following composition in the wherein said Cortex Eucommiae extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each purposes of second aspect present invention, wherein said geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4,2 '; 4 '-the trihydroxy dihydrochalcone and from the total amount of pine gum acid account for said extract gross weight 1-90% (w/w, i.e. percetage by weight, down with); Preferred 2-90%, preferred 5-80%, preferred 5-60%; Preferred 5-50%, preferred 5-30%, preferred 5-20%.
According to each purposes of second aspect present invention, the dosage of wherein said medicine is that said mammal every kg body weight every day is equivalent to Cortex Eucommiae medical material 1-40g, preferred 2-20g, more preferably 5-10g.
According to each purposes of second aspect present invention, wherein said Cortex Eucommiae extract obtains as solvent extraction through water, ethanol or ethanol water.In one embodiment, described Cortex Eucommiae extract be through water, ethanol or ethanol water as solvent extraction, pass through then that purification (for example adopting solvent-extracted method) obtains.Said solvent extraction uses such as but not limited to following solvent: petroleum ether, chloroform, ethyl acetate, n-butyl alcohol and combination thereof.
According to each purposes of second aspect present invention, wherein said Cortex Eucommiae extract prepares through following steps: the Cortex Eucommiae is extracted with aquiferous ethanol solution; Make the gained alcohol extract use petroleum ether extraction, discard ether layer concentrates residue, and drying gets extract; Perhaps make the gained alcohol extract use petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, with each extract reclaim respectively behind the solvent solid content, the solid content of combined chloroform part, ethyl acetate part and n-butyl alcohol part, extract.
According to each purposes of second aspect present invention, wherein said Cortex Eucommiae extract prepares through following steps:
A) with the Cortex Eucommiae with 2～20 times of amounts (preferred 5～15 times of amounts; More preferably 8～12 times of amounts; 10 times of amounts for example) 60～98% (preferred 70～98%; More preferably 80～98%, for example 80%, 85%, 90%, 95% or 98%) soak with ethanol 5～24 hours (preferred 5～18 hours, more preferably 8～15 hours);
B) reflux, extract, 0.5～10 hour (preferred 0.5～8 hour, more preferably 0.5～6 hour, more preferably 0.5～4 hour, more preferably 1～4 hour, for example 1 hour, 2 hours, 3 hours, 4 hours), inclining extracting solution;
C) randomly make the medicinal residues of step b) repeat step a) and step b) 1～3 time (for example 1 time, 2 times, 3 times);
Combining step b) and the extracting solution of step c) d), filter, filtrate recycling ethanol to density at room temperature is about the concentrated solution of 1.15-1.20, extract; Perhaps further
E) make the concentrated solution of step d) use petroleum ether extraction, discard ether layer concentrates residue, and drying gets extract; Perhaps, e) make the concentrated solution of step d) use petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, with each extract reclaim respectively behind the solvent solid content, the solid content of combined chloroform part, ethyl acetate part and n-butyl alcohol part, extract.
The chemical composition of the extract of said method gained can be known through the present invention's purification step hereinafter described; Perhaps directly carrying out chromatography records and comprises at least a following composition that is selected from this extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.Preferably comprise whole above-mentioned 5 kinds of compositions.
Third aspect present invention provide be selected from following ingredients one or more be combined in preparation as the purposes in the medicine of blood vessel protective agent and/or hypotensive agent; Perhaps be used for treating and/or preventing the purposes of the medicine of vascular proliferative disease: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4 in preparation; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each purposes of third aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.According to each purposes of third aspect present invention, wherein said vascular proliferative disease is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.According to each purposes of third aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
According to each purposes of third aspect present invention; It is that following ingredients is preparing as the purposes in the medicine of blood vessel protective agent and/or hypotensive agent: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each purposes of first aspect present invention, wherein said medicine gives mammal, and its dosage is that said mammal every kg body weight every day is equivalent to Cortex Eucommiae medical material 1-40g, preferred 2-20g, more preferably 5-10g.
According to each purposes of third aspect present invention, wherein said medicine gives mammal, with said mammal every kg weighing machine every day, wherein said each composition separately or the dosage during combination in any can be respectively independently of each other:
| Composition | Dosage (mg/kg body weight/day) |
| Geniposide | (0.01-4mg preferred 0.1-1mg) |
| Wogonin | (0.05-2mg preferred 0.2-1mg) |
| Chiba element A | (0.05-2mg preferred 0.2-1mg) |
| α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-the trihydroxy dihydrochalcone | (0.05-2mg preferred 0.2-1mg) |
| Betulic acid | (0.01-4mg preferred 0.1-1mg) |
According to each purposes of third aspect present invention; Wherein one or more combination of following ingredients is from the Cortex Eucommiae, to extract to obtain: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each purposes of third aspect present invention, wherein from the Cortex Eucommiae, extract geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-step of trihydroxy dihydrochalcone and betulic acid is following:
The Cortex Eucommiae is extracted with aquiferous ethanol solution;
Make the gained alcohol extract use petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, with each extract reclaim respectively behind the solvent the solid content of each extractant part;
After chloroform partly passes through silica gel column chromatography (petroleum ether-acetone eluting); 33～36 fractions are through obtain betulic acid respectively behind the silica gel column chromatography repeatedly; 43～56 fractions carry out silica gel column chromatography with chloroform-acetone, and the gained fraction obtains wogonin and Chiba element A respectively behind polyamide column chromatography;
Ethyl acetate is taked chloroform partly through silica gel column chromatography: methanol solvate system gradient elution, and repeatedly behind the silica gel column chromatography, obtain yellow powder shape material is EUE-3 to gained 49～55 fractions through ethyl acetate-methanol, chloroform-methanol solvent system;
After n-butyl alcohol partly passed through the D101 macroporous adsorbent resin, 30% ethanol water solvent eluting was partly crossed silica gel column chromatography (ethyl acetate-methanol gradient elution), and 18～20 flow points are with chloroform: methanol: H
2The O system carries out silica gel column chromatography, and separating out white powder in gained 8～14 flow points is geniposide (EUB30-1).
Fourth aspect present invention provides a kind of and has been used for as blood vessel protective agent and/or hypotensive agent or is used to treat and/or prevent the pharmaceutical composition of mammal (particularly people) vascular proliferative disease, wherein comprises the Cortex Eucommiae extract that treats and/or prevents effective dose and the optional acceptable excipient of pharmacy.
According to each pharmaceutical composition of fourth aspect present invention; Comprise at least a following composition that is selected from the wherein said Cortex Eucommiae extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each pharmaceutical composition of fourth aspect present invention; Comprise following composition in the wherein said Cortex Eucommiae extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each pharmaceutical composition of fourth aspect present invention, wherein said geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4,2 '; 4 '-total amount of trihydroxy dihydrochalcone and betulic acid account for the 1-90% of said extract gross weight (w/w, i.e. percetage by weight, down with); Preferred 2-90%, preferred 5-80%, preferred 5-60%; Preferred 5-50%, preferred 5-30%, preferred 5-20%.
According to each pharmaceutical composition of fourth aspect present invention, it is that said mammal every kg body weight every day is equivalent to Cortex Eucommiae medical material 1-40g that wherein said pharmaceutical composition gives said mammiferous dosage, preferred 2-20g, more preferably 5-10g.
According to each pharmaceutical composition of fourth aspect present invention, wherein said Cortex Eucommiae extract prepares through following steps: the Cortex Eucommiae is extracted with aquiferous ethanol solution; Make the gained alcohol extract use petroleum ether extraction, discard ether layer concentrates residue, and drying gets extract; Perhaps make the gained alcohol extract use petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, with each extract reclaim respectively behind the solvent solid content, the solid content of combined chloroform part, ethyl acetate part and n-butyl alcohol part, extract.The further preferred step for preparing said extract is mentioned referring to second aspect present invention.
Fifth aspect present invention provides a kind of and has been used for as blood vessel protective agent and/or hypotensive agent or is used to treat and/or prevent the pharmaceutical composition of mammal (particularly people) vascular proliferative disease; Wherein comprise at least a active constituents of medicine that treats and/or prevents effective dose and the optional acceptable excipient of pharmacy; Described active constituents of medicine is selected from: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each pharmaceutical composition of fifth aspect present invention; Wherein comprise the geniposide, wogonin, Chiba element A, the α-oxygen-β-D-glucopyanosyl base-4 that treat and/or prevent effective dose; 2 '; 4 '-trihydroxy dihydrochalcone and betulic acid, and the optional acceptable excipient of pharmacy.
Sixth aspect present invention provides a kind of method of in the mammal (particularly people) that needs is arranged, carrying out vascular protection and/or abrupt antihypertensive therapy and/or prevention; Perhaps, the mammal (particularly people) that needs treats and/or prevents the method for vascular proliferative disease in being arranged; Said method comprises the Cortex Eucommiae that treats and/or prevents effective dose to said administration; Perhaps administering therapeutic and/or the prevention effective dose Cortex Eucommiae extract; Perhaps administering therapeutic and/or at least a of prevention effective dose are selected from following composition: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
According to each method of sixth aspect present invention, wherein said vascular proliferative disease is selected from: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache.According to each method of sixth aspect present invention, wherein said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
Arbitrary characteristic that the arbitrary aspect of the present invention or this arbitrary aspect each (embodiment) had is equally applicable to other arbitrary embodiment of the arbitrary aspect of the present invention; As long as they can be not conflicting; Certainly at where applicable each other, necessary words can be done suitably to modify to individual features.In the present invention, when for example, mentioning " first aspect present invention each ", should " each " be meant the arbitrary sub-aspect (embodiment) of first aspect present invention; When others are mentioned in a similar manner, also has identical meanings.
Detailed Description Of The Invention:
Do further to describe with characteristics to various aspects of the present invention below.
All documents that the present invention quoted from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition; Various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art; Nonetheless; The present invention still hopes at this more detailed explanation and explanation to be done in these terms and phrase, and term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.
As described herein, term " Cortex Eucommiae " meets the regulation under the version Pharmacopoeia of the People's Republic of China corresponding entry in 2005.
The term " about " of using among this paper, for example when modifying the productive rate of preparation product, it typically refers to the range of error that this area allows, for example ± 10%, for example ± 5%, for example ± 2%.
The phrase that uses among this paper " vascular proliferative disease " has and well known to a person skilled in the art one type of disease or symptom; And generally include but be not limited to: with blood vessel injury diseases associated or disease; The vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, or the like.In one embodiment, blood vessel protective agent according to the invention is used to treat and/or prevent and is selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
Although the present invention has made detailed description to related active component; The inventor still is willing to be intended to this and stresses; (it includes, but not limited to geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4 to these active component; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid) have chemical constitution well known by persons skilled in the art, physicochemical properties.
As described herein, term " effective dose " is meant the dosage that can in the experimenter, realize treating, prevent, alleviate and/or alleviating disease according to the invention or disease.
As described herein, term " pharmaceutical composition " is meant to be used in the material of realizing treating, prevent, alleviate and/or alleviating disease according to the invention, disease, symptom among the experimenter.
As described herein; Term " experimenter " can refer to that patient or other accept the present composition and extract to treat, to prevent, to alleviate and/or to alleviate the animal of disease according to the invention, disease, symptom; Particularly mammal, for example people, Canis familiaris L., monkey, cattle, horse etc.
As described herein, term " disease or symptom " is meant a kind of condition of said experimenter, this condition is relevant with disease according to the invention or symptom.
As described herein, " % ", as do not specialize, generally be meant the percentage ratio of w/w when being solid for total material, generally be meant the percentage ratio of weight/volume when being liquid for total material.Certainly, be liquid and solute when being liquid for total material, the percentage ratio that characterizes this liquid solute generally is meant the percentage ratio of volume.
" the acceptable excipient of pharmacy " that use in the pharmaceutical composition of the present invention can be the excipient of any routine in the field of pharmaceutical preparations.The selection of particular excipient will be depended on administering mode or disease type and the state that is used to treat particular patient.The suitable drug preparation of compositions method that is used for the specific administration pattern is fully in drug world technical staff's the ken.For example, can be used as diluent, carrier, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier and the lubricant etc. that the acceptable excipient of pharmacy comprises that pharmaceutical field is conventional.In case of necessity, can also in pharmaceutical composition, add flavouring agent, antiseptic and sweeting agent etc.
Pharmaceutical composition of the present invention can be processed tablet, powder, granule, capsule, oral liquid, unguentum, cream, injectable emulsion various ways such as (sterile powder for injection pins).The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The inventor finds some chemical constituent and the Cortex Eucommiae extract in the Cortex Eucommiae, and they for example have the physiology's advantage as blood vessel protective agent.For example, in some embodiments, the inventor finds chemical constituent Chiba element A in the Cortex Eucommiae, wogonin can receptor modulators property Ca
2+Passage is brought into play quick vasodilatory effect, and Chiba element A, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide can pass through regulating and controlling voltage property Ca
2+Passage suppresses the interior calcium ion concentration of VSMC and raises and quick vasodilator, points out above-mentioned five kinds of chemical compounds to have vascular protection effect and hypotensive activity.More surprisingly wherein wogonin and betulic acid can also suppress vascular smooth muscle cell proliferation; The hyper-proliferative of VSMC (VSMC) is one of the common cell pathology of diseases such as atherosclerosis, hypertension and postangioplasty restenosis basis; Suppressing the VSMC abnormality proliferation is one of important channel of treatment of vascular proliferative disease, and result of study prompting The compounds of this invention therefore of the present invention or extract can be used to prepare the medicine of diseases such as preventing and treating atherosclerosis, hypertension and vascular restenosis.
Description of drawings
Fig. 1 has shown that Chiba element A can reduce the rising of the intracellular calcium concentration that high potassium causes.This figure is that Chiba element A is to 60mM K
+The time-histories figure of the interior calcium ion concentration influence of the rat chest aorta VSMC (A7r5) that causes.That represent among the figure is 60mM K
+The rising of ability significant stimulation A7r5 intracellular calcium concentration; Chiba element A (0.001mM, 0.01mM, but 0.1mM) concentration relies on the rising that ground suppresses the post-stimulatory A7r5 intracellular calcium concentration of high potassium; This effect is not by estrogen receptor antagon (ICI-182780 (Faslodex); Can be abbreviated as ICI or ICI-182780 or ICI182780 in this article) institute's antagonism, 0.01mM Chiba element A acts on the A7r5 cell separately, does not cause the rising of calcium ion concentration.Data are represented with the % that the fluorescence intensity of every each time point of porocyte accounts for the fluorescence intensity of this porocyte in the time of 0 second.
*Relatively there is significant difference p＜0.01 group with matched group.The control that occurs among Fig. 1 and other each figure representes contrast." control " curve representation does not add potassium, not dosing, does not add ICI among the figure; " 60mM K
+" shown in curve representation add 60mM K
+But do not add other material, " 60mM K
++ 0.001mM " shown in curve representation add 60mM K
+Also add the 0.001mM medicine, " 60mM K
++ ICI+0.01mM " shown in curve representation add 60mM K
+Also add ICI and also add the 0.01mM medicine, curve representation shown in " 0.01mM " adds the 0.01mM medicine but does not add potassium and also do not add ICI; Also has similar meaning among the result of the test figure of other correlation method.
Fig. 2 shown α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-the trihydroxy dihydrochalcone can reduce the rising of the intracellular calcium concentration that high potassium causes.This figure be α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-the trihydroxy dihydrochalcone is to 60mM K
+The time-histories figure of the interior calcium ion concentration influence of the rat chest aorta VSMC (A7r5) that causes.That represent among the figure is 60mM K
+The rising of ability significant stimulation A7r5 intracellular calcium concentration, α-oxygen-β-D-glucopyanosyl base-4,2 '; 4 '-trihydroxy dihydrochalcone (0.001mM; 0.01mM, but 0.1mM) concentration relies on the rising that ground suppresses the post-stimulatory A7r5 intracellular calcium concentration of high potassium, and this effect is not by estrogen receptor antagon (ICI) institute antagonism; 0.01mM Chiba element A acts on the A7r5 cell separately, does not cause the rising of calcium ion concentration.Data are represented with the % that the fluorescence intensity of every each time point of porocyte accounts for the fluorescence intensity of this porocyte in the time of 0 second.
*P＜0.05 group is more variant with matched group,
*Relatively there is significant difference p＜0.01 group with matched group.
Fig. 3 has shown that betulic acid can reduce the rising of the intracellular calcium concentration that high potassium causes.This figure is that betulic acid is to 60mM K
+The time-histories figure of the interior calcium ion concentration influence of the rat chest aorta VSMC (A7r5) that causes.60mM K
+The rising of ability significant stimulation A7r5 intracellular calcium concentration; Betulic acid (0.001mM, 0.01mM, but 0.1mM) concentration relies on the rising that ground suppresses the post-stimulatory A7r5 intracellular calcium concentration of high potassium; 0.01mM betulic acid acts on the A7r5 cell separately, does not cause the rising of calcium ion concentration.Data are represented with the % that the fluorescence intensity of every each time point of porocyte accounts for the fluorescence intensity of this porocyte in the time of 0 second.
Fig. 4 has shown that geniposide can reduce the rising of the intracellular calcium concentration that high potassium causes.This figure is that geniposide is to 60mM K
+The time-histories figure of the interior calcium ion concentration influence of the rat chest aorta VSMC (A7r5) that causes.60mM K
+The rising of ability significant stimulation A7r5 intracellular calcium concentration; Geniposide (0.001mM, 0.01mM, but 0.1mM) concentration relies on the rising that ground suppresses the post-stimulatory A7r5 intracellular calcium concentration of high potassium; 0.01mM geniposide acts on the A7r5 cell separately, does not cause the rising of calcium ion concentration.Data are represented with the % that the fluorescence intensity of every each time point of porocyte accounts for the fluorescence intensity of this porocyte in the time of 0 second.
But Fig. 5 a, Fig. 5 b have shown the rat chest aorta that Chiba element A diastole norepinephrine causes and have shunk.Fig. 5 a and Fig. 5 b show with final concentration 10
-6The complete vasoconstriction of the norepinephrine of M (NE) stimulating endothelial, accumulative total adds Chiba element A makes its final concentration be respectively 10
-6, 10
-5, 10
-4M, but the vasoconstriction that Chiba element A concentration dependent diastole norepinephrine (NE) causes have compared significant difference (P＜0.05 or P＜0.01) with contrast.The Channel that occurs among coordinate and other figure among Fig. 5 b representes passage.
But Fig. 6 a, Fig. 6 b have shown the rat chest aorta that wogonin diastole norepinephrine causes and have shunk.Fig. 6 a and Fig. 6 b show with final concentration 10
-6The complete vasoconstriction of the norepinephrine of M (NE) stimulating endothelial, accumulative total adds wogonin makes its final concentration be respectively 10
-6, 10
-5, 10
-4M, wogonin have vasodilatory trend, and concentration is 10
-4The pre-shrunk vascular ring that can obviously relax during M has been compared significant difference (P＜0.01) with contrast.
Fig. 7 a, Fig. 7 b have shown the Cortex Eucommiae 70% ethanol extraction (extract 2) but rat chest aorta that the diastole norepinephrine causes shrinks.Figure a and figure b show with final concentration 10
-6The complete vasoconstriction of the norepinephrine of M (NE) stimulating endothelial; Accumulative total adds the Cortex Eucommiae 70% ethanol extraction makes its final concentration be respectively 0.4; 0.8; 1.2mg/ml, but significant difference (P＜0.01) has been compared in the vasoconstriction that the Cortex Eucommiae 70% ethanol extraction concentration dependent diastole norepinephrine (NE) causes with matched group.
Fig. 8 a, Fig. 8 b have shown component 6 (extract 3) but rat chest aorta that the diastole norepinephrine causes shrinks.Figure a and figure b show with final concentration 10
-6The complete vasoconstriction of the norepinephrine of M (NE) stimulating endothelial, accumulative total adds component 6, makes its final concentration be respectively 0.4; 0.8; 1.2,1.6,2.0mg/ml; But significant difference (P＜0.01) has been compared in the vasoconstriction that the Cortex Eucommiae 70% ethanol extraction concentration dependent diastole norepinephrine (NE) causes with matched group.
Fig. 9 has shown that E2 and wogonin can suppress the inductive A7r5 vascular smooth muscle cell proliferation of platelet derived growth factor-BB (n=6).Wherein show the influence (n=6) of wogonin to the inductive A7r5 cell proliferation of PDGF-BB.Wogonin (10
-7, 10
-6, 10
-5M) but concentration dependent suppresses the A7r5 cell proliferation that platelet derived growth factor-BB stimulates, with matched group significant difference is arranged relatively.Vertical coordinate among the figure " cell viability (%of control) " expression " cell survival rate (% of contrast) ", wogonin representes wogonin.
Figure 10 has shown that betulic acid can suppress the inductive A7r5 VSMC of platelet derived growth factor-BB cell proliferation (n=6).Wherein show the influence (n=6) of betulic acid to the inductive A7r5 cell proliferation of PDGF-BB.Platelet derived growth factor-BB (10ng/ml) ability obvious stimulation A7r5 cell proliferation, betulic acid (10
-7, 10
-6, 10
-5M) but concentration dependent suppresses the A7r5 cell proliferation that platelet derived growth factor-BB stimulates, with matched group significant difference is arranged relatively.Vertical coordinate among the figure " cell viability (%of control) " expression " cell survival rate (% of contrast) ", betulinic acid representes betulic acid.
Figure 11 shown in the langendorff perfusion system, 70% ethanol extraction (extract 2) but 1mg/ml, 10mg/ml concentration dependent reduce isolated rat heart perfusion pressure, 10
-7The SNP of M/L is as contrast; Vertical coordinate is represented Coronary Perfusion Pressure; Abscissa is represented the group with Cortex Eucommiae extract dosage (DUZHONG (mg/ml)) sign; Before " before medicine " expression administration, after " after medicine " expression administration, also has similar sign among other correlation test method gained result's the figure among the figure.
Figure 12 shown in the langendorff perfusion system, component six (extract 3) but 0.1mg/ml, 1mg/ml, 10mg/ml concentration dependent reduce isolated rat heart perfusion pressure, 10
-7The SNP of M/L is as contrast, and vertical coordinate is represented Coronary Perfusion Pressure, and abscissa is represented the group with Cortex Eucommiae extract dosage (DUZHONG extraction (mg/ml)) sign.
The specific embodiment
Can further describe the present invention through following embodiment, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that are used in the test.Though for realizing that employed many materials of the object of the invention and operational approach are well known in the art, the present invention still does to describe in detail as far as possible at this.
Embodiment 1: the preparation of 5 chemical compounds in the Cortex Eucommiae
Get Cortex Eucommiae medical material (available from Xinyang, Henan medical material company) 31.5Kg; The ethanol extraction of 95% ethanol extraction twice, 60% once, merge extractive liquid; Be concentrated into and do not have the alcohol flavor; Be scattered in the water, use petroleum ether, chloroform, ethyl acetate and n-butanol extraction successively, four solvent extracts get solid content after reclaiming solvent respectively.The result is following: Cortex Eucommiae petroleum ether extraction part 153g, Cortex Eucommiae chloroform extraction part 384g, Cortex Eucommiae ethyl acetate extraction part 126g, Cortex Eucommiae n-butanol extraction part 520g.Wherein, Cortex Eucommiae n-butanol extraction partly adopts the D101 macroporous adsorbent resin column chromatography, through obtaining 140g, 50g respectively behind the ethanol water system gradient elution such as 30%, 50%.
Cortex Eucommiae ethanol extraction chloroform extraction partial purification process: Cortex Eucommiae chloroform extract 350g, carry out silica gel column chromatography and separate, use petroleum ether: acetone system gradient elution, common 105 flow points.33～36 flow points are used petroleum ether: the acetone system is labeled as EUC-2 respectively behind silica gel column chromatography repeatedly.After the literature value comparison, confirm that EUC-2 is a betulic acid.43～56 flow points are with chloroform: the acetone system carries out silica gel column chromatography, and the gained flow point obtains wogonin and Chiba element A behind twice polyamide column chromatography.
The ethyl acetate extraction part of Cortex Eucommiae ethanol extraction through silica gel column chromatography, is taked chloroform: methanol solvate system gradient elution; 19～27 flow points utilize silica gel column chromatography again; Pass through petroleum ether: ethyl acetate solvent system gradient elution obtains the white powder material, called after EUE-1.After the literature value comparison, confirm the EUE-1 genipin.49～55 flow points pass through ethyl acetate again: methanol, chloroform: behind the methanol solvate system repeatedly silica gel column chromatography, obtain yellow powder shape material EUE-3.With after the literature value comparison, confirm EUE-3 be α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-the trihydroxy dihydrochalcone.
After Cortex Eucommiae n-butyl alcohol part 500g crossed the D101 macroporous adsorbent resin, 30% part 130g carried out silica gel column chromatography and separates ethyl acetate: methanol solvate system gradient elution, 18～20 flow points are with chloroform: methanol: H
2The O system carries out silica gel column chromatography, and the adularescent powder is separated out in gained 8～14 flow points, is labeled as EUB30-1, after the literature value comparison, confirms that EUB30-1 is a geniposide.
Embodiment 2: the preparation of Cortex Eucommiae extract
Get Cortex Eucommiae medical material (available from Xinyang, Henan medical material company) 31.5Kg, the ethanol extraction of 95% ethanol extraction twice, 60% once, merge extractive liquid, is condensed into thick paste, drying under reduced pressure obtains extract, can be described as " extract 1 " in the present invention.
In addition, get Cortex Eucommiae medical material (available from Xinyang, Henan medical material company) 1kg, add 70% ethanol extraction twice, each 2h merges extracted twice liquid, reclaims ethanol and vacuum drying and gets the Cortex Eucommiae 70% ethanol extraction, also can be described as " extract 2 " in the present invention.
Get Cortex Eucommiae medical material 1kg, water reflux, extract, twice, each 2h; Merge extracted twice liquid, be evaporated to relative density 1.15 (50 ℃), equal-volume ethyl acetate extraction 4 times; The combined ethyl acetate extract; Reclaim ethyl acetate and concentrate vacuum drying, get extract, can be described as " extract 3 " or " component 6 " or " component 6 samples " or " component six " in the present invention.
Embodiment 3: the composition analysis of Cortex Eucommiae extract
Measure the composition in three kinds of Cortex Eucommiae extract that embodiment 2 obtains with the UPLC method.
Sample is with 50% methanol ultrasonic dissolution,
Reference substance comprises: Chiba element A, wogonin, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide, process standard solution with methanol respectively.
Acetonitrile-0.1% formic acid water system, wavelength switching: 237nm is between 0-3min; 277nm is between 3-6min; 237nm is between 6-9.2min; 227nm is between 9.2-12min; And 275nm is between 12-16min.
Chromatographic column: UPLCTM BEH C18 post (100mm * 3.0mm, granularity 1.7 μ m, Waters)
The result:
Extract 1: detect Chiba element A, wogonin, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide.In the extract gross weight, their amounts separately are between 0.01～4 weight %, and for example the content of geniposide in this extract is about 3.86%, and the content of Chiba element A in this extract about 0.018%.Five kinds of composition summations account for about 7.11% of this extract weight.It will be appreciated by those skilled in the art that and adopt conventional method for concentration, can also further obtain the higher extract of purity from extract 1.
Extract 2: detect Chiba element A, wogonin, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide.In the extract gross weight, their amounts separately are between 0.01～5 weight %, and for example the content of geniposide in this extract is about 4.56%, and the content of Chiba element A in this extract about 0.021%.Five kinds of composition summations account for about 7.92% of this extract weight.
Extract 3: detect Chiba element A, wogonin, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone, betulic acid, geniposide.In the extract gross weight, their amounts separately are between 0.01～7 weight %, and for example the content of geniposide in this extract is about 7.57%, and the content of Chiba element A in this extract about 0.018%.Five kinds of composition summations account for about 11.45% of this extract weight.
Embodiment 4: the structural identification of 5 chemical compounds in the Cortex Eucommiae
5 chemical compounds that embodiment 1 is obtained have carried out structural confirmation (entrusting Institute of Analysis of University Of Tianjin to detect the 500MHz nuclear-magnetism).Analysis result shows that the structure of these chemical compounds is consistent with the structure of bibliographical information, specific as follows.
Geniposide (EUB30-1), white powder:
1HNMR (DMSO-d
6, 400MHz): δ 7.47 (1H, s, H-3), 5.68 (1H, s, H-7), 5.12 (1H, d; J=6.8Hz, H-1), 4.53 (1H, d, J=7.6Hz, H-1 '), 4.14 (1H, brd, J=14.4Hz; H-10a), 3.97 (1H, br d, J=14.4Hz, H-10b), 3.66 (3H, s ,-OCH
3), 3.15 (1H, m, H-5), 2.70 (1H, m, H-6a), 2.64 (1H, m, H-9), 2.05 (1H, br d, J=14.4Hz, H-6b).
Wogonin (EUC-4), yellow needle;
1HNMR (DMSO-d
6, 500MHz): δ 12.50 (s), 8.05 (2H, m, H-2 ', 6 '), 7.60 (3H, m, H-3 ', 4 ', 5 '), 6.99 (1H, s, H-6), 6.30 (1H, s, H-3), 3.84 (3H, s).
13CNMR (DMSO-d
6, 125MHz): δ 182.7 (C-4), 163.7 (C-2), 158.0 (C-7), 157.0 (C-5); 150.3 (C-9), 132.8 (C-1 '), 131.5 (C-4 '), 130.0 (C-3 '; 5 '), 128.4 (C-8), 127.0 (C-2 ', 6 '); 105.8 (C-3), 104.4 (C-10), 99.9 (C-6), 61.7 (OCH
3).
Chiba element A (EUC-6), yellow needle;
1HNMR (DMSO-d
6, 500MHz): δ 12.92 (s), 10.82 (s), 8.06 (2H, m, H-2 ', 6 '), 7.56 (3H, m, H-3 ', 4 ', 5 '), 6.97 (1H, s, H-8), 6.63 (1H, s, H-3), 3.74 (3H, s).
α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone (EUE-3), yellow powder;
1HNMR (CD
3OD, 500MHz): δ 7.43 (2H, d, J=9.0Hz, H-2,6), 7.14 (2H, d, J=9.0Hz, H-3; 5), 5.46 (1H, dd, J=12.5,13Hz, H-α), 2.73 (1H, m, β-H), 3.04 (1H; M, H-β), 6.37 (1H, d, J=2.0Hz, H-3 '), 6.52 (1H, dd, J=9.0,2.0Hz; H-5 '), 7.74 (1H, d, J=9.0Hz, H-6 '), 4.95 (1H, d, J=7.0Hz, H-1 ").
13C-NMR(CD
3OD，125MHz)：δ193.2(C＝O)，80.7(C-α)，45.0(C-β)，129.9(C-1)，128.8(C-2，6)，117.8(C-3，5)，159.2(C-4)，115.0(C-1′)，165.4(C-2′)，103.8(C-3′)，166.8(C-4′)，111.8(C-5′)，134.4(C-6′)，102.2(C-1″)。
Betulic acid (EUC-2), white powder;
1HNMR (C
5D
5N-d
5, 500MHz): δ 4.90 (1H, s, H-29a), 4.72 (1H, s, H-29b), 3.49 (1H, m, H-3), 1.74 (3H, s, H-30), 1.18 (3H, s, H-26), 1.02 (3H, s, H-27), 1.01 (3H, s, H-23), 0.96 (3H, s, H-25), 0.77 (3H, s, H-24).
13CNMR(C
5D
5N-d
5，125MHz)：δ178.8(C-28)，151.3(C-20)，109.9(C-29)，78.1(C-3)，56.6(C-17)，55.9(C-5)，50.9(C-9)，49.7(C-18)，47.7(C-19)，42.8(C-14)，41.1(C-8)，39.5(C-4)，39.2(C-13)，38.5(C-1)，37.6(C-22)，37.5(C-10)，34.8(C-7)，32.8(C-16)，31.2(C-21)，30.2(C-15)，28.6(C-2)，28.3(C-23)，26.1(C-12)，21.1(C-11)，19.4(C-6)，18.8(C-26)，16.4(C-30)，16.4(C-25)，16.3(C-24)，14.9(C-27)。
Embodiment 5: Ca in the A7r5 cell that 5 chemical compounds and Cortex Eucommiae extract stimulate high potassium in the Cortex Eucommiae
2+
Concentration
Influence
Test method:
The no phenol red conventional A7r5 cell (rat breast large artery trunks smooth muscle cell) of cultivating of DMEM culture fluid that contains 10%CS-FBS is with 5 * 10
4Cells/well is inoculated in 96 orifice plates, cultivates 24h, and every hole adds 100 μ L
Behind Calcium 4 fluorometric reagents (Molecular Devices, the U.S.), in 37 ℃ of incubators, hatch 1h.After the taking-up, in every hole, add 50 μ L respectively and contain/do not contain KCl (60mmol/L) and variable concentrations medicine, at excitation wavelength 485nm, detect the fluorescent value of every porocyte under the emission wavelength 525nm condition in real time, detection time, 5min read corresponding fluorescent value.Data with every porocyte dosing after the fluorescent value percentage ratio that accounts for fluorescent value before the corresponding cell dosing (promptly 0 second time fluorescent value) represent.
Result of the test:
The high potassium solution of 60mM can significantly promote the rising of intracellular calcium concentration.Chiba element A (10
-6M, 10
-5M, 10
-4M), α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone (10
-6M, 10
-5M, 10
-4M), betulic acid (10
-6M, 10
-5M, 10
-4M), geniposide (10
-6M, 10
-5M, 10
-4M) the ability concentration dependent reduce the intracellular calcium concentration rising that high potassium causes equally, compares with high potassium group to have statistical significance (P＜0.01).Referring to Fig. 1, Fig. 2, Fig. 3 and Fig. 4.
Through above measurement of test method, find that wogonin also has and result (not shown) like the Chiba element category-A, the ability concentration dependent reduce the intracellular calcium concentration rising that high potassium causes, compares with high potassium group to have statistical significance (P＜0.01).
Through above measurement of test method; Find that extract 1 (0.3g/L, 3g/L, 30g/L), extract 2 (0.3g/L, 3g/L, 30g/L), extract 3 (0.3g/L, 3g/L, 30g/L) three also have and the similar result of geniposide (not shown); The ability concentration dependent reduce the intracellular calcium concentration rising that high potassium causes, compares with high potassium group to have statistical significance (P＜0.01).
Those skilled in the art can expect; Can suppress the material that intracellular calcium concentration raises due to the high potassium; It can be used as effective blood vessel protective agent, for example is used to treat and/or prevent be selected from following disease or disease: syndrome, central serous chorioretinopathy, thrombophlebitis, vascular permeability raise and cause before vascular disease (for example atherosclerosis), hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache, obliterated cerebral vascular disease (and the hemiplegia that causes, aphasia), the coronary heart disease infarction edema, lymphedema, burn and traumatic edema arteriosclerosis, diabetic machine nethike embrane pathological changes, microangiopathy, varicosis.
Test method:
1.. and the experiment buffer (Kreb ' s liquid) configuration, (fresh is best, logical 95%O to transfer pH=7.4
2/ 5%CO
2Balance).
2.. open experimental apparatus, passage is set, the zeroing of antiotasis baseline.
3.. put to death animal, original position is peeled off tremulous pulse (must not tractive, in order to avoid destroy endothelial function), gets in Kreb ' the s liquid that appropriate length is placed on pre-cooling.
4.. further peel off blood vessel, the arterial ring of scalpel intercepting 3mm length (must not tractive), all the other blood vessels still are placed in Kreb ' the s liquid of pre-cooling and continue step experiment down.
5.. the arterial ring of handling well is placed on the tension pick-up, and arterial ring should be in relaxed state, fixes back adjustment arterial ring tension force to 0.5g.
6. .1-1.5h internal program property adjustment arterial ring tension force changes liquid twice (20min each) to 3.0g, treats the formal experiment in stable back.
7.. add at first that accumulative total concentration is respectively 15,30,60, the high K+ solution (each mass action time 2-4min) of 120mM, stimulate vasoconstriction, make amount effect curve.
8.. eluting when treating the vascular ring tension adjustment to 3.0g, adds accumulative total concentration and is respectively 10
-8, 10
-7, 10
-6, 10
-5The norepinephrine of M (NE) solution (each mass action time 4min) stimulates vasoconstriction, makes amount effect curve.
9.. eluting, when treating the vascular ring tension adjustment, at first use NE (10 to 3.0g
-6M) stimulate vasoconstriction, add accumulative total concentration then and be respectively 10
-8, 10
-7, 10
-6, 10
-5The acetylcholine of M (Ach) solution (each mass action time 4min) stimulates vasoconstriction, makes amount effect curve.
10.. eluting, when treating the vascular ring tension adjustment, at first use NE (10 to 3.0g
-6M) stimulate vasoconstriction, add accumulative total concentration then and be respectively 10
-8, 10
-7, 10
-6, 10
-5M receives reagent thing solution (each mass action time 8-10min), stimulates vasoconstriction, makes amount effect curve.
Result of the test:
Chiba element A (10
-6, 10
-5, 10
-4M) but the vasoconstriction that concentration dependent diastole norepinephrine (NE) causes, diastolic rate is respectively 89.84%, 58.57%, 28.48%, has compared significant difference (P＜0.05 or P＜0.01) with contrast.The result sees Fig. 5 a, Fig. 5 b.
Wogonin (10
-6, 10
-5M) vasodilatory trend is arranged, concentration is 10
-4The pre-shrunk vascular ring diastolic rate that can obviously relax during M is 48.09%, has compared significant difference (P＜0.01) with contrast.The result sees Fig. 6 a, Fig. 6 b.
Extract 2 (0.4,0.8,1.2mg/ml) but vasoconstriction that concentration dependent diastole norepinephrine (NE) causes, diastolic rate is respectively 85.09%, 61.59%, 28.32%, has compared significant difference (P＜0.01) with the blank group.The result sees Fig. 7 a, Fig. 7 b.
Extract 3 (0.4,0.8,1.2,1.6,2.0mg/ml) but vasoconstriction that concentration dependent diastole norepinephrine (NE) causes; Diastolic rate is respectively 96.52%, 89.63%, 79.78%, 70.92%, 59.72%, has compared significant difference (P＜0.01) with the blank group.The result sees Fig. 8 a, Fig. 8 b.
But the vasoconstriction that the norepinephrine stimulating endothelial is complete, the rat chest aorta that causes shrinks.Having rat chest aorta that the diastole norepinephrine causes, to shrink active material be a kind of potential blood vessel protective agent.Present embodiment indicates that chemical compound provided by the present invention or extract can be used for treating and/or preventing and is selected from following disease or disease: vascular disease (for example atherosclerosis); Hypertension; Postangioplasty restenosis; Coronary heart disease; Cerebrovascular; Renal glomerular disease; Pulmonary hypertension; Diabetic angiopathy becomes; Vasculitis; Migraine; Vascular headache; Obliterated cerebral vascular disease (and the hemiplegia that causes; Aphasia); Syndrome before the coronary heart disease infarction; Central serous chorioretinopathy; Thrombophlebitis; The edema that vascular permeability raises and causes; Lymphedema; Burn and traumatic edema arteriosclerosis; Diabetic machine nethike embrane pathological changes; Microangiopathy; Varicosis.
Embodiment 7: chemical component of eucommia bark used and extract are to the influence of the A7r5 cell proliferation of PDGF-BB stimulation
Test method:
The A7r5 cell is cultivated so that the DMEM culture fluid that contains 10%FBS is conventional, when 80% merges, uses 0.25% trypsinization, with 4 * 10
3The every hole of individual cell is inoculated in 96 orifice plates.Place 37 ℃, 5%CO
2After cultivating 24h in the incubator, cell attachment is good, sprawls growth.Inhale this moment and remove culture fluid, use D-Hank ' s liquid 200 μ l every hole washing 1 time, add no phenol red serum-free DMEM culture fluid and cultivate 24h, make cell be still in G
0/ G
1Phase.Experiment is divided into solvent control group (0.1%DMSO), estradiol (E
2) group (0.01 μ M), E
2(0.01 μ M)+estrogen receptor antagon (ICI) (0.1 μ M) group, each concentration group of medicine and medicine+ICI (0.1 μ M) group, except that matched group, it is 10ng.ml that every porocyte adds final concentration
-1Platelet derived growth factor-BB (PDGF-BB), continue to cultivate 24h, kit detection cell propagation.
Result of the test:
PDGF-BB (10ng.ml
-1) can obvious stimulation A7r5 cell proliferation.Wogonin (10
-7, 10
-6, 10
-5M) but concentration dependent suppresses the A7r5 cell proliferation that PDGF-BB stimulates, and its depression effect can be by ICI institute antagonism, show that wogonin can see Fig. 9 through activating the effect that the ER performance suppresses vascular smooth muscle cell proliferation.In addition, the active component betulic acid (10 in the Cortex Eucommiae
-7, 10
-6, 10
-5M) but concentration dependent suppresses the A7r5 cell proliferation that PDGF-BB stimulates, see Figure 10.
The inventor also finds through above test method, Chiba element A (10
-7, 10
-6, 10
-5M), extract 3 (0.4,2,10mg/ml) has respectively and is similar to result shown in Figure 9; α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone (10
-7, 10
-6, 10
-5M), betulic acid (10
-7, 10
-6, 10
-5M), geniposide (10
-7, 10
-6, 10
-5M), extract 1 (0.4,2,10mg/ml) has respectively and is similar to result shown in Figure 10 (not shown).
Present embodiment is a kind of test model that is used for the effect of detection of active material vascular protection of classics.The result of present embodiment shows that extract of the present invention and active component have effective vascular protection effect.
Embodiment 8: the effect coronarius of chemical component of eucommia bark used expansion isolated rat heart
Test method:
Choose healthy rat, and carry out random packet, rat is put to death the back and takes out heart rapidly; Place 4 ℃ of K-H liquid, discharge remained blood gently, heart is connected to langendorff isolated heart perfusion device; Fix with silk thread; The beginning perfusion, perfusate is kept 37 ℃, and feeds the mist of 95% oxygen and 5% carbon dioxide.Irrigation flow is adjusted in carries out the constant flow rate perfusion about 9.00 ± 0.1ml/min; Indexs such as coronary perfusion pressure, heart rate before the record administration are injected the medicinal liquid that the 0.2ml/ heart prepares through dosing holes, the above-mentioned each item index of immediate record after each administration finishes after stable; Continue to observe 15 minutes; Indexs such as record injection pressure, heart rate are calculated injection pressure decline percentage rate after the administration, reflect the protective effect to cardiac muscle and blood vessel of rat coronary artery degrees of expansion and medicine with this.
Result of the test:
The Cortex Eucommiae 70% ethanol extraction 1mg/ml, 10mg/ml can significantly reduce the isolated rat heart perfusion pressure, with relatively there were significant differences (P＜0.01) before the administration, see Figure 11; Component six 0.1mg/ml, 1mg/ml, 10mg/ml can significantly reduce the isolated rat heart perfusion pressure, with relatively there were significant differences (P＜0.01) before the administration, see Figure 12.The result shows that the Cortex Eucommiae 70% ethanol extraction, component six can significantly be expanded the isolated rat heart coronary artery, thereby performance is to the protective effect of cardiac muscle and blood vessel.
The present embodiment result is indicating that on the one hand extract of the present invention can be used as effective blood vessel protective agent, is indicating that on the other hand extract of the present invention has the effect of blood pressure lowering.
Visible from the present invention, in some embodiments, Chiba element A can produce quick diastole effect to the pre-shrunk vascular ring of NE, and can reduce that calcium ion concentration raises in the VSMC that high potassium causes through non-ER dependence approach; Wogonin both can be brought into play vasorelaxation action fast, can suppress vascular smooth muscle cell proliferation through the ER mediated pathways again; α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-the trihydroxy dihydrochalcone can reduce that calcium ion concentration raises in the VSMC that high potassium causes through the non-dependence approach of ER.Betulic acid can reduce the interior calcium ion concentration rising of VSMC that high potassium causes, also has the effect that suppresses vascular smooth muscle cell proliferation simultaneously; Geniposide reduces the interior calcium ion concentration rising of VSMC that high potassium causes, performance vasodilator effect.The Cortex Eucommiae extract isolated rat heart coronary flow that can raise produces quick diastole effect to the pre-shrunk vascular ring of NE.Generally speaking, the present invention finds that some chemical constituent and Cortex Eucommiae extract in the Cortex Eucommiae can be used as the blood vessel protective agent use.
Claims (10)
1. the Cortex Eucommiae is used for the purposes as the medicine of blood vessel protective agent and/or hypotensive agent in preparation, perhaps is used for treating and/or preventing the purposes of the medicine of vascular proliferative disease in preparation.
2. Cortex Eucommiae extract is used for the purposes as the medicine of blood vessel protective agent and/or hypotensive agent in preparation, perhaps is used for treating and/or preventing the purposes of the medicine of vascular proliferative disease in preparation.
3. according to the purposes of claim 2, it is characterized in that,
Said vascular proliferative disease is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache; And/or
Said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: vascular disease (for example atherosclerosis); Hypertension; Postangioplasty restenosis; Coronary heart disease; Cerebrovascular; Renal glomerular disease; Pulmonary hypertension; Diabetic angiopathy becomes; Vasculitis; Migraine; Vascular headache; Obliterated cerebral vascular disease (and the hemiplegia that causes; Aphasia); Syndrome before the coronary heart disease infarction; Central serous chorioretinopathy; Thrombophlebitis; The edema that vascular permeability raises and causes; Lymphedema; Burn and traumatic edema arteriosclerosis; Diabetic machine nethike embrane pathological changes; Microangiopathy; Varicosis.
4. according to each purposes of claim 2 to 3; Comprise at least a following composition that is selected from the wherein said Cortex Eucommiae extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
5. according to the purposes of claim 4; Wherein said geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-total amount of trihydroxy dihydrochalcone and betulic acid accounts for the 1-90% (w/w) of said extract gross weight.
6. according to each purposes of claim 2 to 5, wherein said Cortex Eucommiae extract obtains as solvent extraction through water, ethanol or ethanol water.
7. be selected from following ingredients one or more be combined in preparation as the purposes in the medicine of blood vessel protective agent and/or hypotensive agent; Perhaps be used for treating and/or preventing the purposes of the medicine of vascular proliferative disease: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4 in preparation; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
8. according to the purposes of claim 7, it is characterized in that,
Said vascular proliferative disease is selected from following disease or disease: the vascular disease includes but not limited to atherosclerosis, hypertension, postangioplasty restenosis, coronary heart disease, cerebrovascular, renal glomerular disease, pulmonary hypertension, diabetic angiopathy change, vasculitis, migraine, vascular headache; And/or
Said blood vessel protective agent is used to treat and/or prevent and is selected from following disease or disease: vascular disease (for example atherosclerosis); Hypertension; Postangioplasty restenosis; Coronary heart disease; Cerebrovascular; Renal glomerular disease; Pulmonary hypertension; Diabetic angiopathy becomes; Vasculitis; Migraine; Vascular headache; Obliterated cerebral vascular disease (and the hemiplegia that causes; Aphasia); Syndrome before the coronary heart disease infarction; Central serous chorioretinopathy; Thrombophlebitis; The edema that vascular permeability raises and causes; Lymphedema; Burn and traumatic edema arteriosclerosis; Diabetic machine nethike embrane pathological changes; Microangiopathy; Varicosis.
9. according to the purposes of claim 7 or 8; Wherein one or more combination of following ingredients is from the Cortex Eucommiae, to extract to obtain: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
10. one kind is used for as blood vessel protective agent and/or hypotensive agent or is used to treat and/or prevent the pharmaceutical composition of mammal blood vessel proliferative disease, wherein comprises the Cortex Eucommiae extract that treats and/or prevents and the optional acceptable excipient of pharmacy; Perhaps; A kind ofly be used for as blood vessel protective agent and/or hypotensive agent or be used to treat and/or prevent the pharmaceutical composition of mammal (particularly people) vascular proliferative disease; Wherein comprise at least a active constituents of medicine that treats and/or prevents effective dose and the optional acceptable excipient of pharmacy; Described active constituents of medicine is selected from: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4; 2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.In one embodiment, comprise at least a following composition that is selected from the described Cortex Eucommiae extract: geniposide, wogonin, Chiba element A, α-oxygen-β-D-glucopyanosyl base-4,2 ', 4 '-trihydroxy dihydrochalcone and betulic acid.
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| CN115671124B (en) * | 2022-08-25 | 2023-07-04 | 海南医学院第一附属医院 | Application of aucubin in preparation of anoxic pulmonary arterial high pressure vascular remodeling drugs |
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| CN103191174B (en) | 2016-06-29 |
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| CN103191174A (en) | 2013-07-10 |
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Address after: 343100 Innovation Avenue 278, Jinggangshan Economic and Technological Development Zone, Ji'an City, Jiangxi Province Patentee after: Jiangxi Puzheng Pharmaceutical Co., Ltd. Address before: 343100 Xiajiang Industrial Park, Ji'an, Jiangxi Patentee before: Jiangxi Pozin Pharmaceutical Co., Ltd. |