CN102218054A - Use of apigenin in preparing drugs for preventing and/or treating cardiovascular and cerebrovascular diseases - Google Patents
Use of apigenin in preparing drugs for preventing and/or treating cardiovascular and cerebrovascular diseases Download PDFInfo
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- XADJWCRESPGUTB-UHFFFAOYSA-N apigenin Natural products C1=CC(O)=CC=C1C1=CC(=O)C2=CC(O)=C(O)C=C2O1 XADJWCRESPGUTB-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 229940117893 apigenin Drugs 0.000 title claims abstract description 83
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Abstract
The invention discloses new uses of apigenin. The new uses comprise: on the one hand, a use of the apigenin in preparing drugs or health care products for preventing and/or treating cardiovascular and cerebrovascular diseases; on the other hand, a use of the apigenin in preparing the drugs with vasodilation functions or health care products with the vasodilation functions. Experiments provided by the present invention show that the apigenin has characteristics of obvious cardiac and cerebral vasodilation, effective absorption, blood brain barrier permeating, little influence on vascular endothelial cells, definite therapeutic effect, safety, convenience, no toxic and side effect and low price. The drugs provided by the present invention has obvious effects for preventing and treating the cardiovascular and cerebrovascular diseases, and can be applicable for preventing and treating the cardiovascular and cerebrovascular diseases induced by a plurality of reasons.
Description
Technical field
The present invention relates to apigenin and prevent and/or treat application in the cardiovascular and cerebrovascular diseases medicament in preparation.
Background technology
Apigenin (apigenin) belongs to Polyphenols chromocor compound (flavonoids), is widespread in nature.Apigenin is present in leaf, flower and the fruit of plant more, and wherein the content in Herba Apii graveolentis is the highest.The chemical name of apigenin is 4 ', 5,7-trihydroxyflavone, 3 hydroxyls of its 4 ', 5,7 positions and C
2=C
3Two keys have determined physicochemical characteristics and the pharmacological effect that it is unique.Multiple pharmacological effect such as that apigenin has is antibiotic, antiviral, infection, antitumor enjoy numerous scholars' favor.
For a long time, cardiovascular and cerebrovascular disease is a kind of serious threat mankind, the commonly encountered diseases that particularly in middle-aged and elderly people, takes place, and cardiovascular and cerebrovascular disease has become the highest No.1 killer of the human dead cause of disease, also is " the noiseless demon " who influences health of people.The modern medicine scientific research confirms that the cardiovascular and cerebrovascular disease of serious harm human health takes place mainly from vascellum endometrial hyperplasia, vascular endothelium dysfunction, vasospasm etc. at present, thereby makes cerebral ischemia, even blood supply interrupts causing cardiovascular and cerebrovascular disease.And take place for the disease of cardiovascular and cerebrovascular vessel, vascular lesion, vasospasm are the most direct factors.Therefore, the vasodilator activity of increase medicine has direct influence for the prophylactic treatment of this type of disease.
Summary of the invention
The new purposes that the purpose of this invention is to provide apigenin.
The new purposes of apigenin provided by the present invention: on the one hand being it prevents and/or treats application in cardiovascular and cerebrovascular diseases medicament or the health product in preparation; Being it on the other hand has application in vasodilation function medicament or the health product in preparation.
Above-mentioned medicine that prevents and/or treats cardiovascular and cerebrovascular disease and vasodilator drug can directly oral or injections, perhaps mix to make to use medicinal dosage form more, comprising: drop pill, soft capsule, hard capsule, oral liquid, electuary, unguentum, sublimed preparation, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch, nanometer formulation, slow releasing preparation and controlled release preparation etc. with pharmaceutically acceptable carrier auxiliary material.
(this dosage was the conversion dosage of the oral maximum tolerated dose of mice (MTD), MTD＞20g/kg of mice, so its LD below the dosage of apigenin can be the 2.2g/kg body weight among the present invention
50＞20g/kg).
Oral formulations is a kind of important form of administration of apigenin medicine, and the main absorption site of oral drugs is the important means of estimating drug bioavailability at small intestinal and absorb.The present invention utilizes the Caco-2 cell model to study the degree of absorption of apigenin, and the result shows that apigenin has very high apparent infiltration coefficient (P by the Caco-2 cell membrane time
App), and along with its penetrating rate of variation of time presents good time-dependent relation.Blood brain barrier is one of main obstruction that influences the cerebrovascular drug curative effect, the present invention adopts rat brain capillary endothelium and spider cell associating cultured method to set up the transmembrane transport that external blood brain barrier model is studied apigenin, the result shows that apigenin has very high apparent infiltration coefficient (P by blood brain barrier the time
App), and along with its penetrating rate of variation of time also presents good time-dependent relation.Cell viability influence test by to the brain micro blood vessel endothelium cell shows that apigenin has the characteristics safe, that toxic and side effects is low.Above-mentionedly experiment showed, that medicine of the present invention can effectively prevent and treat cardiovascular and cerebrovascular disease, and safety is very high, has that determined curative effect, toxic and side effects are low, safe ready and cheap characteristics.
Description of drawings
Fig. 1 is transhipment permeability and the time relation of apigenin in Caco-2 cell monolayer model among the embodiment 2; Administration concentration is apigenin 20 μ M, and experimental result is represented (n=3-5) with mean ± S.D..
Fig. 2 is transhipment permeability and the time relation of apigenin in the BBB cell model among the embodiment 3; Administration concentration is apigenin 20 μ M, and experimental result is represented (n=3-5) with mean ± S.D..
Fig. 3 be among the embodiment 4 apigenin to the effect of vigor of RBMECs; Experimental result is represented with mean ± S.D., compares with the normal control group, and * P＜0.05, * * P＜0.01, C represents the normal cell matched group.
The specific embodiment
The present invention will be described below by specific embodiment, but the present invention is not limited thereto.
Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and biomaterial if no special instructions, all obtain from commercial channels.Apigenin solution used among the embodiment 2-4 is with embodiment 1.
Embodiment 1, apigenin are to the vasorelaxation action of isolated rat thoracic aorta
1, material and method
1.1 laboratory animal
Male SD rat, body weight restrains at 250-280, is provided by Military Medical Science Institute.The animal quality certification number is SCXK (capital) 2007-0001.The free diet of animal is raised in clean room.
1.2 reagent preparation
The standard substance apigenin is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Apigenin is a yellow powder, and precision takes by weighing apigenin 2.7mg and places the 1ml volumetric flask, fully dissolves the mother solution that is mixed with 10mM with DMSO.All administration concentration all form the mother solution dilution with tri-distilled water, and desired concn is respectively 0.8 μ M, 4 μ M, 20 μ M.
1.3 method
After dislocation is put to death behind the SD rat anesthesia, open the thoracic cavity rapidly, take out thoracic aorta and put into 4 ℃ KH liquid.Careful dialling removed fat and connective tissue around the blood vessel, is divided into the vascular ring that is about 3-5mm, is suspended on to place 5ml, 37 ℃ KH liquid on two three parallel square rings, and continues to pass to 95%O
2-5%CO
2Gaseous mixture.With Powerlab bio signal processing system record vascular ring tension force, stablize after vascular ring hangs and added 1g rest tension balance 1h in 10 minutes, change 1 time KH liquid during this period every 20 minutes, and constantly adjust tension force about 1g.The KCl 100 μ l that add 3M behind the 1h stimulate continuous three times of vasoconstriction, wash to platform with KH liquid to add 2.5 * 10
-3The phyenlephrinium of M (PE) 50 μ l add 10 after reaching and shrinking platform
-2The acetylcholine of M (ACh) is to check whether blood vessel endothelium is complete.If make the pre-shrunk vascular ring diastole of PE more than 60% after adding ACh, can think that endothelium is complete; Otherwise, then think vessel endothelium and induced endothelial.The blood vessel of endothelium-denuded fully is with not producing diastole behind the ACh.
Get the complete blood vessel of endothelium, adding PE, to make final concentration be 10
-6M, reach the maximum collapse platform after, successively add apigenin, make final concentration reach 0.8 μ M, 4 μ M, 20 μ M, record tension force change curve in time reaches maximum to stretching reaction, calculates diastole percentage rate (Rex%).With add concentration be 20 μ M normal saline solution the blank group relatively.
2, data statistics is handled
All data represent that with mean ± S.D. statistical analysis adopts SPSS 11.5 analysis software.With PE 10
-6It is 100% that M brings out maximum shrinkage amplitude, brings out the variation of the ratio reflection antiotasis between the maximum shrinkage amplitude with antiotasis amplitude behind the adding medicine and PE.All data are all by test of normality, adopt the independent sample t check to analyze, with P＜0.05 for significant difference is arranged.The computing formula of diastole percentage ratio is:
3, experimental result
Apigenin has different diastole percentage rate to PE preshrinking blood vessel when variable concentrations, and present must linear relationship (y=2.074x+27.23).The results are shown in Table 1.
The apigenin of table 1 variable concentrations is to the vasorelaxation action (n=6) of PE preshrinking thoracic aortic ring
(* * compares with the normal control group, P＜0.01)
4, conclusion
The result shows that under this experiment condition, but apigenin equal dose dependent ground on the blood vessel of complete endothelium reduces PE preshrinking antiotasis.And when its concentration is the low dosage of 0.8 μ M, can also maintain more than 20%, so the explanation apigenin has vasorelaxation action, to having significant prevention and therapeutical effect by caused by factors cardiovascular disease such as vasospasms to the diastole of blood vessel.
The transhipment in external Caco-2 cell monolayer model of embodiment 2, apigenin absorbs
1, material and reagent
1.1 cell source
The Caco-2 cell is purchased in consonance cell centre (coming from U.S. ATCC company)
1.2 reagent preparation and administration
Precision takes by weighing apigenin 2.7mg and places the 1ml volumetric flask, fully dissolves the mother solution that is mixed with 10mM with DMSO.HBSS solution dilution with freshly prepared pH ≈ 7.4 becomes 20 μ M to carry out administration.
2, method
2.1 the foundation of external Caco-2 cell monolayer model
The AP side of 43 generation Caco-2 cell inoculations in poly carbonic ester film 12 hole Transwell culture ponds that state is very good, density is 6～8 * 10
4/ hole adds the 0.8ml culture medium in supply pool, accepting pond adding 1ml culture medium culturing 21 days, change liquid the last week every other day, changes liquid later every day.The cell monolayer picture and the positive drug verapamil P of TEER value, phenol red leak test and the scanning electron microscope of record
AppIt is the index of identifying the model success or not.
2.2 the transhipment of apigenin in Caco-2 cell monolayer model absorbs
From incubator cell plates are taken out, the TEER value of measuring each hole (reaches 600 Ω 2cm
2Below then satisfy condition).The AP in each hole, the culture medium of BL face are all siphoned away, with before after the good HBSS of incubator preheating (pH ≈ 7.4) washes 2 times, the HBSS that adds 0.8ml, 1ml respectively in AP, BL side put into constant temperature shaking table balance 30min (37 ℃, 55rpm).
1) in order to obtain the apparent permeability P of apigenin
App, then discarding this HBSS, the AP side in a part of cell culture pond adds apigenin solution (concentration the is 20 μ M) 0.5ml that is prepared, and the BL side adds HBSS 1ml.Press 90min from BL side-draw liquid 100 μ l, the sample of gained is put in-20 ℃ of refrigerators and preserves to be measured.
2) permeability and the time relation in order to measure apigenin then discards this HBSS, and the AP side (villous surface) in another part cell culture pond adds apigenin solution (concentration the is 20 μ M) 0.5ml that is prepared, and the BL side adds HBSS 1ml.By 15,30,60,90,120,180min gets liquid 100 μ l from BL side (basal surface), and adds 100 μ l HBSS immediately, surveys the TEER value behind the 180min.The sample of gained is put in-20 ℃ of refrigerators and preserves to be measured.
Before the HPLC sample introduction, add the methanol 100 μ l of equal volume in the above sample, after the vortex vibration, (13,000g 5min), gets supernatant 50 μ l sample introductions to high speed centrifugation.The chromatographic condition of HPLC sample detection is as follows: XDB-C18 chromatographic column (250mm * 4.6mm, i.d.5 μ m, U.S. Agilent company), mobile phase is that (from 30: 70to 40: 60 for methanol and 1% glacial acetic acid aqueous solution, v/v), flow velocity is 1.0ml/min, and column temperature is 59 ℃, and sample size is 50 μ l.The detection wavelength of apigenin is 270nm.
3, date processing and analytical method
The computing formula of apparent infiltration coefficient is:
The transhipment amount (μ M/s) of Δ Q/ Δ t representation unit time medicine, representative are subjected to the speed of medicine at the BL side joint; C
0Represent initial concentration (the 20 μ M/cm of the medicine AL side of giving
3); A is the surface area (1.13cm of poly carbonic ester film
2).When the experiment of the permeability of doing apigenin and time relationship, owing to all want fluid infusion after each sampling, the permeability of medicine has been produced diluting effect, thereby the penetrating concentration of the accumulation of medicine can be used following formula correction:
The detection data of each index all adopt SPSS 13.0 statistics softwares to carry out one factor analysis of variance (ANOVA) check.Experimental result is represented with mean ± S.D..
4, experimental result
According to the standard curve (y=128.1029x+5.4771) of above apparent infiltration coefficient formula and apigenin, the gained apigenin is apparent permeability (P in external Caco-2 cell monolayer model
App=3.20 * 10
-6Cm/s, n=3-5).Apigenin the results are shown in Figure 1 in the permeability and the time relation of this model.
5, conclusion
It is generally acknowledged P
App＞1 * 10
-6The medicine of cm/s be considered to absorptance more completely medicine (Artursson P, Palm K.Luthman K.Caco-2 monolayers in experimental and theoretical predictions of drugtransport[J] .Adv Drug Delivery Rev 2001; 46 (1-3): 27-43.).Apigenin has very high P in external Caco-2 cell monolayer model
App, this may with the chemical constitution of apigenin with and corresponding fat water distribution coefficient higher relevant.Simultaneously, permeability and the time of apigenin in this model also presents certain dependence.So can infer that apigenin has good absorption in vivo, this has direct directive function for apigenin oral absorption or direct injection when prevention and the treatment cardiovascular and cerebrovascular disease.
(1) separation and Culture of former generation brain micro blood vessel endothelium cell (RBMECs)
1, material and method
1.1 laboratory animal
Male Sprague-Dawley rat, three weeks, available from Military Medical Science Institute, quality certification SCXK (army) 2007-004.
1.2 primitive cell culture method
After the dislocation of SD rat is put to death, the cerebral tissue that aseptic condition takes out is down put into the culture dish that fills cold PBS, dissect and remove cerebellum, diencephalon (comprising Hippocampus), pia mater encephali, only keep cerebral cortex behind meninges trunk and the cerebral white matter, brain cortex is shredded the back move into homogenate in the homogenizer, 600 * g, 10 ℃, the centrifugal back of 10min abandoning supernatant, centrifugal (4000 * g behind the adding 20%BSA suspension mixing, 20min, 4 ℃) remove nervous tissue and trunk at the middle and upper levels, keep bottom precipitation, 37 ℃ of water-baths digest 1h after adding 2ml 0.1% collagenase II suspension mixing, centrifugal (600 * g, 10 ℃, 10min), remove supernatant, add 2ml ECM (endothelial cell medium, endotheliocyte culture medium) and contain 20%FBS, after the culture fluid of 1%ECGF suspends, the blood capillary section that 74 μ m filter screens filter gained purification after 2 times once more.Be inoculated in the 25cm of coating bio-matrix
2The disposable plastic culture bottle places 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, change liquid behind 12～24h, change liquid subsequently every other day.Have the brain micro blood vessel endothelium cell to swim out of around the visible blood capillary section of 3-4d, the cell refractivity is strong, and form is mainly fusiformis, also sees polygon, and monolayer growth also has the contact inhibition phenomenon, meets the endothelial cell growth characteristic.7-10d left and right sides cell grows to and presents shop cobble shape when merging.
2, identified by immunofluorescence RBMECs cell purity
Adopt VIII factor related antigen immunocyte histochemistry authentication method (model is auspicious. the foundation of blood brain barrier external model and Borneolum Syntheticum are to the research [D] of its influence. Tianjin: Tianjin College of Traditional Chinese Medicine, 2004.) identify the purity of former generation RBMECs.Get the very good RBMECs of state, 4% paraformaldehyde room temperature is fixed 1 hour, PBS wash 3 times each 5 minutes, add the lowlenthal serum sealing 1 hour that contains 0.3%Triton X-100, drip rabbit anti-people VIII factor related antigen antibody (1: 100) 4 ℃ and spend the night, PBS wash 3 times each 5 minutes, dripping FITC hatched 2 hours for 37 ℃, PBS washed 3 times each 5 minutes, added Hochest and dyed nuclear after 2 minutes, utilized Laser Scanning Confocal Microscope to observe.Cell is cytoplasm through the dyeing of VIII factor related antigen more than 95% painted, shows that the cell that adopts above method to obtain is the brain micro blood vessel endothelium cell, the purity height.2 substitute the external BBB scale-model investigation of cultivating in altogether.
(2) separation and Culture of former generation astrocyte (AS)
1, material and method
1.1 laboratory animal
Male Sprague-Dawley rat, 1-2 days, available from Military Medical Science Institute, quality certification SCXK (army) 2007-004.
1.2 primitive cell culture method
Newborn 1-2 days SD rat neonatal rats, aseptic condition are removed meninges and trunk down.Separate the both sides cerebral cortex and be cut into 1mm
3Size adds 37 ℃ of air bath concussions of 0.25% trypsin digestion 10min, the centrifugal supernatant that goes, with the resuspended precipitation of DMEM culture medium that contains 10%FBS,, collect filtrate through 75 μ m screen filtrations twice, centrifugal collecting precipitation is resuspended with the DMEM culture medium that contains 10%FBS, plants in 25cm
2Culture bottle after 1 hour differential sticks the removal fibroblast, is transferred to a new culture bottle with cell suspension and is continued to cultivate, and changes liquid once in two days.After 7 days culture bottle put into horizontal shaking table, (260rpm, 2h, 37 ℃) after, change liquid reject microglia, put into the incubator balance 1 hour, place horizontal shaking table again, (260rpm, 18h, 37 ℃) after, change liquid reject oligodendrocyte, wash 2 times with fresh culture, add 1: 1 mixed liquor of 0.25% trypsin and 0.02%EDTA and digest, go down to posterity standby.
2, identified by immunofluorescence astrocyte purity
Adopt glial fibrillary acidic protein (GFAP) immunocyte histochemistry authentication method.Get the astrocyte that satisfies condition, 4% paraformaldehyde room temperature is fixed 1 hour, PBS wash 3 times each 5 minutes, add the lowlenthal serum sealing 1 hour that contains 0.3%Triton X-100, drip glial fibrillary acidic protein antibody (1: 50) 4 ℃ and spend the night, PBS wash 3 times each 5 minutes, dripping FITC hatched 2 hours for 37 ℃, PBS washed 3 times each 5 minutes, added Hochest and dyed nuclear after 2 minutes, utilized Laser Scanning Confocal Microscope to observe.Cell is cytoplasm through anti-GFAP dyeing more than 95% painted, shows that the cell that adopts above method to obtain is an astrocyte, the purity height.2 substitute the external BBB scale-model investigation of cultivating in altogether.
(3) transhipment of apigenin in external BBB model absorbs
1, the foundation of external blood brain barrier model
With the star spongiocyte digestion of former generation obtain 2 generation cell, adjust cell density to 8 * 10
4Plant in wrapping by the downside 100 μ l of the cell culture pond poly carbonic ester film of bio-matrix in advance in/hole, relies on surface tension to cultivate 1.5h, then culture pond is put into 24 well culture plates, places 5%CO
2, 37 ℃ of incubators continue to cultivate, and change liquid once in 2 days, treat that star spongiocyte merged to 80% o'clock, at culture pond inoculation RBMECs altogether, latter's density is 1 * 10 after promptly 3-5 days
5/ hole is planted to wrap by the upside of the cell culture pond poly carbonic ester film of bio-matrix in advance and is carried out the common cultivation of two kinds of cells.Every day, iuntercellular resistance (TEER) striden in record.When becoming monolayer growth in poly carbonic ester film film both sides respectively to two kinds of cells after 3-5 days, TEER value, phenol red leak test and scanning electron microscope detect the compactness of its monolayer.After external BBB modelling is finished, can carry out the transport experiment research of apigenin on the BBB cell model.The selected positive, the negative mark post medicine of BBB model administration experiment is respectively chloromycetin and ketoconazole.
2, the transhipment of apigenin in external BBB model absorbs
To be total to the cultured cells plate from incubator and take out, the TEER value of measuring each hole (reaches 300 Ω cm
2, satisfy condition).The AP in each hole, the culture medium of BL face are all siphoned away, with before after the good HBSS of incubator preheating (pH ≈ 7.4) washes 2 times, the HBSS that adds 0.4ml, 0.6ml respectively in AP, BL side put into constant temperature shaking table balance 30min (37 ℃, 55rpm).1) in order to obtain the apparent permeability P of apigenin
App, then discarding this HBSS, the AP side in a part of cell culture pond adds apigenin solution (it is 20 μ M that the 10mM mother solution of preparing in embodiment 1 is diluted to concentration with the HBSS) 0.4ml that is prepared, and the BL side adds HBSS 0.6ml.Press 90min from BL side-draw liquid 60 μ l.The sample of gained is put in-20 ℃ of refrigerators and preserves to be measured.2) transport velocity and the time relation in order to measure apigenin then discards this HBSS, and the AP side in another part cell culture pond adds apigenin solution (concentration the is 20 μ M) 0.4ml that is prepared, and the BL side adds HBSS 0.6ml.By 15,30,60,90,120,180min is from BL side-draw liquid 60 μ l, and adds 60 μ l HBSS immediately.Survey the TEER value behind the 180min.The sample of gained is put in-20 ℃ of refrigerators and preserves to be measured.
Before the HPLC sample detection, add the methanol 60 μ l of equal volume in the above sample, after the vortex vibration, (13,000g 5min), gets supernatant 50 μ l sample introductions to high speed centrifugation.The HPLC chromatographic condition is with embodiment 2.
(4) date processing and analytical method
The computing formula of apparent infiltration coefficient is:
The transhipment amount (μ M/s) of Δ Q/ Δ t representation unit time medicine, representative are subjected to the speed of medicine at the BL side joint; C
0Represent initial concentration (the 20 μ M/cm of the medicine AL side of giving
3); A is the surface area (0.6cm of poly carbonic ester film
2).Do apigenin permeability and time relation when experiment, owing to all want fluid infusion after each sampling, the permeability of medicine has been produced diluting effect, thereby the penetrating concentration of the accumulation of medicine can used following formula correction:
The detection data of each index all adopt SPSS 13.0 statistics softwares to carry out one factor analysis of variance (ANOVA) check.Experimental result is represented with mean ± S.D..
(5) experimental result
According to the standard curve (y=128.1029x+5.4771) of above apparent infiltration coefficient formula and apigenin, the gained apigenin is apparent permeability (P in external BBB model
App=5.28 * 10
-6Cm/s, n=3-5).Apigenin the results are shown in Figure 2 in the transport velocity and the time relationship of this model.
(6) conclusion
Apigenin has very high P in external BBB cell model
App, show that promptly the apigenin ratio is easier to see through blood brain barrier, this may with the chemical constitution of apigenin with and corresponding fat water distribution coefficient higher relevant.Simultaneously, permeability and the time of apigenin in this model also presents certain dependence.And the P of apigenin
AppAll higher in Caco-2 with identical time intrinsic permeability than it.So can infer that good transhipment is arranged when apigenin penetrates blood brain barrier in vivo, this has established theoretical basis for the extensive use of apigenin when preventing and treat cerebrovascular disease.
1, materials and methods
1.1 cell source
From embodiment 3 Central Plains be commissioned to train rat BMECs foster 2 generation cell.
1.2 reagent preparation and administration
Precision takes by weighing apigenin 2.7mg and places the 1ml volumetric flask, fully dissolves the mother solution that is mixed with 10mM with DMSO.DMEM solution dilution with freshly prepared pH ≈ 7.4 becomes 0.8 μ M, 4 μ M, 20 μ M, 40 μ M, 50 μ M to carry out administration.
1.3 method
Adopt mtt assay to measure the BMECs cell viability.Its principle is that the succinate dehydrogenase in the living cells can make MTT decompose, and produces water-fast blue first a ceremonial jade-ladle, used in libation granule, and the formation amount of first a ceremonial jade-ladle, used in libation and viable count and cell viability are proportional.Get the good 2-3 of growth conditions for BMECs, make 6 * 10 with trypsinization
4The cell suspension of individual/ml is planted in 96 orifice plates, every hole 100 μ l.After cultivating 24h, be replaced by the DMEM culture fluid of serum-free, continue to cultivate 24h, make the growth of cell synchronous.Apigenin concentration is (0.8,4,20,40,50 μ M).After cultivating 48h, add 5mg/ml, 10%MTT 200 μ l are hatched 4h, add 150 μ l DMSO, vibrate 3 minutes, and full-automatic microplate reader 570nm wavelength place surveys absorbance (OD).
2, statistical procedures
The detection data of each index all adopt SPSS 13.0 statistics softwares to carry out one factor analysis of variance (ANOVA), relatively adopt Least Significant Difference Procedure (LSD) check between group in twos.Experimental result represents that with mean ± S.D. P＜0.05 explanation difference has the significance meaning.
3, experimental result
Adopt MTT experimentation apigenin that rat brain capillary endothelium effect of vigor be the results are shown in Figure 3.
4, conclusion
Under this experiment condition, to compare with the normal control group, apigenin presents certain dose-dependence to the vigor inhibitory action of rat BMECs cell.When concentration was 40 μ M, its activity inhibition rate was 10.39%, and when concentration was 50 μ M, its activity inhibition rate was 27.28%.In general, the brain micro blood vessel endothelium cell is the main cell that forms BBB, and the tight connection that forms between BMECs is the architecture basics that constitutes BBB.Thereby investigation variable concentrations apigenin is to the cell viability influence of BMECs, thereby judge the comparatively safe dosage of variable concentrations apigenin to blood brain barrier, and then can obtain the transhipment absorption data of apigenin in BBB cell in vitro model more accurately, this safe medication for the clinical treatment cardiovascular and cerebrovascular disease provides important assurance.The result shows that apigenin has the advantages that safety is very high, toxic and side effects is low.The computing formula of activity inhibition rate is:
Claims (4)
1. apigenin prevents and/or treats application in the cardiovascular and cerebrovascular disease product in preparation.
2. application according to claim 1 is characterized in that: described product is medicine or health product.
3. apigenin has application in the vasodilation functional product in preparation.
4. application according to claim 3 is characterized in that: described product is medicine or health product.
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| CN110713503A (en) * | 2018-07-12 | 2020-01-21 | 北京恒润泰生医药科技有限公司 | Fresh celery leaf water extract powder, preparation, vasodilatation effect and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1284492A (en) * | 1999-08-17 | 2001-02-21 | 钱少华 | Method of producing mixture of apium essence and apiin |
| CN100473415C (en) * | 2005-12-21 | 2009-04-01 | 张恩及 | Health-care product for treating cardiovascular and cerebralvascular disease |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1284492A (en) * | 1999-08-17 | 2001-02-21 | 钱少华 | Method of producing mixture of apium essence and apiin |
| CN100473415C (en) * | 2005-12-21 | 2009-04-01 | 张恩及 | Health-care product for treating cardiovascular and cerebralvascular disease |
Non-Patent Citations (1)
| Title |
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| 《卫生研究》 20101130 隋海霞等 芹菜素对自发性高血压大鼠的降压作用及对肾脏血管紧张素转化酶2表达的影响 第696页右栏 1-4 第39卷, 第6期 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713503A (en) * | 2018-07-12 | 2020-01-21 | 北京恒润泰生医药科技有限公司 | Fresh celery leaf water extract powder, preparation, vasodilatation effect and application thereof |
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