CN105030756A - Application of EGCG structure modified derivatives in preparation of blood vessel regulating and controlling medicine - Google Patents
Application of EGCG structure modified derivatives in preparation of blood vessel regulating and controlling medicine Download PDFInfo
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Abstract
The invention relates to application of EGCG structure modified derivatives in preparation of blood vessel regulating and controlling medicine. The EGCG structure modified derivatives have stability and pharmacological activity higher than those of EGCG, and the blood vessel growth regulating and control functions can be achieved by regulating and controlling the expression of VEGF and CBR1 and restraining the growth of vascular endothelial cells.
Description
Technical field
The present invention relates to pharmacology and medical science, relate generally to a class EGCG structural modification derivant and preparing the application in blood vessel regulation and control class medicine.
Background technology
Angiogenesis is all significant in the growth of tumor cell and transfer, ocular angiogenesis are to the new vessels relevant disease such as infringement, diabetes new vessels pathological changes, renal blood vessels proliferative lesion, coronary heart disease of eye structure and function.Suppress new vessels to generate, the nutrition supply of tumor can be cut off, impel the degeneration of tumor, atrophy; lower eye blinding chance, reduce diabetic complication, protection kidney normal physiological function; developing of Prevention and Curation coronary heart disease, these viewpoints are by a large amount of results of study is confirmed.All along with microvascular induction and growth in the growth of kinds of tumors and the process of transfer.When new vessels is to tumor tissues perfusion, the speed of growth and the infiltration metastasis energy of tumor also can be strengthened greatly.Therefore angiogenesis is to the growth of solid tumor with shift most important.Experimental results demonstrate, the generation of kinds of tumors, transfer, recurrence and prognosis are all closely related with tumor-blood-vessel growth.Therefore block angiogenesis and become novel drugs target spot.The medicine of current research Cancer therapy has become a new focus, and existing multiple vasoinhibitor enters clinical, extracts the research direction that anti-angiogenesis active substances also becomes new from plant.
EGCG is the main active substances in green tea, and its chemical constitution is clear and definite, composition is single, is a kind of non-protein Polyphenols micromolecule, belongs to flavanol compound.EGCG mainly contains biological effect and the pharmacodynamics effects such as anti-angiogenesis activity, antioxidation, scavenging free radicals, antiinflammatory, antiviral, mutation and antitumor formation, has the effect promoting tumor and endothelial cell apoptosis, Antineoplastic angiogenesis, reduction microvessel density.Epidemiological study discloses, and takes in the ability that EGCG has anti-angiogenesis, thus can reduce the sick incidence rate such as diabetic proliferative neovascular pathological changes, coronary heart disease and tumor.Find in antihepatocarcinoma effect research, EGCG suppresses the growth of hepatoma carcinoma cell and the generation of hepatocarcinoma new vessels by the increase of suppression HIF-1 α albumen and the activity of VEGF/VEGFR.EGCG obviously can suppress the propagation of HepG2 hepatoma carcinoma cell, effectively suppresses the protein expression of two important factor HIF-1 α, VEGF in hypoxia response, also has obvious inhibitory action to the genetic transcription of VEGF.
In tumor growth, anoxia is considered to one of basic feature of parenchyma physics microenvironment always, hypoxia inducible factor (hypoxia-induciblefactor1, HIF-1) in Tumor Growth, key is played a part, participate in the main regulate factors of tumor-blood-vessel growth, the vascular endothelial cell factor (vascularendothelialgrowthfactor, VEGF) gene expression.HIF-1 is made up of HIF-1 α and HIF-1 β two subunits, and HIF-1 α is main functional unit.Anoxia, as one of the initiating agent of tumor growth and transfer, first activates HIF-1 α, causes vegf expression to raise, induced tumor angiogenesis; Assist tumor cell to enter vascular system, increase the probability of neoplasm metastasis, this theory is universally accepted.HIF-1 α, as a kind of transcription factor be extensively present in mammalian cell, experiences the change of tissue oxygen content, and the target gene of regulation and control relates to tumor cell proliferation, energy metabolism, angiogenesis, neoplasm metastasis, ion metabolism and catechol metabolism etc.HIF-1 α participates in mediation to be had: under (1) anaerobic condition, the HIF-1 α of process LAN regulates and controls the multiple factor with associated angiogenesis in its downstream and receptor thereof, as VEGF (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) etc., promote the formation of new vessels in tumor tissues by activating multiple tyrosine kinase.As can be seen here, suppress the activity of HIF-1 α can reduce the sensitivity of tumor cell to anoxia, reduce the formation of new vessels, inhibition tumor cell transfer and multidrug resistance.
Inquire the following patent applied in medicine about EGCG:
Chinese patent, application number: 201210009960.5, applicant: Xi'an Communications University, the application of denomination of invention: EGCG in preparation treatment type Ⅱdiabetes mellitus medicine, summary: the present invention is the application of EGCG in preparation treatment type ii diabetes medicine, relate to biology and pharmaceutical field, its object is to provide the purposes of EGCG in treatment type ii diabetes, its technical characteristic is mainly, EGCG can effectively prevention and therapy type ii diabetes and adjoint myocardial cell mitochondrial dysfunction thereof, autophagy increases and oxidative stress raises, and point out EGCG may be by suppressing FoxO1 to realize for the regulation and control of autophagy for the inhibitory action of insulin resistant, prompting FoxO1 may be the target spot for the treatment of diabetes.
Chinese patent, application number: 201410024805.X; Applicant: Xi'an Communications University; Denomination of invention: EGCG prevents and treats the food of energy metabolism of myocardial obstacle and the application of medicine in preparation; Summary: EGCG prevents and treats the food of energy metabolism of myocardial obstacle and the application of medicine in preparation, the action target spot of EGCG is FoxO1 signal path in cell, the myocardial cell autophagy that energy metabolism of myocardial obstacle causes can be suppressed, alleviate myocardial mitochondria to lose, the useful effect concentration of EGCG to myocardial cell is 20uM; Or be 5-10mg/kg/ day by the intake of whose body weight, energy metabolism of myocardial ability can be regulated, recover the myocardial cell functional defect caused by energy metabolism impairment; Also for suppressing the autophagy that myocardial cell H9c2 metabolism obstacles of blood glucose is relevant to raise; Also for suppressing the oxidative stress that myocardial cell H9c2 metabolism obstacles of blood glucose is relevant; Also for reducing the insulin resistant that myocardial cell H9c2 metabolism obstacles of blood glucose is relevant; The present invention namely can food function factor, also can be used for prevention and the treatment of the diseases such as factor myocardial injury such as energy metabolism of myocardial obstacle, myocardium insulin resistant, hyperglycemia by medicament forms.
Chinese patent, application number: 201410359356.4; Applicant: Sheng Dakang bio tech ltd, Wuhan; Denomination of invention: the application of EGCG cetylate in preparation treatment or prevention enterovirns type 71 infection medicine; Summary: the present invention relates to new medical use technical field, specifically discloses the application of a kind of EGCG cetylate in preparation treatment or prevention enterovirns type 71 infection medicine.Present invention demonstrates EGCG cetylate and in the test of enterovirns type 71 infection cell, there is good antivirus action in vitro, the infection of enterovirns type 71 can be prevented, its action effect is more more obvious than positive control drug Pu Kenali, and disclosing this medicine has the prospect being developed to anti-enterovirns type 71 medicine.
Chinese patent, application number: 201410359357.9; Applicant: Sheng Dakang bio tech ltd, Wuhan; Denomination of invention: the application of EGCG cetylate in preparation treatment or prevention of hepatitis C infection medicine; Summary: the present invention relates to new medical use technical field, specifically discloses the application of a kind of EGCG cetylate in preparation treatment or prevention of hepatitis C infection medicine.Present invention demonstrates EGCG cetylate and in infection with hepatitis C virus test cell line, there is good antivirus action in vitro, can the infection of prevention and therapy hepatitis C virus, its action effect is suitable with positive control drug IFN-α 1, and disclosing this medicine has the prospect being developed to anti hepatitis C virus drug.
The application of the application of above-mentioned patent respectively in treatment type Ⅱdiabetes mellitus medicine, the food preventing and treating energy metabolism of myocardial obstacle and medicine, the application in treatment or prevention enterovirns type 71 infection medicine, the application in treatment or prevention of hepatitis C infection medicine, also do not have bibliographical information EGCG structural modification derivant preparing the application in blood vessel regulation and control class medicine at present.
Summary of the invention
The object of the invention is to there are provided a kind of EGCG structural modification derivant and preparing the application in blood vessel regulation and control class medicine, this medicine mainly contains biological effect and the pharmacodynamics effects such as anti-angiogenesis activity, antioxidation, scavenging free radicals, antiinflammatory, antiviral, mutation and antitumor formation, has the effect promoting tumor and endothelial cell apoptosis, Antineoplastic angiogenesis, reduction microvessel density.
EGCG structural modification derivant is preparing the application in blood vessel regulation and control class medicine, and the formula of the type EGCG structural modification derivant is:
Further, the structure of EGCG structural modification derivant is:
The present invention adopts quantitative real-time PCR and detected by Western blot research EGCG structural modification derivant on the impact of HIF-1 α, vegf expression in the alpha mediated signal transduction pathway of HIF-1, from the mechanism of action of gene and protein level checking EGCG modulating vascular.
Real-timePCR and Westernblot testing result display of the present invention, compare with normal oxygen matched group, under anaerobic environment, in SMMC-7721 hepatoma carcinoma cell, on the alpha mediated signal transduction pathway of HIF-1, the genetic transcription of HIF-1 α, VEGF and protein expression all significantly raise, thus promote that new vessels is formed.For SMMC-7721 hepatoma carcinoma cell, EGCG and EGCG ethylization derivant all significantly lowers HIF-1 α, VEGF genetic transcription and protein expression level.Result of the present invention confirms, the signal transduction pathway that EGCG structural modification derivant can regulate HIF-1 alpha mediated, by suppressing HIF-1 α and VEGF genetic transcription and protein expression, suppresses new vessels to be formed and the transfer of tumor cell.
Human umbilical vein endothelial cells is the vascular endothelial cell with collagenase digesting umbilical vein gained, has purity high, active good, can cultivate the characteristic in seven generations of also can going down to posterity more than in vitro.The regulatory factor that it can synthesize, secrete the activity factor of blood coagulation and fibrinolytic system and inhibitive factor, affect platelet adhesion and gathering; Endotheliocyte is release control cell proliferation and the molecule etc. regulating blood vessel wall tensity also, is the external model of very excellent Quality Research angiogenesis.
In the Newborn Process of fetal development and tissue, there is (vasculogenesis) and angiogenesis (angiogenesis) by blood vessel and combine and work and produce in blood vessel.In the effect by chemotactic and somatomedin of the generating process of chick embryo development medium vessels and tumor vascular Newborn Process, all experience the propagation of vascular endothelial cell, migration and become pipe process, so have dependency.Chick chorioallantoic membrane (chickenchorioallantoicmembrane, CAM) be a skim outside Embryo Gallus domesticus, major function is to provide the surface of carrying out gas exchange, the enforcement of CAM function simultaneously also depends on the support of fine and close vasoganglion, just because of the formation of chorioallantoic membrane blood vessel a large amount of in chick embryo development process, for the generation of research blood vessel and formation provide good experimental model.The early stage developing immune system of Embryo Gallus domesticus is still incomplete, can not produce rejection to added medicine, be the angiopoietic a kind of classical model of research.The intervention result in this period is convenient to adopt microscopic examination shooting and use image processing software to quantize result.The angiogenic growth of Embryo Gallus domesticus has its evident characteristic: Embryo Gallus domesticus 3rd ~ 5 days of hatching and 7th ~ 10 days angiogenic growths the most active, after the 12nd day, blood vessel starts atrophy gradually, therefore, within 8 days ~ 12 days, is pharmaceutically-active preferably period.
Mtt assay testing result of the present invention shows, EGCG and EGCG ethylization derivant all has comparatively high inhibition effect to Human umbilical vein endothelial cells, there is doses dependency, illustrate that EGCG structural modification derivant is all strong than parent compound EGCG to the inhibitory action of cell proliferation of human umbilical vein, especially the strongest with Y6 effect, the effect that EGCG derivant can generate by suppressing vascular endothelial cell to play modulating vascular is described.The present invention is again using chick chorioallantoic membrane as experimental model, select to hatch to instar chicken embryo dosing on the 7th and observe, found that EGCG structural modification derivant has significant suppression Embryo Gallus domesticus angiogenesis function, and have certain dose dependent, confirm the effect that EGCG derivant modulating vascular generates.
Good effect of the present invention is:
1, the EGCG in EGCG structural modification derivant is the main composition of Folium Camelliae sinensis, and output is large, convenience of drawing materials.
2, by experiment of the present invention, confirming the signal transduction pathway that EGCG structural modification derivant can regulate HIF-1 alpha mediated, by suppressing HIF-1 α and VEGF genetic transcription and protein expression, suppressing new vessels to be formed and the transfer of tumor cell.For SMMC-7721 hepatoma carcinoma cell, EGCG and EGCG ethylization derivant all significantly lowers HIF-1 α, VEGF genetic transcription and protein expression level.
3, mtt assay testing result of the present invention shows, EGCG and EGCG ethylization derivant all has comparatively high inhibition effect to Human umbilical vein endothelial cells, there is doses dependency, illustrate that EGCG structural modification derivant is all strong than parent compound EGCG to the inhibitory action of cell proliferation of human umbilical vein, especially the strongest with Y6 effect, the effect that EGCG derivant can generate by suppressing vascular endothelial cell to play modulating vascular is described.
4, this several ECGC structural modification derivant has the stability higher than EGCG and pharmacologically active, and can by regulation and control VEGF, CBR1 expression, suppress vascular endothelial cell growth and realize modulating vascular growth function.
Accompanying drawing explanation
Fig. 1 EGCG structural modification derivant is on the impact (40X) of CAM blood vessel.
Detailed description of the invention
Below in conjunction with specific embodiment, illustrate technical scheme of the present invention further.Should be understood that these embodiments are only not used in the scope of restriction request protection of the present invention for illustration of the present invention.
In following examples: medicine EGCG structural modification derivant used is the following EGCG structural modification derivant of a kind of structural formula:
embodiment 1
1. real-time fluorescence quantitative PCR (Real-timePCR) method detects EGCG structural modification derivant to the impact of hepatoma carcinoma cell HIF-1 α, VEGF gene expression
1.1 human liver cancer cells Hep G2 are cultivated
Get well-grown SMMC-7721 hepatoma carcinoma cell, be divided into two large groups: normal oxygen matched group
(not pastille culture medium) and anoxia group.Anoxia group is divided into again: anoxia matched group (not pastille culture medium), EGCG high dose group (EGCG-H, 50 ~ 100 μm of ol/L), dosage group (EGCG-M in EGCG, 25 ~ 50 μm of ol/L), EGCG low dosage (EGCG-L, 1 ~ 25 μm of ol/L), EGCG ethylization derivant Y1 ~ 6 high dose group (25 ~ 50 μm of ol/L), dosage group (10 ~ 25 μm of ol/L), EGCG ethylization derivant Y1 ~ 6 low dose group (1 ~ 10 μm of ol/L) in EGCG ethylization derivant Y1 ~ 6.Put normal oxygen after each group of cell dosing respectively, hypoxia culture box cultivates 16h.Normal oxygen condition of culture: 21% oxygen, 5% carbon dioxide, 37 DEG C, the standard incubator of saturated humidity; Anoxia condition of culture: 3% oxygen, 5% carbon dioxide, 92% nitrogen, 37 DEG C, three gas incubators of saturated humidity.
total serum IgE extracting
(1) by culture fluid sucking-off in culture bottle, clean 1 time with the PBS of pre-cooling, add the RNAisoPlus solution of 1ml pre-cooling, weak vibrations, guarantee to make lysate be uniformly distributed in cell surface.Interior celliferous lysate is transferred to 1.5ml centrifuge tube, with in liquid-transfering gun repeatedly pressure-vaccum to lysate without obvious sediment.Room temperature leaves standstill 5min.
(2) add 200 μ l chloroforms, cover tightly centrifuge tube lid, be mixed to emulsifying soln and become milky.Standing at room temperature 5min.
(3) 12,000g4 DEG C of centrifugal 15min.Careful absorption about 400 μ l supernatant, is transferred in new RNase-free1.5mlEppendorf pipe, adds the isopropyl alcohol of 400 μ l, and the centrifuge tube that softly turns upside down mixes, and ambient temperatare puts 10min.12,000g4 DEG C of centrifugal 10min.
(4) RNA washing of precipitate: carefully discard supernatant, adds 1ml75% ethanol, washing of turning upside down gently, 12,000g4 DEG C of centrifugal 5min.Carefully discard supernatant.
(5) RNA dissolves: in super-clean bench, open centrifuge tube lid, and drying at room temperature RNA precipitates.Add appropriate RNase-free water dissolution.
(6) RNA purity and concentration determination: measure RNA purity and concentration with BioSpec-nano ultraviolet-visible spectrophotometer.Pass through OD
260/ OD
280the purity of ratio in judgement RNA, OD
260/ OD
2801.8 ~ 2.2, ratio illustrates that RNA quality is better.
reverse transcription cDNA
By Reverse Transcriptase kit, (PrimeScript is described
rrTreagentKitwithgDNAEraser, TaKaRa company) synthesize cDNA.
reaction
1.4.1PCR primer sequence
According to the sequence of Genebank, design and synthesize primer by precious biological engineering (Dalian) company limited, wherein GAPDH is house-keeping gene.Each gene primer sequence is in table 1.
reaction system
1.4.3Real-timePCR reaction condition
Real-timePCR reaction condition: 1. 95 DEG C of 30s; 2. 95 DEG C of 5s, 60 DEG C of 31s,
Totally 40 circulations; 3. 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s.
result calculates
After reaction terminates, adopt compare threshold method (2 according to Ct value
-△ △ Ctmethod) calculate the mRNA relative expression quantity of each gene.In formula, △ Ct represents the amplification cycles number difference of target gene and self reference gene GAPDH, and △ △ Ct represents amplification cycles number difference between drug treating group and matched group.
experimental result
Quantitative real-time PCR result shows, with anoxia matched group for reference, the mrna expression content of normal oxygen matched group HIF-1 α, VEGF and CBR1 gene lower (
p﹤ O.Ol), to show under anaerobic environment that in SMMC-7721 hepatoma carcinoma cell, HIF-1 α, VEGF and CBR1 gene expression significantly increases (
p﹤ O.Ol).
Compare with anoxia matched group, SMMC-7721 hepatoma carcinoma cell gives EGCG and EGCG ethylization derivant under anaerobic environment, each dosage group to HIF-1 α, VEGF gene expression all occur lowering in various degree (
p﹤ 0.01 or
p﹤ 0.05), there is certain dose dependent.The results are shown in Table 2, illustrate that EGCG derivant is by these two angiogenic growth related protein genes of regulation and control HIF-1 α, VEGF, plays the angiogenesis regulating and controlling effect similar with EGCG.
embodiment 2
2. protein immunoblot (Westernblot) method detects the impact that EGCG structural modification derivant is expressed hepatoma carcinoma cell HIF-1 α, vegf protein
2.1 cell culture and grouping are with 1.1.
After hepatoma carcinoma cell dosing, normal oxygen, anoxia cultivate 16h respectively, discard culture fluid, rinse 2 times, add PBS1ml in culture bottle, scrape collecting cell with cell with PBS, are transferred to Eppendorf pipe, the centrifugal 5min of 12,000g.Discard supernatant, add the protein lysate of 500 μ l, after turbula shaker vibration, put 30min on ice.The centrifugal 10min of 12,000g, collects supernatant BCA kit measurement protein concentration.SDS-PAGE albumen buffer is mixed in proportion, and boils 5min, subpackage, put-20 DEG C for subsequent use.
gel
With the SDS-PAGE gel of 8%-12%, albumen applied sample amount is 15ug, concentrated glue 80V30min, separation gel 110V50min.With absolute methanol process pvdf membrane 30s, be then immersed in 15min in transferring film buffer.Cut glue transferring film according to molecular weight of albumen size, wet transfer printing is to pvdf membrane.Film is put into 5% skim milk, under room temperature, slowly sway closed 2h.Add primary antibodie 4 DEG C to spend the night.
washing film
TBST washs pvdf membrane 5min × 3 time, and according to the situation of primary antibodie, the IgG of 1:2000-1:5000 variable concentrations dilution horseradish peroxidase labelling, sways with film under room temperature and hatch 1.5h.Two anti-hatch end after, with TBST post rinse film 4 times, each 5min.Chemiluminescence agent ECL develops.With ImageJ software Bian collection band gray value, get object band gray value and internal reference band gray value ratio is relative expression quantity.
result
Detected by Western blot result shows, and compares with normal oxygen matched group, the expression of anoxia matched group HIF-1 α, vegf protein is all raised (
p﹤ 0.01), to show under anaerobic environment that HIF-1 α in SMMC-7721 hepatoma carcinoma cell, vegf protein are expressed and significantly increased (
p﹤ 0.01).
Compare with anoxia matched group, SMMC-7721 hepatoma carcinoma cell gives EGCG and EGCG ethylization derivant Y1 ~ 6 under anaerobic environment, each dosage group is expressed HIF-1 α and vegf protein and all occurred lowering in various degree (
p﹤ 0.01 or
p﹤ 0.05), there is certain dose dependent.The results are shown in Table 3.Illustrate that EGCG derivant can reduce the expression of blood vessel Regulated Proteins, play the angiogenesis regulating and controlling effect similar with EGCG.
embodiment 3
3MTT method detects the impact that EGCG structural modification derivant grows Human umbilical vein endothelial cells
3.1 experimental technique
Get form normal exponential phase HUVEC cell, digestion, centrifugal, remove supernatant, collecting cell precipitate.Add the high sugared culture fluid piping and druming of appropriate DMEM evenly, form single cell suspension, counted under microscope.Cell suspension density is adjusted to 1 × 10
4/ mL, is inoculated in 96 well culture plates, and 100 μ L are inoculated in every hole, are placed in 5%CO
2incubator, cultivates about 24h by 37 DEG C under the condition of saturated humidity.EGCG, AcEGCG and EGCG ethylization derivant Y6 DMEM is high, and sugared culture fluid is diluted to 5 concentration respectively as medicine group, wherein the drug dose of EGCG is 200 μ g/ml, 150 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the drug dose of Y1 ~ 6 is respectively 120 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/ml, 10 μ g/ml, the each concentration of medicine group establishes 5 multiple holes, and cell blank matched group establishes 5 multiple holes.Every hole adds medicinal liquid 100 μ L, mixes with 100 μ L culture fluid in original hole, forms final concentration, and in every hole, the percent by volume of DMSO is less than 0.1%.Cell blank contrasts every hole and adds the high sugared culture fluid of 100 μ LDMEM.After dosing, cell continues to cultivate 48h in the incubator of the same terms, and then every hole adds the MTT solution 20 μ L that concentration is 5mg/mL.Continue to cultivate 4h, stop cultivating, inhale and abandon mixed liquor, every hole adds DMSO150 μ L, is placed in microplate reader and vibrates, and measures absorbance A value in 490nm wavelength place.According to inhibitory rate of cell growth (%)=(1-experimental group A value/control group A value) × 100%, calculate EGCG and derivant thereof to the growth inhibition ratio of cell, and make curve with medicine variable concentrations and inhibitory rate of cell growth, ask and calculate half-inhibition concentration (IC
50).
result
Mtt assay testing result shows, EGCG and EGCG ethylization derivant Y1 ~ 6 pair Human umbilical vein endothelial cells all has inhibitory action.The results are shown in Table 4.Illustrate that EGCG derivant can by suppressing the growth of human vascular endothelial and modulating vascular.
embodiment 4
4EGCG structural modification derivant is on the impact of CAM angiogenesis
4.1 Embryo Gallus domesticus are hatched
Buy hatching to the Embryo Gallus domesticus of the 5th day, with 75% alcohol wipe eggshell surface sterilization, determine and labelling air chamber position with candler.Keep chick embryo air sac upward, major axis hatches horizontal by 45° angle.Incubator temperature is 37.5 DEG C, and humidity is 60%, every day turning egg(s) 3 times.
pharmaceutical formulations and grouping
Using EGCG(250 μ g/ml) as positive control, the middle dosage of EGCG ethylization derivant Y1 ~ 6 is equal with the molal quantity of EGCG.Y1 ~ 6 height (Y1 ~ 6-H), in (Y1 ~ 6-M), low (Y1 ~ 6-L) dosage be respectively: 500 μ g/ml, 350 μ g/ml, 200 μ g/ml; With sodium chloride injection dilution (whole solution is containing 1%DMSO) after each medicinal appropriate DMSO dissolves.Solvent control group (Control): 1%DMSO is dissolved in sodium chloride injection.Medicine prepared before use.Often organize each 10 Embryo Gallus domesticus.
inoculation hatching
When hatching was by the 7th day, at superclean bench, carefully get out 1 1mm by dental burr at a slow speed in air chamber centre
2aperture, with 75% cotton ball soaked in alcohol by after eggshell powder wiped clean, then open 1.5cm × 1.5cm breach with the careful tweezer of ophthalmic tweezers, expose air chamber, open the egg film in air chamber with ophthalmic tweezers, expose chick chorioallantoic membrane (Chickenchorioallantoicmembrane, CAM).Be placed on CAM by the sterilizing qualitative filter paper disk being cut into diameter 5mm in advance, experimental group adds 100 μ l variable concentrations medicines on filter paper, and solvent control group adds equal-volume sodium chloride injection (containing 1%DMSO) on filter paper.Ensure chick embryo air sac opening after dosing just upward, close chorion opening with the adhesive tape of sterilizing, put into incubator, continue to hatch by former condition of culture.
prepared by specimen
Within after dosing the 3rd day, tear adhesive tape off, then the mixed liquor 1.5ml of equivalent methanol and acetone is dripped at opening part, 15min is fixed under room temperature, after blood coagulation, remove chorion above CAM plane and membrana putaminis with ophthalmic tweezers, centered by filter paper carriers, cut chorioallantoic membrane with eye scissors, put into the surface plate that distilled water is housed, chorioallantoic membrane is launched, is laid on microscope slide, dries in the shade.Under 40 power microscopes, observe the growing state of Embryo Gallus domesticus blood vessel and take pictures.
vascular counts
Choose centered by dosing filter paper dick, the big and small vessel of radius 5mm inner region counts.Press blood vessel thickness difference counting.According to blood vessel diameter, blood vessel is divided three classes: 1. > 0.2mm is I grade of blood vessel; 2. 0.05mm ~ 0.2mm is II grade of blood vessel; 3. < 0.05mm is III grade of blood vessel, counts respectively to blood vessel at different levels.
statistical procedures
Adopt the process of SPSS13.0 statistical software.Data result with ±
srepresent.Employing one factor analysis of variance is compared between group, with
p﹤ 0.05 has statistical significance for difference.
structural modification derivant is on the impact of CAM angiogenesis
During Embryo Gallus domesticus CAM blood vessel normal growth, present natural vein shape, big and small vessel quantity is certain proportion, is naturally dispersed grow many thin vessels by trunk, and form the radial vasoganglion that dense degree is different, vessel boundary is high-visible.Observe and add each group of chick chorioallantoic membrane blood vessel of different pharmaceutical, can find, compared with solvent control group, each medicine all can cause blood vessel mean at different levels to reduce, but on I level blood vessel affect no significant difference (
p﹥ 0.05).Each medicine each dosage group to II, III level blood vessel all have remarkable inhibitory action (
p<0.05 or
p<0.01), EGCG and EGCG ethylize derivative object height, middle dosage group to II level blood vessel have remarkable inhibitory action (
p<0.05), the high, medium and low dosage group of each medicine to the effect of III level vascular study significantly (
p<0.01 or
p<0.05).Basis of microscopic observation visible vessels is distributed growth blood capillary and is reduced, and blood vessel slight deformation, blood engorgement reduces.The results are shown in Table 5, Fig. 1.
Claims (3)
1. a class EGCG structural modification derivant is preparing the application in blood vessel regulation and control class medicine.
2. application according to claim 1, is characterized in that, the formula of described EGCG structural modification derivant is:
。
3. the formula of EGCG structural modification derivant according to claim 2, is characterized in that: the structure of EGCG structural modification derivant is further:
。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109432083A (en) * | 2018-12-29 | 2019-03-08 | 中国医科大学附属第医院 | EGCG improves the application in endothelial cell damage drug caused by blood glucose fluctuation in preparation |
| CN112138002A (en) * | 2020-09-23 | 2020-12-29 | 深圳大学 | Inhibitor for resisting miR483-3p mediated tumor metastasis and application thereof |
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| CN103833719A (en) * | 2014-03-11 | 2014-06-04 | 广西医科大学 | Epigallocatechin gallate alkylation derivatives and antitumor application thereof |
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| 赵丽萍等: "茶叶中EGCG 功效研究进展", 《中国农学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109432083A (en) * | 2018-12-29 | 2019-03-08 | 中国医科大学附属第医院 | EGCG improves the application in endothelial cell damage drug caused by blood glucose fluctuation in preparation |
| CN112138002A (en) * | 2020-09-23 | 2020-12-29 | 深圳大学 | Inhibitor for resisting miR483-3p mediated tumor metastasis and application thereof |
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