CN101095673B - Application of 3-benzyl-5-(2-nitrophenoxymethyl)-gamma-butyrolactone - Google Patents
Application of 3-benzyl-5-(2-nitrophenoxymethyl)-gamma-butyrolactone Download PDFInfo
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- CN101095673B CN101095673B CN200710016184A CN200710016184A CN101095673B CN 101095673 B CN101095673 B CN 101095673B CN 200710016184 A CN200710016184 A CN 200710016184A CN 200710016184 A CN200710016184 A CN 200710016184A CN 101095673 B CN101095673 B CN 101095673B
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Abstract
The invention discloses the application of 3-benzyl-5-(2-nitro phenoxy)-gamma-butyrolactone as medicine that can inhibit cytoplasm vacuolization induced by chloroquinine. The medicine can inhibit cytoplasm vacuolization for vascular endothelial cells and smooth muscle cell, and its concentration is 60-180 um.
Description
Technical field
The present invention relates to the new purposes of 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton, relate in particular to of the application of 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton as the Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation.
Background technology
The gamma-butyrolacton structural formula is:
Molecular formula: C
4H
6O
2
Molecular weight: 86
Character: gamma-butyrolacton is that nontoxic transparent oily liquids and water can dissolve each other fully, dissolves in ethanol, ether, benzene and acetone, can dissolve many organic and inorganic compound.Be that a kind of boiling point height, dissolubility are strong, the solvent of electrical property and good stability.
Butyrolactone derivative obtains extensive use in the organic synthesis field, be very important intermediate of a class and end-product.The butyrolactone structure is the very important construction unit of a class, all contains this construction unit in many natural products and the synthetic drug.Now, owing to replace the pharmacology of gamma-butyrolactone derivative and medicinal property, economic worth, it still is the emphasis of research worker research.But, the report of butyrolactone derivative research at present mostly be to butyrolactone derivative the biological activity of synthetic this analog derivative test, show that this analog derivative has effects such as antitumor, antiulcer, anti-inflammation and sterilization, sedation-analgesia, spasmolytic, inhibition platelet aggregation, inhibition nervus centralis.And utilize it to carry out pharmacological research and application as the Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation, and look into newly through authoritative institution's retrieval, do not appear in the newspapers as yet both at home and abroad at present.
Summary of the invention
The object of the present invention is to provide the new purposes of a kind of butyrolactone derivative 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton, promptly 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton is as the application of the Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation.
Gamma-butyrolactone derivative 3-arylmethyl of the present invention-5-aryloxy methyl-gamma-butyrolacton is represented with following general formula (I):
Wherein: R
1Represent H, C
1-5Alkyl, C
1-5One of alkoxyl, halogen, nitro, hydroxyl;
R
2Represent H, C
1-5One of alkyl, phenyl, substituted-phenyl, naphthyl, substituted naphthyl.
In 3-benzyl-5-of the present invention (2-nitro Phenoxymethyl)-gamma-butyrolacton:
R
1The preferred nitro of representing,
R
2The preferred phenyl of representing.
The present invention relates to of the application of 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton as the Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation.
Be specially:
3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton is as the application of the Cytoplasm vacuolation medicine that suppresses the generation of chloro-quinine inducement vascular endothelial cell.
Wherein: the described drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular endothelial cell generation is 60 μ M～180 μ M.
The above-mentioned drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular endothelial cell generation is preferably 110 μ M～140 μ M.
The above-mentioned drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular endothelial cell generation most preferably is 120 μ M.
3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton is as the application of the Cytoplasm vacuolation medicine that suppresses the generation of chloro-quinine inducement vascular smooth muscle cell.
Wherein: the described drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular smooth muscle cell generation is 60 μ M～180 μ M.
The above-mentioned drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular smooth muscle cell generation is preferably 110 μ M～140 μ M.
The above-mentioned drug level that can effectively suppress the Cytoplasm vacuolation of chloro-quinine inducement vascular smooth muscle cell generation most preferably is 120 μ M.
In order to understand essence of the present invention better, pharmacological evaluation and the result with 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton illustrates that it is in vascular endothelial cell that suppresses chloro-quinine inducement and the application in the vacuolation of smooth muscle cell Cytoplasm below.
The preparation of vascular endothelial cell and smooth muscle cell:
Cultivate human umbilical vein endothelial cell and human umbilical artery vascular smooth muscle cell with conventional method, it is standby to choose good and vascular endothelial cell that be in exponential phase of growth conditions and smooth muscle cell.
Adopt the method for Celluar and Molecular Biology, carry out following experiment: the application of research 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton in the Cytoplasm vacuolation that suppresses the chloro-quinine inducement generation.
1, inverted phase contrast microscope observation of cell morphological change:
Human umbilical vein endothelial cell is inoculated in the 24 porocyte culture plates, matched group is set: under the condition that contains serum and somatomedin, add 8 μ M chloro-quinines and cultivate; Experimental group: adding concentration under the condition of culture that contains serum and somatomedin is that 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton that 8 μ M chloro-quinines and concentration are respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M is cultivated.37 ℃, CO
2Cultivate the morphological change that inverted phase contrast microscope is observed vascular endothelial cell down in the incubator.
The human umbilical artery vascular smooth muscle cell is inoculated in the 24 porocyte culture plates, normal group is set: under the normal condition that contains serum and somatomedin, cultivate; Solvent control group: under the condition that contains serum and somatomedin, add dimethyl sulfoxide (DMSO) and cultivate; Chloro-quinine processed group: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and cultivate; 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group: contain and add 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton that concentration is respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M under the condition of serum and somatomedin and cultivate; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation that concentration is respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M.37 ℃, CO
2After cultivating 24 hours in the incubator, inverted phase contrast microscope is observed the morphological change of vascular smooth muscle cell down.
The result shows: be 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton of 60 μ M～180 μ M with concentration, can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement, the drug level that wherein effectively suppresses the Cytoplasm vacuolation of chloro-quinine inducement vascular endothelial cell and vascular smooth muscle cell generation most preferably is 120 μ M.
2, through the variation of acridine orange dyeing with acid film bubble in the fluorescence microscope Cytoplasm:
Human umbilical vein endothelial cell is inoculated in the 24 porocyte culture plates, matched group is set: cultivate containing under the normal condition of culture of serum and somatomedin; The chloro-quinine processed group: the chloro-quinine that adds 8 μ M under the condition of culture that contains serum and somatomedin is cultivated; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 8 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation that concentration is respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M.37 ℃, CO
2After cultivating 6 hours in the incubator, through acridine orange dyeing, acid film bubble is dyed orange red, observes the variation of acid film bubble under fluorescence microscope.
The human umbilical artery vascular smooth muscle cell is inoculated in the 24 porocyte culture plates, normal group is set: under the normal condition that contains serum and somatomedin, cultivate; Solvent control group: under the condition that contains serum and somatomedin, add dimethyl sulfoxide (DMSO) and cultivate; Chloro-quinine processed group: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and cultivate; 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group: contain and add 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton that concentration is respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M under the condition of serum and somatomedin and cultivate; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation that concentration is respectively 40 μ M, 60 μ M, 100 μ M, 120 μ M, 140 μ M, 180 μ M and 200 μ M.37 ℃, CO
2After cultivating 24 hours in the incubator, through acridine orange dyeing, acid film bubble is dyed orange red, observes the variation of acid film bubble under fluorescence microscope.
The result shows: 60 μ M～180 μ M 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolactons, can effectively suppress the vascular endothelial cell of chloro-quinine inducement and the vacuolation of vascular smooth muscle cell.
3, dimethyl diaminophenazine chloride dyeing detects the variation of acid compartment cumulative volume in the cell:
Dimethyl diaminophenazine chloride dyes through being usually used in lysosome, and is used for the acid compartment cumulative volume of quantitative assay cell.
The exponential phase vascular endothelial cell is inoculated in the culture dish of sub-diameter 6cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, calculate the acid compartment cumulative volume of different disposal group through the dimethyl diaminophenazine chloride staining.
The exponential phase vascular smooth muscle cell is inoculated in the culture dish of diameter 6cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, calculate the acid compartment cumulative volume of different disposal group through the dimethyl diaminophenazine chloride staining.
The result shows: 3-benzyl-5-of 60 μ M～180 μ M (2-nitro Phenoxymethyl)-gamma-butyrolacton can significantly suppress vascular endothelial cell and the long-pending increase of the acid film foam of vascular smooth muscle cell that chloro-quinine causes.
4, Na
+K
+-atpase activity detects:
Vascular endothelial cell is inoculated in the culture dish of diameter 10cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, peptic cell is respectively organized cellular enzymes liquid through the ultrasonication extraction, passes through Na
+K
+-ATP enzyme reagent kit detects respectively organizes Na
+K
+-atpase activity.
Vascular smooth muscle cell is inoculated in the culture dish of diameter 10cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, peptic cell is respectively organized cellular enzymes liquid through the ultrasonication extraction, passes through Na
+K
+-ATP enzyme reagent kit detects respectively organizes Na
+K
+-atpase activity.
The result shows: 3-benzyl-5-of 60 μ M～180 μ M (2-nitro Phenoxymethyl)-gamma-butyrolacton can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement, and suppresses the Na that chloro-quinine causes
+K
+-atpase activity rising phenomenon.
Above-mentioned experimental data statistical procedures:
Experimental data is represented with mean+/-standard error, checks through t: P＜0.05 expression has significant difference; P＜0.01 expression has utmost point significant difference.
By above-mentioned experiment and result thereof, can draw as drawing a conclusion:
The present invention finds that first chloro-quinine can induction of vascular endothelial cell and vascular smooth muscle cell vacuolation as anti-malaria medicaments widely, and this process is accompanied by Na
+K
+The reduction of-atpase activity.
Concentration is that 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton of 60 μ M～180 μ M can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement, and the drug level that wherein effectively suppresses the Cytoplasm vacuolation of chloro-quinine inducement vascular endothelial cell and vascular smooth muscle cell generation most preferably is 120 μ M.
Therefore, the 3-benzyl-5-that the present invention relates to (2-nitro Phenoxymethyl)-gamma-butyrolacton is laid a good foundation for developing the relevant Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation.
Description of drawings
Fig. 1 inverted phase contrast microscope is observed the vascular endothelial cell morphological change.
Wherein: A, D, G are respectively the chloro-quinine of 8 μ M and handled 6 hours, and 12 hours, 24 hours vascular endothelial cell; B, E, H is respectively the chloro-quinine of 8 μ M and 3-benzyl-5-of 120 μ M (2-nitro Phenoxymethyl)-gamma-butyrolacton was handled 6 hours jointly, and 12 hours, 24 hours vascular endothelial cell; C, F, I are respectively the chloro-quinine of 8 μ M and 3-benzyl-5-of 180 μ M (2-nitro Phenoxymethyl)-gamma-butyrolacton was handled 6 hours jointly, and 12 hours, 24 hours vascular endothelial cell.
Fig. 2 inverted phase contrast microscope is observed the vascular smooth muscle cell morphological change.
Wherein: A is a normal group; B is the DMSO processed group; C is the chloro-quinine processed group; D is 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group; E is chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton.
Fig. 3 is through the variation of acridine orange dyeing with the acid film bubble of fluorescence microscope vascular endothelial cell.
Wherein: A is a matched group; B is the chloro-quinine processed group; C is chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton.
Fig. 4 is through the variation of acridine orange dyeing with the acid film bubble of fluorescence microscope vascular smooth muscle cell.
Wherein: A is a normal group; B is the DMSO processed group; C is the chloro-quinine processed group; D is 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group; E is chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton.
Fig. 5 dimethyl diaminophenazine chloride staining detects the variation of acid compartment cumulative volume in the vascular endothelial cell.
Wherein: CQ represents chloro-quinine; On behalf of 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton * *, 3BDO represent to compare P＜0.01 with the chloro-quinine processed group.
Fig. 6 dimethyl diaminophenazine chloride staining detects the variation of acid compartment cumulative volume in the vascular smooth muscle cell.
Wherein: CQ represents chloro-quinine; 3BDO represents 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton
The Na of Fig. 7 vascular endothelial cell
+K
+-atpase activity detects.
Wherein: CQ represents chloro-quinine; 3BDO represents 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton
Expression is compared with matched group, P＜0.05; * represents to compare with matched group, P＜0.01; # represents to compare with the chloro-quinine group, P＞0.05 ， ﹠amp; Expression is compared P＜0.05. with the chloro-quinine group
The Na of Fig. 8 vascular smooth muscle cell
+K
+-atpase activity detects.
Wherein: CQ represents chloro-quinine; 3BDO represents 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton
The specific embodiment
The preparation of embodiment 1 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton
0.235 gram (10.22 mM) sodium metal is added in 10 milliliters of dehydrated alcohol, treat that the sodium reaction finishes after, begin to stir.Be warming up to 50 ℃, slowly drip ethanol (2 milliliters) solution of 2.503 gram (10.01 mM) diethyl benzyl malonates then.Be cooled to 37 ℃, add ethanol (2 milliliters) solution of 1.953 gram (10.02 mM) 3-(2-nitro-phenoxy)-1,2 epoxy prapanes.55 ℃ of reactions were cooled to below 15 ℃ after 3.5 hours, added 1 milliliter of acetic acid.Decompression steams ethanol, and residue adds 10 ml waters, with chloroform extraction three times (each 20 milliliters).Merge organic facies, use 5%NaHCO
3Solution is given a baby a bath on the third day after its birth time (each 6 milliliters), washes twice (each 6 milliliters), uses anhydrous magnesium sulfate drying, filters concentrating under reduced pressure.Residue adopts silica gel column chromatography to separate (developing solvent is a petrol ether/ethyl acetate=3: 1, volume ratio), obtains cis-and trans-3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton mixture (0.721 gram), yield 22%.
Structural formula is as follows:
3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton is as the application of the Cytoplasm vacuolation medicine that suppresses the chloro-quinine inducement generation.
The preparation of vascular endothelial cell and smooth muscle cell:
Cultivate human umbilical vein endothelial cell and human umbilical artery vascular smooth muscle cell with conventional method, it is standby to choose good and vascular endothelial cell that be in exponential phase of growth conditions and smooth muscle cell.
Adopt the method for Celluar and Molecular Biology, carry out following experiment: the application of research 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton in the Cytoplasm vacuolation that suppresses the chloro-quinine inducement generation.
1, inverted phase contrast microscope observation of cell morphological change:
Human umbilical vein endothelial cell is inoculated in the 24 porocyte culture plates, matched group is set: under the condition that contains serum and somatomedin, add 8 μ M chloro-quinines and cultivate; Experimental group: adding concentration under the condition of culture that contains serum and somatomedin is that 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton that 8 μ M chloro-quinines and concentration are respectively 120 μ M and 180 μ M is cultivated.37 ℃, CO
2Cultivate the morphological change that inverted phase contrast microscope is observed vascular endothelial cell down in the incubator.(seeing accompanying drawing 1)
The human umbilical artery vascular smooth muscle cell is inoculated in the 24 porocyte culture plates, normal group is set: under the normal condition that contains serum and somatomedin, cultivate; Solvent control group: under the condition that contains serum and somatomedin, add dimethyl sulfoxide (DMSO) and cultivate; Chloro-quinine processed group: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and cultivate; 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group: contain 3-benzyl-5-(2-nitro the Phenoxymethyl)-gamma-butyrolacton that adds 120 μ M under the condition of serum and somatomedin and cultivate; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation of 120 μ M.37 ℃, CO
2After cultivating 24 hours in the incubator, inverted phase contrast microscope is observed the morphological change of vascular smooth muscle cell down.(seeing accompanying drawing 2)
The result shows: with concentration is 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton of 120 μ M, 180 μ M, can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement.
2, through the variation of acridine orange dyeing with acid film bubble in the fluorescence microscope Cytoplasm:
Human umbilical vein endothelial cell is inoculated in the 24 porocyte culture plates, matched group is set: cultivate containing under the normal condition of culture of serum and somatomedin; The chloro-quinine processed group: the chloro-quinine that adds 8 μ M under the condition of culture that contains serum and somatomedin is cultivated; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 8 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation of 120 μ M.37 ℃, CO
2After cultivating 6 hours in the incubator, through acridine orange dyeing two minutes, inhale and abandon acridine orange, acid film bubble is dyed orange red, observes the variation of acid film bubble under fluorescence microscope.(seeing accompanying drawing 3)
The human umbilical artery vascular smooth muscle cell is inoculated in the 24 porocyte culture plates, normal group is set: under the normal condition that contains serum and somatomedin, cultivate; Solvent control group: under the condition that contains serum and somatomedin, add dimethyl sulfoxide (DMSO) and cultivate; Chloro-quinine processed group: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and cultivate; 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton processed group: contain 3-benzyl-5-(2-nitro the Phenoxymethyl)-gamma-butyrolacton that adds 120 μ M under the condition of serum and somatomedin and cultivate; Chloro-quinine and 3-benzyl-5-(2-nitro Phenoxymethyl)-common processed group of gamma-butyrolacton: contain the chloro-quinine that adds 16 μ M under the condition of serum and somatomedin and 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton co-cultivation of 120 μ M.37 ℃, CO
2After cultivating 24 hours in the incubator, through acridine orange dyeing two minutes, inhale and abandon acridine orange, acid film bubble is dyed orange red, observes the variation of acid film bubble under fluorescence microscope.(seeing accompanying drawing 4)
The result shows: 3-benzyl-5-(2 nitro Phenoxymethyl)-gamma-butyrolacton can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement.
3, dimethyl diaminophenazine chloride dyeing detects the variation of acid compartment cumulative volume in the cell:
Dimethyl diaminophenazine chloride dyes through being usually used in lysosome, and is used for the acid compartment cumulative volume of quantitative assay cell.
The exponential phase vascular endothelial cell is inoculated in the culture dish of diameter 6cm, the old culture fluid of sucking-off after 24 hours, packet transaction is after 24 hours, and the sucking-off culture fluid is washed ware 2 times with PBS, each 1 minute.(0.5% dimethyl diaminophenazine chloride storage liquid is diluted in 1 * PBS) for 10 times, places incubator to hatch 4 minutes to add the dimethyl diaminophenazine chloride Incubating Solution.The sucking-off Incubating Solution is washed 3 times with PBS, each 1 minute.Add sour ethanol (containing 50% ethanol, 1% acetic acid, 49% water) 3ml, extract the dimethyl diaminophenazine chloride that the acid film bubble of cell is absorbed.The sour alcoholic solution of dimethyl diaminophenazine chloride is surveyed the OD value of its 540nm wavelength on Cintra 5UV-visibleSpectrometer spectrophotometer.VAC with matched group is 100%, the relative VAC of experiment with computing group, and formula is as follows: the relative VAC=(A of experimental group
The 540nm experimental group/ experimental group protein concentration)/(A
540nm Matched group/ matched group protein concentration) * 100%.(seeing accompanying drawing 5)
The exponential phase vascular smooth muscle cell is inoculated in the culture dish of diameter 6cm, the old culture fluid of sucking-off after 24 hours, packet transaction is after 24 hours, and the sucking-off culture fluid is washed ware 2 times with PBS, each 1 minute.(0.5% dimethyl diaminophenazine chloride storage liquid is diluted in 1 * PBS) for 10 times, places incubator to hatch 4 minutes to add the dimethyl diaminophenazine chloride Incubating Solution.The sucking-off Incubating Solution is washed 3 times with PBS, each 1 minute.Add sour ethanol (containing 50% ethanol, 1% acetic acid, 49% water) 3ml, extract the dimethyl diaminophenazine chloride that the acid film bubble of cell is absorbed.The sour alcoholic solution of dimethyl diaminophenazine chloride is surveyed the OD value of its 540nm wavelength on Cintra 5UV-visibleSpectrometer spectrophotometer.VAC with matched group is 100%, the relative VAC of experiment with computing group, and formula is as follows: the relative VAC=(A of experimental group
540 experimental grouies/ experimental group protein concentration)/(A
540nm Matched group/ matched group protein concentration) * 100%.(seeing accompanying drawing 6)
The result shows: 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton can significantly suppress the long-pending increase of acid film foam that chloro-quinine causes.
4, Na
+K
+-atpase activity detects:
Vascular endothelial cell is inoculated in the culture dish of diameter 10cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, peptic cell is respectively organized cellular enzymes liquid through the ultrasonication extraction, passes through Na
+K
+-ATP enzyme reagent kit detects respectively organizes Na
+K
+-atpase activity.(seeing accompanying drawing 7).
Vascular smooth muscle cell is inoculated in the culture dish of diameter 10cm packet transaction cell, 37 ℃, CO
2After cultivating 24 hours in the incubator, peptic cell is respectively organized cellular enzymes liquid through the ultrasonication extraction, passes through Na
+K
+-ATP enzyme reagent kit detects respectively organizes Na
+K
+-atpase activity.(seeing accompanying drawing 8)
The result shows: 3-benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton, can effectively suppress the vacuolation of the vascular endothelial cell and the smooth muscle cell of chloro-quinine inducement, and suppress the Na that chloro-quinine causes
+K
+-atpase activity rising phenomenon.
Claims (2)
1.3-the application of benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton in the Cytoplasm vacuolation medicine that preparation inhibition chloro-quinine inducement vascular endothelial cell produces.
2.3-the application of benzyl-5-(2-nitro Phenoxymethyl)-gamma-butyrolacton in the Cytoplasm vacuolation medicine that preparation inhibition chloro-quinine inducement vascular smooth muscle cell produces.
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| CN1857257A (en) * | 2006-03-21 | 2006-11-08 | 山东大学 | Application of 3-benzyl-5-(2-nitrophenoxymethyl)-gamma-butyrolactone in preparing medicine for inhibiting vascular endothelial senility and apoptosis |
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| CN1857257A (en) * | 2006-03-21 | 2006-11-08 | 山东大学 | Application of 3-benzyl-5-(2-nitrophenoxymethyl)-gamma-butyrolactone in preparing medicine for inhibiting vascular endothelial senility and apoptosis |
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