CN111759832B - Application of ginkgolic acid in preparation of medicine for treating vascular restenosis disease - Google Patents

Application of ginkgolic acid in preparation of medicine for treating vascular restenosis disease Download PDF

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CN111759832B
CN111759832B CN202010722910.6A CN202010722910A CN111759832B CN 111759832 B CN111759832 B CN 111759832B CN 202010722910 A CN202010722910 A CN 202010722910A CN 111759832 B CN111759832 B CN 111759832B
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ginkgolic acid
drying
etoh
water content
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CN111759832A (en
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姚文娟
梁秀瑛
祁妍
陈云
刘兆国
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Nantong University
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • A61K2236/30—Extraction of the material
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Abstract

The invention discloses an application of ginkgolic acid in preparation and treatment of a medicament for treating vascular restenosis disease, wherein ginkgo episperm is peeled, the first drying temperature is 50 ℃, the drying time is 50min, the water content is reduced to 20%, the second drying temperature is 65 ℃, the drying time is 45min, the water content after drying is 6-10%, the ginkgo episperm is crushed into fine particles with the particle diameter of 0.1-0.5 mm, 80% EtOH is extracted for 3 times under reflux at 70 ℃ to obtain a crude extract, the crude extract is dissolved into a saturated solution by 96% EtOH, the saturated solution is filtered by macroporous resin, and the 20%, 40%, 80% and 95% EtOH are used for eluting sequentially; evaporating and drying the eluate until the water content is 1-2% to obtain ginkgolic acid C17:1 final product.

Description

Application of ginkgolic acid in preparation of medicine for treating vascular restenosis disease
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of ginkgolic acid in preparation of a medicine for treating vascular restenosis diseases.
Background
Vascular remodeling is considered to be the pathophysiological basis for cardiovascular diseases such as hypertension, atherosclerosis, and restenosis. Vascular Smooth Muscle Cells (VSMCs) are the major cellular components of arteries and are also key determinants of vascular disease. VSMCs can respond to various stimuli to retain phenotypic changes from a differentiated phenotype (contractile phenotype) to a dedifferentiated phenotype (synthetic phenotype). In many vascular pathologies, VSMCs undergo a rapid reversible change from a quiescent contractile phenotype to a proliferative and secretory phenotype characterized by increased proliferation, migration, and extracellular matrix (ECM) production. This synthetic VSMC phenotype is closely associated with cardiovascular disease, including atherosclerosis and hypertension. Therefore, the excessive proliferation and migration of VSMC play an important role in the development of cardiovascular diseases (such as hypertension). Although clinical antihypertensive or heart failure drugs ACEI (angiotensin converting enzyme inhibitor) can reverse vascular or myocardial hypertrophy, at present, no drug specially aiming at vascular remodeling exists, so that the drugs for vascular remodeling can be developed or enriched to effectively protect target organs.
Rho-associated coiled-coil protein kinase (ROCK) is an effector of the small GTP-binding protein Rho. ROCK plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and migration, cell division and gene expression. ROCK activation is involved in many cardiovascular diseases, such as hypertension and atherosclerosis. Clinically, the ROCK inhibitor fasudil shows satisfactory effects on aspects of controlling blood pressure, regulating arteriosclerotic coronary artery diseases, treating heart failure and the like, but the clinically available ROCK inhibitors have fewer varieties, especially selective ROCK inhibitors.
The ginkgo is a plant fruit with very rich nutritive value, contains ginkgolic acid, ginkgol, reducing sugar, riboflavin and other components, can nourish the human body, and has very high edible value, medicinal value and health care value. The content of ginkgolic acid, bilobalide and other substances in the ginkgo episperm is 2-3 times that in ginkgo fruit or ginkgo leaf. Has good effects on preventing and treating hyperlipemia and cardiovascular and cerebrovascular diseases, and can be used for preventing cardiovascular and cerebrovascular diseases. At present, the research on the effective ingredients and the treatment efficacy of the traditional Chinese medicine is rarely reported.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems, if no medicine specially aiming at vascular remodeling exists at present, the variety of clinically available ROCK inhibitors is less, and the invention aims to provide the application of ginkgolic acid in preparing and treating the medicines for treating vascular restenosis diseases. Provides a new medicinal application of ginkgolic acid in ginkgo episperm, in particular to an application of ginkgolic acid (C17:1) in preparing a medicament for treating vascular restenosis diseases. The ginkgolic acid (C17:1) can inhibit the proliferation and migration capacity of VSMC by inhibiting ROCK activation, and can be used for preparing medicines for treating diseases such as hypertension vestibular stenosis, atherosclerotic plaque, vascular restenosis caused by surgery or trauma, and the like.
The technical scheme is as follows:
application of ginkgolic acid in preparation of medicine for treating vascular restenosis disease, wherein the structural formula of ginkgolic acid is shown in the specification
Figure 759701DEST_PATH_IMAGE001
Molecular formula C 24 H 38 O 3 And the molecular weight is 374.5612.
Further, the preparation method of the ginkgolic acid comprises the following steps: peeling off the ginkgo episperm, wherein the first drying temperature is 50 ℃, the drying time is 50min, the water content is reduced to 20%, the second drying temperature is 65 ℃, the drying time is 45min, the dried water content is 6-10%, the ginkgo episperm is crushed into fine particles with the particle diameter of 0.1-0.5 mm, 80% EtOH is refluxed and extracted for 3 times at 70 ℃ to obtain a crude extract, the crude extract is dissolved into a saturated solution by 96% EtOH, the saturated solution is filtered by macroporous resin, and the 20%, 40%, 80% and 95% EtOH are sequentially used for elution; evaporating and drying the eluate until the water content is 1-2% to obtain ginkgolic acid C17:1 final product.
Further, the experimental steps of the ginkgolic acid for inhibiting VSMC proliferation and migration in vitro are as follows:
the first step is as follows: constructing an aorta VSMC proliferation and migration model;
the second step is that: aortic VSMC was pretreated with 0, 20, 40, 60, 100 μ M ginkgolic acid for 6h, PDGF-BB was used to stimulate cell proliferation and migration, EdU was used to detect cell proliferation, and Transwell was used to detect cell migration.
Further, aortic VSMC were stored in the laboratory and treated with 10 ng/ml PDGF-BB for 24 h to construct a proliferative migration model.
Further, ginkgolic acids inhibit ROCK activity: respectively treating aorta VSMC of over-expression ROCK with 0, 20, 40, 60 and 100 μ M ginkgolic acid, and detecting ROCK activation by Western blot method; ROCK activity is manifested by the detection of phosphorylated myosin phosphatase targeting subunit 1 (MYPT 1).
Further, ginkgolic acid inhibits vascular restenosis in rats: a rat vascular restenosis model was constructed, the drug-treated group was given 10, 30, 60, 100mg/kg intragastrically to rats daily, the left common carotid artery was isolated after 14 days, fixed with 4% paraformaldehyde, OCT-embedded, cryosectioned at a thickness of 5 μm with a cryomicrotome, and then observed for vascular morphology by HE staining.
Further, the EdU detection cell proliferation step is as follows:
first, EdU labeling: complete cell culture media were used at 1000: 1, preparing 50 mu M EdU culture medium;
the second step is that: adding 200 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and removing the culture medium;
the third step: washing the cells with PBS for 1-2 times, 5 minutes each time;
fourthly, cell immobilization: adding 200 mu L of cell fixing solution into each hole, incubating for 30 minutes at room temperature, wherein the cell fixing solution is PBS containing 4% paraformaldehyde, and removing the fixing solution;
the fifth step: adding 200 mu L of 2 mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution;
and a sixth step: adding 200 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;
the seventh step: adding 200 mu L of penetrant into each hole, incubating for 10 minutes by a decoloring shaking table, washing for 1 time by PBS and washing for 5 minutes, wherein the penetrant is 0.5 percent TritonX-100 PBS;
eighth step, Apollo dyeing red: adding 200 mu L of 1 XApollo dyeing reaction liquid into each hole, incubating for 30 minutes in a light-proof, room temperature and decoloring shaking table, and discarding the dyeing reaction liquid;
the ninth step: adding 200 mu L of penetrant, decoloring and shaking the mixture for 2-3 times, and washing the mixture for 10 minutes each time, and discarding the penetrant, wherein the penetrant is 0.5% TritonX-100 PBS;
the tenth step: adding 200 mu L of methanol into each hole for washing for 1-2 times, 5 minutes each time, then washing for 1 time by PBS, and washing for 5 minutes;
the tenth step, DNA staining blue: deionized water was added according to a 100: 1, diluting Hoechst33342, preparing a proper amount of 1 Xhoechst 33342 reaction solution, and storing in dark place;
the twelfth step: adding 200 mu L of 1 × Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the dyeing reaction solution;
the thirteenth step: adding 200 mu L PBS into each hole for washing for 1-3 times;
the fourteenth step is that: adding 100 mu L PBS into each hole for storage;
the fifteenth step: and acquiring and analyzing pictures.
Further, the Transwell detection cell migration step is as follows:
the first step is as follows: adding 200 μ L of pre-warmed medium into upper chamber of transwell on super clean bench, standing at room temperature for 20min, and discarding;
the second step is that: resuspending the cells in 0.5% BSA, adding to the upper chamber of a transwell, adding to the lower layer 10% FBS, and standing at 37 deg.C and 5% CO 2 Adherent growth in a 95% air cell culture box;
the third step: dripping 10 mu g/mL MMC for 1 h;
the fourth step: according to the non-added medicine group; 10 ng/ml PDGF-BB treatment group; 20 μ M GA +10 ng/ml PDGF-BB treated group; 40 μ M GA +10 ng/ml PDGF-BB treated group; 20 μ M GA +60 ng/ml PDGF-BB treated group; adding corresponding drugs into a 100 mu M GA +10 ng/ml PDGF-BB treatment group for continuous culture;
the fifth step: taking out the transwell chamber, discarding the culture medium, washing once with PBS and discarding, and wiping the upper layer of non-migrated cells with a cotton swab;
and a sixth step: after fixing with 4% paraformaldehyde for 30min, washing with PBS for three times;
the seventh step: after the crystal violet staining solution is stained for 15min, PBS is used for washing for three times;
eighth step: the photographs were observed under an inverted microscope in 5 fields selected at random per well.
Further, the ROCK activity detection comprises the following specific steps:
the first step is as follows: cleaning the mould for preparing the glue, and putting the mould into an oven for drying for later use;
the second step is that: pouring separation glue from one end of the glass plate by using a liquid transfer device, and sealing by using double-distilled water;
the third step: standing at room temperature for 40min, separating glue and water to form a line, solidifying, removing upper water layer, and drying with filter paper;
the fourth step: pouring concentrated glue between the glass plates, inserting a Telfon comb, and standing at room temperature for more than or equal to 30 min;
the fifth step: assembling a glass plate, fixing an electrophoresis tank, filling electrophoresis liquid into an inner tank to drive bubbles in the tank, pulling out a Telfon comb, loading the sample according to the required protein amount, and adding the electrophoresis liquid into an outer tank to a half position;
sixth step, running electrophoresis: firstly, keeping the constant voltage at 80V, keeping the constant voltage at 100V until the bottom of the separation gel when the separation gel runs to the position of the separation gel, and turning off the power supply;
the seventh step: preparing sponge, filter paper and NC membrane in advance, soaking in buffer solution, and precooling in a refrigerator at 4 ℃;
eighth step, film transfer: cutting separation gel, assembling in the order of white board-sponge-filter paper-NC membrane-gel-filter paper-sponge-blackboard, loading into a tank, rotating the membrane for 90min at 260mA, performing ice bath in the whole process, and cutting corresponding membrane strips after the membrane rotation is finished;
ninth, sealing: 5% skim milk was prepared in advance, and the membrane was placed in it and sealed at room temperature for 2 h. Washing the membrane with 1 × TBST for three times (5 min each time) after sealing;
tenth step, primary antibody incubation: putting the protein band into corresponding diluted MYPT-1 and phor-MYPT-1 primary antibody solution, incubating overnight on a shaking table at 4 ℃, and washing the membrane for three times by 1 × TBST after finishing, wherein each time is 5 min;
tenth step, secondary antibody incubation: diluting the secondary antibody with 1% skimmed milk, placing the membrane in the secondary antibody, incubating for 2h in a shaking table at room temperature, and washing the membrane with 1 × TBST for three times, each time for 5 min;
the twelfth step, development: soaking the film in the developer solution, and developing in a BIO-RAD scanner.
Further, the ginkgolic acid experiment for inhibiting rat vascular restenosis specifically comprises the following steps:
step one, constructing a vascular restenosis model, wherein the operation equipment comprises the following steps: 2F Forgaty balloon catheter, a bent disc, a 2mL sterile syringe, a shaver, a scalpel, tissue forceps, ophthalmological forceps, hemostatic forceps, a miniature drag hook, an artery clamp, a triangular suture needle, a needle holder, a No. 7 suture thread, normal saline, a sterile cotton ball and sterile gauze;
step two, the operation method comprises the following steps: injecting 4% pentobarbital sodium solution into abdominal cavity at a ratio of 1mL/kg for anesthesia, shaving hair at the operation position by a hair shaver, sterilizing neck skin by using 2% iodine tincture, deiodinating by using 75% alcohol, making an incision of about 1-1.5 cm in the middle of the neck, separating a left common carotid artery by about 1cm, ligating a far-center end, clamping and closing a blood vessel at the near heart end by an artery clamp, cutting a small opening at the ligation position near the far-center end, reversely introducing a 2F balloon catheter to an aortic arch, filling the balloon, pulling back to the position near the incision, evacuating the gas in the balloon, repeatedly performing 3 times to achieve the purpose of injury, withdrawing the balloon catheter, ligating the left common carotid artery, and suturing the skin;
step three, specimen acquisition: anesthetizing the experimental animal 14 days after molding, separating the left common carotid artery, cutting a carotid artery injury section of 0.9-1.1cm for each, cleaning the blood vessel with precooled normal saline, and storing in a refrigerator at-80 ℃;
step four, HE staining: the frozen section is stained with hematoxylin for 5min, washed with double distilled water for 1 min, differentiated with hydrochloric acid for 30 s, stained with eosin staining solution for 2 min, dehydrated with ethanol, and then transparent cut with xylene, and photographed by microscope observation.
Has the advantages that:
1. provides a new medicinal application of ginkgolic acid in ginkgo episperm, in particular to an application of ginkgolic acid (C17:1) in preparing a medicament for treating vascular restenosis diseases. The ginkgolic acid (C17:1) can inhibit the proliferation and migration capability of VSMC by inhibiting ROCK activation, and can be used for preparing medicines for treating diseases such as hypertension and vestibular stenosis, atherosclerotic plaque, vascular restenosis caused by operation or trauma, etc.
2. 60, 100 μ M Ginkgolic Acid (GA) significantly inhibited VSMC proliferation.
3. 60, 100 μ M Ginkgolic Acid (GA) significantly inhibited VSMC migration.
4. 40, 60 and 100 mu M Ginkgolic Acid (GA) can obviously inhibit MYPT1 phosphorylation, namely ROCK activation.
Drawings
FIG. 1 is a graph of ginkgolic acid inhibiting VSMC proliferation and migration in vitro;
FIG. 2 is a graph of the inhibition of VSMC migration by ginkgolic acid in vitro inhibition of VSMC proliferation and migration;
FIG. 3 is a graph of ginkgolic acid inhibiting ROCK activity;
FIG. 4 is a graph of ginkgolic acids inhibiting vascular restenosis in rats.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The invention is described in further detail below with reference to specific examples and with reference to data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1
Application of ginkgolic acid in preparation of medicine for treating vascular restenosis disease, wherein the structural formula of ginkgolic acid is shown in the specification
Figure 270317DEST_PATH_IMAGE001
Molecular formula C 24 H 38 O 3 And the molecular weight is 374.5612.
The preparation method of the ginkgolic acid comprises the following steps: peeling off the ginkgo episperm, wherein the first drying temperature is 50 ℃, the drying time is 50min, the water content is reduced to 20%, the second drying temperature is 65 ℃, the drying time is 45min, the dried water content is 6-10%, the ginkgo episperm is crushed into fine particles with the particle diameter of 0.1-0.5 mm, 80% EtOH is refluxed and extracted for 3 times at 70 ℃ to obtain a crude extract, the crude extract is dissolved into a saturated solution by 96% EtOH, the saturated solution is filtered by macroporous resin, and the 20%, 40%, 80% and 95% EtOH are sequentially used for elution; evaporating and drying the eluate until the water content is 1-2% to obtain ginkgolic acid C17:1 final product.
The experimental steps of the ginkgolic acid in vitro VSMC proliferation and migration inhibition are as follows:
the first step is as follows: constructing an aorta VSMC proliferation and migration model, storing the aorta VSMC in a laboratory, treating the aorta VSMC with 10 ng/ml PDGF-BB for 24 h, and constructing a proliferation and migration model;
the second step is that: aortic VSMC was pretreated with 0, 20, 40, 60, 100 μ M ginkgolic acid for 6h, PDGF-BB was used to stimulate cell proliferation and migration, EdU was used to detect cell proliferation, and Transwell was used to detect cell migration.
The method for detecting the cell proliferation by the EdU comprises the following steps:
first, EdU labeling: cell-free medium was used at a volume ratio of 1000: 1 (reagent A) to prepare 50. mu.M EdU medium (ready to use);
the second step is that: adding 200 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and removing the culture medium;
the third step: the cells were washed 1-2 times with PBS for 5 minutes each time. The cleaning purpose is to elute the EdU which is not doped with DNA, the cleaning mode is determined according to different cell types, and the cleaning strength is reduced for cells which are not firmly adhered to the wall;
fourthly, cell immobilization: adding 200 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30min, and discarding the fixing solution;
the fifth step: adding 200 mu L of 2 mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution;
and a sixth step: adding 200 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;
the seventh step: adding 200 μ L of penetrant (0.5% TritonX-100 PBS) per well, decolorizing and shaking for 10 min; washing with PBS for 1 time and 5 minutes;
eighth step, Apollo staining (red): adding 200 mu L of 1 XApollo dyeing reaction liquid into each hole, incubating for 30 minutes in a light-proof, room temperature and decoloring shaking table, and discarding the dyeing reaction liquid;
the ninth step: adding 200 mu L of penetrating agent (PBS of 0.5 percent TritonX-100) to decolor and wash by a shaking table for 2-3 times, each time for 10 minutes, and discarding the penetrating agent;
the tenth step: adding 200 mu L of methanol into each hole for cleaning for 1-2 times, and each time for 5 minutes; PBS washing for 5 minutes for 1 time;
tenth step, DNA staining (blue): deionized water was added according to a 100: 1, diluting Hoechst33342, preparing a proper amount of 1 Xhoechst 33342 reaction solution, and storing in dark place;
the twelfth step: adding 200 mu L of 1 × Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the dyeing reaction solution;
and a thirteenth step of: adding 200 mu L PBS into each hole for washing for 1-3 times;
the fourteenth step is that: adding 100 mu L PBS into each hole for storage;
the fifteenth step: and acquiring and analyzing pictures.
As shown in fig. 1, 60, 100 μ M Ginkgolic Acid (GA) significantly inhibited VSMC proliferation.
The Apollo dyeing reaction liquid is prepared in the following sequence:
Figure DEST_PATH_IMAGE003
the Transwell detection cell migration step comprises the following steps:
the first step is as follows: adding 200 μ L of pre-warmed medium into upper chamber of transwell on super clean bench, standing at room temperature for 20min, and discarding;
the second step is that: resuspending the cells in 0.5% BSA, adding to the upper chamber of the transwell, adding to the lower layer 10% FBS (note that no air bubbles can be present between the lower layer and the chamber), and standing at 37 deg.C with 5% CO 2 Adherent growth in a 95% air cell culture box;
the third step: dropwise adding 10 mu g/mL MMC to act for 1h to remove the influence of cell proliferation on the experiment;
the fourth step: according to the non-added medicine group; 10 ng/ml PDGF-BB treatment group; 20 μ M GA +10 ng/ml PDGF-BB treated group; 40 μ M GA +10 ng/ml PDGF-BB treated group; 20 μ M GA +60 ng/ml PDGF-BB treated group; adding corresponding drugs into a 100 mu M GA +10 ng/ml PDGF-BB treatment group for continuous culture;
the fifth step: taking out the transwell chamber, discarding the culture medium, washing once with PBS and discarding, and lightly wiping the upper layer of non-migrated cells with a cotton swab;
and a sixth step: after fixing with 4% paraformaldehyde for 30min, washing with PBS for three times;
the seventh step: after the crystal violet dye solution is dyed for 15min, washing for three times by PBS;
eighth step: the photographs were observed under an inverted microscope in 5 fields selected at random per well.
As shown in fig. 2, 60, 100 μ M Ginkgolic Acid (GA) significantly inhibited VSMC migration.
Ginkgolic acid inhibits ROCK activity: respectively treating aorta VSMC of over-expression ROCK with 0, 20, 40, 60 and 100 μ M ginkgolic acid, and detecting ROCK activation by Western blot method; the ROCK activity is embodied by detecting phosphorylated myosin phosphatase targeting subunit 1 (MYPT 1), and the ROCK activity detection comprises the following specific steps:
the first step is as follows: cleaning a mould for preparing the glue, and putting the mould into an oven for drying for later use;
the second step is that: preparing separation glue according to the specification, pouring the glue (adding the glue from one end of the glass plate by using a liquid transfer device), and slowly sealing the glue by using double distilled water to prevent air bubbles from entering;
the third step: standing at room temperature for 40min, separating glue and water to form a line, solidifying, removing upper water layer, and drying with filter paper;
the fourth step: preparing concentrated glue, pouring the concentrated glue between glass plates, quickly inserting a Telfon comb, taking notice that bubbles cannot be generated, and standing at room temperature for at least 30 min;
the fifth step: assembling glass plates, fixing an electrophoresis tank, pouring the inner tank with electrophoresis liquid to remove bubbles in the tank, pulling out a Telfon comb, slowly loading the sample according to the required protein amount, and adding the electrophoresis liquid in the outer tank to a half part;
sixth step, running electrophoresis: firstly, keeping the constant voltage at 80V, keeping the constant voltage at 100V until the bottom of the separation gel when the separation gel runs to the position of the separation gel, and turning off a power supply;
the seventh step: preparing sponge, filter paper and NC membrane in advance, soaking in buffer solution, and precooling in a refrigerator at 4 ℃;
eighth step, film transfer: cutting separation gel with corresponding molecular weight according to requirements, assembling the separation gel in the order of white board-sponge-filter paper-NC membrane-gel-filter paper-sponge-blackboard, loading the separation gel into a tank, rotating the membrane for 90min at 260mA, carrying out ice bath in the whole process, and cutting corresponding membrane strips after the membrane rotation is finished;
ninth, sealing: 5% skim milk was prepared in advance, and the membrane was placed in it and sealed at room temperature for 2 h. Washing the membrane with 1 × TBST for three times (5 min each time) after sealing;
tenth step, primary antibody incubation: putting the protein band into correspondingly diluted MYPT-1 and phor-MYPT-1 primary antibody solution, incubating overnight on a shaking table at 4 ℃, and after finishing, washing the membrane by 1 × TBST for three times, wherein each time is 5 min;
tenth step, secondary antibody incubation: diluting the secondary antibody with 1% skimmed milk, placing the membrane in the secondary antibody, incubating for 2h in a shaking table at room temperature, and washing the membrane with 1 × TBST for three times, each time for 5 min;
the twelfth step, development: soaking the film in a developing solution, and developing in a BIO-RAD scanner.
As shown in fig. 3, 40, 60, 100 μ M Ginkgolic Acid (GA) was able to significantly inhibit MYPT1 phosphorylation, i.e., ROCK activation.
Ginkgolic acid inhibits vascular restenosis in rats: a rat vascular restenosis model was constructed, and the drug-treated group was gavaged daily to rats at 10, 30, 60, 100mg/kg, and 14 days later the left common carotid artery was isolated and fixed with 4% paraformaldehyde. OCT embedding was carried out, cryo-sectioning was carried out with a cryomicrotome at a thickness of 5 μm, and then the morphology of the blood vessels was observed by HE staining. The method comprises the following specific steps:
step one, constructing a vascular restenosis model, wherein the operation equipment comprises the following steps: 2F Forgaty balloon catheter, a bent disc, a 2mL sterile syringe, a shaver, a scalpel, tissue forceps, ophthalmological forceps, hemostatic forceps, a miniature drag hook, an artery clamp, a triangular suture needle, a needle holder, a No. 7 suture thread, normal saline, a sterile cotton ball and sterile gauze;
step two, the operation method comprises the following steps: injecting 4% sodium pentobarbital solution into abdominal cavity at a ratio of 1mL/kg for anesthesia, shaving hair at the operation position by a hair shaver, sterilizing neck skin by using 2% iodine tincture, deiodinating by using 75% alcohol, making an incision of about 1-1.5 cm in the center of the neck, separating a left common carotid artery by about 1cm, ligating a telecentric end, clamping a blood vessel at the proximal cardiac end by an artery clamp, cutting a small opening at the ligation position close to the telecentric end, reversely guiding a 2F balloon catheter to an aortic arch, filling the balloon, slowly pulling back to the proximal incision position, evacuating the gas in the balloon, and repeating the steps for 3 times to achieve the purpose of injury. Slowly withdrawing the balloon catheter, ligating the left common carotid artery, and suturing the skin;
step three, specimen acquisition: anesthetizing the experimental animal 14 days after molding, quickly separating the left common carotid artery, cutting a 1cm left and right carotid artery injury section for each animal, cleaning the blood vessel by using precooled normal saline, and storing in a refrigerator at the temperature of minus 80 ℃;
step four, HE staining: the frozen sections were stained with hematoxylin for 5min, washed with double distilled water for 1 min, differentiated with hydrochloric acid for 30 s, and stained with eosin staining solution for 2 min. The slices were dehydrated with ethanol, then transparent sliced with xylene and photographed by microscopic observation.
As shown in FIG. 4, the 60, 100mg/kg Ginkgolic Acid (GA) gavage treatment significantly inhibited intimal thickening and stenosis of the vessels.

Claims (2)

1. The application of ginkgolic acid as a unique active ingredient in preparing a medicament for treating vascular restenosis is characterized in that: the ginkgolic acid has a structural formula of
Figure DEST_PATH_IMAGE001
Molecular formula C 24 H 38 O 3 Molecular weight is 374.5612; the vascular restenosis is particularly caused by atherosclerotic plaque and surgery or trauma.
2. Use of ginkgolic acids as the sole active ingredient in the preparation of a medicament for the treatment of restenosis according to claim 1, characterized in that: the preparation method of the ginkgoic acid comprises the following steps: peeling off the ginkgo episperm, wherein the first drying temperature is 50 ℃, the drying time is 50min, the water content is reduced to 20%, the second drying temperature is 65 ℃, the drying time is 45min, the dried water content is 6-10%, the ginkgo episperm is crushed into fine particles with the particle diameter of 0.1-0.5 mm, 80% EtOH is refluxed and extracted for 3 times at 70 ℃ to obtain a crude extract, the crude extract is dissolved into a saturated solution by 96% EtOH, the saturated solution is filtered by macroporous resin, and the 20%, 40%, 80% and 95% EtOH are sequentially used for elution; evaporating and drying the eluate until the water content is 1-2% to obtain ginkgolic acid C17:1 final product.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101595983A (en) * 2009-01-15 2009-12-09 孔赟荣 A kind of preparation method of gingko healthy tea

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101595983A (en) * 2009-01-15 2009-12-09 孔赟荣 A kind of preparation method of gingko healthy tea

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* Cited by examiner, † Cited by third party
Title
Egb诱导体外培养的胎儿血管平滑肌细胞凋亡的实验研究;梁新剑;《中国知网》;20050115(第1期);第1、32-39页 *

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