CN116549433A - Application of luteolin in preparation of platelet glycoprotein VI inhibitor - Google Patents

Application of luteolin in preparation of platelet glycoprotein VI inhibitor Download PDF

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Publication number
CN116549433A
CN116549433A CN202310385361.1A CN202310385361A CN116549433A CN 116549433 A CN116549433 A CN 116549433A CN 202310385361 A CN202310385361 A CN 202310385361A CN 116549433 A CN116549433 A CN 116549433A
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luteolin
gpvi
platelet
inhibitor
platelet aggregation
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孟照辉
叶雨佳
杨理宏
冷敏
孙贵虎
杨晓娜
华欢
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First Affiliated Hospital of Kunming Medical University
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First Affiliated Hospital of Kunming Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention provides application of luteolin in preparation of a platelet glycoprotein VI inhibitor, and belongs to the technical field of medical biology. The luteolin anti-platelet is an anti-GPVI specific activator induced platelet aggregation reaction, and the result of optical turbidimetry detection shows that the luteolin has a remarkable inhibition effect on the GPVI specific activator induced human platelet aggregation. The interaction of luteolin and GPVI shows that the luteolin directly interacts with GPVI through a surface plasmon resonance detection result. Luteolin can be used for not only the development of GPVI inhibitors and medicines, but also monitoring the existing antiplatelet therapy.

Description

Application of luteolin in preparation of platelet glycoprotein VI inhibitor
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to application of luteolin in preparation of a platelet glycoprotein VI inhibitor.
Technical Field
Platelets play an important role in arterial thrombosis, and classical antiplatelet aspirin and novel drug which blocks alpha IIb beta 3 from combining with fibrinogen, such as Acximab, play an important clinical treatment role, but potential risks such as gastrointestinal reaction and hemorrhage exist. The development of antiplatelet medicines with better safety and effectiveness has important clinical and social values.
Vascular endothelial cells are damaged or plaque ruptured and subendothelial collagen exposure initiates acute thrombosis. Collagen, together with platelet surface glycoprotein VI (GPVI) and integrin- α2β1, activates the integrin αiibβ3 activation pathway, causing platelet activation and aggregation. The activated substances such as thrombin released in the platelet activation process further promote platelet aggregation, and finally lead to thrombus formation. The currently reported small molecule inhibitors of GPVI are few and none are in clinical use.
Luteolin is a major component of flavonoids in erigeron breviscapus, and is also widely found in vegetables, fruits and herbs. Many researches show that luteolin has anti-inflammatory and anti-oxidative stress activities and plays a positive role in treating cardiovascular diseases such as coronary heart disease, diabetes and the like. Although the prior art reports the antiplatelet and antithrombotic functions of luteolin, the activity of luteolin in a platelet glycoprotein VI inhibitor is not reported.
The invention comprises the following steps:
the invention aims to provide application of luteolin in preparation of platelet glycoprotein VI inhibitor. The luteolin related by the invention is a main component of flavonoids in erigeron breviscapus, and also widely exists in vegetables, fruits and Chinese herbal medicines. Experiments show that luteolin selectively resists platelet aggregation induced by GPVI specific inhibitors. Thus, luteolin can be used in antiplatelet drugs. Luteolin may also be used in the preparation of GPVI specific inhibitors in combination with a composition of one or more pharmaceutically acceptable carriers.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
application of luteolin in preparing platelet glycoprotein VI inhibitor is provided.
According to the application of luteolin in preparing platelet glycoprotein VI inhibitor, wherein the luteolin interacts with GPVI to form K D =4.13μM。
The application of luteolin in preparing antiplatelet medicines, wherein the antiplatelet effect of luteolin is to achieve the antiplatelet aggregation effect by the antiplatelet aggregation effect induced by the GPVI specific inhibitor.
The application of luteolin in preparing antiplatelet medicines, wherein the antiplatelet effect of the luteolin is platelet aggregation induced by an antiplatelet GPVI specific inhibitor, and the inhibition efficiency is dose-dependent, so that the antiplatelet aggregation effect is achieved.
Application of luteolin in preparing anti-platelet medicine for inhibiting platelet aggregation induced by GPVI specific activator is provided.
According to the application of the luteolin in preparing a platelet glycoprotein VI inhibitor, the luteolin has the effects of resisting platelet aggregation induced by a GPVI specific inhibitor, and the inhibition efficiency is dose-dependent.
When the luteolin is used as a medicament, the luteolin can be directly used or used in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99% of luteolin compound, preferably 0.5-90% of luteolin as effective component, and pharmaceutically acceptable adjuvants or carriers for human and animals. The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example by mixing the active ingredient with one or more carriers and then forming it into the desired dosage form.
The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, for example: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate and magnesium stearate, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
Compared with the prior art, the invention has the advantages that:
1. the invention discovers a new medical application for luteolin, the clinical existing medicine does not have an inhibitor of small molecular platelet membrane glycoprotein VI, the luteolin can be used as the inhibitor of the target point, and a new application field is developed.
2. The luteolin product of the invention has rich source, low cost, no toxic or side effect, simple preparation process, convenient use, and can be prepared into oral dosage forms, injection dosage forms, tablets and the like.
3. The luteolin product can obviously inhibit platelet aggregation induced by GPVI specific inhibitor, and the inhibition efficiency is dose-dependent. Can effectively inhibit platelet aggregation and can be effectively used as an antiplatelet drug.
Drawings
FIG. 1 is a graph and bar graph of the effect of luteolin on activator-induced platelet aggregation;
FIG. 2 is a graph showing the interaction of luteolin with GPVI by surface plasmon resonance analysis.
Detailed Description
The following examples are provided to illustrate the present invention in more detail with reference to the accompanying drawings. But are not intended to limit the invention.
Example 1
Luteolin selectively inhibits GPVI-specific inhibitor Convulxin-induced human platelet aggregation in vitro.
(1) Healthy volunteers of experimental human platelet origin sign informed consent for blood donation and are given nutritional supplements. Venous whole blood was collected, and the apheresis platelet was isolated from Kunming blood centers and collected in the department of Hematology, university of Kunming medical science.
(2) The specific implementation mode is as follows:
step one, dissolving luteolin by using DMSO.
Step two, washing the platelets. Taking 1mL of human single blood collection platelets stored by constant-temperature shaking at 25 ℃ in a 1.5mL centrifuge tube, adding EDTA with a final concentration of 5mM and Apyrase (prepared by normal saline to prevent platelet aggregation in the centrifugation process) with a final concentration of 0.1U/mL, and centrifuging at 400g at room temperature for 10 minutes; the supernatant was discarded from the centrifuged human apheresis platelets, and 1mL Tyrode's Buffer B was added to the cell pellet (137mM NaCl,27mM KCl,1mM MgCl 2 ，0.42mM NaH 2 PO 4 5.5mM Glucose,5.55mM HEPES,0.25% Bovine Serum Albumin, pH 6.5), 5mM EDTA,0.1U/mL Apyrase were gently blown up, and centrifuged again at 400g for 10min at room temperature; with Tyrode's Buffer A (137mMNaCl,27mM KCl,1mM MgCl) 2 ,0.42mM NaH 2 PO 4 5.5mM Glucose,5.55mM HEPES,0.25% Bovine Serum Albumin, pH 7.4) the centrifuged platelets were resuspended and the platelet count was adjusted to 150-250X 10 9 and/L. 70rpm,25 ℃ vibration storage, washing platelets in 1 hours.
Step three, 400. Mu.L of the washed platelet resuspension was aspirated and preheated at 37℃for 5min. Luteolin was added to the experimental group at a final concentration of 2.5-25. Mu.M, and an equal volume of DMSO was added to the positive control group (Vehicle) and incubated at 37℃for 10min. Opening a platelet aggregation instrument, setting parameters according to requirements, putting a magnetic rod into the incubated washed platelets, inserting a platelet reaction cup with the magnetic stirring rod into a detection hole of a machine, placing the platelet reaction cup in a test area of the platelet aggregation instrument, zeroing, adding collagen, and observing the influence of luteolin on platelet aggregation caused by different activators at 37 ℃ and 1200rpm for 10min. And drawing a corresponding scatter diagram according to a platelet aggregation curve recorded by the platelet aggregation instrument. Experimental data were statistically analyzed using GraphPadPrism 9.0 software. The comparison was performed using analysis of variance. p <0.05 then the difference is considered statistically significant.
As shown in fig. 1 a of the specification: human washed platelets were incubated with luteolin or DMSO at various concentrations for 10min at 37 ℃, activated with the GPVI specific activator Convulxin (5 ng/mL), and platelet aggregation curves were plotted and recorded using a platelet aggregation instrument. The abscissa is the time depicted by the aggregation curve, and the ordinate is the real-time aggregation rate of platelets depicted by the platelet aggregation meter. Presented in fig. 1 a is a platelet aggregation rate curve representative of the aggregation experiment. At the beginning of the aggregation test, the platelet aggregation rate was 0%, convulxin was added immediately, and the platelets of the positive control (Vehicle) test group began to aggregate when the test was carried out for about 10 seconds. When the aggregation test was carried out until about 2 minutes, the aggregation rate of the Vehicle group reached about 80% peak, the aggregation rate of the 2.5. Mu.M luteolin group reached about 70% peak, and the aggregation rate of the 100. Mu.M luteolin group reached about 0%. Description the diagram B in fig. 1 shows: comparison of platelet aggregation peaks for Vehicle and luteolin (2.5-100. Mu.M) groups at different concentrations. C-F in FIG. 1 of the specification: effects of 25 μm luteolin on platelet aggregation induced by different activators (ADP, thrombin and U46619) aggregation graphs and bars. The experiments of Vehicle and luteolin with different concentrations are repeated four times, the peak value of platelet aggregation is expressed by mean ± standard error, wherein the difference is statistically significant, and p is less than or equal to 0.0001 compared with the Vehicle group. As can be seen from fig. 1: luteolin can obviously inhibit platelet aggregation induced by GPVI specific activator Convulxin, and its inhibition efficiency is dose dependent, IC 50 =8.66 μΜ. Although luteolin also has a certain inhibition effect on platelet aggregation induced by U46619, the inhibition effect of luteolin on platelet aggregation induced by different activators is selective only at a high concentration of 100 mu M, and luteolin mainly inhibits platelet aggregation mediated by GPVI receptors.
Example 2
Surface plasmon resonance plot of luteolin interaction with GPVI.
This experiment was used to verify the direct interaction of luteolin with GPVI in vitro and calculate the equilibrium constants of both.
The specific implementation mode is as follows:
GPVI protein (human recombinant GPVI is from MCE company, product number HY-P77952) is immobilized on a chip, luteolin with different concentrations is sequentially added to analyze affinity with GPVI, reaction signals within 90 seconds are recorded, a kinetic curve is made, and relevant parameters are calculated.
As can be seen from fig. 2, the affinity of GPVI to luteolin (K D ) 4.13. Mu.M. In recent years, a variety of small molecules have been reported in the literature to inhibit GPVI mediated platelet aggregation, but there is evidence that small molecules that interact directly with GPVI are fewer and have generally not high affinity, for example: honokiol, K D =289μM（Lee TY, Chang CC, Lu WJ, et al., Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies. Sci Rep, 2017;7:40002.），Losartan K D =170μM（Ono K, Ueda H, Yoshizawa Y, et al., Structural basis for platelet antiaggregationby angiotensin II type 1 receptor antagonist losartan (DuP-753) via glycoprotein VI. J Med Chem, 2010;53(5):2087-93.），GSK669 K D =14.4 μm (Pan G, chang L, zhang J, et al, GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet gpvi, biochem Pharmacol,2021; 183:114315), and the like. From this, the affinity of luteolin to GPVI (K D ) 4.13. Mu.M, is the higher affinity of the small molecules reported to date. The luteolin is used as a template for molecular transformation, and a small molecular inhibitor with higher affinity is synthesized to prepare a novel high-efficiency anti-GPVI small molecular preparation.
Example 3:
tablet: luteolin 10mg, lactose 180 mg, starch 55 mg, magnesium stearate 5 mg.
The preparation method comprises the following steps: mixing luteolin, lactose and starch, wetting with water, sieving the wetted mixture, drying, sieving again, adding magnesium stearate, tabletting the mixture, each tablet having weight of 250 mg and compound content of 10 mg.
Example 4:
ampoule agent: luteolin, or a pharmaceutically acceptable salt thereof 2 mg, sodium chloride 10 mg.
The preparation method comprises the following steps: dissolving luteolin and sodium chloride in appropriate amount of injectable water, filtering to obtain solution, and packaging into ampoule under aseptic condition.
Example 5:
freeze-dried preparation for injection: luteolin 10mg, sodium bicarbonate 2 mg, mannitol 252 mg.
The preparation method comprises the following steps: dissolving sodium bicarbonate and mannitol in water for injection, adding activated carbon for adsorption for 30 min to remove pyrogen, filtering to remove activated carbon, adding luteolin into the filtrate, performing ultrasonic treatment to dissolve, adjusting pH to 5.0-7.0 with 1N hydrochloric acid, filtering with microporous membrane, adding water for injection, packaging, freeze drying, plugging, and capping.
Example 6:
the capsule comprises the following components: luteolin 10mg, lactose 187 mg, magnesium stearate 3mg.
The preparation method comprises the following steps: mixing luteolin with cosolvent, sieving, mixing, and encapsulating into hard gelatin capsules with weight of 200 mg and active ingredient content of 10 mg.
The scope of the present invention is not limited to the above-described embodiments, which are provided only for the purpose of aiding in explaining and explaining the present invention, but not limiting the scope of the present invention, as long as the design is identical to the design of the present invention or is equivalent thereto, and falls within the scope of the present invention as claimed.

Claims (5)

1. Application of luteolin in preparing platelet glycoprotein VI inhibitor is provided.
2. Use of luteolin according to claim 1 for the preparation of an inhibitor of platelet glycoprotein VI, characterized in that wherein said luteolin interacts with GPVI at K D =4.13μM。
3. Use of luteolin in the manufacture of an antiplatelet agent for use in the treatment of GPVI-specific inhibitor-induced platelet aggregation.
4. The luteolin is applied to the preparation of platelet aggregation induced by the anti-GPVI specific inhibitor, and the inhibition efficiency of the luteolin is dose-dependent, so that the anti-platelet aggregation effect is achieved.
5. Application of luteolin in preparing anti-platelet medicine for inhibiting platelet aggregation induced by GPVI specific activator is provided.
CN202310385361.1A 2023-04-12 2023-04-12 Application of luteolin in preparation of platelet glycoprotein VI inhibitor Pending CN116549433A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101352416A (en) * 2006-10-10 2009-01-28 上海格鲁奥丽生物医药技术有限公司 Luteolin solution composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101352416A (en) * 2006-10-10 2009-01-28 上海格鲁奥丽生物医药技术有限公司 Luteolin solution composition

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