JPH04297418A - Blood platelet agglutination inhibitor and leukocytic activation inhibitor - Google Patents

Abstract

PURPOSE:To obtain a medicine, especially a blood platelet agglutination inhibitor and a leukocytic activation inhibitor useful for treating and preventing adult diseases caused by a change in eating habits and immunological abnormal diseases accompanying allergy due to a change in the eating habits and living environment. CONSTITUTION:The aforementioned medicine containing licochalcone A expressed by formula I and/or licochalcone B expressed by formula II which are flavonoid ingredients in Licorice as an active ingredient. The above- mentioned medicine is useful as a medicinal material or food material having excellent blood platelet agglutination inhibitory action useful for treating and preventing adult diseases such as thrombotic diseases, e.g. arteriosclerosis, complications (angitis, renal insufficiency or retinopathy) in diabetes, hyperlipemia, various thromboses and coronary arterial diseases or allergic diseases such as rheumarithrits, asthma, dermatitis, gastroenteropathy or renopathy and inhibitory action on leukocytic activation accompanying immunological abnormality and allergic inflammations.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a drug effective for the treatment and prevention of various adult diseases and allergic inflammatory diseases, particularly to a platelet aggregation inhibitor and a leukocyte activation inhibitor.

0002

[Prior art] In recent years, due to changes in dietary habits, animal fat,
The intake of salt and sugar has increased significantly, and the number of adult diseases associated with it has increased. In particular, the number of patients suffering from arteriosclerosis, hyperlipidemia, various thromboses, coronary artery disease, non-insulin dependent diabetes and its complications such as vasculitis, diabetic retinopathy, and diabetic renal failure is increasing. ing. In these adult diseases, the blood is in a procoagulant state, and several platelet aggregation inhibitors have been developed as therapeutic agents to improve this state. Typical drugs include platelet cyclooxygenase inhibitors such as aspirin and indomethacin [J. R. Vane, Natu
re, 231, 232 (1971); H. Ha
Mberg & B. Samuelsson, Proc.
．． Natl. Acad. Sci. USA, 7
2, 2944 (1975)], platelet thromboxane synthase inhibitors such as U-51605 [R. R. Gor
Man et al., Proc. Natl. Acad. Sc
i. USA, 74, 4007 (1979)], and cyclic AMP phosphodiesterase inhibitors such as dipyridamole [D. C. B. Mills & J.
B. Smith, Biochem. J. ,121,
185 (1971)]. All of these are enzyme inhibitors for suppressing the production of thromboxane A2, a potent platelet aggregation factor. In addition, platelet aggregation inhibitors consisting of a new substance contained in licorice (Japanese Unexamined Patent Publication No. 64-50885) and isoliquiritigenin (Japanese Unexamined Patent Publication No. 1-117827) are also known.

[0003] Furthermore, due to changes in dietary habits and living environments, such as the combined effects of high-calorie foods, environmental pollution, and food additives, the number of allergic inflammatory diseases associated with these changes has significantly increased. Diseases associated with allergic inflammation include rheumatoid arthritis, asthma, dermatitis, gastrointestinal disorders, kidney disorders, and vasculitis due to vascular endothelial disorder as a complication of diabetes. As therapeutic agents for improving the conditions of these allergic inflammatory diseases, steroids, prostaglandin synthesis inhibitors, antihistamines, histamine release inhibitors, leukotriene production inhibitors, and the like have been developed so far. As a typical drug, prostaglandin synthesis inhibitors (indomethacin, aspirin) are used as a treatment for arthritis [J. R. Vane,
Nature, 231, 232 (1971);
Yuichi Shiokawa, Therapeutics-Special Inflammation-, 8, 387
(1982)]. Antihistamines (diphenhydramine, tripelenamine, chlorpheniramine, meclizine, promethazine), histamine release inhibitors (Intal), and leukotriene production inhibitors are used as anti-asthmatic and anti-allergic agents.

Isoliquiritigenin [Japanese Patent Publication No. 1-52363] contained in licorice and a licorice hydrophobic flavonoid preparation [Japanese Patent Application Laid-Open No. 204417/1999] have been proposed as antiallergic agents. Also, abioisoriquiritin, which is also contained in licorice [Japanese Patent Application Laid-open No. 60-18
No. 4017] or Abioliquiritin [Unexamined Japanese Patent Publication No. 6
No. 0-184016] is also known as an air supplement for improving intractable diseases such as rheumatism and neuralgia.

[0005]

[Problems to be Solved by the Invention] The above-mentioned conventional technology regarding platelet aggregation inhibitors has been developed to reduce thromboxane A in platelets.
Development centered on platelet aggregation inhibitors, which are inhibitors of cyclooxygenase, thromboxane synthase, and cyclic AMP phosphodiesterase, which are enzymes involved in the production of platelet aggregation factor 2 (platelet aggregation factor). Furthermore, drugs that have been developed as therapeutic agents for the above-mentioned allergic inflammatory diseases are mainly steroids, prostaglandin synthesis inhibitors, antihistamines, histamine release inhibitors, and leukotriene production inhibitors. Also,
The above medicines, especially supplementary medicines, are made of ingredients contained in licorice.
Its effect is unclear.

The present invention aims to treat adult diseases caused by changes in dietary habits such as excessive intake of fat, salt, and sugar, and changes in dietary habits and living environments such as high-calorie foods, environmental pollution, and the combined effects of food additives. The present invention aims to provide a drug useful for the treatment and prevention of the above-mentioned immunological disorders associated with allergies, particularly a platelet aggregation inhibitor and a leukocyte activation inhibitor.

[0007]

[Means for Solving the Problems] The present inventors have discovered that among the many types of flavonoid components contained in licorice, licochalcone A and lycochalcone B not only have an inhibitory effect on thromboxane A2-forming enzyme, but also have an intracellular release effect. It was discovered that it is useful as a platelet aggregation inhibitor because it has a high platelet aggregation inhibitory effect due to the effect of suppressing the increase in Ca concentration, and the present invention was completed. Furthermore, the present inventors have discovered that licochalcone A and/or licochalcone B have an excellent leukocyte activation inhibitory effect, and the activation of leukocytes is involved in the above-mentioned immunological disorders associated with allergies and arteriosclerosis. As a result of paying attention to this, it was discovered that these compounds function as leukocyte activation inhibitors useful for the prevention and treatment of immune disorders associated with allergies and arteriosclerosis, and the present invention was completed.

The gist of the present invention is that licochalcone A and/or
Alternatively, there are platelet aggregation inhibitors containing licochalcone B as an active ingredient, and leukocyte activation inhibitors containing licochalcone A and/or licochalcone B as active ingredients. This leukocyte activation inhibitor is useful as an anti-allergic and inflammatory agent.

[0009] Here, leukocyte activation inhibitors are used to treat immune disorders caused by allergic and inflammatory substances produced in the body, such as rheumatoid arthritis, asthma, dermatitis, gastrointestinal disorders, kidney disorders, and diabetes complications. This term refers to a drug that has the effect of suppressing vasculitis caused by vascular endothelial damage as well as leukocyte activation that occurs in arteriosclerotic lesions. In addition, leukocyte activation refers to the induction or aggravation of symptoms such as immune disorders and arteriosclerosis due to the production of lysosomal enzyme-free leukotrienes from polynuclear leukocytes due to allergy and inflammation-inducing substances.

[0010] Licochalcone A and licochalcone B used in the present invention are flavonoids contained in licorice;
Each can be extracted from licorice. for example,
There are the following extraction methods. First, licorice is extracted with a 70% acetone aqueous solution, and this is concentrated under reduced pressure. The obtained concentrated extract was suspended in water and mixed with ether, acetic acid ethyl ester,
A crude flavonoid fraction is obtained from the ether, ethyl acetate and n-butanol soluble fractions by sequential extraction with n-butanol. When the crude flavonoid fraction is subjected to silica gel column chromatography and eluted with a chloroform-methanol mixture (98:2 V/V), licochalcone A and licochalcone B are obtained. Licochalcone A and lycochalcone B may be further obtained as separate components by silica gel column chromatography, or may be used as a mixture.

Regarding other flavonoid components in licorice, lycochalcone A and lycochalcone B were eluted from the crude flavonoid fraction as described above, and then eluted with a chloroform-methanol mixture (40:1 V/V). Tigenin and liquiritigenin can be obtained, and a chloroform-methanol mixture (4:
Isoliquiritin and liquiritin can be obtained by elution at 1 V/V).

The method for extracting licochalcone A and licochalcone B from licorice is not limited to the above method. Furthermore, since the compounds used in the present invention have been identified and their structures have been clarified, they may be produced by synthesis or plant biotechnology. The structural formula of each compound is as follows. Licochalcone A

[0013]

[Chemical formula 1]

[0014] Licochalcone B

[0015]

[Case 2]

[0016]

[Action] Licochalcone A and Licochalcone B suppress the production of thromboxane A2 from arachidonic acid by inhibiting enzymes in platelets involved in the metabolism of thromboxane A2, a powerful platelet aggregation factor. It also has an antiplatelet aggregation effect against thrombin, which is released and increases during inflammation, and an effect of inhibiting the increase in platelet free Ca concentration.

[0017] The compound of the present invention exhibits an extremely excellent platelet release inhibiting effect because it inhibits the production of platelet aggregation factors, inhibits platelet aggregation upon thrombin stimulation, and inhibits the increase in intracellular free Ca concentration. Effective as a platelet aggregation inhibitor.

Furthermore, licochalcone A and licochalcone B have an inhibitory effect on 12-lipoxygenase, which is an intraplatelet enzyme. 12- produced by this enzyme
Hydroperoxy-5,8,10-14-eicosatetraenoic acid (12-HPETE) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are effective in leukocyte attraction and migration. Action [R. M
．． J. Palmer et al., Prostagrandi
nes, 20, 411 (1980)] and the progression of arteriosclerosis from thickening of the vascular wall due to the migration of vascular media smooth muscle cells (J. Nakano et al., Atherosclerosis).
erosis, 43, 143 (1982)]. Therefore, the compounds of the present invention can inhibit leukocyte attraction, migration, and thickening of blood vessel walls caused by enzyme products, and are expected to inhibit allergic inflammation and prevent arteriosclerosis as a result.

Furthermore, licochalcone A and licochalcone B inhibit platelet activating factor, which is an allergy and inflammation-inducing biological substance.
ng Factor, PAF), leukotriene B
4 (has leukocyte migration effect, leukocyte aggregation effect, histamine release effect from mast cells, etc.) and leukocyte activation synthetic peptide (N-Formyl-Methyo
nyl-Leucyl-Phenylalanine,
fMLP) inhibits the release of lysosomal enzymes from polynucleated leukocytes, and leukocyte activation calcium ionophore (Calcium Ionophore A2)
3187, Ionomycine) has the effect of inhibiting the release of lysosomal enzymes from polynuclear leukocytes. It also has the effect of suppressing the increase in intracellular free calcium concentration due to the effect of suppressing the influx of calcium ions from outside the cell and suppressing mobilization from intracellular calcium storage sites.
Has excellent leukocyte activation inhibitory effects.

Lysosomal enzymes released from leukocytes upon leukocyte activation are factors that cause inflammation in various tissues of the body and destruction of bone morphogenetic proteins. Therefore, this lysosomal enzyme causes rheumatoid arthritis, asthma, dermatitis,
Symptoms such as gastrointestinal disorders, kidney disorders, vasculitis due to vascular endothelial disorders of diabetic complications, and arteriosclerosis are induced or worsened. Conventional anti-inflammatory agents and anti-allergic agents have not been developed based on the function of white blood cells, and their mechanism of action is not clear.

As described above, the compound of the present invention has an excellent platelet aggregation inhibitory effect, and therefore can be used as a platelet aggregation inhibitor to treat thrombotic diseases such as arteriosclerosis and diabetic complications (vasculitis, renal failure, retinopathy). It can be used as a pharmaceutical material or food material for the treatment and prevention of adult diseases such as hyperlipidemia, various thrombosis, and coronary artery disease. Also produced during allergies,
It has a leukocyte activation inhibitory effect on platelet activating factor and leukotriene B4, which are known to increase, so it is used as a leukocyte activation inhibitor to suppress leukocyte activation associated with immune disorders and allergic inflammation. It can be used as a medicine for the prevention and treatment of diseases such as rheumatoid arthritis, asthma, dermatitis, gastrointestinal disorders, kidney disorders, vasculitis due to intravascular disorders during diabetic complications, and the progression of arteriosclerosis.

[0022] When administering licochalcone A and/or licochalcone B of the present invention as a pharmaceutical, it varies depending on the type of disease, severity of symptoms, patient's age, weight, etc.
Normally, for adults, it is sufficient to administer about 60 to 600 mg per day. This can be administered once a day or in several divided doses. Administration methods include parenteral administration via intravenous injection, subcutaneous injection, intramuscular injection, etc., oral administration via tablets, capsules, granules, fine granules, powders, etc., or suppositories,
Local administration may be performed using external solutions, ointments, etc., and is not particularly limited.

The compound of the present invention can be formulated and used in various forms depending on the administration method using additives commonly used in formulation. Examples include dosage forms such as emulsions, wettable powders, aqueous solutions, tablets, capsules, powders, granules, and pills. Additives used in formulation include excipients, disintegrants,
These include lubricants, binders, dispersants, plasticizers, fillers, carriers, etc. For these, commonly used additives may be used. The effects of the platelet aggregation inhibitor and leukocyte activation inhibitor of the present invention will be explained below using experimental examples.

(Experimental example) 1. Extraction of flavonoids from licorice, isolated licorice 1kg
was extracted with a 70% acetone aqueous solution (5 liters x 3 times) and concentrated under reduced pressure. The obtained concentrated extract was suspended in 1 liter of water, and ether, acetic acid ethyl ester, n-
The crude flavonoid fraction was obtained from the ether, ethyl acetate and n-butanol soluble fractions by sequential extraction with butanol. This crude flavonoid fraction was analyzed using silica gel (manufactured by Merck) column chromatography using a chloroform-methanol mixture (98:2 V/V).
) lycochalcone A and lycochalcone B
A mixture of
and 2.15 g of licochalcone B were obtained.

2. Platelet aggregation inhibitory effect (1) Isolation of human platelets Human platelets were collected from the cubital vein of a normal person who had not taken drugs such as aspirin and indomethacin for 10 days and isolated. 3.8 as an anticoagulant in the collected blood
% sodium citrate solution was added in an amount of 1/10 and mixed well. This blood was centrifuged at 220 × g for 10 minutes at room temperature, and the supernatant (platelet-rich plasma) was further centrifuged at 1.50 × g.
Platelets were obtained by centrifugation at 0 xg and 4°C for 10 minutes. The platelets thus isolated were mixed with Hepes saline buffer (25 mM Hepes, 135 mM Na) containing 2 mM EDTA.
Cl, 5mM KCl, 5.5mM glucose, pH
7.4) twice and suspended so that the platelet count was 5 x 105 cells/μl.

(2) Effect of each flavonoid compound in licorice on human platelet arachidonic acid metabolism The human platelet suspension prepared in (2) above (5 x 105
Each flavonoid component (licochalcone A and licochalcone B) isolated in (1) above was added to 130 μl (cells/μl) and preincubated at 37° C. for 5 minutes. Next, radiolabeled [1-14C] arachidonic acid (specific activity: 54.5 mCi/mmol=201
6.5MBq/mmol, manufactured by New England Nuclear Co.) 50μl (0.05μCi = 1.85KBq/
(test tube) and reacted for 5 minutes in a final volume of 200 μl. Add 0.5N formic acid (200 μl) to the reaction solution.
was added to stop the reaction. Then, 8 times the amount of acetic acid ethyl ester was added to extract arachidonic acid metabolites. The extract was dried in a nitrogen stream. 50μl of this dry matter
The mixture was dissolved in ethyl acetate and quantitatively spotted on a silica gel plastic sheet (manufactured by Merck & Co.) and developed. The developing solvent is acetic acid ethyl ester/2,2
,4-trimethylpentane/acetic acid/distilled water (100:5
0:20:100 v/v, upper layer) was used. Radioactive metabolites were detected by autoradiography. The results are shown in Figure 1.

Furthermore, each spot was cut out and each radioactivity was measured using a liquid scintillation counter, and the effects of licochalcone A and licochalcone B on arachidonic acid metabolites were summarized in a graph [FIGS. 2(a) and (b)].

In this experimental system, mainly three spots can be detected. Namely, thromboxane B2 (TXB2) (a stable compound of thromboxane A2), 12-hydroxy-
5,8,10-heptadecatrienoic acid (HHT), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Thromboxane B2 and HHT are metabolized from arachidonic acid via cyclooxygenase, and 12-HET
E is metabolized via 12-lipoxygenase. From Figures 1 and 2, licochalcone A and licochalcone B are thromboxane B2, HHT, 12-
It was found that it suppresses the production of HETE. For comparison, similar experiments were conducted with isoliquiritigenin and liquiritigenin, and the results are shown in Figures 2(c) and (d).
) is shown. In this case, thromboxane B2, HHT
The production of 12-HETE is suppressed, but the production of 12-HETE increases.

From the graph of FIG. 2, the concentration of each flavonoid component that inhibits HHT and troboxane B2 production by 50% was calculated and summarized in Table 1. Also, 12-HETE
The 50% inhibitory concentration on production is also shown in Table 1. Lycochalcone A is 3.97 x 10-6M, lycochalcone B is 2.3 x 10-5M, and isoliquiritigenin is 3.97 x 10-6M.
78 x 10-6 M and liquiritigenin >10-3 M
inhibits thromboxane B2 production by 50%. Thus, licochalcone A and licochalcone B inhibit the production of thromboxane B2 at low concentrations. On the other hand, licochalcone A is 8.23×10-5M, and licochalcone B is 1.34×
10-5M inhibits 12-HETE production by 50%, and inhibition is possible at low concentrations, whereas isoliquiritigenin and liquiritigenin inhibit 12-HETE production by 50%.
M increased the production of 12-HETE by about 2 times.

[0030]

[Table 1]

[0031] Thus, licochalcone A and licochalcone B inhibit the enzyme involved in the production of thromboxane A2, and inhibit the enzyme 12-HETE involved in the production of 12-HETE.
It is clear that it inhibits lipoxygenase.

(3) Effect of each flavonoid component in licorice on human platelet aggregation induced by thrombin Human platelets (2 x 108 cells) were added to a cuvette, and incubated at 37°C.
Add each flavonoid component while stirring at 1000 rpm, preincubate for 1 minute, and then add 0.1
Units/ml of thrombin was added, and platelet aggregation was monitored for 5 minutes using a platelet aggregometer (NBS HEMA aggregometer, manufactured by Niko Biosciences). The results are shown in Figures 3(a), (b) and (c), where lycochalcone A is 2 x 10-5 to 2 x 10
Thrombin-induced platelet aggregation was significantly inhibited at -4M concentration. Licochalcone B is also 2×10-7 to 2×10
Platelet aggregation was strongly inhibited at -4M concentration. In contrast, isoliquiritigenin does not inhibit thrombin-induced platelet aggregation.

(4) Effect of flavonoid components in licorice on the free Ca concentration in human platelets (4-1) Free Ca measurement reagent for human platelets (Fur
a2) Introduction method 3 μM of 1-[2-(5'-amino-carboxyoxazol-2'-yl)-6-aminobenzofuran-
5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid penta-acetoxyl methyl ester (Fura 2-
After adding AM) to platelet-rich plasma (PRP) and incubating at 37°C for 30 minutes, unreacted Fura
2-AM in Hepes saline buffer (25mM Hepes,
135mM NaCl, 5mM KCl, 5.5mM
Wash twice with Mglucose, pH 7.4). Then,
It was suspended in Hepes-saline buffer (same components as above) containing 1mM ethylene glycol-bis(β-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or 1mM CaCl2.

(4-2) Effect of each flavonoid component in licorice on free Ca concentration in human platelets upon thrombin stimulation Each flavonoid component was added to human platelets labeled with Fura 2 prepared by the above method and incubated for 1 minute. After pre-incubation, 0.1 unit/ml
of thrombin and the intracellular free Ca concentration measuring device (
JACO CAF-100, Ca2+ analyzer)
The free Ca concentration within platelets was monitored by fluorescence intensity. Hepes saline buffer containing 1mM CaCl2 (25mM Hepes, 135mM NaCl, 5mM
Stimulation with thrombin (KCl, 5.5 mM glucose, pH 7.4) increases intraplatelet free Ca concentration. When each flavonoid component in licorice was applied to this system, the results shown in Figures 4(a) and (b) were obtained. That is, lycochalcone A and lycochalcone B
inhibited the increase in intraplatelet free Ca concentration under thrombin stimulation at a concentration of 2×10 −7 M to 2×10 −4 M.

Furthermore, when human platelets were suspended in Hepes saline buffer (component as above) containing 2mM EGTA and treated with thrombin, the free Ca concentration in the platelets increased. This increase in intraplatelet free Ca is due to the mobilization of Ca2+ from intracellular calcium storage sites. Figure 5
As is clear from (a) and (b), in this system as well, lycochalcone A and lycochalcone B are
At concentrations of x10-7M to 2x10-4M, the increase in intraplatelet free Ca concentration under thrombin stimulation was suppressed.

3. Leukocyte activation inhibitory effect (1) Isolation of human polynucleated leukocytes Add 5 ml of 6% dextran to 35 ml of healthy human heparinized peripheral venous blood, mix and let stand for 45 minutes. Take the supernatant fraction and add an equal amount of 0. .9% NaCl and layered gently onto Ficoll/hypaque. This was centrifuged at 1500 rpm for 30 minutes at 4°C, the sediment was collected, and the mixed red blood cells were removed.
．． Hemolyzed with 3% NaCl and added to Dulbecco's phosphate-saline buffer (Dulbecco's PBS; 1
．． 0mM CaCl2, 2.68mM KCl, 1.
47mM KH2PO4, 0.5mM MgCl2,
137mM NaCl, 8.06mM Na2PO
4, pH 7.4). The cells prepared in this way contained 97% or more neutrophils, 1% or less platelets and eosinophils, and no basophils, lymphocytes, or monocytes were observed. The cell survival rate was over 95%. The leukocytes prepared in this manner were designated as polynuclear leukocytes.

(2) Effect of each flavonoid compound in licorice on the release of lysosomal enzymes from human polynucleated leukocytes Human polynucleated leukocytes (2.2 × 106 cells) were injected with each flavonoid compound in licorice obtained in 1 above. (Lycochalcone A and Lycochalcone B) at 37℃
pre-incubated for 5 minutes at 37°C, then added calcium ionophore A23187 (2 μM)
The mixture was allowed to react for 10 minutes. On the other hand, leukotriene B4 (L
The release of lysosomal enzymes from polynuclear leukocytes using platelet activating factor (PAF), platelet activating factor (PAF), and leukocyte activation synthetic peptide (fMLP) was performed as follows. 3 human polynucleated leukocytes (2.2 x 106 cells)
Pre-incubated with each flavonoid compound in licorice (lycochalcone A and lycochalcone B) for 5 minutes at 7°C, and further incubated with cytochalasin B (Cyto.B) (5μ
g/ml) and incubated for 3 minutes. Next, 2.5 m of bovine serum albumin (BSA) was added to the reaction solution.
LTB4 (125 ng/ml) dissolved in Dulbecco's PBS containing g/ml, 200 nM P
AF or 50 nM fMLP was added and reacted at 37°C for 5 minutes. The reaction was stopped by placing each test tube in ice water and centrifuged for 10 minutes at 4°C. Measurement of lysozyme and β-glucuronidase activities released into the supernatant was performed by Takamori & Yamash
[Infection and Immuni
ty: 29, 395 (1980)] and Avil
a & Convit [Biochem. Biop
hys. Acta: 293, 397 (1973)
] The method was followed. In addition, the intracellular enzyme lactate dehydrogenase (LDH) is an indicator of cytotoxicity.
The measurement of the activity of Bergmeyer & Berne
t [In H. U. Bergmeyer (ed.)
, Method of enzyme
ysis, 2nd ed. Academic Pr
ess, Inc. New York pp. 574-
579, 1974]. The activity of all enzymes in polynucleated leukocytes was determined by applying 0.1% Triton X-100 and determining the total released amounts of lysozyme, β-glucuronidase, and LDH according to the method described above.

The effects of licochalcone A and licochalcone B on the release of lysosomal enzymes from human polynuclear leukocytes are summarized graphically (FIGS. 6 and 7). As shown in Figure 6, licochalcone A ranges from 0.5 μM to 100
At μM concentration, calcium ionophore A2318
7. Suppressed the release of lysosomal enzymes from polynuclear leukocytes by LTB4, PAF and fMLP. Lycochalcone B also inhibits calcium ionophore A23187, LTB4, PAF at concentrations of 10 μM to 1000 μM.
and fMLP-induced release of lysosomal enzymes from polynucleated leukocytes (Figure 7). Under the above experimental conditions, lycochalcone A and lycochalcone B are
Does not cause DH release. This indicates that licochalcone A and licochalcone B do not cause cell damage.

(3) Effect of each flavonoid compound in licorice on free calcium concentration in human polynuclear leukocytes (
3-1) Introduction of free calcium measurement reagent (Fura2) into human polynucleated leukocytes (4.5 x 107 cells) 2.
5 μM of 1-[2-(5'-amino-carboxyoxazol-2'-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane- N,N,N',N'-tetraacetic acid penta-acetoxyl methyl ester (Fura 2
-AM) was added and reacted at 34.5°C for 20 minutes. After the reaction was completed, centrifugation was performed at 650 × g to remove excess Fura 2-AM in the medium, and Hepes saline buffer (25mM Hepes, 135mM NaCl, 5mM
KCl, 5.5 mM glucose, pH 7.4)
Washed twice. Then 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N'-tetraacetic acid (EGTA, calcium chelator) or 1
Suspended in Hepes saline buffer (components as above) containing mM CaCl2.

(3-2) Calcium ionomycin,
Effect of each flavonoid component in licorice on free calcium concentration in polynuclear leukocytes upon LTB4, PAF and fMLP stimulation Fura 2 prepared by the above method
Add each flavonoid compound (licochalcone A and licochalcone B) to a suspension of human polynucleated leukocytes (2.5 × 106 cells) labeled with or react with buffer alone at 37°C for 1 minute, and then add 2 μM calcium ionomycin. , LTB4 (125ng/ml), 200
Add nM PAF or 50 nM fMLP and use an intracellular free calcium concentration measuring device (JACO CAF-
The influence of licochalcone A and licochalcone B on the free calcium concentration in leukocytes was investigated by monitoring the free Ca concentration in leukocytes by fluorescence intensity using a Ca2 + analyzer).

Hepes saline buffer containing 1mM CaCl2 (25mM Hepes, 135mM NaCl, 5mM
KCl, 5.5 mM glucose, pH 7.4) along with 2 μM calcium ionomycin, LTB4 (12
5ng/ml), 200nM PAF or 50n
Stimulation with M fMLP increases intraleukocyte free calcium concentration. When each flavonoid component in licorice was applied to this system, the results shown in FIGS. 8(a) and 8(b) (representative example: PAF stimulation; similar trends apply) were obtained. That is, licochalcone A and licochalcone B at concentrations of 1 to 100 μM inhibited the increase in free calcium concentration in leukocytes stimulated by calcium ionomycin, LTB4, PAF, and fMLP.

[0042] Human polynuclear leukocytes were also treated with 1mM EGTA.
When the cells were suspended in Hepes saline buffer (same components as above) and treated with calcium ionomycin, the concentration of free calcium in polynuclear leukocytes increased. This increase in free calcium concentration within polynuclear leukocytes is due to the mobilization of Ca2+ from intracellular calcium storage sites. As is clear from FIGS. 9(a) and (b), in this system as well, lycochalcone A and lycochalcone B are
At a μM concentration, the increase in free calcium concentration within polynuclear leukocytes under stimulation with calcium ionomycin was suppressed.

[0043]

【Example】

Example 1 Tablet ■Corn starch
65g
■Crystalline cellulose
20g ■Carboxymethyl cellulose calcium
3.5g
■Light silicic anhydride
0.5g ■Magnesium stearate 1g
■Compound of the present invention (Lycochalcone A)
10g
Total
100g According to the above recipe, ① to ① were mixed uniformly and compressed using a tablet machine to obtain tablets each weighing 200mg. One tablet contains 20 mg of the compound of the present invention, and adults should take 3 to 30 tablets a day in several divided doses.

[0044] Application example 2 Granule ■Corn starch
84g
■Carboxymethyl cellulose calcium 5g
■Light silicic anhydride
0.5g
■Magnesium stearate
0.5g ■Compound of the present invention (
Ricochalcone B) 10g

Total 100g According to the above recipe, mix ■～■ uniformly, and after compression molding with a compression molding machine,
The mixture was crushed using a crusher and sieved to obtain granules. 1 g of this granule contains 100 mg of the compound of the present invention, and adults should take 1 to 6 g per day in several divided doses.

Usage Example 3 Capsule ■Corn starch
94.5g
■Light silicic anhydride
0.5g ■Compound of the present invention (licochalcone A) 2.5g
(
Ricochalcone B) 2.5g

Total: 100g According to the above recipe, ① to ② were mixed uniformly, and 200mg was filled into No. 2 capsules. One capsule contains 10 mg of the compound of the present invention, and adults should take 6 to 60 capsules a day in several divided doses.

Example 4 Injection ■ Distilled water for injection
Appropriate amount
■Glucose
10mg ■Macrogol 400
25mg ■Sodium chloride
9mg
■Compound of the present invention (Lycochalcone A)
10mg
Whole amount
Dissolve ■ to ■ in 1 ml of distilled water for injection, adjust the pH to around 7.0, fill in a 1 ml ampoule, seal, and heat at 121°C.
It was autoclaved for 15 minutes.

[0047]

Effects of the Invention As explained in detail above, among the flavonoid components in licorice, lycochalcone A and lycochalcone B
Because it has a platelet aggregation inhibitory effect, it is used as a platelet aggregation inhibitor that improves the procoagulant state of blood.
It can be effectively used for adult diseases such as complications of diabetes, hyperlipidemia, various thromboses, and thrombotic diseases such as coronary artery disease. Furthermore, licochalcone A and licochalcone B have an extremely excellent effect of suppressing white blood cell activation associated with immune abnormalities and allergic inflammation, and are effective in suppressing immune abnormal diseases such as rheumatoid arthritis, asthma, dermatitis, gastrointestinal disorders, renal disorders, and diabetes complications. It is a useful leukocyte activation inhibitor for vasculitis caused by vascular endothelial damage and arteriosclerosis.

[Brief explanation of drawings]

FIG. 1 shows the results of autoradiography of arachidonic acid metabolites in human platelets.

[Figure 2] Figures 2 (a), (b), (c) and (d)
) is a graph showing the influence of flavonoid components in licorice on arachidonic acid metabolites.

FIG. 3 (a), (b) and (c) are graphs showing the influence of flavonoid components in licorice on human platelet aggregation ability upon thrombin stimulation.

[Figure 4] Figures 4 (a) and (b) show that the flavonoid components in licorice are responsible for the release of free C in human platelets upon thrombin stimulation.
It is a graph showing the influence on a concentration.

[Figure 5] Figures 5 (a) and (b) show that the flavonoid components in licorice are responsible for the release of free C in human platelets upon thrombin stimulation.
It is a graph showing the influence on a concentration.

FIGS. 6(a) and 6(b) are graphs showing the effect of licochalcone A in licorice on the release of lysosomal enzymes from human polynuclear leukocytes.

FIGS. 7(a) and 7(b) are graphs showing the influence of licochalcone B in licorice on the release of lysosomal enzymes from human polynuclear leukocytes.

[Figure 8] Figures 8(a) and (b) show that licochalcone A and licochalcone B in licorice are 1mMCa2 +
2 is a graph showing the effect on free calcium concentration in human polynucleated leukocytes upon stimulation with platelet activating factor in the presence of PBS.

FIGS. 9(a) and 9(b) are graphs showing the influence of licochalcone A and licochalcone B in licorice on the free calcium concentration in human polynucleated leukocytes upon stimulation with calcium ionomycin in the presence of 1 mM EGTA.

Claims (3)

[Claims] 1. A platelet aggregation inhibitor containing licochalcone A and/or licochalcone B as an active ingredient. 2. A leukocyte activation inhibitor containing licochalcone A and/or licochalcone B as an active ingredient. 3. An anti-allergic inflammatory agent containing licochalcone A and/or licochalcone B as an active ingredient.