Platelet aggregation inhibitor

Abstract

To provide a novel platelet aggregation inhibitor.SOLUTION: The present invention has the following features to solve the above problem. 1. A collagen-induced platelet aggregation inhibitor, comprising an extract of yellow strawberry guava (Psidium littorale) fruit as an active ingredient. 2. An adenosine diphosphate-induced platelet aggregation inhibitor, comprising an extract of Psidium littorale fruit as an active ingredient. 3. An induced platelet aggregation inhibitor, comprising an extract of Psidium littorale fruit as an active ingredient. 4. The agent described in any one of (1) to (3), wherein the extract of Psidium littorale fruit comprises adenosine. 5. An extract of Psidium littorale fruit, comprising adenosine.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel platelet aggregation inhibitor, which can be widely used in medicines, foods, health foods (including functional foods, foods for specified health uses, etc.), etc. [Background technology]

[0002] Yellow strawberry guava is a tropical fruit of Psidium littorale, also known as yellow pomegranate. There is no significant difference between the edible fruit of Psidium littorale, a member of the Myrtaceae family, and the Banjirou (guava), a member of the same genus, apart from the color of the skin. Therefore, when the word "guava" is used in the fruit market, it often includes the fruit of this species, but it is a different species from Banjirou (guava). In Japan, seedlings are sold for home use, but they are only cultivated on a commercial scale in a limited area of ​​Minamiizu. In addition to being eaten raw, they are used as ingredients in jams, fruit vinegars, and local beers. There are few academic reports on Yellow Strawberry Guava, and in component studies, it has only been announced that phloroglucinols have been isolated from the leaves (Non-Patent Document 1) and fruits (Non-Patent Document 2), and megastigman glycoside (Non-Patent Document 3) has been isolated from the leaves. In addition, in functional studies, the only report has been an anti-glycation effect (Non-Patent Document 4) (see Non-Patent Documents 1 to 4).

[0003] Platelet aggregation on the vascular wall is also caused by factors such as collagen. For example, when vascular endothelial cells are damaged, collagen at the bottom of the epithelial cells is exposed to the bloodstream and activates platelets. Platelet activation induces a series of reactions on the vascular wall, which can cause serious circulatory system diseases such as atherosclerosis, stroke, and coronary thromboembolism (Patent Document 1).

[0004] It is also known that the general agonist adenosine diphosphate (ADP) plays an important role in the initiation and progression of arterial thrombus formation (Bernat et al., Thromb. Haemostas., vol. 70, pp. 812-826, 1993; Maffrand et al., Thromb. Haemostas., vol. 59, pp. 225-230, 1988; Herbert et al., Arterioscl. Thromb., vol. 13, pp. 1171-1179, 1993). ADP is involved in platelet aggregation, morphological changes, secretion, Ca 2+ ADP induces the influx and intracellular translocation of ADP and inhibition of adenylyl cyclase. Binding of ADP to platelet receptors is required to elicit ADP-induced platelet responses. There are at least three P2 receptors expressed on human platelets: the cation channel receptor P2X1, the G protein-coupled receptor P2Y1, and the G protein-coupled receptor P2Y12 (also called P2Yac or P2T). The P2X1 receptor is involved in rapid calcium influx and is activated by ATP. The role of the P2X1 receptor in the process of platelet aggregation is not fully understood. However, it has been suggested that the P2X1 receptor is involved in platelet shape changes (Rolf et al., Thromb Haemost. 85:303-308, 2001) and in platelet thrombus formation under high shear forces (Hechler et al., J. Exp. Med. 198:661-667, 2003). The P2Y1 receptor is involved in calcium mobilization, morphological changes, and initiation of aggregation. The P2Y12 receptor is involved in adenylyl cyclase inhibition and is required for efficient aggregation. (Hourani et al., "Platelet ADP Receptor Conference", La Thuile, Italy, March 29-31, 2000)

[0005] Antiplatelet agents have also been the subject of extensive research over the years as potential candidates for inhibiting thrombosis. Several drugs, including aspirin and dipyridamole, have come to be used as prophylactic antithrombotic agents. However, aspirin has no effect on ADP-induced platelet aggregation, so its effect on platelet aggregation is limited. Furthermore, aspirin is well known to often have side effects on the stomach, so it is not effective in many patients (Patent Document 2).

[0006] [Non-Patent Document 1] Hui-Lin Zhu, Yun-Wei Hu, Wei Qu, Jie Zhang, Er-Yan Guo, Xue-Yang Jiang, Wen-Yuan Liu, Feng Feng, Jian Xu. Littordial F, a novel phloroglucinol meroterpenoid from the leaves of Psidium littorale. Tetrahedron Letters, 60, 28, 1868-870, 2019. [Non-Patent Document 2] Ji-Cheng Shu, Hang-Qing Cui, Yin-Zheng Huang, Jian-Qun Liu, Hui-Lian Huang. A novel phloroglucinol and two new phenolic glycosides from Psidium littorale. Journal of Asian Natural Products Research, 19: 9, 854-861, 2017. [Non-Patent Document 3] Peng CY, Liu JQ, Shu JC, Zhang R. Study on megastigmane glycosides and lignan constituents from leaves of Psidium littorale. Zhong Yao Cai, 37, 12, 2201-2203, 2014. [Non-Patent Document 4] Yan C, Lee J, Kong F, Zhang D. Anti-glycated activity prediction of polysaccharides from two guava fruits using artificial neural networks. Carbohydr Polym, 98, 1, 116-121, 2013. [Patent Document 1] Special Publication No. 2003-507486 [Patent Document 2] Special Publication No. 2007-531737 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0007] Against this background, the inventors evaluated the platelet aggregation inhibitory effect in order to investigate the physiological activity of extracts from yellow strawberry guava fruit, and found that it has strong platelet aggregation inhibitory effects, in particular, inhibitory effects on both collagen-induced platelet aggregation and adenosine diphosphate-induced platelet aggregation. Furthermore, they searched for active ingredients using activity as an indicator, and found that the purine base adenosine is the active ingredient, thus completing the present invention. That is, the present invention aims to provide a novel use of yellow strawberry guava fruit extract and to provide a novel platelet aggregation inhibitor. [Means for solving the problem]

[0008] The features of the present invention for solving the above problems are as follows. 1. A collagen-induced platelet aggregation inhibitor containing yellow strawberry guava fruit extract as the active ingredient. 2. An inhibitor of adenosine diphosphate-induced platelet aggregation, the active ingredient of which is an extract of yellow strawberry guava fruit. 3. An inhibitor of induced platelet aggregation, the active ingredient of which is an extract of yellow strawberry guava fruit. 4. The agent according to any one of 1. to 3. above, wherein the extract of yellow strawberry guava fruit contains adenosine. 5. An extract of yellow strawberry guava fruit, characterized by containing adenosine. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the process of isolating active ingredients of yellow strawberry guava extract using the inhibitory effect on collagen-induced platelet aggregation as an indicator, and the evaluation results of their respective inhibitory effects on collagen-induced platelet aggregation. [Diagram 2] 1 is a graph showing the results of an evaluation of the inhibitory effect of a component (Fr. 22-27-3) isolated from an extract of yellow strawberry guava fruit on collagen-induced platelet aggregation in subject A. [Diagram 3] 1 is a graph showing the results of evaluating the inhibitory effect of a component (Fr. 22-27-3) isolated from an extract of yellow strawberry guava fruit on collagen-induced platelet aggregation in subject B. [Figure 4] 1 is a graph showing the results of evaluating the adenosine diphosphate-induced platelet aggregation inhibitory effect in subject C of a component (Fr. 22-27-3) isolated from an extract of yellow strawberry guava fruit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described in detail. The present invention is characterized in that it contains an extract of yellow strawberry guava fruit as an active ingredient.

[0011] As the extraction solvent, polar solvents such as water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, etc. Two or more of these solvents may be mixed. Preferably, active ingredients can be extracted efficiently by using aqueous ethanol or a mixture of these, that is, aqueous ethanol, as the extraction solvent.

[0012] When water is used as the extraction solvent, the type of water is not particularly limited, and tap water, distilled water, mineral water, alkaline ionized water, deep sea water, etc. can be used.

[0013] When using aqueous ethanol as the extraction solvent, the concentration of ethanol is not particularly limited, but it is preferable that the ethanol concentration is 10 to 90% (wt / wt), and preferably 20 to 80% (wt / wt). The reason for setting the ethanol concentration at 90% (wt / wt) or less is that if the ethanol concentration is too high, oil will easily dissolve in the aqueous ethanol.

[0014] The extraction temperature should be 20 to 80° C., preferably about 40 to 50° C. If the extraction temperature is too low, it becomes difficult to extract the active ingredients, and if the extraction temperature is too high, the active ingredients decompose, decreasing their physiological activity (health functionality).

[0015] As the extraction method, any method such as stirring extraction, continuous extraction, immersion extraction, countercurrent extraction, and supercritical extraction can be adopted, and any device can be used at room temperature or under reflux heating.

[0016] The specific extraction method is as follows: the raw strawberry guava fruit is placed in a processing tank filled with the extraction solvent, and the active ingredients are dissolved while stirring. For example, when using aqueous ethanol as the extraction solvent, about 2 to 100 times the amount (by weight) of the extraction solvent as the raw material is used, and extraction is carried out for about 30 minutes to 2 hours. After the active ingredients are dissolved in the solvent, the extract is obtained by filtering to remove the extraction residue.

[0017] The extract is then diluted, concentrated, purified, dried, and other processes according to conventional methods to produce the agent according to the present invention.

[0018] Moreover, the above-mentioned extract of yellow strawberry guava fruit preferably contains adenosine, since adenosine is the main active ingredient for inhibiting platelet aggregation. Here, the extract of the yellow strawberry guava fruit may be used as it is, or the adenosine may be concentrated and purified before use. In this case, the content of adenosine in the extract of the yellow strawberry guava fruit is not particularly limited as long as it functions as an active ingredient, but it is preferably, for example, 0.0001 to 0.05%, or more preferably 0.005% to 0.02%, because the adenosine functions as an active ingredient when it contains such a content.

[0019] In this case, the purification method includes, but is not limited to, a method in which the active ingredient is adsorbed by passing the extract through a synthetic adsorption resin, gel filtration resin, etc., and then eluted with methanol, ethanol, etc. to concentrate adenosine.

[0020] The platelet aggregation inhibitor of the present invention can be used as an ingredient in various foods and beverages. Examples of foods and beverages include general foods such as confectionery (gum, candy, caramel, chocolate, cookies, snacks, jellies, gummies, tablet candies, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, black tea, tea, carbonated drinks, sports drinks, etc.), health foods (tablets, capsules, etc.), nutritional supplements (nutritional drinks, etc.), functional foods, and foods for specified health uses. The agent of the present invention may be appropriately blended into these foods and beverages.

[0021] These foods and beverages can contain a variety of ingredients depending on their type, for example, the following food ingredients can be used: glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, flavorings, and preservatives.

[0022] As a specific manufacturing method, the agent of the present invention can be easily incorporated into food and beverages (instant foods, etc.) by spray-drying or freeze-drying the agent together with powdered cellulose and forming it into a powder, granules, tablets, or solution. The agent of the present invention can also be dissolved in, for example, fats and oils, ethanol, glycerin, or a mixture of these to form a liquid, which can then be added to beverages or solid foods. If necessary, it can also be mixed with a binder such as gum arabic or dextrin to form a powder or granules, which can then be added to beverages or solid foods.

[0023] When the agent of the present invention is applied to food and drink, the amount of active ingredient added is preferably 0.1% or less in total, since the main purpose is disease prevention and health maintenance.

[0024] The platelet aggregation inhibitor of the present invention may be used as a material for medicines (including pharmaceuticals and quasi-drugs). Pharmaceutical preparations can be produced by appropriately mixing the agent of the present invention with raw materials for pharmaceutical preparations. Examples of pharmaceutical raw materials that can be incorporated into the agent of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cocoa butter, hardened vegetable oil, kaolin, talc, etc.), binders (distilled water, saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrants (sodium alginate, agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, starch, lactose, powdered gum arabic, gelatin, ethanol, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption enhancers (quaternary ammonium bases, sodium lauryl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearates, polyethylene glycol, etc.), etc.

[0025] The platelet aggregation inhibitor of the present invention can generally be administered orally in the form of tablets, pills, soft or hard capsules, fine granules, powders, granules, liquids, etc., but may also be administered parenterally. When administered parenterally, it may be administered in the form of a solution or with the addition of a dispersant, suspending agent, stabilizer, etc., by local tissue administration, intradermal, subcutaneous, intramuscular, or intravenous injection. It may also be in the form of a suppository, etc. Furthermore, it may be administered as an eye drop.

[0026] The dosage may vary depending on the method of administration, the condition, the age of the patient, etc., but for adults, it is usually 1 to 1,000 mg of the extract of active ingredient per day (0.01 to 5 μg of adenosine), and for children, it is usually about 0.5 to 500 mg of the extract (0.005 to 2 μg of adenosine). The mixing ratio of the platelet aggregation inhibitor can be changed appropriately depending on the dosage form, but in general, when administered orally or by mucosal absorption, it is recommended to set the extract at approximately 0.1 wt% or less (0.0001 to 0.001 wt% as adenosine). Note that the dosage varies depending on various conditions, so in some cases a smaller dosage than the above is sufficient, and in other cases it may be necessary to administer more than the range. EXAMPLES

[0027] Examples of the present invention are described below. Note that the examples shown below are described to confirm the various actions and effects of the agent of the present invention obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods. Furthermore, in this example, sugar is removed and the active ingredient is isolated in order to identify the active ingredient, but any extract that contains the main active ingredient, adenosine, at the required concentration (for example, 0.001% to 0.01%) as the active ingredient will be effective.

[0028] Example: Preparation of Yellow Strawberry Guava Extract and Fractions The preparation of component fractions from yellow strawberry guava fruit was carried out using the method shown in Figure 1. That is, yellow strawberry guava fruit (4.61 kg) was crushed, 99% ethanol (14.25 kg) was added, and extraction was carried out with stirring at room temperature (24 hours). After filtration at room temperature, the filtrate was concentrated to dryness under reduced pressure to obtain an ethanol extract (540.7 g, yield: 11.7%). The ethanol extract (270 g) was applied to an XAD4 resin (1.2 L) column, washed with 5 times the volume of water, and eluted with methanol (5 times the volume of the column). This procedure was repeated twice, and the mixture was concentrated to dryness under reduced pressure to obtain the XAD4 methanol eluate (12 g). The methanol eluate (12 g) of XAD4 was subjected to ODS-based flash column chromatography (2 L) and gradient (water → methanol) fractionation was performed. The weight of the obtained fraction (Fr.)-1 (10% methanol eluate) was 9 g. The above Fraction 1 (8.5 g) was applied to HP-20 resin column (85 g) and eluted with water (500 mL), 50% methanol (500 mL), and methanol (500 mL), respectively, and concentrated to dryness under reduced pressure. The weights of the eluted fractions were 3.87 g for the water eluate, 1.89 g for the 50% methanol eluate, and 0.43 g for the methanol eluate. The 50% methanol eluate (1.5 g) was subjected to aminopropyl flash column chromatography (M) and gradient fractionation (90% acetonitrile → 70% acetonitrile) was performed. The yield of the 85-80% acetonitrile eluate (Fr. 22-27) was 100 mg. Fr. 22-27 (90 mg) was purified using three types of HPLC columns [(1) Aminopropyl (GL Science) 85% acetonitrile, (2) Cosmosil PAQ (Nacalai Tesque), 50% methanol, (3) Inertsil ODS-HL (GL Science) 40% methanol] to obtain a white powder, Fr. 22-27-3 (9.1 mg). The resulting white powder, Fr. 22-27-3, was found to be adenosine, a purine base compound, by comparing the NMR and MS spectral data with the values ​​described in the following literature. Literature: Cho BP Structure of oxidatively damaged nucleic acid adducts: pH dependence of the 13 C NMR spectra of 8-oxoguanosine and 8-oxoadenosine. Magnetic Resonance in Chemistry, 31, 1048-1053 (1993).

[0029] Test example: Evaluation of platelet aggregation inhibitory effect 1. Platelet aggregation inhibition test method (1) Collection of PRP and PPP Venous blood was collected from volunteers in the company. Blood was collected from the median vein of the forearm using a disposable syringe with a 22 G needle, and 4.5 mL was placed in a centrifuge tube containing 3.8% sodium citrate (0.5 mL) in advance. The blood was thoroughly mixed with the anticoagulant and left to stand at room temperature for 20 minutes. Platelet-rich plasma (PRP) was prepared by centrifuging the tubes at room temperature for 10 minutes at 120 G without using the brake. After centrifugation, the supernatant was collected to obtain PRP. PPP (platelet poor plasma) was obtained by centrifuging the sediment after collection of PRP (platelet rich plasma) at 2000 G for 10 minutes.

[0030] (2) Platelet aggregation inhibition test Platelet aggregation tests were performed using a TAIYO platelet aggregation analyzer PRP313M. PRP (200 μL) was heated at 37°C for 30 seconds while rotating with a magnetic stirrer. The sample solution (22 μL) of this example was added and reacted for 1 minute, after which 20 μg / mL collagen solution (20 μL) or 50 μM adenosine diphosphate (ADP) solution was added to induce aggregation. After that, aggregation was recorded for 10 minutes using PPP (300 μL) as a control.

[0031] 2. Measurement results and effects of the embodiment (1) Isolation of components based on their platelet aggregation inhibitory activity Figure 1 shows the results of evaluating the maximum aggregation rate of collagen-induced platelet aggregation using the strawberry guava extract fraction as the sample solution in this example. Strong activity was observed in the methanol (MeOH) eluate from which the XAD-treated water eluate (carbohydrate fraction) had been removed. Next, as shown in Figure 1, this was subjected to ODS flash chromatography, and the fractions fractionated with water / methanol were similarly evaluated, and the activity was found to be concentrated in the fraction (Fr. 1).Fractions were then fractionated with HP-20 and similarly evaluated, and a strong concentration of activity was found in the 50% methanol fraction. The 50% methanol fraction was further purified by flash chromatography and HPLC, revealing that Fr. 22-27-3 (ie, adenosine) was the only active component.

[0032] (2) Effect of isolated component Fr. 22-27-3 on collagen-induced platelet aggregation 1) Subject A's blood In this example, a test was performed on the blood of subject A using Fr. 22-27-3 as the sample solution. The results are shown in Table 1 and Figure 2. As shown in Table 1 and Figure 2, 100 μg / mL of the component (Fr. 22-27-3) showed the same inhibition of collagen-induced platelet aggregation as aspirin (200 ug / mL). [Table 1]

[0033] 2) Blood of Subject B In this example, a test was conducted on the blood of subject B using Fr. 22-27-3 as the sample solution. The results are shown in Table 1 and Figure 2. The results for subject B are shown in Table 2 and Figure 3 below. As shown in Table 2 and Figure 2, a similar test was conducted on a different subject, and the component (Fr. 22-27-3) showed stronger inhibition of coagulation. [Table 2]

[0034] (3) Effect of isolated component Fr. 22-27-3 on ADP-induced platelet aggregation 1) Blood of Subject B In this example, ADP-induced aggregation was examined using platelets obtained from the blood of subject B using Fr. 22-27-3 as the sample solution. The results are shown in Table 3. As shown in Table 3, Fr. 22-27-3 (200 μg / mL) was found to have a stronger platelet aggregation inhibitory effect than aspirin. [Table 3] 2) Blood of Subject C In this example, Fr. 22-27-3 was used as the sample solution to similarly examine the effect of Fr. 22-27-3 on ADP-induced platelet aggregation using platelets from a third subject, C. The results are shown in Table 4 and Figure 4. As a result, as shown in Table 4 and Figure 4, aspirin hardly inhibited aggregation, but the component Fr. 22-27-3 inhibited platelet aggregation at 50 to 200 μg / mL. [Table 4]

[0035] These results indicate that the yellow strawberry guava extract has a strong platelet aggregation inhibitory effect, and that the active ingredient is Fr. 22-27-3, i.e., adenosine.

[0036] The following are examples of formulations of the platelet aggregation inhibitor according to the present invention (adenosine-containing yellow strawberry guava extract, recommended intake amount 100 mg). Note that the following formulation examples do not limit the present invention. Formulation example 1: Chewing gum Sugar 53.0wt% Gum base 20.0 Glucose 10.0 Starch syrup 16.0 Fragrance 0.5 Platelet aggregation inhibitor 0.5 100.0wt%

[0037] Mixture Example 2: Gummies Reduced starch syrup 40.0wt% Granulated sugar 20.0 Glucose 20.0 Gelatin 4.7 Wednesday 9.68 Yuzu juice 4.0 Yuzu flavor 0.6 Dye 0.02 Platelet aggregation inhibitor 1.0 100.0wt%

[0038] Mixture example 3: Candy Sugar 50.0wt% Syrup 33.0 Wednesday 14.4 organic acid 2.0 Fragrance 0.2 Platelet aggregation inhibitor 0.4 100.0wt%

[0039] Mixture example 4: Yogurt (hard / soft) Milk 41.5wt% Skim milk powder 5.8 Sugar 8.0 Agar 0.15 Gelatin 0.1 Lactic acid bacteria 0.005 Platelet aggregation inhibitor 0.4 fragrance trace amount water residue 100.0wt%

[0040] Formulation example 5: Soft drink Fructose glucose liquid sugar 30.0wt% Emulsifier 0.5 Platelet aggregation inhibitor 0.02 Fragrance (appropriate amount) Purified water remainder 100.0wt%

[0041] Formulation example 6: Tablet confectionery Sugar 71.9wt% Glucose 19.0 Sucrose fatty acid ester 0.2 Platelet aggregation inhibitor 5.0 Purified water 3.9 100.0wt%

[0042] Formulation example 7: Soft capsule Brown rice germ oil 37.0wt% Yuzu seed oil 40.0 Emulsifier 12.0 Platelet aggregation inhibitors 10.0 100.0wt%

[0043] Formulation Example 8: Tablets Lactose 35.0wt% Crystalline cellulose 30.0 Starch decomposition product 10.0 Glycerin fatty acid ester 5.0 Platelet aggregation inhibitors 20.0 100.0wt% [Industrial Applicability]

[0044] As described above, the present invention can provide a novel platelet aggregation inhibitor.

Claims

1. A collagen-induced platelet aggregation inhibitor containing yellow strawberry guava fruit extract as an active ingredient.

2. An inhibitor of adenosine diphosphate-induced platelet aggregation, the active ingredient of which is an extract of yellow strawberry guava fruit.

3. A platelet aggregation inhibitor containing yellow strawberry guava fruit extract as the active ingredient.

4. The agent according to any one of claims 1 to 3, characterized in that the extract of the yellow strawberry guava fruit contains adenosine.

5. An extract of yellow strawberry guava fruit characterized by containing adenosine.

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