KR102254421B1 - Pharmaceutical composition comprising the extracts from the flesh and seed of passion fruit as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an antithrombotic composition containing a mixed extract of passion fruit (Passion Fruit, Passiflora edulis ) pulp and seeds as an active ingredient, and more particularly, ethanol extract from a mixture of pulp and seeds of passion fruit selected with water It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and containing it as an active ingredient, and a health functional food. The mixed extract of passionfruit pulp and seeds as active ingredients of the pharmaceutical composition for preventing or treating thrombosis of the present invention and health functional food, as demonstrated through the examples of the present specification, inhibits thrombosis-related enzymes and blood coagulation It exhibits strong antithrombotic activity by inhibition of factor, does not show hemolytic activity against human red blood cells at all, has excellent thermal stability, and has the effect of inhibiting blood clotting factors and enzymes related to thrombogenesis even in acidic conditions of pH 2 and in plasma. Since there is no loss, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation.The active ingredient is processed into various forms such as extract, powder, pills, tablets, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared in this possible form.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACTS FROM THE FLESH AND SEED OF PASSION FRUIT AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention is Passion Fruit, Passiflora edulis ) Relates to an antithrombotic composition containing a mixed extract of pulp and seeds as an active ingredient, and more specifically, an ethanol extract is prepared from a mixture of pulp and seeds of passion fruit selected by washing with water, and contains it as an active ingredient. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation, and a health functional food.

Blood as a component of the human body has various important functions such as transporting and buffering oxygen, nutrients and waste products, maintaining body temperature, maintaining osmotic pressure and maintaining ionic equilibrium, maintaining moisture schedule, controlling liquid properties, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation by complementarily regulating the blood coagulation system and the thrombosis dissolution system in the body. Among them, the mechanism of the blood coagulation system is after platelets adhere and aggregate on the blood vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombus is formed around the platelet aggregate.

On the other hand, the formation of fibrin thrombi leads to activation of thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, and fibrin monomers are polymerized by calcium, platelets and endothelium. It binds to the cell and forms a fibrin polymer that is cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a pivotal role in the formation of blood clots by activating platelets, factors V, and VII to promote blood clotting reactions. Therefore, the substance inhibiting the activity of thrombin can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation. On the other hand, in the endogenous thrombogenic pathway, it is known that sequential activation of factor XII, factor XI, factor XII, factor IX, and factor X, followed by activation of prothrombin, ultimately activates thrombin. It has become a target for the development of sex-disease therapeutics, and in the case of exogenous thrombus generation pathways, thrombus formation is known by activation of factor II (prothrombin), factor V, factor VII, and factor X. To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also bleeding side effects, gastrointestinal disorders and irritability. Its use is limited due to reactions and the like.

On the other hand, Passion Fruit, Passiflora edulis ) is one of the most common tropical fruits with a lot of skins, and is a vine perennial plant in the dicotyledonous plant of the Passionaceae family, and is also called Passionfruit, Passionflower, Maracas, or Pascalaceae. The origin is known as Brazil, the size of the fruit is about 5cm, and the fruit, skin and seeds are used for food (Korea Food and Drug Administration's Food Ingredients Database).

Passionfruit has excellent efficacy in relieving anxiety and reducing stress (Klein N et al., Phytother Res. 2014, 28: 706-713; Barbosa, Paulo R et al., 2008. J. Medicinal Food 11: 282-288), and antioxidant activity. (Zeraik ML et al. 2011, Food Chem. 128: 259-265), melanogenesis inhibitory activity (Jorge, AT S et al., 2012, Int. J. Cosmetic Sci. 34: 435-440) is known.

Peptide components isolated from pulp juice did not inhibit thrombin, which plays a central role in blood coagulation, and did not affect thrombin time, while increasing prothrombin time due to inhibition of prothrombin and increased aPTT due to inhibition of blood coagulation factors. Anticoagulant activity is known (Sato, Ana Claudia et al., 2012, Protein Peptide Lett. 19: 501?508). However, the anticoagulant active ingredient of the juice is difficult to use as an effective anticoagulant because there is no thrombin inhibition, and it is known that the anticoagulant activity rapidly decreases during heat treatment.

On the other hand, piceatannol, which has strong antibacterial activity and antidiabetic activity, is known in passionfruit seeds (Maruki-Uchida H et al., 2013, Biol Pharm Bull 36:845-849), and digestive enzyme trypsin inhibitory activity (Botelho- Junior S et al., 2008, J. Agric. Food Chem. 56: 9404-9409), anti-inflammatory activity (Vargas AJ et al., Fitoterapia. 2007 78: 112-119), antioxidant activity (Sunitha, M. 2009. Indian J. Pharma.Sciences 71: 310-311) has been reported.

In addition, studies on passion fruit bark extract are very limited, and until now, the effect of bark extract on preventing ulcerative colitis (Cazarin, Cinthia Bb, etc., 2014, Exp. Biology Medicine 239: 542-551), antioxidant activity (Kandandapani S, etc.) , Chin J Nat Med. 2015 13: 680-686; da Silva, JK et al., 2014. LWT-Food Sci. Tech. 59: 1213-1219) and antihypertension (Lewis, BJ et al., 2013, The J. Nutr) Biochem. 24: 1359?1366) is known, and the effect of promoting microbial growth in the colon by dietary fiber of the skin and enhancing the synthesis of short chain fatty acid (da Silva, JK et al., 2014. LWT-Food Sci. Tech. 59: 1252-1257) has been reported.

However, until now, the strong antithrombotic activity of the mixed extract of passionfruit pulp and seed has not been reported, and the anticoagulant activity by inhibiting thrombin, prothrombin, and blood coagulation factor while having excellent heat resistance has not been known at all.

As a patent related to passion fruit known to date, Korean Patent Laid-Open No. 10-2015-0064314 discloses [a cosmetic composition containing apple mango extract, passion fruit extract, dragon fruit extract, red kiwi extract and atemoya extract as active ingredients] , Patent Publication No. 10-2016-0059271 [composition for improving scalp condition comprising a flower extract of Paciflora edulis], Patent Publication No. 10-2016-0058832 [lipid extract obtained from Passiflora seeds] Is disclosed, and US Patent Publication No. US-0074312 discloses [extracts of passionflower seeds and cosmetic, pharmaceutical, dermatological and nutritional compositions containing the same], and US-0076769 discloses passionfruit for cancer treatment. Herbal compositions containing seed extracts have been disclosed, and the anti-inflammatory effect of the bark extract in Japanese Patent No. JP-0278762 [Anti-caries composition: Cariostatic agent], JP-0350433 as a blood pressure lowering agent and JP-0302659 Is known. However, until now, the antithrombotic effect of the mixed extract of passionfruit pulp and seed by inhibiting the strong thrombogenic enzyme and blood coagulation factor has not been known.

JP 2007-302659 A

Sato, Ana Claudia et al., 2012, Protein Peptide Lett. 19: 501-508

The present invention was conceived to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to prepare an extract obtained by mixing the pulp and seeds of passionfruit, and prepare an antithrombotic composition containing the same as an active ingredient. I want to provide.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a mixed extract of Passiflora edulis pulp and seeds as an active ingredient.

The mixed extract as an active ingredient of the pharmaceutical composition is preferably an ethanol extract.

In addition, the present invention is Passiflora edulis ) Provides a blood coagulation inhibitor containing a mixed extract of pulp and seeds as an active ingredient.

The mixed extract as an active ingredient of the blood coagulation inhibitor is preferably an ethanol extract.

In addition, the present invention is Passiflora edulis ) Provides a health functional food for preventing or improving thrombosis containing a mixed extract of pulp and seeds as an active ingredient.

The mixed extract as an active ingredient of the health functional food is preferably an ethanol extract.

The mixed extract of passionfruit pulp and seeds as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and a health functional food, as demonstrated through the examples of the present specification, inhibits thrombosis-related enzymes and blood coagulation It exhibits strong antithrombotic activity by inhibition of factor, does not show hemolytic activity against human red blood cells at all, has excellent thermal stability, and has the effect of inhibiting blood clotting factors and enzymes related to thrombogenesis even in acidic conditions of pH 2 and in plasma. Since there is no loss, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is processed into various forms such as extract, powder, pills, tablets, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared in this possible form.

1 shows the passionfruit fruit used in the experiment,
2 shows a cross section of a passion fruit fruit,
3 shows a mixture of passionfruit pulp and seeds,
Figure 4 is a photograph showing a passion fruit peel.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of the passionfruit fruit extract, the inventors of the present invention prepared an extract and a peel extract of a mixture of passionfruit flesh and seeds prepared by a certain method, and evaluated the antithrombotic activity of the extract. The mixed extract of fruit flesh and seeds was recovered as an antithrombotic component, and the mixed extract did not show any hemolytic activity against human red blood cells, but had excellent thermal stability and acid stability. It was intended to be used as a preventive or therapeutic/improving pharmaceutical composition and health functional food.

Specifically, in order to develop a pharmaceutical composition and a health functional food for preventing or treating/improving thrombosis by using passion fruit, which is known to have various physiological activities in the private sector, the present inventors target a mixture of passion fruit and a mixture of seeds and skin. Each of the ethanol extracts was prepared, and the antithrombotic activity of these extracts was inhibited by thrombin (Thrombin Time), prothrombin (Prothrombin Time), and blood coagulation factor (activated part thromboplastin time, activated Partial Thromboplastin Time: aPTT). ) Was evaluated, and it was confirmed that the mixed extract of passionfruit pulp and seed has excellent antithrombotic activity due to strong anticoagulation inhibition.

The pulp and seeds of passion fruit are difficult to separate substantially separately and are used for food as they are, so an ethanol extract was prepared by extracting a mixture of pulp and seeds with ethanol, and 11.7 g per 100 g can be recovered. The ethanol extract exhibited 2.51 times extended thrombin time, 1.87 times extended prothrombin time and 1.24 times extended AP time compared to the no additives at a concentration of 5 mg/ml. , Ana Claudia et al., 2012, Protein Peptide Lett. 19: 501?508). In addition, it was confirmed that the active extract did not show hemolytic activity against human red blood cells and thus did not induce acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, comprising a mixed extract of the pulp and seeds of passionfruit as an active ingredient.

The mixed extract of passionfruit pulp and seed, which is an active ingredient of the pharmaceutical composition, may be a hot water extract or an ethanol extract, and is preferably an ethanol extract.

In addition, the present invention provides a blood coagulation inhibitor containing a mixed extract of the pulp and seeds of passionfruit as an active ingredient.

The mixed extract of passionfruit pulp and seed, which is an active ingredient of the blood coagulation inhibitor, may be a hot water extract or an ethanol extract, and is preferably an ethanol extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, comprising a mixed extract of the pulp and seeds of passion fruit as an active ingredient.

The mixed extract of passionfruit pulp and seed, which is an active ingredient of the health functional food, may be a hot water extract or an ethanol extract, and is preferably an ethanol extract.

Hereinafter, a method for preparing a mixed extract of passionfruit pulp and seeds of the present invention and an efficacy experiment will be described in more detail.

The inventors of the present invention in order to achieve the object of the present invention, separating the peel, pulp and seeds from the selected, washed passionfruit fruit; Preparing an extract from the mixture of the separated pulp and seeds; Experiments of the antithrombotic activity evaluation and stability evaluation steps of the extract were performed.

The "mixed extract of passion fruit pulp and seeds" included in the composition of the present invention includes the steps of recovering pulp and seeds from passion fruit fruits, extracting the mixture of pulp and seeds with a solvent, and using a filter net of 0.06 mm or less in the extract. It can be obtained by filtering using and concentrating under reduced pressure.

The solvent used in the present invention includes water (cold water, hot water), alcohol, anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), and a mixed solvent of the lower alcohol and water. Hot water, or 95% ethanol extraction is most preferred.

As a preferred embodiment, the mixed extract of passionfruit pulp and seeds of the present invention may be used after extraction with hot water or ethanol. Here, the hot water or ethanol extract may be sequentially or fractionated with an organic solvent of hexene, ethyl acetate and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time by using the mixed extract of the fruit flesh and seeds of passionfruit at a concentration of 5 mg/ml, it was extended by 2.51 times, 1.87 times, and 1.24 times, respectively, compared to the non-added group. Time was shown to show excellent thrombin inhibition and prothrombin inhibition. This inhibition is different from the fact that the existing Peptide anticoagulant of Passionfruits did not affect thrombin time and prolonged AP time (Sato, Ana Claudia et al., 2012, Protein Peptide Lett. 19: 501 to 508). This is a contrasting result. The anticoagulant activity of the mixed extract of the pulp and seeds of the passionfruit fruit of the present invention was concentration-dependent, and at the concentration of 7mg/ml, the thrombin time and prothrombin prolonged 7.56 times, 15 times or more, and 2.45 times, respectively, compared to the non-added group. Time and AP time are shown.

These results indicate that the mixed extract of passionfruit pulp and seeds exhibited strong thrombin, prothrombin, and blood coagulation factor inhibition, so that it can be used as a thrombosis treatment or specifically an anticoagulant (blood coagulation inhibitor) that can replace aspirin, which has a high risk of side effects such as gastrointestinal disorders. It means that it can be developed.

The mixed extract of passionfruit pulp and seeds of the present invention may be prepared as a powder through a conventional powdering process such as vacuum drying, freeze drying, or spray drying. They are not decomposed by various degrading enzymes in plasma, and they maintain their activity even at a heat treatment of 100°C and a pH of 2 in the human stomach.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. Such diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis. , Deep vein thrombosis, lower extremity swelling, pain, and acute peripheral arterial atresia. As venous thrombosis, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention is formulated according to a conventional method for each purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injectable solutions. It may be formulated and used in various forms such as, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, and the like, in the pharmaceutical composition. It is formulated by mixing sucrose, lactose, gelatin, and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

As a preferred embodiment, as a liquid formulation for oral use, a suspension, a liquid solution, an emulsion, a syrup, etc. may be exemplified, and various excipients other than water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweetening agents, Fragrances, preservatives, and the like may be included.

As a preferred embodiment, the preparation for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, a suppository, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyloleate. Injectables may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

As a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and is generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kilogram of body weight daily. Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

In the present invention, the "subject" is not particularly limited, but includes, for example, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a mouse, a rabbit, or a guinea pig. And, preferably, it means a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways such as foods and beverages effective in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. .

In general, the active ingredient of the present invention may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in an amount of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The health functional food of the present invention may contain the compound as an essential component in the indicated ratio as an additional component, as well as food supplementary additives acceptable for food, such as natural carbohydrates and various flavoring agents.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As the flavoring agent, natural flavoring agents such as stevia such as taumatin, rebaudioside A, or glysirhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes a variety of nutrients, vitamins, minerals, synthetic flavors and natural flavors, and other flavoring agents, coloring agents and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, a specific method of the present invention will be described in more detail through examples. The following examples are only one preferred specific example of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example One: Passion Fruit Preparation of mixed extract of pulp and seeds

After purchasing the passion fruit fruit from Vietnam, foreign substances were removed, and separated into a mixture of pulp and seeds and a skin (FIGS. 1 to 4), respectively, and used to prepare an extract. When preparing an ethanol extract of a mixture of pulp and seeds, 10 times ethanol (95%, Deoksan, Korea) was added to the sample, extracted once at room temperature, collected and filtered, and concentrated under reduced pressure to prepare a powder. The extraction efficiency and component analysis results of the ethanol extract are shown in Table 1. On the other hand, ethanol was extracted from the skin, which is the non-edible part of passionfruit, and this was used as a control. The contents of total polyphenols, total flavonoids, total sugars and reducing sugars were measured by component analysis of the prepared extracts and fractions. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. Tannic acid was used as a standard reagent. For the total flavonoid content, each sample was extracted with methanol for 18 hours, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1N NaOH was added again, and the absorbance was measured at 420 nm after 1 hour reaction at 37°C. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Passion Fruit Analysis of Yield and Components of Mixed Extract of Pulp and Seed and Peel Extract

As a result, as shown in Table 1, the ethanol extraction yield of the mixture of the pulp and seeds of the passionfruit fruit was 11.7%, which was 4 times higher than the extraction yield of the peel, and the total polyphenol and total flavonoid content was 0.7mg/g. And 0.5 mg/g, which was very low. On the other hand, the skin showed high total polyphenol and total flavonoid contents of 26.5mg/g and 12.9mg/g, and the total sugar content was 51% of the mixture of pulp and seeds.

Example 2: Passion Fruit Evaluation of Blood Coagulation Inhibitory Activity of Mixed Extract of Pulp and Seed and Bark Extract

The blood coagulation inhibitory activity (thrombogenicity inhibitory activity) of the mixed extract of passionfruit pulp and seed prepared in Example 1 and the peel extract were evaluated, and the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928). Tea time was measured and evaluated. Plasma was used as a commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time, and AP time were measured by the following procedure.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA) at 37°C, 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was mixed. After addition, the time until plasma coagulation was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 30.5 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Prothrombin time ( prothrombin time)

70μl of standard plasma (MD Pacific Co., China) and 10μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A(Japan), heated at 37℃ for 3 minutes, and then 130μl of PT reagent was added and plasma coagulated. The time until completion was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 16.7 seconds. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37°C for 3 minutes, 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and then again at 37°C for 3 minutes. Incubated for minutes. Thereafter, after 50 μl CaCl ₂ (35 mM) was added, the time until plasma coagulation was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 58.1 seconds. The results of aPTT were expressed as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 2] Passion Fruit Evaluation of Blood Coagulation Inhibitory Activity of Mixed Extract of Pulp and Seed and Bark Extract

As a result, as shown in Table 2, the mixed extract of the fruit flesh and seeds of passionfruit showed superior anticoagulant activity than the peel extract, and specifically, in the case of the mixed extract of flesh and seeds, no addition at 7mg/ml concentration. The thrombin time is 7.56 times longer than that of the sphere. Prothrombin time extended by 15 times and aPTT extended by 2.45 times showed strong anticoagulant activity. These results suggest that the mixed extract of passionfruit pulp and seeds can be developed as a powerful antithrombotic agent that can replace aspirin, which indicates gastrointestinal disorders.

Example 3: Passion Fruit Evaluation of Human Red Blood Cell Hemolytic Activity of Mixed Extracts of Pulp and Seed

Passionfruit pulp and seeds are used for edible use, and are registered as edible in the Food Ingredient Database of the Korean Food and Drug Administration. Therefore, it is judged that the mixed extract of pulp and seed has no specific toxicity. In order to evaluate the potential acute toxicity of the mixed extract of passionfruit pulp and seeds, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 3. At this time, the hemolytic activity was evaluated according to the previous report (Ho-Yong Son, 2014, Korean J. Microbiol. Biotechnol. 42: 285~292), and simply 100 μl of human red blood cells washed three times with PBS was added to a 96-well microplate. 100 μl of sample solution of various concentrations was added and reacted for 30 minutes at 37°C. After that, the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. It was measured. DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for hemolysis of red blood cells. The hemolytic activity was calculated using the following formula.

( % ) Hemolysis = [( Abs .S- Abs .C)/( Abs .T- Abs .C)]×100

Abs. S: absorbance of the sample addition port

Abs. C: absorbance of the DMSO addition port,

Abs . T: Triton X-100 Addition Absorbance.

[Table 3] Passion Fruit Hemolytic Activity of Human Red Blood Cells of Mixed Extracts of Pulp and Seed

First, it was confirmed that DMSO and distilled water used as control cells had no red blood cell hemolytic activity, and Triton X-100 hemolytic 100% red blood cells at a concentration of 1 mg/ml. On the other hand, hemolytic activity was not shown in the extract of the mixture of passionfruit pulp and seeds.

Example 4: Passion Fruit Plasma, acid and thermal stability evaluation of mixed extracts of pulp and seeds

Plasma stability, thermal stability, and acid stability against anticoagulant activity were confirmed for the mixed extract of passionfruit pulp and seeds obtained in Example 1 above. The mixed extract maintained excellent activity without loss of anticoagulant activity even when heat treatment at 100° C. for 1 hour, treatment at pH 2 (0.01M HCl) for 1 hour, and treatment in plasma for 1 hour. Therefore, when considering the component analysis results of Example 1 and the above stability results, the active substance of the mixed extract of passionfruit pulp and seeds is expected to be a polysaccharide substance. The above results suggest that the mixed extract of passionfruit pulp and seed can be practically used as an antithrombotic agent, and it is believed that it can complement and replace aspirin, which has reported side effects such as gastrointestinal disorders.

Claims (4)

Passiflora ( Passiflora edulis ) A pharmaceutical composition for the prevention or treatment of thrombosis containing ethanol extract of pulp and seeds as an active ingredient. delete A blood coagulation inhibitor comprising the active ingredient according to claim 1. Health functional food for preventing or improving thrombosis comprising the active ingredient according to claim 1.

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