KR20210047781A - Pharmaceutical composition comprising the extract of prune 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 a pharmaceutical composition and a health functional food for preventing or treating thrombosis, containing an extract of Prunus domestica as an active ingredient, and more specifically, to a pharmaceutical composition and a health functional food for preventing or treating/alleviating thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, comprising Prunus domestica, a Big Purple variety, as an active ingredient. The extract of Prunus domestica of the present invention as the pharmaceutical composition and the health functional food for preventing or treating/alleviating thrombosis exhibits strong antithrombotic activity due to an aggregation inhibitory effect of platelets that play a role in initiating thrombus formation along with inhibition of thrombus formation-related enzymes and blood coagulation factors, while showing no hemolytic activity against human red blood cells at the same time. In addition, the Prunus domestica extract of the present invention has excellent thermal stability and no loss of blood coagulation factor inhibitory effect and thrombus formation-related enzyme inhibitory effect even under acidic conditions of pH 2 and in plasma, and thus is expected to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation. The active ingredient may be easily processed into various forms such as extract, powder, pill, tablet, etc., has an excellent effect of being formulated in a form that can be taken at all times, and thus can be very advantageously used in the pharmaceutical industry and the food industry.

Description

Pharmaceutical composition and health functional food for preventing or treating thrombosis containing furun extract as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PRUNE AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing a prune (Prunus domestica ) extract as an active ingredient, and more specifically, a Prune of the Big Purple variety as an active ingredient It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, 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.

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. Finally, fibrin monomers are produced from fibrinogen. Fibrin monomers are polymerized by calcium, and platelets and endothelial cells are formed. It binds and forms a fibrin polymer that is cross-linked by factor XIII, resulting in 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, thrombin activity inhibitors can be used as very useful prophylactic and therapeutic agents for various thrombotic diseases caused by excessive blood coagulation. On the other hand, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by prothrombin activation, is known to ultimately activate thrombin. Being a target.

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. It is a situation in which its use is limited to such.

On the other hand, Prunus domestica is a fruit of a shrub belonging to the Rosaceae family, and is also called "sheep plum", and has excellent taste and aroma, and its origin is known as the Caucasus Mountains near the Caspian Sea in Western Asia. Prunes are externally distinguished from Asian plums (Prunus salicina , Prunus triflora ), which are called Japanese or Chinese plums, in shape and size, and are also clearly differentiated from rosacea plums (Prunus mume ) and apricots ( Prunus armeniaca).

Prunes are mainly harvested before maturation, dried, processed into dried plums, and sold. This is because, unlike plums, the fruit surface is often cracked during maturity, which makes it difficult to harvest high-quality prunes. In addition, prunes do not rot easily, unlike plums, even if dried in the presence of seeds after harvesting. Potassium sorbate is made on the surface during the drying process, making it appear as if white powder was sprinkled on the surface, showing antibacterial activity, so it is easily rotted. There is a characteristic that does not work. Prune contains a large amount of protein, lipids, vitamins, and minerals, and has been used as a constipation improvement agent since the past, especially because it has a significant amount of dietary fiber, and has the effect of improving eye health, osteoporosis, heart disease prevention, and hypertension. It is known from.

Studies related to prunes are insignificant compared to plums, and research on oriental plums ( Prunus salicina ) has been intensively conducted. In various research literature, Plum (Oriental Plum) has been reported to have antioxidant, antibacterial, and anti-inflammatory activities (Ye-Na Heo, 2007, Daegu University Master's Thesis), and nutritional and physicochemical components (Sung Yoon-Jeong, 2002, J. Kor. Soc. Agric. Chem. Biotechnol. 45, 134-137). Recently, platelet aggregation inhibitory activity has been reported in a mixture of bamboo leaf extract and plum ( Prunus mume ) extract (Son E et al., 2017. BMC Complement Altern Med. 2017 17:541. doi: 10.1186/s12906-017-2032) -5; Jin WY et al., 2013. Integr Med Res. 2:70-75).

In addition, furun has been reported to induce growth inhibition and death of human colon cancer cells (Fujii Takashi et al., 2006, Nutr. Sci. Vitamonol. 52, 389-391), antibacterial (Yaqeen Zahra, 2013, Pakistan Journal of pharmaceuticals) sciences 26: 409-414) and cancer cell killing effect (Miljic, Uros, 2016, Journal of the Institute of Brewing 122: 342-349), antioxidant activity (Usenik Valentina et al., 2013, J. Science of Food and Agriculture 93: 681 -692) is known. In addition, potent antioxidant activity has also been reported in the leaves of furun (Stierlin Emilie et al., 2018, J. Sci. Food Agricul. 98, 726-736), and the extract oil from furun leaves harvested in August has potent antioxidant activity and butyrylcholinesterase inhibitory activity. It has been reported (Marco Bonsi, 2019, Antioxidant, 8, 2; doi:10.3390/antiox8010002), and it has been suggested that the prevention of neurodegenerative diseases caused by inflammation and death of neurons may be helpful. In addition, there have been reports of Purunusides with α-Glucosidase inhibitory activity from shoots of furun (Shaheen Kosar et al., 2009, Purunusides AC, α-Glucosidase Inhibitory Homoisoflavone Glucosides from Prunus domestica. The sprouts of the furun were dried and then extracted with ethanol, the extract was sequentially fractionated with an organic solvent, and then the butanol fraction was obtained through silica-gel chromatography to obtain a prunuside material, and the antithrombotic effect of the furun fruit has been reported so far. none.

Among the patents related to platelet aggregation, among plants of the Prunus family, the platelet aggregation inhibitory activity of the extract of Prunus lannesiana is disclosed as International Patent WO-0039249, and is disclosed in Japanese Patent JP-0029710, but other Prunus family Plant platelet aggregation inhibitory activity is unknown. Patents related to prunes include "Prune juice production using cellulase" in Korean Patent No. 10-1731232, "Prune juice production using cellulase" in US-4371552, Japanese patent JP- 0238079 Tea manufacturing (Leaf tea and beverage therefrom and product from its extract evaporation to dryness), JP-0002967 tyrosinase activity inhibitor and cosmetic containing the same (Tyrosinase activity inhibitor and cosmetic containing the same) and JP-0002964 The use of cosmetics through hyaluronidase activity inhibitor and humectant and cosmetic containing the same, and the manufacturing method and use of furun juice as a urease inhibitor are registered in JP-0193477.

In addition, Korean Patent Application Publication No. 10-2007-0065665 "whitening cosmetic composition", No. 10-2003-0009739 "a pharmaceutical composition for the treatment of constipation", No. 10-2016-0089117 "Prune extraction and filtration method" , No. 10-2016-0089118 discloses "a feed additive for fisheries using residues extracted from prunes", and No. 10-2003-0096615 discloses "a composition of a functional food with excellent constipation improvement effect and a method for manufacturing the same". US Patent Publication No. US-0360062 discloses "prune-based nutrient-rich materials and related processes", and Japanese Patent Application Publication No. JP-0008938 discloses "antitumor agent containing extract of leaves of plants belonging to rosaceae prunus". However, to date, no studies on the antithrombotic activity of furun have been known.

Son E et al., 2017. BMC Complement Altern Med. 2017 17:541. doi: 10.1186/s12906-017-2032-5. Jin WY et al., 2013. Integr Med Res. 2:70-75.

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 provide a pharmaceutical composition for preventing or treating thrombosis containing a furun extract as an active ingredient and a health functional food It is to do.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, containing a furun extract of a big purple variety as an active ingredient.

It is preferable that the prune of the big purple cultivar is a mature fruit of 130 days or more after flowering.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing the prune extract of the big purple variety as an active ingredient.

It is preferable that the prune of the big purple cultivar is a mature fruit of 130 days or more after flowering.

Prune extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention is due to the inhibitory effect of platelet aggregation, which plays a role in the initiation of thrombus formation, as well as inhibition of blood clotting factors and enzymes related to thrombus formation. At the same time, it exhibits strong antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and shows a loss of blood coagulation factor inhibitory effect and blood clotting-related enzyme inhibitory effect even in acidic conditions of pH 2 and plasma. Therefore, 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., so that it can be taken at all times. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared with.

FIG. 1 is a diagram showing the mature fruits of the President, Big Purple, Black King, Stanley and Mountain varieties used in the present invention from the left,
Figure 2 shows the cultivars of big purple harvested from the left at 100 days after flowering, 114 days after flowering, 121 days after flowering, and 134 days after flowering,
3 is a cross-sectional view of a big purple variety prunes harvested from the left at 100 days after flowering, 114 days after flowering, 121 days after flowering, and 134 days after flowering,
Figure 4 shows the human platelet aggregation inhibitory activity of various varieties of furun extract: 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: solvent control (water), 4: President furun hot water extract (0.25mg/ml), 5: Big Purple Prune Hot Water Extract (0.25mg/ml), 6: Black King Prune Hot Water Extract (0.25mg/ml), 7: Stanley Prune Hot Water Extract (0.25mg/ml), 8: San Purun hot water extract (0.25mg/ml).
Figure 5 shows the human platelet aggregation inhibitory activity of the furun extract of the big purple variety harvested at various times: 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: solvent control (water), 4: Prune hot water extract (0.25mg/ml) of Big Purple cultivar harvested 100 days after flowering, 5: Prune hot water extract of Big Purple cultivar harvested 114 days after flowering (0.25mg/ml), 6: 121 days after flowering Prune hot water extract (0.25mg/ml) of harvested big purple cultivar, 7: Prune hot water extract (0.25mg/ml) of big purple cultivar harvested 134 days after flowering.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared various varieties of purun extracts by a certain method in order to test the antithrombotic efficacy of puruns, and recovered the purun extracts of the big purple varieties as antithrombotic active ingredients, and the purun extracts were human red blood cells. The purun extract was intended to be used as a pharmaceutical composition for preventing or treating/improving thrombosis and as a health functional food by confirming that it does not show hemolytic activity at all, but has excellent characteristics of thermal stability and acid stability.

Specifically, the present inventors in order to develop a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis by using a furun known to be effective in various diseases such as vascular and circulatory system, constipation and osteoporosis in the private sector, various varieties A hot water extract was prepared for the puruns of the bacterium, and their antithrombotic activity was directly inhibited in human plasma and human thrombin (Thrombin Time), Prothrombin Time (Prothrombin Time) and activated Partial Thromboplastin (activated Partial Thromboplastin) Time: aPTT) was evaluated to confirm that there was anticoagulant activity in the Prune extracts of Big Purple, Stanley, and Mountain cultivars, and the only strong platelet aggregation inhibition in the mature Prune extract of the Big Purple cultivar was evaluated by evaluating the human platelet aggregation inhibitory activity. Confirmed. In addition, it was confirmed that the mature purun extract of the Big Purple variety did not show hemolytic activity against human red blood cells, and thus could be used in practice.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, containing a furun extract of the big purple variety as an active ingredient.

It is preferable that the prune of the big purple cultivar is a mature fruit of 130 days or more after flowering.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing the prune extract of the big purple variety as an active ingredient.

It is preferable that the prune of the big purple cultivar is a mature fruit of 130 days or more after flowering.

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

The present invention comprises the steps of preparing extracts from various varieties of furun; Preparing an extract from the furun of the Big Purple variety at various times; It includes the step of evaluating the antithrombotic activity of the extract and the step of investigating the stability of the active substance.

The "furun extract" included in the composition of the present invention may be obtained by extracting the furun with an organic solvent and filtering the extract using a filter net of 0.06 mm or less, and concentrating it under reduced pressure.

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

As a preferred embodiment, the furun extract of the present invention may be used by extracting the furun with hot water or ethanol. Here, the hot water or ethanol extract may be sequentially or separately 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 using various varieties of furun extract at a concentration of 5 mg/ml, excellent thrombin inhibitory activity was shown in the order of acid, Stanley, big purple, and blacking. , Stanley, and Prune extracts of the San cultivar were confirmed to prolong prothrombin time and AP time due to inhibition of prothrombin and blood coagulation factors. However, all of the above extracts showed weaker anticoagulant activity than aspirin (1.5mg/ml), which is known as an antithrombotic agent.

However, as a result of confirming the inhibition of platelet aggregation by using various varieties of purun extracts at a concentration of 0.25 mg/ml, most varieties of purun extracts had little effect, whereas the big purple extract showed strong platelet aggregation inhibition. These results suggest that the maturation and extracts of Big Purple Prune can replace anticoagulants and antiplatelet drugs such as aspirin, which are highly likely to have side effects.

The purun extract 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, the present invention will be described in more detail through examples. The following examples are only a 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 1: Characteristics of Prunes Varieties and Harvested Mature Fruits

As for the prunes used in this example, mature Presidents, Big Purple, Black King, Stenly and Prunes of mountain varieties were used, which were grown at the Pulun Farm in Waryong-myeon, Waryong-myeon, Andong-si, Gyeongsangbuk-do, Korea in 2019.In particular, in the case of Big Purple, the maturation and In addition, fruits of 100 days, 114 days, and 121 days after flowering were additionally used. FIG. 1 is a photographic view of used prunes, and FIG. 2 is a photograph of fruits harvested on days 100, 114, 121, and 134 after flowering of Big Purple Prunes, and FIG. 3 is a picture of fruits of Big Purple Prunes by harvest time. It is a cross-sectional view of. The characteristics and physicochemical analysis results of each harvested Prune variety are shown in Tables 1 and 2.

The physicochemical analysis and color difference were measured by taking the juice after grinding the mature fruit. At this time, the color difference analysis was measured using a Hunter Color Difference meter (Super color SP-80 Colormeter, Tokyo Denshoku Co., Japan), and brightness. (White 100~0 black), redness (red 100~-80 green), and yellowness (yellow 70~-80 black) were measured. The chromaticity of the standard white board was set as 92.44 for L value, -0.06 for a value, and 1.35 for b value, and the average value was calculated by measuring three times per sample, and the color difference (△E) was calculated using the following equation. I did.

[Table 1] Weight and Size of Prune (Mature Family) by Variety

[Table 2] Analysis of Physicochemical Characteristics and Color Differences of Prunes (Mature Family) by Variety

As shown in Table 1, the size of the Prune varieties was in the order of Black King> President, Big Bubble> Stanley> Mountain varieties. In particular, the Stanley and Mountain varieties showed an elongated sausage type compared to the original varieties. Their moisture content was 78.4~85.0%, the highest moisture content in Stanley and the lowest moisture content in Big Purple.

On the other hand, as shown in Table 2, all of the Purun varieties exhibited a pH of 3.4, and an acidity of 0.23 to 0.60. On the other hand, brix was varied from 4.8 to 9.2, and the best sugar content was 9.2 in big purple. Therefore, it was judged that the big purple varieties with high sugar content and moderate acidity would have the best sensory properties, and are known as the most tasty varieties. In the case of color difference, they were very similar, from 76.29 to 77.17, and the brightness was similar, whereas in yellowness, Stanley and Big Purple were high, and the redness was the highest in President.

Example 2: Extraction of hot water and analysis of useful components from mature fruits of each cultivar Prune

Distilled water 10 times the weight of the fruits of various varieties harvested in Example 1 was added, heated at 100° C. for 1 hour, and filtered to prepare a purun extract. The prepared purun extract was concentrated under reduced pressure at 60°C to prepare a powder. Each extraction efficiency and component analysis results of the extract are shown in Table 3. The contents of total polyphenols, total flavonoids, total sugars and reducing sugars were measured by component analysis of the hot water extract of furun. 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 then allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. 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 1 N 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 3] Analysis of useful ingredients for preparation of various varieties of purun maturation and hot water extract

As shown in Table 3, the extraction efficiency of various varieties of prunes varied from 8.8 to 13.9%, in the order of Big Purple> President> Black King> Stanley> Mountain varieties, and the total polyphenol content was acid> Stanley> Big Purple = Black King> President. The acid variety with the highest content (27.3mg/g) showed about 2.5 times higher total polyphenol content than the President variety (10.7mg/g). As a result of the total flavonoid content analysis, Stanley> acid> blacking> president> big purple appeared in the order of 2.1~7.1mg/g. On the other hand, the total sugar content was the lowest in Stanley and San cultivar, and the reducing sugar content was significantly lower in President. It was found that most of the hot water extract of Prune (36-55%) was composed of sugar.

Example 3: Evaluation of blood coagulation inhibitory activity of various varieties of furun maturation and extracts

The blood coagulation inhibitory activity was evaluated for the purun extracts of various varieties obtained in Example 2, and the results are shown in Table 4. At this time, the method for evaluating the blood coagulation inhibitory activity of the furun extract was evaluated according to 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), thrombin time, prothrombin time and AP time were measured. 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 32.1 seconds. The thrombin inhibitory effect was expressed as the average value of experiments repeated three or more times, and the thrombin 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.

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 18.1 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 the addition of 50 μl CaCl ₂ (35 mM), the time until plasma coagulation was measured. As a solvent control, DMSO was used instead of the sample, and in this case, the coagulation time was 55.1 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 time at the time of sample addition by the aPTT time of the solvent control.

[Table 4] Anticoagulant Activity of Hot Water Extracts of Prunes Fruits of Various Varieties

As a result, as shown in Table 4, the best prolonged thrombin time (inhibition of thrombin) and prolonged AP time (inhibition of blood coagulation factor) in the acid variety among the furun extract (5 mg/ml), Stanley, Big Bubble, and Black King and President showed blood coagulation inhibitory activity in the order. On the other hand, aspirin (1.5mg/ml), which is currently used as an antithrombotic agent in clinical practice, showed excellent thrombin, prothrombin, and coagulation factor inhibitory activities.

Example 4: Platelet Aggregation Inhibitory Activity of Purun Extract

The human platelet aggregation inhibitory activity of the extracts of various varieties of Example 2 was evaluated, and the results are shown in Table 5 and FIG. 4. Platelets are disk-shaped small cells that circulate in blood vessels with various blood cells. Instead of having a nucleus, platelets have cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentrations. It is an important cell that secretes factors and initiates thrombus formation by binding to collagen exposed due to damage to endothelial cells to form a primary hemostatic plug. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombus formation. to be. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were concentrated human platelets, which were washed once with a washing buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 1mM EDTA, pH 6.5). Thereafter, re-suspended in a suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), and then at 3,000 rpm for 10 minutes After centrifugation, it was resuspended in the suspending buffer again, and the platelet count was adjusted to be ^{4x10 9 /ml.} Thereafter, 2.5 μl collagen was added to the 1 ml suspension and reacted for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 5] Platelet Aggregation Inhibitory Activity of Various Varieties of Prune Fruit Hot Water Extracts

As shown in Table 5 and FIG. 4, first, aspirin exhibited 48.8% aggregation at a concentration of 0.25mg/ml, indicating excellent inhibition of platelet aggregation, indicating the basis for its use as an antithrombotic agent in clinical practice. On the other hand, among the various varieties of prune extract, President, Black King, and Stanley extracts exhibited a degree of aggregation of 97.3 to 99.2% at 0.25 mg/ml, and did not show any effect on platelet aggregation. The acid cultivar extract showed 108.3% of human platelet aggregation at a concentration of 0.25mg/ml, which was found to promote aggregation rather than the additives.

However, the extract of the Big Purple variety showed only 38.8% of aggregation at a concentration of 0.25mg/ml, showing 61.2% of platelet aggregation inhibitory activity compared to the non-added group. This is expected to be able to replace antithrombotic agents such as aspirin, which exhibit side effects such as gastrointestinal disorders, with the extract of the Big Purple variety, as it has a stronger anti-aggregation activity than the same concentration of aspirin.

Example 5: Preparation of Prune Extract of Big Purple Varieties Harvested at Various Times and Analysis of Their Useful Components

Prunes of the big purple cultivar were harvested on the 100th, 114th, 121st, and 134th days after flowering, respectively, to prepare a hot water extract in the same manner as in Example 2, and the analysis results of its components are shown in Table 6. .

[Table 6] Analysis of Useful Components of Prune Extract of Big Purple Varieties Harvested at Various Times

As shown in Table 6, the extraction efficiency of the big purple cultivars harvested at various times increased as the number of cultivation days increased. On the other hand, the highest content of total polyphenols and total flavonoids was 114 days after flowering, and then decreased with maturity. On the other hand, the total sugar and reducing sugar contents were the lowest on the 121st day after flowering, but then increased rapidly as maturity reached. Therefore, the organoleptic performance was the most excellent in the maturity stage big purple.

Example 6: Anticoagulant Activity of Prune Extract of Big Purple Varieties Harvested at Various Times

The anticoagulant activity of the furun extract of the big purple variety prepared in Example 5 was evaluated in the same manner as in Example 3, and the results are shown in Table 7.

[Table 7] Anticoagulant Activity of Prune Extract of Big Purple Varieties by Harvest Time

As shown in Table 7, the anticoagulant activity of the big purple extract (5mg/ml) by harvest time showed the best thrombin inhibition and blood coagulation inhibitory activity at 100 days after flowering, and the activity gradually weakened toward maturity. However, the anticoagulant activity of the big purple extract (5mg/ml) was relatively weak compared to aspirin (1.5mg/ml).

Example 7: Human Platelet Aggregation Inhibitory Activity of Prune Extracts of Big Purple Varieties Harvested at Various Times

The activity of inhibiting human platelet aggregation of the furun extract of the Big Purple variety prepared by harvest time prepared in Example 5 was evaluated in the same manner as in Example 4, and the results are shown in Table 8 and FIG. 5.

[Table 8] Platelet Aggregation Inhibitory Activity of Prune Extract of Big Purple Varieties by Harvest Time

As a result, among the furun extracts of the big purple cultivar prepared by harvest time, only the extracts of the maturation period (134 days after flowering) showed strong platelet aggregation inhibitory activity. Therefore, it was confirmed that the furun extract of the mature big purple variety can complement and replace existing antithrombotic agents such as aspirin, which are limited in use due to hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

Example 8: Human Red Blood Cell Hemolytic Activity of Prune Extracts of Various Varieties and Prune Extracts of Big Purple Varieties of Various Harvest Times

Prune is registered as a food raw material and has secured safety, is edible by itself, and is also used as a subsidiary material and additive for various food processing. In order to evaluate the possibility of acute toxicity of the furun extracts of various varieties and the furun extracts of the big purple varieties at various harvest times, human red blood cell hemolysis activity was evaluated, and the results are shown in Table 9. At this time, the hemolytic activity was evaluated according to the previous report (Ho-Yong Son, 2014 ㆍKorean 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.

[Table 9] Human Red Blood Cell Hemolytic Activity of Prunes Extracts of Various Varieties and Prunes Extracts of Big Purple Varieties at Various Harvest Times

First, it was confirmed that DMSO and water used as control cells had no hemolytic activity, and triton X-100 hemolytic 100% red blood cells at a concentration of 1 mg/ml. In addition, it was confirmed that amphotericin B, which is used as an anticancer agent and antifungal agent, hemolyzes more than 50% of red blood cells at a concentration of 0.025mg/ml. It was confirmed that the purun extracts of various varieties of the present invention and the purun extracts of the big purple varieties at various harvesting periods did not show any red blood cell hemolysis up to a concentration of 1 mg/ml, and thus had no acute toxicity and red blood cell hemolysis activity.

Example 9: Plasma, acid, and heat stability evaluation of the furun extract of the big purple variety

Plasma stability, thermal stability, and acid stability against antithrombotic activity were confirmed for the mature purun extract of the big purple variety obtained in Example 5 above. The mature pruned extract of the Big Purple variety did not show a decrease in blood coagulation inhibition and platelet aggregation inhibitory 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. .

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis, containing a furun extract of the big purple variety as an active ingredient. The pharmaceutical composition of claim 1, wherein the prune of the big purple variety is a mature fruit of 130 days or more after flowering. A health functional food for preventing or improving thrombosis, which contains the prune extract of the Big Purple variety as an active ingredient. The health functional food according to claim 3, wherein the prune of the big purple cultivar is a mature fruit for at least 130 days after flowering.

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