KR102216223B1 - Pharmaceutical composition comprising the mature fruit extract of magnolia denudata 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 health functional food for the prevention or treatment of thrombosis containing a mature fruit extract of Magnolia denudata as an active ingredient, and more specifically, an ethanol extract of mature white magnolia fruit. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation as a component, and a health functional food. Baek magnolia mature fruit extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention has an inhibitory effect on the aggregation of platelets that plays a role in the initiation of thrombus formation along with inhibition of thrombus formation-related enzymes and blood clotting factors At the same time, it exhibits strong anti-thrombotic activity due to antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, has an effect of inhibiting blood coagulation factors even in acidic conditions of pH 2 and plasma, and loss of the inhibitory effect of thrombogenic enzymes. 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 a possible form.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE MATURE FRUIT EXTRACT OF MAGNOLIA DENUDATA AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE MATURE SAME}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing a mature fruit extract of Magnolia denudata as an active ingredient, and more specifically, an ethanol extract of mature white magnolia fruit. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation as a component, 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 as the blood coagulation reaction system and the thrombosis dissolution system are complementarily regulated in the body. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate on the 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, and fibrin monomers are polymerized by calcium, resulting in platelets and endothelial cells. It binds and forms a fibrin polymer that is cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood clotting reactions. Therefore, the thrombin activity inhibitory substance 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, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by prothrombin activation, is known to ultimately activate thrombin, and specific inhibition of blood coagulation factors is also important. 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. The situation is limited to such use.

On the other hand, Magnolia denudata is a deciduous tall tree of the magnolia family, reaching 10-15m in height, and its stem bark is grayish-white, with many hairs pressed by young branches and winter snow. Petiole is 1~2cm long, wide obovate, 8~15cm long, 4~11cm wide, there are hairs on both sides, but gradually disappears. Flowers bloom before leaves, bell-shaped, and white. Sepals and petals are not distinguished, and they are narrow obovate, 7-8cm long, 3-4cm wide, and do not spread out. There are many surgeries, and they are arranged in a spiral shape. It is planted for ornamental purposes in Korea, and flower buds are used for medicinal purposes. Magnolias are classified into various types according to flower color and shape. Representatively, white magnolia, purple magnolia, and star magnolia are known, and most of the domestic landscape trees use white magnolia native to China with excellent pollution resistance and cold resistance.

The buds of magnolia are known as shinyi (辛夷) and are used as a very valuable medicinal material in oriental medicine, and shinyi is known for its astringent, capillary dilatation, anti-inflammatory, blood pressure lowering, pain relief, sedation, and antibacterial properties. Magnolias have a large amount of fruit after harvesting the buds and after the flowers have faded. Until now, this fruit has no special purpose and has been discarded.

To date, studies on magnolia trees have been reported on antibacterial and antioxidant activities of various magnolia trees (Sungsook Lee et al., 2010. Wood Engineering 38: 579~586). Most studies on flower buds and leaves have been reported. , Research on white magnolia, the largest number of landscaping trees in Korea, is insignificant. Studies related to white magnolia include the antioxidant activity, anticancer activity and anti-inflammatory activity of white magnolia flower extract (Jin-Woo No, 2009, Journal of Food and Nutrition Science 38: 1478-1484), and isolation of melanin production inhibitors from Shin-I (辛夷) ( Heo, Gwang-Hwa et al., 2004, Journal of the Korean Society of Herbal Medicine 35: 152~156), Shin-I's Allergy Inhibition Effect (Seungmin Hong, 2013, Master's Thesis at Seoul National University) and anti-obesity effect (Changsuk Kong et al., 2011, J. Agricultural and Food Chemistry 59: 5665~ 5670), Antioxidant and Anticancer Effects of Magnolia Calyx Extract (Park, Hye et al., 2015. Molecules J. Synthetic Chemistry Natural Product Chemistry 20: 12154~12165), Inflammatory cytokines and NO in LPS-activated peritoneal macrophages. Inhibitory effects (Kim Do-yoon et al., 2007. Journal of Dong-Eui Physiological Pathology 21: 916-920), analysis of the components of white magnolia leaves (Du, Jiang et al., 2001, J. Asian Natural Products Res. 3: 313-319) are known. In addition, studies related to the white magnolia fruit include analysis of the lignan component of the white magnolia fruit (Lee Seong-suk, 2010, Wood Engineering 38: 579-586), and the purification of cytotoxic (apoptotic) phenolic compounds from the white magnolia fruit (Noshita Toshiro et al., 2009. Bioscience, Biotechnology Biochemistry 73: 726-728), larval activity of lignan obtained from white magnolia seeds (Wang, Zhang-qian et al., 2016, Pest Management Science 72: 897-906), IL-4 and IL-13 in immune T cells The expression inhibitory activity of (Jin Mirim et al., 2013, In vitro Cellular Developmental Biology. Animal 49: 805-814) has been known.

Regarding the antithrombotic activity of magnolia, only known effects of Obovatol, a biphenolic component isolated from the leaves and bark of Magnolia obovata , inhibits arterial blood clots and platelet aggregation in rats (Park Eun-seok, 2008, Master's thesis, Chungbuk National University). , To date, there are no reports of potent antithrombotic activity of the mature fruit extract of Magnolia denudata .

Patents related to magnolia trees include Korean Patent Registration No. 10-1397188, [Method of manufacturing magnolia flower tea], No. 10-1079980, [Lignan components isolated from Shinyi and its use], and No. 10-0795512. And Skin Protection Composition Containing Magnolia Extract], No. 10-1563802, [Bola branch made using magnolia flower tea, and its manufacturing method], No. 10-1560367, [New neorignan derived from Hwang magnolia and brain containing the same] Cell protection composition], No. 10-1024043 [(Japan) method of sesamin separation from magnolia] and No. 10-0909572 (separated from Japanese magnolia leaves and bark) [Obobatol and its synthetic derivatives as active ingredients Containing a composition for preventing and treating diabetes and metabolic syndrome diseases] is disclosed, and in Korean Patent Publication No. 10-2009-0025645 [An antiseptic cosmetic composition containing magnolia extract], [Magnolia] in No. 10-2018-0093238 Petal tea and its manufacturing method], No. 10-2015-0065341 [fragrance composition that reproduces the scent of magnolia], No. 10-2012-0000243 [Anti-cancer agent composition containing Japanese magnolia fruit extract], No. 10- 2008-0044612 [C-kit protein activity inhibitory composition containing Japanese magnolia extract], 10-2016-0070986 [Pharmaceutical composition having antioxidant effect containing magnolia flowers fermented by microorganisms] , No. 10-2016-0070972 [Functional food composition having antioxidant effect containing magnolia flowers fermented by microorganisms] and No. 10-2005-0111257 [Magnolia extract for improvement of symptoms of degenerative central nervous system disease] Functional food containing a] is disclosed. However, until now, no patent for antithrombotic activity of mature white magnolia fruit extract has been known.

KR 10-1560367 B

Eun-Seok Park, 2008. Master's thesis, Chungbuk National University. Effect of Obovatol, a Biphenolic Component Isolated from Leaves and Bark of Magnolia kobus, Inhibits Arterial Vascular Thrombi and Platelet Aggregation in Rats

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 a pharmaceutical composition for preventing or treating thrombotic diseases containing an extract of mature white magnolia as an active ingredient, and a health function They want to provide food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which contains a magnolia denudata mature fruit extract as an active ingredient.

The white magnolia mature fruit extract is preferably an ethanol extract of the mature white magnolia fruit in which the fruit is opened and the seeds are exposed.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains a magnolia denudata mature fruit extract as an active ingredient.

The white magnolia mature fruit extract is preferably an ethanol extract of the mature white magnolia fruit in which the fruit is opened and the seeds are exposed.

Baek magnolia mature fruit extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention has an inhibitory effect on the aggregation of platelets that plays a role in the initiation of thrombus formation along with inhibition of thrombus formation-related enzymes and blood clotting factors At the same time, it exhibits strong anti-thrombotic activity due to antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, has an effect of inhibiting blood coagulation factors even in acidic conditions of pH 2 and plasma, and loss of the inhibitory effect of thrombogenic enzymes. 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 a possible form.

1 shows the fruit of white magnolia. 1: immature fruit before seed generation, 2: immature fruit immediately after seed generation, 3: mature fruit with seed fully formed and fruit open.
Figure 2 shows the activity of inhibiting human platelet aggregation of the ethanol extract of white magnolia fruit. 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: aspirin (0.125mg/ml), 4: immature fruit extract before seed formation (0.25mg/ml), 5: seeds Immediately after production, immature fruit extract (0.25mg/ml), 6: mature fruit extract (0.25mg/ml) in which seeds have been fully produced and fruit is spread.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of the white magnolia fruit, the inventors of the present invention recovered the white magnolia fruit by maturation period by a certain method, prepared an extract thereof, and evaluated the antithrombotic activity. The extract was recovered as a pharmaceutical composition and health functional food for the prevention or treatment/improvement of thrombosis by confirming that the extract does not exhibit any hemolytic activity against human red blood cells, but has excellent thermal stability and acid stability. I tried to use it.

Specifically, the inventors of the present invention to develop a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis using Baek magnolia, which is known to be effective in various diseases such as cold, fever, hepatitis, and skin disease treatment in the private sector, The fruits of white magnolia, which are discarded without being discarded, are recovered by maturation period, and each ethanol extract is prepared, and their antithrombotic activity is directly inhibited by thrombin against human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time), and By evaluating the activated Partial Thromboplastin Time (aPTT), it was confirmed that in mature fruit extracts with open fruit and exposed seeds, excellent anticoagulant activity and strong platelet aggregation inhibitory activity. In addition, it was confirmed that the white magnolia mature fruit extract did not show any hemolytic activity against human red blood cells, unlike the immature fruit extract in which seeds were formed.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which contains the magnolia denudata mature fruit extract as an active ingredient.

The white magnolia mature fruit extract is preferably an ethanol extract of the mature white magnolia fruit in which the fruit is opened and the seeds are exposed.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains a magnolia denudata mature fruit extract as an active ingredient.

The white magnolia mature fruit extract is preferably an ethanol extract of the mature white magnolia fruit in which the fruit is opened and the seeds are exposed.

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

The present invention comprises the steps of preparing an extract from the fruits of each maturation period of white magnolia; Evaluating the antithrombotic activity of the extract; And it is carried out including the step of investigating the stability of the active material.

In the present invention, the "white magnolia extract" refers to the step of extracting the immature fruit in which the seed of the white magnolia is not produced, the immature fruit immediately after the seed is produced, the mature fruit with the open fruit and the seed exposed with an organic solvent, and the extract is not more than 0.06mm It can be obtained by filtering using a filtration network of, and concentrating 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 white magnolia fruit extract of the present invention can be used by extracting the immature fruit of which the white magnolia seed is not produced, the immature fruit immediately after the seed is produced, and the mature fruit with open and exposed seeds 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 with a concentration of 5 mg/ml of a white magnolia mature fruit extract, good anticoagulant activity extended by 1.29 times, 1.35 times, and 1.38 times, respectively, compared to the non-additional group. Indicated. This activity was similar to that of aspirin (1.5 mg/ml) known as an antithrombotic agent. No seeds were produced, or the immature fruit extract immediately after seed was produced showed little anticoagulant activity.

In addition, after adjusting the fruit extract for each maturation period of Baek Magnolia to a concentration of 0.25mg/ml, as a result of confirming the platelet aggregation inhibitory activity, 58.0% aggregation rate in the immature fruit extract before the seed was produced, the fruit flesh was widened, and the mature seed was The exposed mature fruit extract showed a 65% aggregation rate, confirming excellent platelet aggregation inhibitory activity. On the other hand, the immature fruit extract immediately after seed formation showed an aggregation rate of 159.7%, which showed a rather strong platelet aggregation promotion. Similar to the anticoagulant activity, the platelet aggregation inhibitory activity was also excellent in the mature fruit extract, and it was found that the components and activities of each maturation period were changed. These results indicate that the white magnolia mature fruit extract exhibits very strong antithrombotic activity, and suggests that it can replace anticoagulants such as aspirin (blood coagulation inhibitors) and antiplatelet agents (platelet aggregation inhibitors), which have a high risk of side effects. .

The white magnolia mature fruit 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 maintain their activity even at a heat treatment of 100°C and a pH of 2 in the stomach.

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. The 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.

Pharmaceutical compositions containing the active ingredients of the present invention are oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method for each purpose of use, 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 contain 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 formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid formulations 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 formulation, an emulsion, a syrup, and the like 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, etc. 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" refers to 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 drugs, 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 other therapeutic agents, 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 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 depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected and may be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural 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 "object" is not particularly limited, but includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit, or guinea pig And, preferably, a mammal, more preferably a human.

In addition, the health functional food of the present invention can be variously used for 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.

Taumatin as the flavoring agent; Natural flavoring agents such as stevia, such as rebaudioside A or glycyrrhizin, 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 flavoring agents such as natural flavors, colorants 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 drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is generally selected from 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 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 1: Preparation of white magnolia fruit extract and analysis of useful components thereof

In 2018, white magnolia fruits from Andong, Gyeongbuk were collected and used to prepare ethanol extracts. Specifically, immature fruits before the seeds of white magnolia are produced (recovered on June 10, 2018: immature fruits (represented as 1)), and immature fruits immediately after the seeds are generated (recovered on July 20, 2018: immature fruits) (Represented as (2)), after dividing into mature fruits (recovered on September 10, 2018: marked as mature fruits (3)) with open pulp and exposed mature seeds (Fig. 1), 10 for each sample After adding pear of ethanol, extracting twice at room temperature, collecting the extract, filtering, and then concentrating under reduced pressure to prepare an ethanol extract.

Contents of total polyphenols, total flavonoids, total sugars and reducing sugars were measured by component analysis of the white magnolia fruit extract. 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, and 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, and the absorbance was measured at 420 nm after 1 hour reaction at 37°C. . Rutin was used as a standard reagent. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Analysis of extraction efficiency and useful components of fruit extract by maturation period of Baek magnolia

As shown in Table 1, the ethanol extraction efficiency of the white magnolia fruit was higher as the maturity increased, and the extraction efficiency of the immature fruit (1) before the seed was generated was only 1.2%, but the immature fruit (2) was It increased to 2.7%, and the yield of mature fruit (3) was 4.3%. As a result of analysis of total polyphenol content in fruit extract, the highest content of 232.3mg/g in the extract of immature fruit (1) before seed formation was shown, and it gradually decreased as the fruit matured, and 97.8 in the case of mature fruit (3). It was expressed as mg/g, which was 42% level compared to the initial immature fruit (1). The flavonoid content measurement result also showed the highest 78.2mg/g in the early immature fruit (1) extract, and then 27.2~31.0mg/g in the immature fruit (2) and mature fruit (3) extracts. ) Compared to the 40% level. On the other hand, the total sugar content increased as the fruit matured, showing 117.7 mg/g in the initial immature fruit (1) extract, while 153.7 mg/g and 307.2 mg/g in the immature fruit (2) and mature fruit (3) extract, respectively. g. As a result of evaluating the reducing sugar content, the content of the extract of the immature fruit (2) was increased 1.37 times compared to the initial immature fruit (1), but the extract of the mature fruit showed 27.6 mg/g, which was 42% higher than that of the immature fruit (1). Indicated. Therefore, it is expected that changes in useful physiological functions will occur due to changes in components during the maturation process of white magnolia fruit, and the results of actual antioxidant and antibacterial activity evaluation showed that the immature fruit extract showed stronger activity than the mature fruit extract.

Example 2: Evaluation of blood coagulation inhibitory activity of white magnolia fruit extract

The blood coagulation inhibitory activity of the ethanol extract of Example 1 was evaluated, and the results are shown in Table 2. At this time, the method of evaluating the blood coagulation inhibitory activity of the extract of white magnolia 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 the 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 the result 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 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 a 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 again at 37° C. Incubated for 3 minutes. Thereafter, after adding 50 μl CaCl ₂ (35 mM), 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 55.1 seconds. The result of aPTT was 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] Blood Coagulation Inhibitory Activity of Fruit Extracts by Maturation Period

As a result, the mature fruit (3) extract of white magnolia showed excellent anticoagulant activity by extending thrombin time, prothrombin time and APT time by 1.29 times, 1.35 times, and 1.38 times, respectively, compared to the non-added group at a concentration of 5mg/ml. At /ml concentration, it was extended by 3.71 times, 1.48 times and 1.67 times, respectively, showing strong anticoagulant activity. However, the extracts of immature fruit (1) and immature fruit (2) were not recognized for anticoagulant activity at the concentration of 5 mg/ml. Considering that aspirin, which is currently used as an antithrombotic agent in clinical practice, extends thrombin time, prothrombin time, and APT time by 1.45 times, 1.40 times, and 1.67 times, respectively, compared to the non-additives, at a concentration of 1.5 mg/ml, the mature fruit extract of Baek Magnolia It was found that showed good anticoagulant activity.

Example 3: Platelet Aggregation Inhibitory Activity of Fruit Extracts by Maturation Period of White Magnolia

The activity of inhibiting human platelet aggregation of the white magnolia fruit extract of Example 1 was evaluated, and the results are shown in Table 3 and FIG. 2. 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, and aggregation when damage to the inner wall of blood vessels occurs. It is an important cell that secretes factors and initiates thrombus formation by forming a primary hemostatic plug by binding to collagen exposed due to damage to endothelial cells. Therefore, platelet aggregation inhibition 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 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 to react for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Platelet Aggregation Inhibitory Activity of Fruit Extracts by Maturation Period of Baek Magnolia

As shown in Table 3 and Figure 2, first, aspirin exhibited 33.1% aggregation at 0.25mg/ml concentration and 58.9% aggregation at 0.125mg/ml concentration, indicating a strong inhibition of platelet aggregation in a concentration-dependent manner. The basis for being used as a blood clot was found. On the other hand, white magnolia immature fruit (1) and mature fruit (3) extracts showed an aggregation rate of 58.0% and 65.0% at 0.25mg/ml, respectively, similar to the degree of aggregation at 0.125mg/ml of aspirin. This means that the extracts of the immature white magnolia fruit (1) and the mature fruit (3) have strong anti-aggregation activity. However, it was found that the extract of immature white magnolia fruit (2) showed a degree of aggregation of 159.7% at 0.25mg/ml, and rather exhibited an aggregation promoting effect. Considering the shortened aPTT (blood coagulation factor promoting activity) and increased platelet aggregation capacity of Example 2, it can be predicted that the Baek magnolia immature fruit (2) extract can be used as a hemostatic agent by promoting thrombus formation. The above results suggest that extracts of immature white magnolia fruit (1) and mature fruit (3) can be used as platelet aggregation inhibitors, and in particular, mature white magnolia fruit (3) extract, which has excellent anticoagulant and platelet aggregation inhibitory activity, is an antithrombotic agent. It was confirmed that it is most preferable.

Example 4: Human erythrocyte hemolytic activity of fruit extracts according to maturation period of white magnolia

The buds of white magnolia are used as a medicinal herb called god, but their fruits are being discarded for no special purpose. In addition, it has not been used for food until now. Therefore, in order to evaluate the possibility of acute toxicity of the white magnolia fruit extract, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 4. 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 placed in a 96-well microplate. Then, 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 outflow due to hemolysis was 414 nm. It was measured at. 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. Hemolytic activity was calculated using the following formula.

[Table 4] Human Red Blood Cell Hemolytic Activity of Fruit Extracts by Maturation Period of Baek Magnolia

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 white magnolia mature fruit (3) extract and the immature fruit (1) extract of the present invention 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 hemolytic activity. However, the immature fruit (2) extract showed 82.0% hemolysis at 1mg/ml concentration and 63.8% erythrocyte hemolysis at 0.5mg/ml concentration.

Example 5: Plasma, acid and heat stability evaluation of mature white magnolia fruit extract

Plasma stability, thermal stability, and acid stability against antithrombotic activity were confirmed for the extract of mature white magnolia fruit (3) obtained in Example 1 above. The extract of mature white magnolia fruit (3) showed no decrease in blood coagulation inhibition and platelet aggregation inhibitory activity even after 1 hour heat treatment at 100°C, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. Therefore, it was confirmed that the extract of mature white magnolia fruit (3) contains antithrombotic active substances of various components having acid resistance and heat resistance.

Claims (4)

A blood coagulation inhibitor containing ethanol extract of mature fruit of Magnolia denudata in a state where the fruit is opened and the seed is exposed as an active ingredient. delete delete delete

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