KR101916340B1 - Pharmaceutical composition for prevention or treatment of thrombosis comprising the extraction of lettuce as an effective component and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food product containing a Lactica sativa L. extract as an effective component for preventing or treating thrombosis. More specifically, the pharmaceutical composition contains an ethyl acetate fraction, which is obtained by fractionating an ethanol extract of Lactica sativa L. sequentially with organic solvents of hexane, ethyl acetate, and butanol, a water residue after the fractionation, or a mixture thereof as an effective component, and thus, the composition is capable of preventing or treating thrombosis by inhibiting blood coagulation and inhibiting platelet agglutination. The Lactica sativa L. extract, which is an effective component of the pharmaceutical composition and the health functional food product of the present invention, indicates a strong antithrombotic activity, which has effects of inhibiting thrombus generation-related enzymes and blood coagulation factors as well as inhibiting the agglutination of platelets functioning as a thrombus generation initiator, while not having a hemoclastic activity to human red blood cells at all, having excellent thermal stability, and preventing the effects of inhibiting the blood coagulation factors and thrombus generation-related enzymes from being lost even in a pH 2 acidic condition and blood plasma, and accordingly, the extract is expected to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke. Also, since the effective component is able to be processed into liquid, powder, pills, tablets, and the like to be taken anytime, the present invention is able to be very beneficial for pharmaceutical and food industries.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which contains an extract of lettuce as an active ingredient, and a health functional food.

TECHNICAL FIELD The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis containing Lactica sativa L. extract as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition and health functional food for preventing or treating thrombosis, A pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, comprising an ethyl acetate fraction obtained by successive fractionation with an organic solvent of acetone and butanol, a water residue after fractionation or a mixture thereof, Health functional foods.

As a constituent of human body, blood has various important functions such as oxygen and nutrients, the function and buffering function of waste products, maintenance of body temperature, control of osmotic pressure and maintenance of ion balance, maintenance of moisture, regulation of fluidity, maintenance and regulation of blood pressure, have. Normal blood circulation facilitates blood circulation by complementary regulation of the blood coagulation system and thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere to the blood vessel walls and coagulate to form platelet thrombus , It is reported that the blood coagulation system is activated and fibrin thrombus is formed centering on the platelet aggregation mass.

On the other hand, the production of fibrin thrombus is activated by thrombin involved in fibrin clotting through several steps of many blood coagulation factors to finally produce fibrin monomers from fibrinogen. Fibrin monomers are polymerized by calcium, Cells to form a cross-linked fibrin polymer by factor XIII and produce a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelet, V factor, and Factor VII to promote blood coagulation. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation. On the other hand, prothrombin activation after sequential activation of factor XII, factor XI, factor IX and factor X is known to activate thrombin in the endogenous thrombogenesis pathway, so that specific inhibition of blood coagulation factors is also important. It is becoming a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and europine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants and thrombolytic agents are not only very expensive but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

Lettuce is a perennial herbaceous plant of the dicotyledonous plant, and is cultivated in a wide area such as Korea, China, Japan, the United States, and the United Kingdom. The other names of lettuce are Wagar (food prime herb), Rajeong (Buhui Ward room), Chunggwangchae (Chungil Rock), Wassoon (Chonnam Chongmok), Wajae , The leaves are mainly edible, the edges of the leaves are flat, wrinkled or irregular sawtooth, and there is no bag. Lettuce may be differentiated into various systems, breed group, envoys of lettuce (Lactuca sativa var. Capitata), ipsangchu (Lactuca sativa var. Crispa), cabbage lettuce (Lactuca sativa var. Longifolia), stem lettuce (Lactuca sativa var. Asparagina ). In Korea, it is planted mainly with leaf lettuce. Recently, however, many lettuce plants have been planted, and many are grown in Gyeonggi and Gyeongsang provinces in Korea. On the other hand, lettuce, which is a reproductive vegetable, is mainly used for salads, salsa, and cabbage. It is nutritionally excellent because it contains a large amount of vitamins (especially vitamin A) and minerals and lactoterine contained in the milky juice lactucerin, and lactucin have analgesic or hypnotic effects, and it is known that many of these ingredients cause sleep when you eat a lot of lettuce. It also has a calming effect on sensitive nerves. It has long been known that eating lettuce helps relieve headaches and insomnia. In addition, lettuce is also used as a medicinal herb, and its main effects are known to have therapeutic effects on the bones, blood, and the like. Flavonoids such as quercetin, luteolin and phytol, Diterpene compounds have been reported (Ahn, KH, et al., 2002, Plant Biotechnology, 29: 63-68).

To date, most studies on domestic lettuce have been conducted on the changes in the quality characteristics of lettuce cultivation and fresh slices (Bae et al., 2016. Korean Food Storage and Distribution Society 23: 27-33), antioxidant and antimicrobial activity The presence of coumaric acid, caffeic acid in association with the action of the enzyme is reported (Moon et al., 2009. J. Life Sci. 19: 206-212). It has been reported that the useful activity of lettuce is antioxidant activity and tyrosinase inhibitory activity (Jang Jin-hee, Master's thesis of Sunchon National University, 2013. 34.) Lactic erasure efficacy and tyrosinase inhibitory activity of extracts and fractions of 34 kinds of medicinal plants, (Shihoon Song, Chonbuk National University Master's thesis, 2014. Effect of high fat diet supplemented with lettuce on serum lipid composition and enzyme activity in rats). In addition, the 70% methanol extract of the dried lettuce was known to have an insecticidal effect against the spotted mite (Lee, Gil, et al., 2001. The Korean J. Pesticide Science 5: 18-25) Pukyong National University) and the production of phytol, which exhibits anticancer activity from lettuce tissue culture (Ahn KH, et al., 2002, Plant Biotechnology, 29: 63-68). However, the anti-thrombotic activity of the extract of lettuce and its active fractions has not been known to date.

On the other hand, as a patent relating to lettuce, Korean Patent Registration No. 10-0564800 discloses a composition for prevention and treatment of cardiovascular diseases containing a compound isolated from lettuce extract, which inhibits blood cholesterol absorption, -0527877 discloses a composition for preventing and treating convulsive diseases containing phytol as an active ingredient, and a composition for preventing or treating periodontal disease containing the extract of prickly lettuce as an active ingredient in Patent No. 10-1574678 It is known. Korean Patent Publication No. 10-2017-0000113 discloses an antioxidant or anti-aging composition containing [lettuce flower extract or fraction as an active ingredient]. However, no patent has been known on the extract of lettuce and its active fractions, which exhibit potent antithrombotic activity through inhibition of blood coagulation and inhibition of platelet aggregation to date, and which are not toxic.

KR 10-0564800 B

 Jeong Dae Gyun et al., 2007. Food Technology, 20, 75-84. A composition for preventing and treating cardiovascular diseases containing a compound isolated from lettuce extract.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention is directed to provide a pharmaceutical composition and a health functional food for preventing or treating thrombosis comprising an extract of lettuce as an active ingredient .

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising an extract of Lactica sativa L. as an active ingredient.

Preferably, the lettuce extract is the ethyl acetate fraction obtained by successively fractionating the ethanol extract of lettuce with hexane, ethyl acetate and butanol, the water residue after the sequential fractionation or a mixture thereof.

The present invention also provides a health functional food for preventing or ameliorating thrombosis comprising an extract of Lactica sativa L. as an active ingredient.

Preferably, the lettuce extract is the ethyl acetate fraction obtained by successively fractionating the ethanol extract of lettuce with hexane, ethyl acetate and butanol, the water residue after the sequential fractionation or a mixture thereof.

The pharmaceutical composition for the prevention or treatment of thrombosis and the extract of lettuce as an active ingredient of the health functional food of the present invention are effective for inhibiting thrombogenesis-related enzymes and blood coagulation factors as well as for inhibiting the aggregation of platelets Exhibit strong antithrombotic activity, exhibit no hemolytic activity on human erythrocytes, exhibit excellent thermal stability, exhibit an acidic condition of pH 2 and a loss of blood coagulation factor inhibitory effect and thrombogenic enzyme-inhibiting effect even in plasma Therefore, it is expected that it can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. The active ingredient can be processed into various forms such as extract, powder, The pharmaceutical industry and the pharmaceutical industry, Industrially very useful inventions.

Figure 1 shows human platelet aggregation inhibitory activity of lettuce ethanol extract and its sequential organic solvent fraction. Herein, 1: solvent control (DMSO), 2: aspirin (0.5 mg / ml), 3: aspirin (0.25 mg / ml), 4: lettuce ethanol extract (0.25 mg / ml) (0.25 mg / ml), 6: Ethyl acetate fraction of ethanol extract of lettuce (0.25 mg / ml), 7: Butanol fraction of ethanol extract of lettuce (0.25 mg / ml) The residue (0.25 mg / ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic effect of lettuce, the inventors of the present invention prepared a lettuce ethanol extract by a certain method, and from this, a specific fraction of the sequential organic solvent fraction and a water residue after fractionation of the organic solvent as the antithrombogenic active ingredient The extracts and fractions were found to have excellent heat stability and acid stability without showing hemolytic activity against human erythrocytes. Thus, the extracts and fractions were used as pharmaceutical compositions for preventing or treating / improving thrombosis, Health functional foods.

Specifically, the inventors of the present invention found that, in order to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis using lettuce known to have excellent diabetes and blood circulation improvement effect in a private enterprise, , And their antithrombotic activity was evaluated through thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) for human plasma and human thrombin, The anticoagulant activity was confirmed. The hexane fraction and ethyl acetate fraction obtained by sequential fractionation of the ethanol extract with the organic solvent showed very strong coagulation factor inhibitory activity and the inhibition of the platelet aggregation inhibition in the ethyl acetate fraction and water residue was confirmed Respectively. In addition, it was confirmed that the extracts and active fractions did not show hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains extract of Lactica sativa L. as an active ingredient.

Preferably, the lettuce extract is the ethyl acetate fraction obtained by successively fractionating the ethanol extract of lettuce with hexane, ethyl acetate and butanol, the water residue after the sequential fractionation or a mixture thereof.

The present invention also provides a health functional food for preventing or ameliorating thrombosis comprising an extract of Lactica sativa L. as an active ingredient.

Preferably, the lettuce extract is the ethyl acetate fraction obtained by successively fractionating the ethanol extract of lettuce with hexane, ethyl acetate and butanol, the water residue after the sequential fractionation or a mixture thereof.

Hereinafter, the lettuce extract of the present invention, the method for producing each active fraction material, the efficacy experiment, and the like will be described in more detail.

The present invention provides a method for preparing an extract, comprising: preparing an extract from lettuce; Preparing a sequential organic solvent fraction of hexane, ethyl acetate and butanol from the lettuce extract and preparing a water residue obtained therefrom; Evaluating the antithrombotic activity of the extract and the fractions and examining the stability of the active substance.

The "extract of lettuce" contained in the composition of the present invention is prepared by purchasing the ground portion of lettuce cultured in Korea in 2017, washing and crushing the same, extracting it with an organic solvent, and extracting the extract using a filter net of 0.06 mm or less Followed by filtration and concentration under reduced pressure.

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

As a preferred embodiment, the lettuce extract of the present invention can be used by extracting lettuce with hot water or ethanol. Here, the hydrothermal or ethanol extract may be fractionated sequentially or separately with an organic solvent of hexene, ethyl acetate and butanol to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction and a water residue.

In the present invention, thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of ethanol extract of lettuce, and as a result, it showed a good blood coagulation inhibitory activity equivalent to 1.5 mg / ml of aspirin known as an antithrombotic agent. However, the inhibition of platelet aggregation was observed at a concentration of 0.25 mg / ml of ethanol extract of lettuce.

However, the hexane fraction and the ethyl acetate fraction of the lettuce ethanol extract prolonged the thrombin time, the prothrombin time and the apathy time in a concentration dependent manner. Especially, the ethyl acetate fraction had a thrombin Time, and apathy time, indicating very strong thrombogenic inhibition. As a result of the evaluation of the inhibitory activity against the platelet aggregation reaction of human platelets, the ethyl acetate fraction of the ethanol extract of lettuce showed strong inhibition of human platelet aggregation equivalent to 0.25 mg / ml of aspirin at the concentration of 0.13 mg / ml, / ml showed an inhibition of platelet aggregation equivalent to 0.25 mg / ml of aspirin. These results suggest that the ethyl acetate fraction of ethanol extract of lettuce and the water residue after fractionation have very strong antithrombotic activity, suggesting that anticoagulants such as aspirin and anti - platelet agents, which have high risk of side effects, can be substituted.

The extract of lettuce and the active fraction thereof of the present invention can be prepared into powders by a conventional powdering process such as vacuum drying, freeze drying, spray drying and the like. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain, and acute peripheral artery occlusion. Vein thrombosis includes deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

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 will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

As a preferable example, 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 kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , 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 for prevention or improvement of thrombosis. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

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

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

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

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. 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 may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such 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 by way of examples. The following examples are only exemplary embodiments 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 lettuce extract and analysis of its useful components

Lettuce (moisture content 92.0 ± 0.7%) grown in Korea in 2017 was purchased, washed with running water to remove impurities, and then used in the production of ethanol extract. Specifically, 10 times ethanol was added to the lettuce samples, and the extracts were repeatedly extracted twice at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare ethanol extracts. The extracts were prepared as follows: hexane, ethyl acetate, , And finally a water residue was recovered. Total polyphenols, total flavonoids, total sugars and reducing sugar contents were determined by the analysis of components of lettuce ethanol extract, organic solvent fraction and water fraction after fractionation. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. For total flavonoid content, each sample was stirred for 18 hours in methanol, 400 μl of 90% diethylene glycol was added to 400 μl of the filtered extract, and 40 μl of 1 N NaOH was added. After reacting for 1 hour at 37 ° C, absorbance was measured at 420 nm Respectively. 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] Ethanol extract of lettuce and Fraction  Analysis of extraction efficiency and useful components

As shown in Table 1, the ethanol extraction efficiency of lettuce was 3.53%, which means that 44% of the lettuce solid is extracted with ethanol, considering that the moisture content of the fresh lettuce is 92%. Most of the extracts (66.8%) were converted to water residues. The ethyl acetate fraction showed 7.74% of the extract, and 0.27g was recovered per 100g of lettuce. As a result of total polyphenol and total flavonoid contents, the content of polyphenol (21.9 mg / g) and flavonoid (11.1 mg / g) were high in the lettuce extract and the ratio of total sugar to reducing sugar was 1.00 to 0.73 Respectively. On the other hand, the ethyl acetate fraction showed the highest content of polyphenol (284.8 mg / g) and flavonoid content (201.7 mg / g). This was 31.6 times higher polyphenol content and 67.2 times higher flavonoid content than water residue. Therefore, the ethyl acetate fraction of the ethanol extract of lettuce was expected to exhibit various physiological activities. As a result of the analysis, the fraction showed strong antioxidative activity and nitrite scavenging ability.

Example  2: Sequential organic solvent of ethanol extract of lettuce Fraction  Evaluation of blood coagulation inhibitory activity

The ethanol coagulation inhibitory activity of the ethanol extract and the fraction of Example 1 was evaluated. The results are shown in Table 2. At this time, the evaluation method of blood coagulation inhibitory activity of ethanol extract of lettuce 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 apathy time were measured. Plasma was obtained from a commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl ₂ and 10 μl of various concentrations of the sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., After the addition, the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 32.1 sec. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The thrombin inhibitory activity was expressed by the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

Prothrombin time ( prothrombin  time)

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 18.1 sec. The prothrombin inhibitory activity was expressed as the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

aPTT  (activated Partial Thromboplastin  Time)

Add 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) to the tubes of Amelung coagulometer KC-1A (Japan) and incubate for 3 minutes at 37 ° C. Add 100 μl of plasma and 10 μl of various concentrations of sample extract Min. After the addition of 50 μl CaCl ₂ (35 mM), the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the mean value of three repeated experiments. The activity of inhibiting blood coagulation factor was represented by aPTT divided by the aPTT of the solvent control when the sample was added.

[Table 2] Ethanol extract of lettuce and Fraction  Blood coagulation inhibitory activity

As a result, both the ethanol extract and its fractions exhibited excellent blood coagulation inhibitory activity at a concentration of 5 mg / ml, and showed a concentration-dependent inhibition of thrombogenesis. Especially, in the ethyl acetate fraction, thrombin time, prothrombin time, and apitime time, which were more than 15 times longer than those of no - added group, showed the most potent thrombogenic inhibitory activity. Followed by hexane fraction, butanol fraction and water residue. It was confirmed that the lettuce has various antithrombotic active ingredients ranging from fat soluble to water soluble. Thus, it was confirmed that the extracts of hexane fraction, butanol fraction And water residues showed antithrombotic activity of aspirin 1.5 mg / ml. Considering that lettuce is used for edible purposes, lettuce extract and its active fractions may be developed as a commercial antithrombotic agent, and it may be possible to replace aspirin, which is a conventional anti-thrombotic agent, exhibiting side effects such as gastrointestinal disorders . In addition, the above-mentioned fractions of lettuce are expected to exhibit excellent antioxidative and carcinostatic activities and thus contribute to blood circulation improvement and antithrombotic activity additionally.

Example  3: Platelet Aggregation Inhibitory Activity of Lettuce Extract

The human platelet aggregation inhibitory activity of the ethanol extract of Example 1 and fractions thereof was evaluated. The results are shown in Table 3 and FIG. Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. Is an important cell that secretes factors and initiates thrombogenesis by forming a primary hemostatic plug in association with collagen exposed by damage of endothelial cells. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombogenesis to be. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then, 2.5 μl of collagen was added to 1 ml of the suspension, and the mixture was reacted for 5 minutes. Platelet aggregation was measured at 37 ° C using a whole-blood aggregator (Chrono-log, USA).

[Table 3] Ethanol extract of lettuce and its Fraction  Platelet aggregation inhibitory activity

As shown in Table 3, first, aspirin exhibited an aggregation of 41.6% at a concentration of 0.25 mg / ml and an agglutination of 19.1% at a concentration of 0.5 mg / ml, showing strong inhibition of platelet aggregation in a concentration- I was able to know the basis used. On the other hand, ethanol extract of lettuce showed no inhibitory activity on human platelet aggregation at a concentration of 0.25 mg / ml. Among the fractions, the hexane fraction (0.25 mg / ml) exhibited human platelet aggregation promoting activity, and the platelet aggregation was confirmed to be about 1.89 times as compared to the non - platelet fraction. The butanol fraction showed no aggregation inhibitory activity. The water residue was weaker than aspirin but exhibited 63.6% agglutination at 0.25 mg / ml concentration and showed excellent antithrombotic activity. The most potent human platelet aggregation inhibitory activity was found in the ethyl acetate fraction. The concentration of 15.6% at the concentration of 0.25 mg / ml and the concentration of 55.3% at the concentration of 0.13 mg / ml showed IC _{50 values} of 0.145 mg / ml , Which was lower than the IC _{50 of} 0.156 mg / ml of aspirin. Therefore, the ethanol extract of Lettuce and its active fractions, especially the ethyl acetate fraction and the water residue, will exhibit excellent antithrombotic activity through inhibition of platelet aggregation inhibition and blood coagulation inhibition.

Example  4: Lettuce extract and its Fraction  Human erythrocyte hemolytic activity

Lettuce is an edible plant that has been registered as a food ingredient and secured. To evaluate the possibility of acute toxicity of ethanol extract of lettuce and its fractions, human hemolytic hemolytic activity was evaluated, and the results are shown in Table 4. The hemolytic activity was assessed according to the existing report (Jung Ho Jung, 2014, Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS was diluted in 96-well microplates 100 μl of the supernatant was transferred to a new microtiter plate and centrifuged for 10 minutes at 1,500 rpm. Then, the hemoglobin efflux according to hemolysis was measured at 414 nm Respectively. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

[Table 4] Lettuce ethanol extract and its Fraction  Human erythrocyte hemolytic activity

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. The ethanol extract of lettuce and its fractions showed no red blood cell hemolysis up to a concentration of 1 mg / ml, indicating no acute toxicity and erythrocyte hemolytic activity.

Example  5: Lettuce ethanol extract, its ethyl acetate Fraction  ≪ / RTI > and plasma residue, acid and thermal stability of water residues

The ethanolic extracts of lettuce, its ethylacetate fractions, and water residues obtained in Example 1 were tested for plasma stability, thermal stability and acid stability against antithrombotic activity. The ethanol extract of lettuce and its active fractions showed no inhibition of blood clotting and inhibition of platelet aggregation inhibition even after 1 hour heat treatment at 100 ° C, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma , Rather, the antithrombotic activity was partially increased. Therefore, it was confirmed that the ethanol extract of lettuce contains various anti-thrombogenic active ingredients having acid resistance and heat resistance.

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis comprising an ethyl acetate fraction of lettuce ( Lactica sativa L.) ethanol extract as an active ingredient. delete A health functional food for preventing or ameliorating thrombosis comprising an ethyl acetate fraction of lettuce ( Lactica sativa L.) ethanol extract as an active ingredient. delete

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