KR20190021502A - Pharmaceutical composition comprising the zingiber officinale leaf extracts as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an antithrombotic composition containing a Zingiber officinale leaf extract as an effective component and, more specifically, to a pharmaceutical composition and health functional food which contain a Zingiber officinale leaf extract, prepared by collecting washed and carefully selected Zingiber officinale leaves and preparing the same, as an effective component for prevention or treatment/alleviation of thrombosis by inhibiting blood coagulation and platelet aggregation. According to the present invention, a Zingiber officinale leaf extract as an effective component of the pharmaceutical composition for prevention or treatment of thrombosis and the health functional food shows strong antithrombotic activity by inhibition of a thrombus creation-related enzyme, a blood coagulation factor, and human platelet aggregation in a concentration-dependent manner, does not show hemolytic activity for human red blood cells, has excellent thermal stability, and does not show loss of thrombosis-related enzyme and blood coagulation factor inhibition effects even in an acidic condition of pH 2 and in plasma, thereby being used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by blood circulation improvement. The effective component can be processed in various forms such as extract, powder, tablet, and pill and thus can be prepared in forms which can be taken at any time, thereby being very useful in the pharmaceutical and food industries. Since leaves discarded after harvesting Zingiber officinale are used, the present invention can contribute to the reuse of unused resources and environment preservation.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which contains ginger leaf extract as an active ingredient,

The invention ginger (Zingiber The present invention relates to an antithrombotic composition containing leaf extract of an officinale or an officinale as an active ingredient. More particularly, the present invention relates to an antithrombotic composition comprising a ginger leaf extract prepared from the selected ginger leaf, And a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of platelet aggregation and a health functional food.

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 a number of blood coagulation factors to finally produce a fibrin monomer from fibrinogen. The fibrin monomers are polymerized by calcium, Cells, which produce permanent blood clots while forming cross-linked fibrin polymers by factor XIII. 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.

On the other hand, ginger (Zingiber officinale ) is one of the subtropical and tropical perennial herbaceous plants of ginger (Zingiberaceae). Its rootstock has unusual taste and aroma, and it is used as three spices together with garlic and pepper in Korea. Ginger is used as food as it is, and it is used as seasoning in various food industries such as kimchi, seafood, and confectionery. In addition, they are processed into product forms such as dried ginger (health or dry powder), oleoresin (preserved ginger), and ginger essential oil, and are used as food and medicine materials and cosmetic materials (Kim Sunhee 2013. Ph.D. Actual ginger is known to have a wide variety of physiological activities that are said to prevent white spots (100 kinds of diseases) in the main stem of the ginger, and it is known that it has excellent effects on the placebo and digestive disorders, vomiting and analgesia. The generic name of the ginger is [health], and the general person is also called [schan]. In the Goryeo Dynasty, it was used as a royal slave, and in ancient medicine books [Ayurveda] of India, it is introduced as "God's treatment".

The main producer of ginger is India, accounting for more than 50% of global production. It is mainly produced in Andong, Youngju, Gyeongbuk, Seosan, Dangjin, Jeonbuk, and Wonju. Ginger varieties are classified as native varieties, improved varieties, and also classified into early, late, and late-life varieties. They are divided into small ginseng, regenerated ginseng, and large ginseng depending on their size. According to the RDA's Food Analysis DB (2006), ginger is 1.5% protein. Fat 0.2%. It contains 13.9% carbohydrates, 1.1% ash and 83.3% water and contains various vitamins, minerals and essential amino acids. In particular, ginger has 344 mg of potassium per 100 g, which is also beneficial for saline emissions. The effective components of ginger are various distillation components of hydrocarbons and ketones, fragrance components such as zingiberene, oil components such as borneol and gingiberol, 6-ginegerol and 6-shagaol which are spicy. These components have antioxidant activity (Lee, EJ et al., 2011, The Korean Society of Food Science and Technology, 43: 469-474), antimicrobial activity (Lee, Myunghee et al., 2012. J. Fd Hyg. Safety 27: 109-116) (Yonsei University, 2013. Doctoral dissertation, University of Ulsan), hyperactivity, and sedative sedation (Han Hyo-sun, 2016, Ph.D., Konkuk University) ), pancreatic lipase inhibition (Bae JS et al., 2011. Korean J. Food Preserv. 18: 390-396), blood cholesterol lowering effect 2012, J Vet Clin 29: 441-446), and brain cell protective action (Jeong Gyun et al., 2010, Korean Journal of Biochemistry 41: 190-195).

Recently, it has been reported that the extract of ginger underground and 6-shogaol and 6-gingerol exhibit platelet aggregation inhibitory activity (Hibino et al., 2008. Journal of Pharmacological Sciences 108: 89-94) and synthetic derivatives of gingerol and shogaol inhibited platelet aggregation (Shih HC et al., 2014. International Journal of Molecular Sciences 15: 3926-3951). However, there is some doubt about the antithrombotic and platelet aggregation inhibitory activity of ginger, and further research is needed There are also reports of authoritative journals (Marx W et al., 2015. PLoS One. Oct 21; 10 (10): e0141119). However, the inhibition of strong platelet aggregation and antithrombotic activity by anticoagulation activity on ginger leaves have not been known to date.

On the other hand, ginger leaves have been used for tea or herbal medicine for a long time, but they are not yet registered as food raw materials in Korea, and there are few studies on its useful physiological activity. In this study, we investigated the antioxidant activity of ginger leaves from the root of ginger (Lee, Hee-ryun et al., 2014. J Korean Soc Food Sci Nutr 43: 1369-1379) Anti-inflammatory activity and anti-cancer activity. Especially, according to the report of Lee, HyeRyun et al., The crude saponin content was 1.7 times higher than that of the ginger root, and 6-gingerol, 10-gingerols and shagaols were found similar to ginger in leaves and stem. Were detected only in ginger root, and arachidic acid was detected only in leaves and roots. These results suggest that ginger leaves have a great difference in composition and potency with ginger root. However, there has been no report on the strong antithrombotic activity of ginger leaves which have been abandoned without special use to date.

Patents related to ginger include Korea Patent No. 10-1476092 [hangover drink and its preparation method], and Korean Patent No. 10-1195527 [method of producing ginger extract, oral composition containing ginger extract prepared therefrom And a process for producing the same], a registered patent No. 10-1145073 [a cosmetic composition for whitening containing ginger extract and a process for producing the same], a registered patent No. 10-1384633 [containing a ginger extract or its fractions as a toxin plasma However, there is no patent for ginger leaf showing useful activity. In particular, there is no patent for strong antithrombotic activity of ginger leaf. .

 Hibino, Tomoko et al., 2008. Goshuyuto, a Traditional Japanese Medicine for Migraine, Inhibits Platelet Aggregation in Guinea-Pig Whole Blood. J. Pharmacological Sciences 108: 89-94.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide an antithrombotic composition containing the discarded ginger leaf as an extract, I would like to.

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

The ginger leaf extract, which is an active ingredient of the pharmaceutical composition, is preferably ginger leaf ethanol extract.

The present invention is ginger leaf (Leaf of Zingiber The present invention provides a health functional food for prevention or amelioration of thrombosis, which comprises an extract of officinale as an active ingredient.

The ginger leaf extract, which is an active ingredient of the health functional food, is preferably ginger leaf ethanol extract.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the ginger leaf extract as an active ingredient of the health functional food can be used in a concentration-dependent manner to inhibit thrombogenesis-related enzymes, And exhibits strong antithrombotic activity due to inhibition of human platelet aggregation, exhibits no hemolytic activity on human erythrocytes, exhibits excellent thermal stability, exhibits an inhibitory effect on thrombogenesis-related enzymes and blood coagulation factors 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 and the active ingredient is processed into various forms such as extract, powder, ring, tablet, etc. Since there is an excellent effect that can be prepared in a form that can be taken at any time, Industry and food industry and the invention is very useful, because the leaves are discarded after use ginger harvest has an advantage that can contribute to the recycling and conservation of resources insoluble.

Fig. 1 shows a ginger root portion of a peeled ginger,
Figure 2 shows the shell of the ginger root,
3 shows ginger leaves,
4: DMSO, 2: aspirin (0.125 mg / ml), 3: aspirin (0.25 mg / ml) 6: Ginger root extract (0.25 mg / ml), 7: Ginger leaf extract (0.18 mg / ml), 5: Ginger root extract, ml) and 8: ginger leaf extract (0.25 mg / ml), respectively.

Hereinafter, the present invention will be described in detail.

In order to test the anti-thrombogenic effect of ginger, the inventors of the present invention prepared an extract after skin removal, a skin extract and a ginger leaf extract of a ginger underground, The ginger leaf extract was recovered as an anti-thrombogenic component. The ginger leaf extract had excellent hemostatic activity against human erythrocytes and had excellent thermal stability and acid stability. As a pharmaceutical composition for prevention or treatment / improvement and as a health functional food.

In order to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis using a ginger known to have various physiological activities in the private sector, the inventors of the present invention have proposed a method for producing a ginger under the skin, And the extracts of these extracts were tested for antithrombotic activity against thrombin time, prothrombin time and blood coagulation factor inhibition (activated partial thromboplastin time : aPTT) was evaluated, and it was confirmed that the ginger leaf extract had excellent antithrombotic activity following strong anticoagulant inhibition.

In addition, platelet aggregation inhibition activity of human platelets was evaluated, and it was confirmed that ginger leaf extract showed more potent inhibitory activity against platelet aggregation than aspirin (platelet aggregation inhibitor used in clinical use).

In addition, the ginger leaf extract can recover 7.6 g per 100 g of ginger leaf, yielding a yield three times higher than the extraction yield of ginger underground, and considering that most of the ginger leaves are discarded, .

In addition, it was confirmed that the ginger leaf extract exhibiting antithrombotic activity does not show hemolytic activity on human erythrocytes and does not cause acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains ginger leaf extract as an active ingredient.

The ginger leaf extract, which is an active ingredient of the pharmaceutical composition, is preferably an ethanol extract of ginger leaf.

Further, the present invention provides a health functional food for preventing or ameliorating thrombosis which contains ginger leaf extract as an active ingredient.

The ginger leaf extract, which is an active ingredient of the health functional food, is preferably an ethanol extract of ginger leaf.

Hereinafter, the method for producing ginger leaf extract of the present invention and the experiment for efficacy will be described in more detail.

The inventors of the present invention have found that, in order to achieve the object of the present invention, Washing the ginger leaves to remove foreign matter; Preparing an extract from the ginger leaf from which water has been removed; Experiments of the antithrombotic activity evaluation of the extract and the evaluation of the stability of the active substance were carried out.

The "ginger leaf extract" contained in the composition of the present invention comprises a step of recovering ginger leaves, a step of extracting ginger leaves with a solvent, a step of filtering the extract using a filter net of 0.06 mm or less, Can be obtained.

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

In the present invention, when the concentration of ginger leaf extract was adjusted to 5 mg / ml, thrombin time, prothrombin time, and apathy time were measured. As a result, the time was extended by 1.27 times, 1.06 times, and 1,05 times, respectively And prothrombin time, prothrombin time, and apathy time were 1.8 times, 1.31 times, and 1.59 times, respectively, at a concentration of 7 mg / ml. Considering that aspirin (product name: Protect), which is used as an antithrombotic agent in clinical practice, prolongs thrombin time, prothrombin time, and apathy time by 1.41 times, 1.34 times, and 1.48 times at 1.5 mg / ml concentration, Activity.

The ginger leaf extract of the present invention can be prepared into powders through conventional powdering processes such as vacuum drying and 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 agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or 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 ginger extract

In 2016, ginger was grown in Andong, Gyeongbuk province. The ginger root was removed from the ground, and the ginger root, ginger root and ginger root were removed. The ginger root, ginger root, and ginger leaf peeled from the shell used in the experiment are shown in Figs. 1 to 3, respectively. Ethanol extracts were prepared by adding 10 times ethanol (95%, Duksan, Korea) to each sample and extracting the extracts three times at room temperature. The extracts were collected, filtered and concentrated under reduced pressure to prepare powders. Total polyphenols, total flavonoids, total sugars and reducing sugar content were determined by analysis of the constituents of the prepared extracts. 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. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C. for 1 hour. 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] Yield and Composition of Extracts by Ginger Region

As a result, as shown in Table 1, the extraction yield of ginger was in the order of ginger leaf> ginger root, ginger root, and ginger leaf in a yield of 7.6%. The total polyphenol content of the ginger root showed the highest total polyphenol content (53.3 mg / g) in the bottom part of the ginger, and the lowest content of 31.9 mg / g in the ginger root. On the other hand, total flavonoid content was 16.2 mg / g in ginger leaf. Total sugars and reducing sugar content were highest in ginger leaf, bottom ginger root and ginger bottom shell.

Example  2: Evaluation of blood coagulation inhibition activity of extracts of ginger

The blood coagulation inhibitory activity (thrombogenesis inhibitory activity) of the ginger extract prepared in Example 1 was evaluated and compared with the previously reported method (Sohn et al., 2004. Kor J. Pharmacogn 35. 52-61; Kwon Thrombin time, prothrombin time, and apathy time were measured and evaluated in the same manner as in J. et al., 2004. J. Life Science, 14. 509-513; . Plasma was obtained from a commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and the 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 sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., and 100 μl of plasma was added And 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 24.2 seconds. 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 16.8 seconds. 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. Then, 50 μl CaCl ₂ (35 mM) was added and 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 42.2 seconds. The results of aPTT were expressed as the mean value of three repeated experiments. The activity of inhibiting blood coagulation factor was expressed as aPTT divided by the aPTT of the solvent control when the sample was added.

[Table 2] Evaluation of blood coagulation inhibitory activity of extracts by ginger

As a result, as shown in Table 2, the ginger root extract and ginger root extract showed a slight anticoagulant activity, while the ginger leaf extract showed excellent thrombin inhibition at a concentration of 5 mg / ml. In addition, at the concentration of 7 mg / ml, the ginger leaf extract showed very good thrombin inhibition, prothrombin inhibition and blood coagulation inhibition through inhibition of blood coagulation factors. At this time, aspirin used as a control was lengthened by 1.41 times, 1.34 times, and 1.48 times of thrombin time, prothrombin time, and apathy time at a concentration of 1.5 mg / ml. These results suggest that ginger leaf extract can be developed as an antithrombotic agent that can replace gastrointestinal aspirin.

Example  3: Evaluation of Platelet Aggregation Inhibitory Activity of Ginger Extract

The human platelet aggregation inhibitory activity of the ginger extract obtained in Example 1 was evaluated and 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. It is an important cell that secretes factors and binds to collagen exposed by damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombogenesis. 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] Human Platelet Aggregation Inhibitory Activity of Ginger Extracts

As shown in Table 3 and FIG. 4, when DMSO was added as a solvent control, human platelets rapidly and strongly aggregated by the addition of collagen, whereas platelet aggregation was strongly influenced by concentration of aspirin, which is a platelet aggregation inhibitor Respectively. Aspirin did not affect the cohesion at the concentration of 0.125 mg / ml, but the coagulation ratio was 56.3% at the concentration of 0.25 mg / ml and 18.0% at the concentration of 0.5 mg / ml, . On the other hand, the ginger extracts and peel extracts from the ginger extracts showed 194.9% and 159.5% aggregation at the concentration of 0.25 mg / ml, respectively. (Jantan I et al., 2005. Phytomedicine 12: 88-92) and Goshuyuto, a Japanese traditional prescription containing ginger, inhibited platelet aggregation (Hibino, Tomoko et al., 2008. J. Pharmacological Sciences 108: 89-94), but there is a contradictory study on the anti-thrombotic and platelet aggregation inhibitory activity of ginger, (Marx W et al., 2015. PLoS One. Oct 21; 10 (10): e0141119). This difference can be understood as a result of the measurement method of platelet aggregation and the difference in mechanism of action.

On the other hand, ginger leaf extract showed strong inhibition of platelet aggregation in a concentration-dependent manner, exhibiting 48.1% at 0.18 mg / ml concentration and 30.1% at 0.25 mg / ml concentration and exhibiting stronger agglutination inhibition effect than aspirin at the same concentration . The strong inhibitory activity of platelet aggregation of ginger leaf extracts suggests that it is possible to produce anti-thrombotic agent and blood circulation improving health functional food using ginger leaf, and suggest that it can contribute to economical production of high value-added products and reuse of by-products have.

Example  4: Assessment of hemolytic activity of human erythrocyte by extract of ginger

Ginger leaves have long been edible as tea and herbs, but have not yet been registered as domestic food ingredients. However, since it has been used for a long time, it is considered that the ginger leaf extract has no specific toxicity. To evaluate the potential acute toxicity of ginger leaf extract, human hemolytic hemolytic activity was evaluated and the results are shown in Table 4. The hemolytic activity was assessed in accordance with a previous report (Son Ho et al., 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.

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

Abs. S: absorbance of sample added sphere,

Abs. C: absorbance of DMSO-added sphere,

Abs. T: Absorbance of Triton X-100 added sphere.

[Table 4] Human erythrocyte hemolytic activity of ginger leaf extract

First, DMSO and distilled water used as control did not have erythrocyte hemolytic activity, and Triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, ginger leaf extract showed no hemolytic activity up to a concentration of 1 mg / ml.

Example  5: Evaluation of Plasma, Acid and Thermal Stability of Ginger Leaf Extract

The plasma stability, thermal stability and acid stability of the ginger leaf extract obtained in Example 1 were confirmed. The extract retained excellent activity without heat treatment at 100 ° C for 1 hour, treatment with pH 2 (0.01M HCl) for 1 hour, and plasma treatment for 1 hour without loss of platelet aggregation inhibitory activity. Therefore, the active ingredient of ginger leaf extract is expected to maintain antithrombotic activity in general food processing and digestion and absorption stages. These results suggest that ginger leaf extract can be used as an antithrombotic agent and it may be possible to supplement and replace aspirin with adverse effects such as gastrointestinal disorder.

Claims (4)

Leaf of Zingiber officinale ) extract as an active ingredient for the prevention or treatment of thrombosis. The pharmaceutical composition according to claim 1, wherein the ginger leaf extract is ginger leaf ethanol extract. Leaf of Zingiber officinale ) as an effective ingredient for the prevention or amelioration of thrombosis. The health functional food according to claim 3, wherein the ginger leaf extract is ginger leaf ethanol extract.

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