KR101645878B1 - Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents
Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same Download PDFInfo
- Publication number
- KR101645878B1 KR101645878B1 KR1020150062903A KR20150062903A KR101645878B1 KR 101645878 B1 KR101645878 B1 KR 101645878B1 KR 1020150062903 A KR1020150062903 A KR 1020150062903A KR 20150062903 A KR20150062903 A KR 20150062903A KR 101645878 B1 KR101645878 B1 KR 101645878B1
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- thrombosis
- organic solvent
- bellflower
- pharmaceutical composition
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/34—Campanulaceae (Bellflower family)
-
- A23L1/3002—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Abstract
The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis, which comprises an organic solvent fraction of an extract of a rootstock or red sea bream underground as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for preventing or treating thrombosis, The present invention also relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis, which comprises an organic solvent fraction obtained after fractionation of an organic solvent fraction thereof from an extract thereof, as an active ingredient. The pharmaceutical composition for the prevention or treatment / improvement of thrombosis according to the present invention and the organic solvent fraction of bellflower or Hongodoraji underground extract as an active ingredient of the health functional food, And exhibits strong antithrombotic activity by the inhibition effect of platelet aggregation inhibiting the initiation of thrombus formation, and has an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In addition, the organic solvent fraction is excellent in thermal stability and does not show loss of antithrombotic activity even in an acidic condition of pH 2 and plasma, and can be processed into various forms such as extract, powder, It is a very useful invention in the pharmaceutical industry and the food industry because of its excellent effects that can be prepared.
Description
The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis, which comprises an organic solvent fraction of an extract of a rootstock or red sea bream underground as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating thrombosis, The present invention also relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis, which comprises an organic solvent fraction obtained after fractionation of an organic solvent fraction thereof from an extract thereof, as an active ingredient.
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 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 europaine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants are not only very high in price, but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.
Platycodon grandiflorum A. DC) is a perennial plant of Campanulaceae. Its main stem is about 10 ~ 15cm, diameter is 1 ~ 3cm, there is irregular stem spot on the upper part, gray to milky white color, vertical wrinkle is deep, There are skin and wrinkles. The vagina is hard, non-fibrous, easy to break, has a slight odor, and has a taste (Jang Chang-soo et al., Hyundai Genetics, Seoul, Korea, 460-461, 1993).
The root of the bellflower is called "Gyungyung" and it is widely used as a useful medicinal material as well as food (Korean Pharmacopoeia, Ministry of Health and Social Affairs, 2007). Generally, moisture content is 85%, protein 1.8%, lipid 0.2%, carbohydrate 10.4% %, Is well known as an alkaline health food rich in fiber and rich in vitamins and minerals. The bellflower is used as a bellflower, a bell pepper, and a raw beet after removing the bitter taste due to its unique fragrance and bitter taste. (Jangho Park, 2011, Chubu University) The bitter component of bellflower is not yet known, but it is known to be alkaloids and glycosides.
The main pharmacological component of the bellflower is tritiated with about 3% of the roots as platyconsin A, C, D, D2 and D3. Pharm. Bull, 20 (2002), pp. 219-222, 1985). In addition, it has been shown that anti-inflammatory action against acute and chronic inflammation, anti-ulcer and gastric secretion inhibiting action, anticholinergic action to lower blood pressure, blood glucose lowering action and improvement of cholesterol metabolism , 1952, 1972; Chem. Pharm. Bull., 23, 2965, 1975; J. Chem. Soc., Perkin trans I., 661, 1984; J. Pharm. Soc. Kor, 19, 164, 1975). In addition, steroid-based compounds such as alpha-spinasterol, stiquamast-7-enol, alpha-spinasterol glucoside and the like and polysaccharides such as inulin and betulin, (Korean Journal of Food Science and Technology, 39: 701-707, 2007), inhibition of lung cancer cell growth (immunochemistry) 17: 183-189, 2003) 387, 1998), oxidative stress protective effects of hepatocytes (Medicine 46: 466-471, 2002) and mutation inhibitory effects (Korean Food Science 33: 651-655, 2001). In recent years, processed foods using platycodon have been developed. Typical examples include bellflower yanggang (East Asian dietary history 19: 78-88, 2009), production of candy using bellflower (Journal of Agricultural Products Research and Distribution 8: 146-150, 2001) (Korean Journal of Food Science and Nutrition 29: 752-757, 2000), bellflower powder added sulgi cake (Korean Journal of Food Science and Technology 22: 77-82, 2007), and Doraji japon (Korean Patent Registration No. 10-0418853) .
On the other hand, domestic and foreign patents related to bellflower include Korea Patent No. 10-0643877 entitled " Composition for inhibiting alcoholic liver disease containing therapeutic agent of Jang Saeng Doraji as an active ingredient, therapeutic composition for therapeutic use and composition for promoting alcohol metabolism & No. 10-0513125 entitled "Anti-fibrosis agent of soy sauce containing bellflower extract as an active ingredient" and the like are disclosed, and registered patent No. 10-1251589 entitled "Brewer's product having increased purity and effective saponin content from bellflower or bellflower extract A composition for prevention or treatment of atopic dermatitis containing the extract of Platycodon grandiflorum as an active ingredient or a composition for treating or preventing atopic dermatitis ", Registered Patent No. 10-1162710" A pharmaceutical composition for the prevention or treatment of hepatitis C containing a bellflower extract or a borage saponin compound ", Registered Patent No. 10-0830236 "Dora A composition for prevention and treatment of prostate cancer containing an extract, "No. 10-0657017 entitled" L-FABP Enhancer Containing Platine Extract and Its Purification Method ", Registered Patent No. 10-0495315 " &Quot;, Korean Patent No. 10-0315002 "Herbal preparation for treating cancer including Jang Saeng Doraji extract ", Registered Patent No. 10-0315001," Jang Saeng Doraji A composition for inhibiting and treating lung cancer caused by tobacco comprising an extract of Jang Saeng Doraji hot water as an active ingredient ", Registered Patent No. 10-0314999 " A herbal preparation for treating antimicrobial and inflammatory diseases containing Jang Saeng Doraji extract, "No. 10-0315000," a herbal preparation for treating hyperlipidemia including Jang Saeng Doraji extract & 10-0761331 "a composition for external application to the skin containing the topical extract of Doraji, its fractions or a compound isolated therefrom" and Korean Patent Publication No. 10-2015-0007810 entitled " Anticancer, antidiabetic or antimicrobial activity ", which is incorporated herein by reference. However, up to now, no literature or patent has been reported on the inhibition of platelet aggregation of platelets and the antithrombotic effect of platelets following inhibition of human prothrombin blood coagulation enzyme (thrombogenic enzyme).
On the other hand, if the process of melding the bellflower is repeated several times, the white bellflower gradually changes to yellow, red and black to produce red bellflower and black bellflower. In this case, the storage property and the functionality are increased, An increase in functionality is expected. As a result of studies on domestic and foreign red platycodon, the browning substances produced during the repeated dry-drying process of platycodon exhibit antioxidative, antimutagenic and antioxidant activities (Lee, Sang-Hoon et al., 2013. Korean Journal of Food Science and Nutrition 42: 1405-1411; , 2011. Korean Journal of Food Preservation and Distribution, 20: 510-517), antioxidant and anti-diabetic activities are reported to increase in red platycodon and black platycodon (Jong Ho Park, 2011, "Journal of the Korean Society of Food Science and Technology, v.18, no.1, pp.157-169," Effect of Diabetes on the Quality of Diabetic Diabetic Rats ", Journal of the Korean Society of Food Preservation, 20: 510-517).
Patent No. 10-0707345 "Reduced gastrointestinal disturbance, increased functionality and repeated washing and drying step" is known as a patent related to Hongdoraji, and it is known from registered patent No. 10-0807496 In general, "blooming red bellflower" produced by the method of manufacturing red bellflower using yellow clay roots and its preparation method, "common bellflower red roots" was placed in a pot with a yellow mortar for 10 minutes at 100 to 120 ° C. for 3 to 5 hours, And the drying process is repeated three or more times to increase the saponin component and the sensory property. Patent Document No. 10-0966304 entitled " Process for preparing aged red platycodon ", Registered Patent No. 10 -1352373 "Blackrod bellflower extract and its preparation method ", Registered patent No. 10-0644239" Jang Saeng Hong red broth manufacturing method and Jang Saeng Hong red broth manufactured by the manufacturing method ", Registered Patent No. 10-0687 715 "a composition for preventing and treating diabetes mellitus containing a hot water extract of Jangsoo Hongdorad as an active ingredient ", Registered Patent No. 10-0662206" a composition for prevention and treatment of gout, . In addition, Japanese Patent Application Laid-Open No. 10-2010-0012769 discloses a composition for enhancing skin function comprising " black platycodone extract or its fractions ", " 10-2009-0013231 discloses "a composition which is useful for improving asthma, which contains black bellflower and mushroom extract as an active ingredient ". However, until now, there have been no reports or patents related to inhibition of strong platelet aggregation inhibition and inhibition of human prothrombin blood coagulation enzyme (thrombus formation enzyme) in co-cultured platelets such as platycodon as well as red platycodon and black platycodon. There is no known patent for pharmaceutical compositions and health functional foods for preventing or treating thrombosis based on inhibition of blood coagulation and platelet aggregation inhibitory activity associated with platycodon and red platycodon.
DISCLOSURE Technical Problem Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide an ethanol extract prepared from red sea bream prepared by blossoming or repeated wet heat cooking and drying, Ethyl acetate and butanol were used for sequential organic solvent fractionation. After recovering the water residue, each fraction was assayed for inhibitory activity on thrombogenesis-related enzymes and platelet aggregation inhibitory activity. As a result, it was confirmed that the activity of platelet aggregation inhibition and prothrombin inhibition And to provide a pharmaceutical composition and a health functional food for preventing or treating / improving a thrombotic disease containing the fraction as an active ingredient.
In order to solve the above-mentioned problems, the present invention provides a method for producing an extract of Platycodon grandiflorum (DC) ethanol, which comprises fractionating ethyl acetate, butanol fraction or a mixture thereof, A pharmaceutical composition for preventing or treating thrombosis.
The bellflower may be a red bellflower.
The red roots are preferably prepared by boiling and drying the roots.
The pharmaceutical composition of the present invention is preferably used for inhibiting blood coagulation or inhibiting platelet aggregation.
The present invention also provides a health functional food for preventing or ameliorating thrombosis comprising the active ingredient of the pharmaceutical composition of the present invention.
The pharmaceutical composition for the prevention or treatment / improvement of thrombosis according to the present invention and the organic solvent fraction of bellflower or Hongodoraji underground extract as an active ingredient of the health functional food, And exhibits strong antithrombotic activity by the inhibition effect of platelet aggregation inhibiting the initiation of thrombus formation, and has an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In addition, the organic solvent fraction is excellent in thermal stability and does not show loss of antithrombotic activity even in an acidic condition of pH 2 and plasma, and can be processed into various forms such as extract, powder, It is a very useful invention in the pharmaceutical industry and the food industry because of its excellent effects that can be prepared.
Hereinafter, the present invention will be described in detail.
The inventors of the present invention conducted an experiment to determine the anti-thrombogenic effect of red blooded roots prepared by repeated wet heat cooking and drying of platycodon or bellflower, And recovered as thrombocyte active components. These fractions were confirmed to have excellent thermal stability and acid stability, so that the fractions were used as pharmaceutical composition and health functional food for preventing or treating / improving thrombosis.
In order to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis using platycodon and platycodon without antithrombotic activity so far, The antithrombotic activity of these extracts was measured by thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) for human plasma and human thrombin, respectively. As a result of the evaluation, it was confirmed that there was no antithrombotic activity in Platycodon grandiflorum and Hongdoradji extract. However, the ethanol extracts from the bottom of the platycodon and Hongdorajia were sequentially fractionated with an organic solvent to obtain hexane fraction, ethyl acetate fraction, butanol fraction and water residue, and the antithrombotic activity was evaluated. As a result, a strong prothrombin inhibitory activity And human platelet aggregation inhibitory activity. In addition, the ethyl acetate fraction of the ethanol extract of Platycodon grandiflorum and Hongdorajia showed a good thrombogenic activity and was highly likely to be developed as an antithrombotic agent. It was confirmed that the above fractions do not show any loss of antithrombotic activity even when subjected to acid treatment at pH 2, heat treatment at 100 ° C, and plasma treatment, thus completing the present invention.
In order to confirm such an effect, the inventors of the present invention have found that the extracts of the bottom part of Rhododendrons and Hongdorajia and sequential organic solvent fractions therefrom are prepared, the useful components such as total polyphenols and total flavonoids are analyzed, and the extracts and fractions The prolongation of the active thromboplastin time and inhibition of human platelet aggregation by the inhibition of human thrombin direct inhibition, prothrombin inhibition effect, inhibition of blood coagulation factors (Factor VIII, Factor IX, Factor XI and Factor XII) It was confirmed that the organic solvent fraction had superior antithrombotic activity to aspirin (trade name: Protect), which is an antithrombotic agent used commercially, and the plasma, heat and acid stability of the substance were examined to confirm that the food processing suitability and stability were excellent Respectively.
Accordingly, the present invention provides a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis, which comprises ethyl acetate fraction, butanol fraction or a mixture thereof, as an effective ingredient, of ethanol extract from the bottom of red sea bream or red sea bream.
As a preferred example, the extracts of the above-mentioned Doraji and Hongdo-raji extracts can be extracted with various solvents including water or ethanol, but they are preferably ethanol extracts, and the active fractions include platelet aggregation inhibitory activity and / or prothrombin inhibitory activity, Fractions and butanol fractions are preferred.
Hereinafter, the method for producing antithrombotic active fractions of Platycodon grandiflorum and Hongdorajia extract of the present invention and the efficacy experiments will be described in more detail.
The present invention provides a method for preparing an extract, comprising the steps of: preparing an extract from Platycodon sp. Preparing sequential organic solvent fractions of hexane, ethyl acetate and butanol from the bellflower and red sea bream extract and preparing the water residue obtained therefrom; Evaluating the antithrombotic activity of the extract and fraction and examining the stability of the butanol fraction.
The "Doraji underground part extract" contained in the composition of the present invention is prepared by purchasing more than 3 years' long cornflower, removing foreign substances, washing, extracting with an organic solvent, filtering the extract using a filter net of 0.06 mm or less, Followed by concentration under reduced pressure. In addition, the "Hongdoraji underground portion extract" is prepared by purchasing more than 3 years' long bellflower, removing foreign substances, washing, adding a certain amount of water to the cauldron, and then boiling and drying it, Extracting the red bellflower with an organic solvent, filtering the extract using a filter net of 0.06 mm or less, and concentrating it under reduced pressure. The organic solvent used in the present invention 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 95% ethanol extraction is most preferred.
In the present invention, the thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of the ethanol extract in the lower part of the platycodon and Hongdoraji, and it was found that the ethyl acetate fraction The prothrombin time, and the aprotic time were measured to be 1.22 times, 1.46 times, and 1.49 times longer than those of the non-added samples, respectively. The ethyl acetate fraction of the red seabream extract As a result of measuring the thrombin time, prothrombin time and apitime time after the addition, the coagulation time was prolonged by 1.36 times, 1.67 times, and 1.70 times, respectively, Anti-thrombotic effect. In addition, the butanol fraction of Platycodon grandiflorum and Hongdorajia extracts was prolonged by 1.58 times and 1.69 times, respectively, as compared to the non - supplemented group, confirming the prothrombinase inhibitory activity of the butanol fraction. In the case of aspirin (trade name Protect), which is a commercially available anti-thrombotic agent, considering that the thrombin time, prothrombin time, and apathy time are extended by 1.82 times, 1.81 times, and 1.92 times, respectively, And the ethyl acetate fraction of the extract from the underside of Hongdoraji can substitute anticoagulant such as aspirin which has high risk of side effects and the butanol fraction of Hongdoraji underground extract can be developed as a prothrombin inhibitor.
On the other hand, as a result of evaluating the activity of inhibiting aggregation using human platelets, the extracts of Platycodon grandiflorum and Hongdoraji showed no inhibitory activity against platelet aggregation, but the butanol fraction of the extract showed very strong platelet aggregation inhibitory activity. Especially, The fractions showed stronger agglutination inhibitory activity than aspirin, confirming that the fraction can be used as a platelet aggregation inhibitor.
The ethyl acetate fraction and the butanol fraction of the platycodon grandiflorum and the underside of the present invention may be prepared into powders through a conventional pulverization process such as vacuum distillation, freeze drying, or spray drying. 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 ethyl acetate fraction, the butanol fraction or a mixture thereof of the present invention of the present invention can be used for the prevention or treatment of various diseases related to 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 can include deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.
The pharmaceutical composition comprising the ethyl acetate fraction, the butanol fraction or the mixture thereof of the bottom part of the phloem and the red sea bream of the present invention may be formulated into powders, granules, tablets, capsules, suspensions, emulsions , Oral formulations such as syrups and aerosols, injections of sterile injectable solutions, and the like, and they can be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration and the like have.
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 ethyl acetate fraction, the butanol fraction or a mixture thereof of the present invention 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.
In a preferred embodiment of the present invention, the effective amount of the ethyl acetate fraction, the butanol fraction, or the mixture thereof in the bottom portion of Rhododendron and Hongdoraji may vary depending on the age, sex, and body weight of the patient, 1 to 5,000 mg, preferably 100 to 3,000 mg may be administered daily 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 the foods containing the ethyl acetate fraction, the butanol fraction or the mixture thereof of the present invention include a variety of foods, beverages, gums, tea, vitamin complexes and health supplement foods, , Granules, tablets, capsules or beverages.
The ethyl acetate fraction, the butanol fraction, or the mixture thereof of the platycodon grandiflorum and the underside of the present invention may be added in an amount of 0.01 to 15% by weight based on the total weight of the whole food, and the health beverage composition may be added in an amount of 0.02 to 10 g Can be added in a proportion of 0.3 to 1 g.
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 enhancers, pectic acid and its salts, alginic acid and its salts, , 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 ethyl acetate fraction, the butanol fraction, or the mixture thereof, of the bottom portion 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: Produce bellflower and red bellflower
When the bellflower is matured at a high temperature and then repeatedly dried, the white bellflower is changed to yellow, red, and black, which is called red bellflower or black bellflower. Platycodon used in this experiment is as follows. The process of making red platycodon is as follows.
1) Purchase medicinal bellflower (raw materials / 3 years or more).
2) Wash the raw materials with tap water to clean the soil or dirt.
3) Refrigerate in the raw material storage.
4) Wash the bellflower stored in the raw material storage with a washing machine and dehydrate.
5) Fill the bottom of the cauldron with about 5cm of water.
6) Primary steaming: Steamed bellflower washed in cauldron at 80 ℃ for 1 hour.
7) Primary drying: Dry for 6 hours at less than 60 ℃ in the dryer.
8) Second steaming: The first dried steak is melted at 80 ℃ for 30 minutes.
9) Secondary drying: Dry in a dryer for less than 60 ℃ for 3 hours.
10) Tertiary steaming: Secondly dried bellflower is boiled at 80 ° C for 20 minutes.
11) Third drying: Dry for 2 hours at a temperature of less than 60 ℃ in the dryer.
12) Fourth seasoning: Thirdly seasoned bellflower is matured at 80 ℃ for 20 minutes.
13) Fourth drying: Dry for 1 hour at a temperature of less than 60 ℃ in the dryer.
14) Red bellflower production (Finished product)
Example 2: Bellflower and Red bellflower Nutritional analysis and physicochemical characterization
The moisture content, crude protein, crude lipid, ash and carbohydrate content of the red bellflower prepared by the method of Example 1 were evaluated using the method determined in the Food Code. The results are shown in Table 1, and it was confirmed that the components were concentrated as the moisture content of red platycodon was lower than that of platycodon. Therefore, the energy (calories) of red platycodon was 3.13 times higher than that of common platycodon.
[Table 1] Red bellflower Nutritional analysis
On the other hand, bellflower and red bellflower were crushed, 10 times as much water was added to each, and the mixture was stirred for 10 minutes (100 rpm, Japan-Korea incubator, Korea) and centrifuged to recover the supernatant. The results of evaluating the pH, brix and useful components of the supernatant are shown in Table 2. The total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of a saturated solution of Na 2 CO 3 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. To the 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added and 40 μl of 1N NaOH was added. The reaction was carried out at 37 ° C. for 1 hour and then absorbance was measured at 420 nm. 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 2] Red bellflower pH , brix And component analysis comparison
As shown in Table 2, the pH change was slight during the process of changing the platycodon to Hongdoraji, but the Brix increased from 4.0 to 18, the total polyphenol was 12 times, the total flavonoid was 1.35 times, the total was 7.4 times, Was increased by 15.1 times. Therefore, Hongdoraji has an increase in taste and aroma compared to bellflower.
Example 3: Bellflower and Red bellflower Extract preparation and sequential organic solvents Fraction Preparation and analysis of their components
The bellflower and the red bellflower prepared by the method of Example 1 were added 10-fold each of ethanol, left at room temperature for 1 day, and the extract was recovered. After repeating the above procedure once, the extract was recovered. The extract was filtered and concentrated under reduced pressure to prepare an ethanol extract. Table 3 shows the ethanol extraction efficiency and the component analysis of the extract solution for each bellflower.
[Table 3] Comparison of Extraction Efficiency and Component Analysis of Ethanol Extract of Bellflower and Hongdorajia
As shown in Table 3, the ethanol extract of P. doloradis showed a 2.64-fold increase in total polyphenol, 1.25 fold in total flavonoid, 1.28 fold in total sugar, and 25.1 fold in reducing sugar, compared to that of S. rhizome. The extraction efficiency of red sea bream was about twice as high as that of fresh red bream.
Then, to prepare fractions of the bellflower and red sea bream extracts, the respective extracts were suspended in distilled water, followed by organic solvent fractionation using hexane, ethyl acetate and butanol, and then the water residue was recovered. The results of the sequential organic solvent fractionation and component analysis are shown in Table 4.
[Table 4] Fractional Efficiency and Angle of Extracts of Bellflower and Rhododendron Fraction Comparing component analysis
As shown in the fractionation efficiency, the red ginseng contains much lipophilic component than the bellflower, and the fractions contain polyphenol and flavonoid components which are greatly increased compared to the bellflower, and the increase of the reducing sugar content Respectively. The highest polyphenol contents were in the ethyl acetate fraction, and the butanol fraction, water residue and hexene fraction were the highest in order. This result supports the previous report that antioxidant activity is increased by boiling and drying of bellflower. Ethyl acetate fraction and butanol fraction of Rhododendron and Hongdorajia extracts showed excellent antioxidant activity, which is expected to contribute to blood circulation improvement and aging inhibition.
Example 4: Platycodon grandiflorum extract and sequential organic solvent Fraction Evaluation of blood coagulation inhibitory activity
Table 5 shows the results of evaluating the anticoagulant activity of the ethanol extracts and the fractions thereof in the lower part of the platycodon and Hongdorajia as an antithrombotic activity evaluation. The antithrombotic activity of each extract was evaluated in accordance with 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 prepared from the whole blood of the applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000g for 5 minutes at 4 ° C and stored frozen (fresh frozen plasma) and thawed at room temperature if necessary. The thrombin time, prothrombin time, and apathy measurements were performed as follows.
Thrombin time( 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), incubate 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 )
100 μl of plasma and 10 μl of various concentrations of sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), and the mixture was heated at 37 ° C for 3 minutes. Then 50 μl of aPTT reagent (Sigma, ALEXINTM) Lt; / RTI > 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, and the coagulation factor inhibitory activity was expressed as aPTT time divided by the aPTT time of the solvent control.
[Table 5] Drosophila and Hongdorajia extracts and their Fraction Anti-thrombosis activation
As shown in Table 5, the antithrombotic activity evaluation of the ethanol extract of Platycodon grandiflorum and Hongdorajia showed no blood clotting inhibitory activity up to a concentration of 5 mg / ml. However, the ethyl acetate fraction of Platycodon grandiflorum and Rhododendron japonica extract showed strong anticoagulant activity, and inhibition of thrombin and prothrombin associated with thrombus formation as well as inhibition of blood coagulation factors were confirmed. In addition, inhibitory activity against prothrombin was strongly exhibited in the butanol fraction of each of the bellflower extracts, and inhibitory activity comparable to aspirin (1.5 mg / ml) was confirmed. These results suggest that the extracts of Platycodon grandiflora and Rhododendron japonica have weak antithrombotic activity. However, its ethyl acetate fraction has very strong antithrombotic activity and it is considered to be able to replace aspirin, which is a conventional antithrombotic agent showing side effects such as gastrointestinal disorders , Antithrombotics using platelets and red bloodediol butanol fractions with excellent prothrombin inhibitory activity could be developed.
Example 5: Platycodon grandiflorum extract and sequential organic solvent Fraction Evaluation of human platelet aggregation inhibitory activity
The platelet aggregation inhibitory activity was evaluated as an antithrombotic activity evaluation of platycodon grandiflorum extract and fractions thereof. 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 Inhibitory Activity Platelet aggregation inhibition activity )
Platelets were purchased and used with the approval of the Institutional Bioethics Committee of Andong University. Human concentrated platelets were 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) and then suspended 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), centrifuged at 3,000 rpm for 10 minutes and resuspended in the suspending buffer , Platelet count was adjusted to 4x10 < 9 > / ml. Then platelet aggregation was measured at 37 ° C using a whole-blood agarometer (Chrono-log, USA) after 2.5 μl of collagen was added to 1 ml of suspension and reacted for 5 minutes.
[Table 6] Drosophila and Hongdorajia extracts and sequential organic solvents Fraction Platelet aggregation inhibitory activity
As shown in Table 6, aspirin used in clinical use as a platelet aggregation inhibitor inhibited platelet aggregation in a concentration-dependent manner. In this experiment, aspirin showed 81.0% aggregation at a concentration of 0.25 mg / ml and 44.1% at a concentration of 0.5 mg / And exhibited concentration-dependent coagulation inhibitory activity. On the other hand, Platycodon grandiflorum extract did not exhibit platelet aggregation inhibitory activity, but rather promoted coagulation. Among the fractions, the butanol fraction showed the inhibition activity of platelet aggregation. The butanol fraction of the platycodon exhibited a coagulation degree of 55.6% at the concentration of 0.25 mg / ml, showing stronger agglutination inhibitory activity than the aspirin, which is an antiplatelet agent, at the same concentration. The platelet aggregation inhibition activity was remarkably exhibited in the butanol fraction of red flounder, and the platelet aggregation was not observed at the concentration of 0.25 mg / ml. When treated at the concentration of 0.15 mg / ml, the inhibition of agglutination was 47.6% Strong agglutination inhibition activity was exhibited. These results indicate that butanol fractions of Platycodon grandiflorum and Hongdorajia, especially the Hongdoraju butanol fraction, can be used as an antiplatelet agent which can replace aspirin, which is a side effect such as gastrointestinal disorder during long term use.
Example 6: Bellflower and red bellflower Butanol Fraction Chemical properties and stability
(Ethanol extraction efficiency: 5.6%, butanol fractionation efficiency: 30.5%) in the case of dorajoo butanol, 100g in the case of red bellflower butanol, and 2.89 g (ethanol extraction efficiency of 10.7%, butanol fraction efficiency of 27.0%). The active fractions were tested for plasma stability, thermal stability and acid stability against antithrombotic activity. The butanol fraction was found to retain its activity during the various composition processes because it did not show a decrease in platelet aggregation inhibitory activity even after 1 hour heat treatment at 100 ° C, 1 hour treatment with pH 2 (0.01 M HCl), and 1 hour treatment with plasma .
Claims (5)
Wherein the composition is for inhibiting blood coagulation or inhibiting platelet aggregation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150062903A KR101645878B1 (en) | 2015-05-06 | 2015-05-06 | Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150062903A KR101645878B1 (en) | 2015-05-06 | 2015-05-06 | Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR101645878B1 true KR101645878B1 (en) | 2016-08-04 |
Family
ID=56709616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150062903A KR101645878B1 (en) | 2015-05-06 | 2015-05-06 | Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101645878B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210059163A (en) * | 2019-11-14 | 2021-05-25 | 한경수 | A pharmaceutical composition for prevention or treatment of thrombosis comprising korean medicinal mixture as an effective component and helth functional food comprising the same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100807496B1 (en) | 2006-11-10 | 2008-02-25 | 김해진 | Manufacturing method of with use loess resourceful water red platycodon and loess red platycodon thereof |
| KR100966304B1 (en) * | 2009-08-07 | 2010-06-28 | (주)메나리 | Processing method of maturing red bellflower |
| KR20150022251A (en) * | 2013-08-22 | 2015-03-04 | 한국 한의학 연구원 | Composition for prevention or treatment of thrombotic diseases comprising extracts or fractions of Playtcodon grandiflorum |
-
2015
- 2015-05-06 KR KR1020150062903A patent/KR101645878B1/en active IP Right Grant
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100807496B1 (en) | 2006-11-10 | 2008-02-25 | 김해진 | Manufacturing method of with use loess resourceful water red platycodon and loess red platycodon thereof |
| KR100966304B1 (en) * | 2009-08-07 | 2010-06-28 | (주)메나리 | Processing method of maturing red bellflower |
| KR20150022251A (en) * | 2013-08-22 | 2015-03-04 | 한국 한의학 연구원 | Composition for prevention or treatment of thrombotic diseases comprising extracts or fractions of Playtcodon grandiflorum |
Non-Patent Citations (2)
| Title |
|---|
| 이상훈 외, 2013. 숙성온도와 기간에 따른 도라지의 이화학적 특성과 항산화 활성. 한국식품영양과학회지 42: 1405-1411 |
| 이수진 외, 2013. 흑도라지의 항산화 및 항균 활성. 한국식품저장유통학회지, 20: 510-517 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210059163A (en) * | 2019-11-14 | 2021-05-25 | 한경수 | A pharmaceutical composition for prevention or treatment of thrombosis comprising korean medicinal mixture as an effective component and helth functional food comprising the same |
| KR102298129B1 (en) * | 2019-11-14 | 2021-09-06 | 한경수 | A pharmaceutical composition for prevention or treatment of thrombosis comprising korean medicinal mixture as an effective component and helth functional food comprising the same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101469351B1 (en) | A pharmaceutical composition comprising the butanol fraction of raphanus sativus l. leaves extract as an effective component for prevention or treatment of diseases related to thrombosis and a health functional food comprising the same | |
| KR101645855B1 (en) | Pharmaceutical composition comprising the extract of lepiduium meyenii hypocotyl as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101387685B1 (en) | Pharmaceutical composition comprising the extract of schizandra chinensis as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101845160B1 (en) | Pharmaceutical composition comprising the extraction of salicornia spp. seed as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101678301B1 (en) | Pharmaceutical composition comprising the extract of ribes nigrum l. fruit as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR102013312B1 (en) | Pharmaceutical composition comprising the extract of apios americana medikus as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR102084503B1 (en) | 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 | |
| KR101645878B1 (en) | Pharmaceutical composition comprising the organic solvent fractions of platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101583910B1 (en) | Pharmaceutical composition for prevention or treatment of thrombosis comprising the seed extract of cornus officinalis | |
| KR101763714B1 (en) | Pharmaceutical composition comprising the hot water extract of black platycodon grandiflorum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101646003B1 (en) | Pharmaceutical composition for prevention or treatment of thrombosis comprising organic solvent fraction of ehwa nuruk extract as an effective component and health functional food comprising the same | |
| KR101440574B1 (en) | Pharmaceutical composition comprising the extract of nardostachys chinenesis as an effective component for prevention or treatment of diseases related to thrombosis and health functional food comprising the same | |
| KR20160101887A (en) | Pharmaceutical composition comprising the extract of phaseolus radiatus l. for prevention and control of thrombosis | |
| KR101471850B1 (en) | A pharmaceutical composition comprising the hexane fraction of gardenia jasminoides extract as an effective component for anti-platelet aggregation and a health functional food comprising the same | |
| KR102216222B1 (en) | Pharmaceutical composition comprising the extraction of leaf of nelumbo nucifera as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR20170127710A (en) | Pharmaceutical composition comprising the extraction of moringa oleifera root as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR20210047781A (en) | Pharmaceutical composition comprising the extract of prune as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101725623B1 (en) | Pharmaceutical composition comprising the organic solvent fraction of sorbus commixta fruit as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101479816B1 (en) | Pharmaceutical composition comprising the extract of phellinus linteus as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101526066B1 (en) | Pharmaceutical composition comprising the organic solvent fraction yielded from hot water extraction of laminaria japonica as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101624538B1 (en) | Pharmaceutical composition comprising the extract of tricholoma matsudake for prevention and control of thrombosis | |
| KR102037530B1 (en) | Pharmaceutical composition comprising the ethylacetate fraction of okmisoo extract as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR102216221B1 (en) | Pharmaceutical composition comprising the extract of pine pollen as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101404165B1 (en) | Pharmaceutical composition comprising the extract of angelica acutiloba as an effective component for prevention or treatment of thrombosis and health functional food comprising the same | |
| KR101710263B1 (en) | Pharmaceutical composition comprising the extract of hovenia dulcis as an effective component for prevention or treatment of thrombosis and health functional food comprising the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E701 | Decision to grant or registration of patent right | ||
| FPAY | Annual fee payment |
Payment date: 20190809 Year of fee payment: 4 |