KR20200073028A - Composition comprising extract of Ginkgo biloba leaves containing of increased terpene lactones for improving blood flow - Google Patents

Abstract

The present invention relates to a composition for improving blood flow containing an extract of Ginkgo biloba leaves with increased terpene lactone content as an active component, and a method for manufacturing the same. By manufacturing the extract of Ginkgo biloba leaves with increased terpene lactone content, and confirming an effect of inhibiting platelet aggregation and increasing blood flow of the manufactured extract, the extract of Ginkgo biloba leaves with increased terpene lactone content of the present invention can be usefully used in the development of a composition for preventing or treating diseases caused by blood flow abnormalities such as blood clots.

Description

Composition comprising extract of Ginkgo biloba leaves containing of increased terpene lactones for improving blood flow}

The present invention relates to a composition for improving blood flow including an extract of Ginko biloba leaves with increased content of terpene lactones and a method for manufacturing the same.

Blood circulation means that blood moves through blood vessels to each part of the body. Blood supplies oxygen and nutrients to the body's tissues and removes waste products produced by the cells. In addition, it carries the hormones necessary for our body, defends cells from external harmful substances, maintains proper body temperature, and maintains homeostasis in the body, such as hemostatic action. Therefore, smooth blood flow is very important for maintaining body function.

It is known that when the blood flow, that is, the blood flow is impaired, the incidence of cardiovascular diseases such as arteriosclerosis and stroke increases, and cardiovascular diseases are the leading causes of death worldwide in the United States, Europe, and Asia. Is becoming In Korea, cardiovascular disease is the number one cause of death and was reported in various survey results. The main factors affecting blood flow include chronic diseases such as hypertension and diabetes, eating habits, lifestyle, and genetic factors, and the incidence of cardiovascular diseases related to blood flow disorder increases as living standards increase and eating habits become more westernized. Tends.

Blood is composed of plasma and blood cells, and it plays an important role in maintaining homeostasis of each tissue by supplying oxygen and nutrients to each tissue of the body and removing waste products generated by metabolism in the cells. In order to perform this role, the functions of constituent cells and tissues must be maintained normally, and blood circulation must also be maintained smoothly. Plasma is composed of coagulation factors related to blood clotting, and exchanges various interactions with blood cells such as platelets, red blood cells, and immune cells, and maintains an equilibrium state of each cell action.

Of these, platelets are small cells with a diameter of 2 to 4 µm liberated in the blood because the cytoplasm of megakaryocytes, which are giant cells of the bone marrow, have a cell life of about 5 to 7 days. Platelets are cells that are responsible for hemostasis by activating when blood vessels are damaged and aggregating at the site of damage. When blood vessels are damaged, collagen on the inner wall of blood vessels, von Willebrand factor (vWF), and fibronectin are exposed to promote platelet adhesion. Platelets are activated as serotonin (serotonin), calcium ion (Ca ^2+), thromboxane A ₂ (thromboxane A ₂₎ to secrete the like amplifies the aggregation of platelets in a different ambient, reacts with blood coagulation factors present in blood plasma By forming a clot, hemostasis can occur more quickly and effectively.

Hemostatic action is a defense mechanism to minimize blood loss from damaged areas and maintain normal circulation of blood. In normal blood vessels, homeostasis is maintained by balancing the activation reaction of the hemostatic mechanism and the suppression reaction or clot decomposition reaction. However, excessive hemostasis and the formation of blood clots interfere with the flow of blood, cause blood flow abnormalities and cause lesions such as thrombus. When a blood clot is formed, blood circulation disorder occurs in a vein, causing edema or inflammation, and ischemia or infarction in an artery, leading to diseases such as myocardial infarction, stroke, and pulmonary artery infarction. In addition, various blood vessel regulatory factors such as serotonin, thromboxane A _{2, and} platelet activating factor (PAF) are secreted during platelet activation, thereby causing blood vessels to contract. When blood vessels are constricted, the flow rate of blood is changed by blocking the flow of blood flow, and platelet activation is amplified, and vascular endothelial damage progresses, thereby inhibiting the normal functioning of the cardiovascular system. These blood flow abnormalities are known to cause the development of vascular system diseases such as atherosclerosis, myocardial infarction, ischemic heart disease and cerebrovascular disease (Marcus, AJ, et al., 1993; Packham, MA, 1994; Harker, LA, 1994).

Ginkgo biloba is a deciduous tree belonging to the Ginkgoaceae family and has survived since the Mesozoic era and is also called a living fossil. Ginkgo biloba extract is a material that is officially listed as a pharmaceutical raw material in standards such as pharmaceuticals other than the Korean Pharmacopoeia, and is also included in the food and health functional foods.

Ginkgo biloba has so far been found to have a large number of compounds by region. Fruits include ginkgol, bilobol and ginkgolic acid, seeds contain cyanogenic glycosides and 4-O-methylpyridoxine, and heartwood It has been found that there are D-sesamin and bilobanone. Ingredients such as coumaric acid, hydrocarbon, docosanol, and sterols were separated from the pollen. Ingredients of ginkgo biloba include flavonoid-based compounds, terpene-based compounds such as bilobalide, ginkgolide A, B, C, M, and J, polyphenols, and polysaccharides More than 100 compounds, including polysaccharides, have been reported separately (Tang, W., et al., 1992).

In the case of the ginkgo biloba component, the flavonoid component is known as the main component of the ginkgo biloba extract, and the flavonoid component has been found to have many pharmacological effects such as antibacterial, antifungal, antiviral, vascular control, tonic, anti-inflammatory, and anti-cancer effects. . Bipeneride and zinc chloride, terpene lactone compounds, are known to have insecticidal and antibacterial effects (Matsumoto, T., et al., 1987; Eunyoung Yang, et al., 2001; Mazzanti, G., et al., 2000). In particular, flavonoids and terpene lactones are known as major active ingredients in the human body, and have been mainly used as effects and effects of improving blood circulation and blood circulation (Langley, SM, et al., 1999; Xu, JP, et al., 2005). Ginkgo biloba extract currently on the market contains about 22 to 27% flavonoids, 5 to 7% terpene lactones (2.8 to 3.4% zinc chloride A, B and C, and 2.6 to 3.2% bilobaride) (Zhang) , Z., et al., 2016; Akiba, S., et al., 1998; Koch, E., 2005), flavonoids 24%, terpene lactones present in the best activity when present in a ratio of 6% have.

However, it has recently been reported that there are side effects such as gastrointestinal disorders, headache, and skin allergies caused by ginkgo biloba extract (Blumenthal, M., et al., 1998). Cases are being reported. One week old 70-year-old patient was congested in the eyes one week after taking the ginkgo biloba, or a 33-year-old female patient had cerebral hemorrhage as the ginkgo biloba was taken for a long time and the blood was clotting longer. In addition, ginkgoric acid contained in ginkgo biloba extract may cause skin allergies (Hausen, BM, 1998) or may exhibit neurotoxicity (Ahlemeyer, B., et al., 2001). It is recommended that the acid does not exceed 5 ppm. In addition, it has been reported that flavonoids have side effects such as irritation of the gastrointestinal tract, and it was found that gastrointestinal disorders of ginkgo biloba extract are caused by flavonoids in ginkgo biloba extract.

Therefore, it is necessary to develop a method of increasing the physiological effect of the ginkgo biloba extract by preparing the ginkgo biloba extract with an increased content of other active ingredients while maintaining the content of some components that can cause side effects.

Thus, the present inventor prepared a ginkgo biloba extract that increased only the content of the terpene lactone-based compound while maintaining the content of flavonoids in the course of studying the blood flow improving effect using the ginkgo biloba extract, and inhibited the excellent platelet aggregation of the ginkgo biloba extract produced therefrom. And by confirming the effect of improving blood flow, it was possible to complete the present invention.

As a prior art, Korean Registered Patent No. 0175067 describes a method for producing ginkgo biloba concentrate concentrated with active ingredients from ginkgo biloba and suppressing platelet aggregation, and treating the peripheral and cerebral arterial circulation disorders, which is similar to the composition of the present invention, but flavon glyco Ginkgo biloba extract containing 40-60% seeds, 5.5-8% of gongkoride A, B, C and J in total, 5-7% bilovalide, while maintaining the content of flavonoids of the present invention at 18-23% by weight. The composition is different from the ginkgo biloba extract increased so that the content of terpene lactone is 10 to 16% by weight. In addition, Korean Patent Publication No. 1540004 discloses a composition for improving blood flow including ginkgo biloba extract, but the ginkgo biloba extract with increased content of terpene lactone of the present invention and its blood flow improving effect are not described. Japanese Patent Publication No. 2006-328014 has a composition for preventing deterioration of blood flow comprising ginkgo biloba leaf extract containing flavonoids and ginkgoride, Japanese Patent No. 2004-123622 for improving blood flow containing proanthocyanidins derived from ginkgo biloba Although the composition is described, the ginkgo biloba extract with increased content of terpene lactone of the present invention and its blood flow improving effect are not described.

Korean Registered Patent No. 0175067, an active ingredient concentrate from ginkgo biloba and a new active ingredient mixture, a method for preparing them, and a drug containing the active ingredient concentrate or the active ingredient mixture, registered on November 07, 1998. Registered Korean Patent No. 1540004, health functional food composition for improving blood sugar, blood pressure, blood flow and cholesterol, registered on July 22, 2015. Japanese Patent Laid-Open No. 2006-328014, a composition having an action to prevent blood flow deterioration, published on December 7, 2006. Published Japanese Patent No. 2004-123622, external preparation for improving blood flow, 2004. 04. 22. published.

Eun-Yang Yang, et al., Insecticidal activity of bilobalide and its decomposition products from ginkgo biloba, Korean Journal of Pesticide Science, 5(1), 24-29, 2001. Ahlemeyer, B., et al., gingolic acids induce neuronal death and activate protein phosphatase type-2C, Eur. J. Pharmacol., 430(1), 1-7, 2001. Akiba, S., et al., Inhibitory effect of the leaf extract of Ginkgo biloba L. on oxidative stress-induced platelet aggregation, Biochemistry and Molecular Biology International, 46(6), 1243-1248, 1998. Blumenthal, M., et al., The Complete German Commission E Mongraphys. Therapeutic Guide to Herbal Mediciens, Investigative Medicine Communications, Boston, 1998. Harker, L.A., Platelets and Vascular Thrombosis, N. Engl. J. Med., 330, 1006-1007, 1994. Hausen, B.M., The sensitizing capacity of ginkgolic acids in guinea pigs, Am. J. Contact. Dermat., 9(3), 146-148, 1998. Koch, E., Inhibition of platelet activating factor (PAF)-induced aggregation of human thrombocytes by ginkgolides: consideration on possible bleeding complications after oral intake of Ginkgo biloba extracts, Phytomedicine, 12, 10-16, 2005. Langley, S.M., et al., Platelet-activating factor receptor antagonism improves cerebral recovery after ciculatory arrest, Ann. Thorac. Surg., 68(5), 1578-1584, 1999. Marcus, A.J., et al., Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis, FASEB J., 7(6), 516-522, 1993. Matsumoto, T., et al., Antifeedant Activities of Ginko biloba L. Components aginst the Larva of Pieris rapae crucivora, Agric. Biol. Chem., 51(1), 249-250, 1987. Mazzanti, G., et al., Antimicrobial investigation of semipurified fractions of Ginkgo biloba leaves, J. Ethnopharmacol., 71(1-2), 83-88, 2000. Packham, M.A., Role of platelets in thrombosis and hemostasis, Can. J. Physiol. Pharmacol., 72(3), 278-284, 1994. Tang, W., et al., Chinese Drugs of Plant Origin, Springer-Verlag, Berlin, 555-565, 1992. Xu, J.P., et al., Effects of ginkgolide on cerebral blood flow in dogs, Zhong Xi Yi Jie He Xye Bao, 3(1), 50-53, 2005. Zhang, Z., et al., Chapter 62-Assessement of Genotoxic Effects of Selected Herbal Dietary Supplements, Nutraceuticals, 883-892, 2016.

An object of the present invention is to provide a composition for improving blood flow, including an extract of Ginko biloba leaves with increased content of terpene lactone, while maintaining the content of flavonoids as an active ingredient, and a method of manufacturing the same.

The present invention is for preventing or treating thrombotic diseases comprising ginkgo biloba extract containing 18 to 23 wt% of flavonoids and 10 to 16 wt% of terpene lactones as an active ingredient based on the total weight of ginkgo biloba extract It relates to a pharmaceutical composition or a health functional food composition for improvement.

The terpene lactone may be at least one selected from the group consisting of ginkgolide A, B, C, M, J and bilobalide.

The composition may further include at least one extract selected from the group consisting of grape extract, blueberry extract, audi extract, seomoktae extract, blacktae extract and frosttae extract.

The composition may have platelet aggregation inhibitory activity.

The thrombotic disease may be selected from the group consisting of arterial thrombosis, venous thrombosis, portal vein thrombosis, pulmonary embolism, chronic venous ischemia, lower veins, deep vein thrombosis, angina pectoris, cerebral infarction, and brain hemorrhage.

The present invention also, i) a first step of washing and drying the ginkgo biloba powder; ii) a second step of extracting ginkgo biloba powder in step 1 with at least one solvent selected from the group consisting of water, lower alcohols of C1-4 and acetone, and concentrating the filtrate obtained by filtration; iii) after adding hexane to the concentrate of step 2, removing the hexane layer and separating the remaining layer; iv) After removing the hexane layer of step 3 and adding saturated ammonium sulfate to the remaining layer, butanone (ethyl methyl ketone) is added to separate the butanone layer, and the separated butanone layer is dried to dry the butanone layer. Four steps to obtain a fraction; v) After adding ethanol to the butanone fraction of step 4, the filtrate obtained by filtration is concentrated and dried to obtain a ginkgo biloba extract; vi) step 6 to further secure the ginkgo biloba extract through steps 1-5; vii) adding an aqueous methanol solution to the additional ginkgo biloba extract obtained in step 6, and then concentrating under reduced pressure to evaporate methanol and secure a water layer; viii) an eight step of concentrating and drying the ethyl acetate fraction obtained by adding ethyl acetate to the water layer of step 7 to obtain a ginkgo biloba fraction; And ix) step 9 of mixing the ginkgo leaf fraction of step 8 with the ginkgo leaf extract of step 5; Ginkgo biloba extract consisting of 18 to 23% by weight based on the total weight and terpene lactone (terpene lactone) relates to a method for producing ginkgo biloba extract containing 10 to 16% by weight.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for the prevention or treatment of thrombotic diseases comprising ginkgo biloba extract with an increased content of terpene lactone as an active ingredient.

Ginkgo biloba extract of the present invention includes flavonoids, terpene lactones, ginkgoric acid, and the like, and has an effect of improving blood circulation and blood circulation, and on the market, ginkgo biloba extracts on average contain 24% of flavonoids and 6% of terpene lactones. This is the ratio that is known to have the best physiological activity. On the other hand, in the case of flavonoids contained in ginkgo biloba extract, it may irritate the gastrointestinal tract and cause gastrointestinal disorders, and ginkgolic acid may cause skin allergies or show neurotoxicity, and extract the extract to increase the content of active ingredients in the ginkgo biloba extract. There may be health and industrial problems for concentration.

The ginkgo biloba leaf extract with an increased content of terpene lactones increases the content of the terpene lactone while maintaining the content of the active ingredient excluding the terpene lactone of the ginkgo biloba extract, preferably the level of flavonoids based on the total weight of the ginkgo biloba extract. While maintaining, increase the content of terpene lactone.

The flavonoid has an antioxidant effect, and maintains an unsaturated fatty acid and arachidonic acid to help cell health, and has the effect of reducing the aggregation ability of platelets and strengthening blood vessels and strengthening capillaries, preferably flavone glycosides (flavone glycoside). More preferably, it may be at least one selected from the group consisting of quercetin glycoside, isorhamnetin glycoside, and kaempherol glycoside, but is not limited thereto.

The flavonoid may be contained at least 18% by weight based on the total weight of ginkgo biloba extract. When the content of the flavonoid is less than 18% by weight, the effect of improving the blood flow by mixing with other active ingredients may not be sufficiently exhibited by the flavonoid in the blood flow improving effect of the ginkgo biloba extract. It is not desirable to appear. Thus, the flavonoid content may be preferably 18 to 23% by weight. However, when ingested with ingredients that can promote the intestinal absorption of flavonoids, such as plant seeds or fruit mucus, even if the content of the flavonoids is small, the drug efficacy may be improved, and even if the content is high, gastrointestinal disorders may be reduced. Can.

The terpene lactone may contain more than 9% by weight based on the total weight of ginkgo biloba extract. It is more preferably 9 to 20% by weight, and most preferably 10 to 16% by weight. When the content of the terpene lactone is less than 9% by weight, it is difficult to obtain an improved blood flow improving effect compared to the ginkgo biloba extract currently on the market, and when the content of the terpene lactone exceeds 20% by weight, blood is continuously taken Side effects such as delay of coagulation and speeding up of blood flow are concerned, and it is not preferable in the manufacturing process due to the difficulty and high cost in the manufacturing process to secure a large amount of the ginkgo biloba fraction of the present invention containing terpene lactone in a high concentration.

The terpene lactones of the present invention include bilovalide and zinc chloride, and the zinc chloride is divided into zinc chloride A, B, C, M and J. The terpene lactone may be at least one selected from the group consisting of zinc chloride A, B, C, M, J and bilovalide. It is preferably one or more selected from the group consisting of zinc chloride A, B, C, and bilovalide.

The ginkgo biloba extract having an increased content of terpene lactone is preferably a ginkgo biloba extract containing 18 to 23 wt% of flavonoids and 10 to 16 wt% of terpene lactone based on the total weight of ginkgo biloba extract.

Ginkgo biloba extract containing 18 to 23% by weight of flavonoids and 10 to 16% by weight of terpene lactone based on the total weight of the ginkgo biloba extract, i) a first step of washing and drying the ginkgo biloba to powder; ii) a second step of extracting ginkgo biloba powder in step 1 with at least one solvent selected from the group consisting of water, lower alcohols of C1-4 and acetone, and concentrating the filtrate obtained by filtration; iii) after adding hexane to the concentrate of step 2, removing the hexane layer and separating the remaining layer; iv) After removing the hexane layer of step 3 and adding saturated ammonium sulfate to the remaining layer, butanone (ethyl methyl ketone) is added to separate the butanone layer, and the separated butanone layer is dried to dry the butanone layer. Four steps to obtain a fraction; v) After adding ethanol to the butanone fraction of step 4, the filtrate obtained by filtration is concentrated and dried to obtain a ginkgo biloba extract; vi) step 6 to further secure the ginkgo biloba extract through steps 1-5; vii) adding an aqueous methanol solution to the additional ginkgo biloba extract obtained in step 6, and then concentrating under reduced pressure to evaporate methanol and secure a water layer; viii) an eight step of concentrating and drying the ethyl acetate fraction obtained by adding ethyl acetate to the water layer of step 7 to obtain a ginkgo biloba fraction; And ix) step 9 of mixing the ginkgo leaf fraction of step 8 with the ginkgo leaf extract of step 5; It may be manufactured through a manufacturing method consisting of.

The first step of drying is 30 to 90 minutes at 90° C., followed by drying at 60° C. for 72 hours, to completely remove the ginkgo leaves. In addition, the treatment at 90° C. for 90 minutes is to inactivate glycosidase present in ginkgo biloba.

The solvent of the second step may be one or more solvents selected from the group consisting of water, lower alcohols of C1-4 and acetone, but is not limited thereto. The lower alcohol of C1-4 may be methanol, ethanol, propanol, isopropanol, butanol, etc., and the solvent in the second step is preferably methanol, and more preferably 60% [v/v] methanol aqueous solution.

The filtrate of the second step can be used by mixing the second filtrate obtained by filtering the extracted extract by putting the same solvent in the remaining residue after filtration to the primary filtrate obtained by filtering the extract extracted with the solvent of the second step. That is, to increase the content of the active ingredient.

The ammonium sulfate in the 4th step removes the hexane layer in the 3rd step and precipitates the active ingredient of the ginkgo biloba in the remaining layer, so that the active ingredient of the ginkgo biloba is contained in the butanone layer in the process of fractionation using butanone. Can handle it.

The butanone of the above 4 steps dissolves flavonoids, such as flavone glycosides and terpenes, which are effective ingredients showing the effect of improving the blood circulation of ginkgo biloba, while quercetin, isorhamnetin, kaempherol, etc. The flavone glycosides and terpenes of ginkgo biloba can be treated to separate them from other components by precipitating flavonoid aglycons and other polymers.

Steps 6 to 8 are for securing a fraction of ginkgo biloba containing only terpene lactone at a high concentration from ginkgo biloba extract.

In the ninth step, the ginkgo biloba leaf extract containing the ginkgo biloba leaf extract of the fifth step is maintained while mixing the ginkgo biloba fraction containing the terpene lactone of the eighth step with a high concentration of the ginkgo biloba extract of the fifth step, while increasing the content of the terpene lactone. It is intended to manufacture.

The content of the terpene lactone increased according to the mixing ratio of the fraction of the ginkgo biloba extract containing the ginkgo biloba extract of the 5th step and the terpene lactone of the 8th step at a high concentration can be adjusted.

The composition may further include a plant extract having an effect of improving blood circulation.

The plant extract having the effect of improving blood circulation has a large amount of anthocyanins and catechins, and thus has excellent antioxidant action, preferably grape extract, blueberry extract, audi extract, seomok extract, blacktae extract And one or more selected from the group consisting of frost extract, but is not limited thereto.

The plant extract may be in the form of a concentrate or powder.

The grapes contain a large amount of various nutrients and anthocyanins, such as sugars, mucus, and vitamins. Grape seeds contain catechins and polymers, especially dimers.

The blueberries contain anthocyanins (mainly delphinidins) and catechins in large quantities, and thus are consumed in large quantities as health functional foods. Moreover, mucus is abundantly contained.

The audi is a fruit of a mulberry, and contains anthocyanins, morin, mucus, and the like.

The Seomok-tae, Heuk-tae, and Seo-ri-tae are representative black beans and contain a large amount of saponins and anthocyanins.

The grape extract, blueberry extract, audi extract, seomoktae extract, blacktae extract and frosttae extract increase the physiological activity by adding the active ingredient of each extract including anthocyanins to the ginkgo biloba extract with increased content of terpene lactone It is to let.

In addition, the grape extract, blueberry extract, audi extract, seomoktae extract, blacktae extract and frosttae extract contain mucus, which may serve as an excipient to increase the solubility of the terpene lactone, and intestinal absorption of the active ingredient of ginkgo biloba extract Can promote and reduce gastrointestinal disorders.

The composition may exhibit an inhibitory effect on platelet aggregation, and through this inhibition of platelet aggregation, an effect of improving blood circulation (blood flow) may be obtained.

The composition may increase blood flow, such as blood flow rate.

The blood circulation is the movement of blood through blood vessels, and the production of blood clots due to excessive aggregation of platelets hinders the flow of blood, causing abnormal blood flow and causing lesions such as thrombus. Due to lesions such as thrombus, circulatory disorders in the veins cause edema or inflammation, and ischemia or infarction in the arteries, leading to diseases such as myocardial infarction, stroke, and pulmonary infarction.

In addition, the present invention relates to a pharmaceutical composition for the prevention and treatment of thrombotic diseases, including the ginkgo biloba extract with increased content of the terpene lactone and a pharmaceutically acceptable excipient.

The ginkgo biloba extract in which the content of terpene lactone is increased is preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, most preferably 0.001 to 30% by weight relative to the total weight of the entire pharmaceutical composition. Can.

The pharmaceutical composition may be formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. Carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, for example starch, in ginkgo biloba extract with an increased content of terpene lactone of the present invention. It is prepared by mixing calcium carbonate, sucrose or lactose, and gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents, suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween-61, cacao butter, laurin butter, and glycerogelatin may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and is generally in the range of 0.01-2000 mg/kg/day. A more preferable dosage is 0.1 to 500 mg/kg/day. Administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to various mammals, such as rats, livestock, and humans. All modes of administration can be expected, for example, oral, rectal or intravenous, intraperitoneal, intramuscular, subcutaneous, intrauterine dura or cerebral vascular injection.

In addition, the present invention relates to a health functional food for the improvement of thrombotic diseases, including a ginkgo biloba extract with an increased content of the terpene lactone and a food-acceptable food supplement additive.

The ginkgo biloba extract with an increased content of terpene lactone is preferably added in an amount of 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 10% by weight, based on the total weight of the health functional food. Can be.

The health functional food of the present invention includes tablets, capsules, pills or liquids, etc. As foods to which the extract of the present invention can be added, for example, various foods, beverages, gums, teas, and vitamin complexes , Health functional foods, etc.

The thrombosis is a disease caused by thrombi, and can be selected from the group consisting of arterial thrombosis, venous thrombosis, hepatic vein thrombosis, pulmonary embolism, chronic venous ischemia, lower veins, deep vein thrombosis, angina, cerebral infarction, and cerebral hemorrhage. However, it is not limited thereto.

The thrombus appears due to excessive hemostatic action and formation of blood clots, and may be generated when blood stagnation and blood clotting rate are increased.

The present invention is a composition for improving blood flow comprising an extract of Ginko biloba leaves with increased content of terpene lactone, more specifically 18-23% by weight of flavonoids and terpene based on the total weight of ginkgo biloba extract A composition for improving blood flow comprising ginkgo biloba extract containing 10 to 16% by weight of lactone as an active ingredient and a method for manufacturing the same, the ginkgo biloba extract having an increased content of terpene lactone has a remarkably excellent inhibitory effect on platelet aggregation and blood flow It was confirmed to increase.

Through this, it is expected that the ginkgo biloba extract having an increased content of terpene lactone of the present invention can be usefully used to develop a composition for preventing or treating diseases caused by blood flow abnormalities such as thrombus.

Figure 1 shows the results confirming the effect of inhibiting platelet aggregation of ginkgo biloba extract with increased content of terpene lactone of the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the contents introduced herein are thorough and complete, and are provided to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Ginkgo biloba extract (GKLE-1) production>

Fresh ginkgo biloba leaves were washed and then completely dried at room temperature. 5 kg of washed and dried ginkgo leaves were placed in a dryer at 90°C, dried for 90 minutes, and dried at 60°C for 72 hours. The dried ginkgo leaves were crushed into 40-70 mesh to obtain ginkgo biloba powder. After adding 5ℓ of 60%[v/v] methanol to 1kg of ginkgo biloba powder, refluxing for 7 hours, filtering, storing the filtrate separately, and filtering the remaining 5% of 60%[v/v] methanol It was repeated twice in the same manner as in the above extraction method to obtain 15 l (3 x 5 l) of filtrate. The obtained filtrate was concentrated to about 3 l using a vacuum concentrator. To the concentrate, 2 l of hexane was added and stirred for 10 minutes, then the hexane layer was removed and the remaining layer was separated. After adding saturated ammonium sulfate to the separated layer, 3 l of butanone (ethyl methyl ketone) was added, stirred and extracted for 3 hours, and left for 5 hours to separate the butanone layer. The butanon layer was separated and 3 l of butanon was added to the remaining layer to further secure the butanon layer. The obtained butanone layers were combined and dried to obtain a butanone fraction in a dry form. 600 ml of 98% [v/v] ethanol was added to the obtained butanone fraction, shaken, and allowed to stand for 2 to 3 hours. Then, the filtrate was separated using filter paper, and 100 ml of 98% of the remaining precipitate after filtration. [v/v] Ethanol was added, and the filtrate was separated through the same precipitation and filtration method as above. The separated filtrates were combined, concentrated and dried to obtain 252 g of dried ginkgo biloba leaf extract (hereinafter referred to as GKLE-1).

The content of terpene lactones (Zingkorides A, B, C, M, J and bilovalide) and flavonoids of ginkgo biloba extract (GKLE-1) obtained above were analyzed.

To analyze the content of terpene lactone, take GKLE-1, add 3 ml of methanol for HPLC, dissolve by stirring, then add 7 ml of distilled water in small portions while stirring continuously, add 20 ml ethyl acetate, and further stir for 20 minutes. . Thereafter, the mixture was transferred to a small fraction filter and left to stand, and then the ethyl acetate fraction was separated. The remaining water layer was put in the stirrer again, 10 ml of ethyl acetate was added, and the mixture was stirred for 20 minutes, and then transferred to the fraction head to separate the ethyl acetate fraction. The separated ethyl acetate fractions were combined and dried to obtain a dry matter. The obtained dried matter was dissolved in 4 ml of methanol for HPLC, and then filtered through a 0.45 µm filter to obtain an analysis sample. High-performance liquid chromatography (HPLC) using 20 μl of analytical sample in a Nova-pak C18 column and using a methanol: distilled water (23:77 [v/v]) isocratic as a mobile phase at a flow rate of 1.5 ml/min. Was performed. At this time, zinc chloride A, B, C, and bilovalide separated by a standard curve were used.

The flavonoid content was analyzed using a method known in the Korean Pharmacopoeia.

As a result, it was confirmed that the terpene lactone of GKLE-1 contained 6% by weight on average and 25% by weight on flavonoids.

<Example 2. Ginkgo biloba fraction (GKLE-ea) production>

800 ml of 60% [v/v] methanol was added to 252 g of GKLE-1 obtained through the manufacturing process of Example 1 and mixed, and then methanol was evaporated using a reduced pressure concentrator to obtain a water layer. After adding 300 ml of ethyl acetate to the secured water layer, shaking for 10 minutes, the ethyl acetate layer was separated, and the ethyl acetate layer was added again to the remaining water layer, and the ethyl acetate layer separation process was repeated once more to obtain ethyl. The acetate fraction was secured. The obtained ethyl acetate fractions were collected and concentrated, and the concentrate was dehydrated and dried using a desiccator for drying to obtain 30.7 g of ginkgo biloba fraction (hereinafter referred to as GKLE-ea) in a dry state.

The content of terpene lactone and flavonoids in the ginkgo biloba fraction (GKLE-ea) obtained above was analyzed in the same manner as in Example 1, and as a result, flavonoids were not detected and terpene lactones averaged 48% by weight It was confirmed to contain a high concentration of.

<Example 3. Preparation of ginkgo biloba extract (GKLE-2) with increased content of terpene lactone>

Ginkgo biloba extract (hereinafter referred to as GKLE-2) with increased content of terpene lactone as shown in Table 1 below by mixing GKLE-1 obtained through the manufacturing process of Example 1-1 and GKLE-ea obtained in Example 2 as shown in Table 1 below. ).

division Mixing amount (g) Content (% by weight) GKLE-1 GKLE-ea Terpene lactone Flavonoids GKLE-2-1 10 One 10.1 22.7 GKLE-2-2 10 2 13 20.8 GKLE-2-3 10 3 15.7 19.2 GKLE-2-4 10 4 18 17.9 GKLE-2-5 10 5 20 16.7 GKLE-2-6 10 10 27 12.5

< Example 4. The content of terpene lactone Increased Preparation of a composition comprising ginkgo biloba extract and plant extract>

1) Grape( Vitis vinifera ) Extract preparation

Red grapes are used to grind the whole grapes containing seeds into a fine powder with a grinder. 300 g of red grape in powder form was put in a pan, boiled for 10 minutes, cooled, transferred to a blender, and 300 ml of distilled water was added, followed by pulverization to obtain a pulverized product. The obtained pulverized material was centrifuged to recover only the supernatant, and the recovered supernatant was concentrated to about 100 ml to prepare a grape concentrate (hereinafter, referred to as Vit-J).

In addition, 100 ml of the grape concentrate obtained by the same method as described above was dried to prepare a grape extract in the form of powder (hereinafter referred to as Vit-Ex).

2) Blueberry ( Vaccinum spp., blueberry) extract preparation

Blueberries used frozen blueberries. 500 g of frozen blueberries were put in a pot, boiled for 30 minutes, cooled, transferred to a blender, and 400 ml of distilled water was added, followed by pulverization to obtain a pulverized product. The obtained pulverized material was centrifuged to recover only the supernatant, and the recovered supernatant was concentrated to about 100 ml to prepare a blueberry concentrate (hereinafter referred to as Vac-J).

In addition, a blueberry extract (hereinafter referred to as Vac-Ex) in powder form was prepared by drying 100 ml of the blueberry concentrate obtained in the same manner as above.

3) Audi( Morus alba fruit) extract preparation

300 g of frozen Audi was put in a pot, boiled for 20 minutes, cooled, transferred to a blender, and 500 ml of distilled water was added, followed by pulverization at low speed for 30 minutes to obtain pulverized products. The obtained pulverized material was centrifuged to recover only the supernatant, and the recovered supernatant was concentrated to about 100 ml to prepare an Audi concentrate (hereinafter referred to as Mr-J).

In addition, 100 ml of the Audi concentrate obtained in the same manner as described above was dried to prepare a powdery Audi extract (hereinafter referred to as Mr-Ex).

4) Seo Mok-tae( Rhyhnchosia volubilis ) Extract preparation

500 g of Seomoktae was added to 1000 ml of distilled water, soaked for 12 hours, and then ground using a grinder. The pulverized product was concentrated to 400 ml, and 600 ml of 95% [v/v] methanol was added thereto and mixed. The mixture was filtered to separate the filtrate, and 500 ml of 60% [v/v] methanol was added to the remaining plant, refluxed for 3 hours, and then filtered to separate the filtrate, mixed with the previous filtrate and concentrated to dryness. Seomoktae extract (hereinafter referred to as Rhy-Ex) in a solid form was prepared.

5) Black Tae ( Glycine max Merr.) extract preparation

Black Tae refers to black beans, which are harvested before frost.

In the same manner as the method for preparing Seomok-tae extract, black-tae extract (hereinafter referred to as Rhy-Ex1) was prepared.

6) Frost Tae( Glycine max Merr.) extract preparation

Frost-Tae was harvested after frost-falling black Tae-tae, and the inside of the bean is green.

Seomoktae extract (hereinafter, referred to as Rhy-Ex2) was prepared in the same manner as Seomoktae extract production method.

7) Preparation of a composition comprising ginkgo biloba extract and plant extract with increased content of terpene lactone

Ginkgo biloba extract with increased content of terpene lactone by mixing the extract obtained from the grape, blueberry, audi, seomoktae, blacktae or frosttae with GKLE-2 of Example 3 and grape, blueberry, audi, seomoktae, blacktae or frosttae A composition comprising an extract was prepared.

At this time, in the case of the concentrate, each of the concentrates 100 ml or 100 ml of the concentrate mixture was prepared by mixing 1,200 mg of GKLE-2-1~2-3 of Example 3, and in the case of extracts in powder form, respectively The composition was prepared by mixing 1,200 mg of GKLE-2-1~2-3 of Example 3 with 100 g of powder or 100 g of powder mixture.

<Example 5. Confirmation of the effect of inhibiting platelet aggregation of ginkgo biloba extract with increased content of terpene lactone>

As a method for confirming the effect of improving the blood flow of ginkgo biloba extract with increased content of terpene lactone of the present invention, the degree of platelet aggregation inhibition was analyzed.

50 ml of blood is collected from a healthy man without experience in circulating drug administration, mixed with 5 ml of 1% aqueous sodium citrate solution, centrifuged at 2,500 rpm to separate the supernatant (serum), and used until 4 Stored at °C.

2 ml of the separated serum was adjusted to an optical density of 0.5 using a coagulation analyzer, and then 200 µl was dispensed into a cuvette provided in the coagulation analyzer. 0.05 µg of a sample in which GKLE-2-1 to 2-6 in Table 1, GKLE-1 in Example 1, or GKLE-ea in Example 2 was dissolved in 1% DMSO (dimethyl sulfoxide) in a test tube in which serum was dispensed. It was treated to be ml, and physiological saline was treated as a control. In addition, by using a commercially available ginkgo biloba extract (24% flavonoid, 6% terpene lactone), the platelet aggregation effect was compared by treating it to the same amount. The sampled test tube was shaken at 37° C. and 25 μl/ml of platelet activating factor (PAF) was added in 25 μl and reacted for 5 minutes. After the reaction, the absorbance was measured, the absorbance in the control group treated with physiological saline was set to 100% platelet aggregation rate, and the platelet aggregation rate in each sample treatment was calculated to decrease the platelet aggregation rate compared to the control group. The results are shown in Fig. 1.

As shown in Figure 1, compared to the case of the GKLE-1 treatment, it was confirmed that the platelet aggregation rate was low when the GKLE-2-1 to 2-6 treatment was performed, and the GKLE-ea having the highest terpene lactone content was treated. In the case, it was confirmed that the platelet aggregation rate was the lowest.

On the other hand, when a commercial ginkgo biloba extract containing 24% flavonoids and 6% terpene lactone was treated with the same amount, it was confirmed that platelet aggregation rate similar to that of GKLE-1 appeared.

In addition, although not shown in the present specification, it was confirmed that the inhibition rate of platelet aggregation was similar to that of GKLE-2-2 even in the case of a composition comprising ginkgo biloba extract and plant extract having an increased content of terpene lactone prepared in Example 4 above. .

Through this, it was found that the higher the content of terpene lactone, the greater the effect of inhibiting platelet aggregation.

<Example 6. Confirmation of total carotid blood flow improvement effect of ginkgo biloba extract with increased content of terpene lactone>

Flavonoids contained in ginkgo biloba extract expand blood vessels by increasing the secretion of endothelium-derived relaxing factor (PGI2) and prostaglandin I2 (prostacyclin) from vascular epithelial cells, and increase their contractility. In addition, it prevents the accumulation of lipids, such as cholesterol, in the blood vessels due to its antioxidant action, thereby protecting blood vessel health. Another active ingredient in ginkgo biloba extract, terpene lactone, prevents platelet aggregation and lowers the viscosity of the blood, thus facilitating blood flow. Thus, the total carotid blood flow rate of a person was measured in order to confirm the effect of promoting the blood flow of the ginkgo biloba extract having an increased content of terpene lactone of the present invention.

Ginkgo biloba extract with an increased content of terpene lactone prepared in 7) of Example 4 to 9 males with a total carotid artery average blood flow rate of 19 to 22 cm/s, 19 to 22 years old, who did not agree to participate in the experiment And 10 ml of a composition containing a blueberry extract (Vac-J) in the form of a concentrate in a composition containing a plant extract as a single dose, orally administered twice a day over 5 days (10am, 5pm) (3 divided doses for each composition). Participant blood flow was measured for 3 days before administration of the composition, blood flow was measured at 20 minute intervals after administration of the composition for 5 days, and the results are shown in Table 2 below.

For blood flow measurement, participants were laid flat on a bed, rested for 20 minutes, and then measured by using a blood flow meter (Transcranial Ultrasonic Doppler Flowmeter, TCD500 VTCD, USA).

division Blood flow (cm/s) Blood flow growth rate
(%) Before administration After administration 1 time Episode 2 3rd time 1 time Episode 2 3rd time Commercial Ginkgo Leaf Extract
& Blueberry Extract Participant 1 20.2 21.1 19.8 19.8 20.8 21.1 1.0 Participant 2 19.4 19.6 20 19.9 20.2 20.4 2.5 Participant 3 19.7 20.1 20.3 20.8 20.9 20.8 4.0 Average 19.8 20.3 20.0 20.2 20.6 20.8 2.5 GKLE-2-1
& Blueberry Extract Participant 1 21.2 18.5 20.0 22.7 21.6 20.5 8.5 Participant 2 22.2 20.1 19.5 20.7 20.9 22.5 3.7 Participant 3 18.8 19.4 20.7 21.0 20.2 20.0 3.9 Average 20.7 19.3 20.1 21.5 20.6 21.0 5.4 GKLE-2-2
& Blueberry Extract Participant 1 20.4 21.4 21.7 23.8 22.3 22.6 8.2 Participant 2 19.8 20.1 20.6 21.9 22.5 22.5 10.6 Participant 3 21.2 21.7 20.5 23.1 22.7 21.8 6.6 Average 20.5 21.1 20.9 22.9 22.5 22.3 8.5 GKLE-2-3
& Blueberry Extract Participant 1 19.2 20.3 20.4 22.2 21.8 21.7 9.7 Participant 2 19.6 19.7 21.8 22.6 21.4 22.9 9.5 Participant 3 19.1 20.4 20.4 23.0 22.1 22.7 13.2 Average 19.3 20.1 20.9 22.6 21.8 22.4 10.8

As shown in Table 2, it was confirmed that the blood flow of the participants increased after administration compared to before the administration of the composition comprising the ginkgo biloba extract and the plant extract having an increased content of terpene lactone, and this increase in blood flow was dependent on the content of terpene lactone. It was confirmed to increase.

Although not shown in the present specification, when the composition containing the GKLE-2-4 and the plant extract was administered, the rate of increase in blood flow was higher than the composition containing the GKLE-2-3 and the plant extract. Therefore, by continuously taking this, it was judged that if the speed of blood flow is increased, side effects such as bleeding, dizziness, and headache may occur, which may be undesirable.

Through this, it was found that in the case of the ginkgo biloba extract having an increased content of terpene lactone of the present invention, as the content of terpene lactone increases, the rate of blood flow improvement increases, but when it is 10% by weight or more, it shows excellent blood flow improvement effect. That is, it was found that the ginkgo biloba extract containing 18 to 23% by weight of flavonoids and 10 to 16% by weight of terpene lactone of the present invention exhibits excellent blood flow improvement effects.

<Formulation Example 1. Pharmaceutical preparation>

Formulation Example 1-1. Preparation of tablets

20 g of GKLE-2-2 of Example 3 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This mixture was dried, and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was purified.

Formulation Example 1-2. Preparation of injection liquid

1 g of GKLE-2-2 of Example 3 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was placed in a bottle and sterilized by heating at 20°C for 30 minutes.

<Formulation Example 2. Preparation of health functional food>

Formulation Example 2-1. Production of health functional food

20 g of GKLE-2-2 of Example 3 of the present invention, an appropriate amount of vitamin mixture, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, vitamin C 10mg, biotin 10µg, nicotinic acid amide 1.7mg, folic acid 50µg, calcium pantothenate 0.5mg, mineral mixture amount, ferrous sulfate 1.75mg, zinc oxide 0.82mg, magnesium carbonate 25.3mg, potassium phosphate 15mg, agent 55 mg of calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed into granules, but can be prepared by modifying them into various formulations depending on the application. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed and prepared according to a conventional health functional food manufacturing method.

Formulation Example 2-2. Production of health functional beverages

GKLE-2-2 1 g of Example 3 of the present invention, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water were mixed and stirred, heated, filtered, sterilized, and refrigerated according to a conventional beverage preparation method to prepare a beverage.

Claims (11)

A pharmaceutical composition for the prevention or treatment of thrombotic diseases comprising ginkgo biloba extract as an active ingredient, characterized in that 18 to 23 wt% of flavonoids and 10 to 16 wt% of terpene lactone are contained based on the total weight of ginkgo biloba extract. According to claim 1,
The terpene lactone is a pharmaceutical composition for the prevention or treatment of thrombotic diseases, characterized in that at least one member selected from the group consisting of zinc chloride A, B, C, M, J and bilovalide. The method according to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of thrombotic diseases, characterized in that it further comprises at least one extract selected from the group consisting of grape extract, blueberry extract, Audi extract, Seomoktae extract, blacktae extract and frosttae extract. . The method according to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of thrombotic diseases, characterized in that it has a platelet aggregation inhibitory activity. The method according to claim 1 or 2,
The thrombotic disease is a pharmaceutical thrombosis disease prevention or treatment characterized in that selected from the group consisting of arterial thrombosis, venous thrombosis, hepatic vein thrombosis, pulmonary artery embolism, chronic venous ischemia, lower veins, deep vein thrombosis, angina pectoris, cerebral infarction and cerebral hemorrhage. Composition. Ginkgo biloba extract based on the total weight of flavonoids 18 to 23% by weight and terpene lactone 10 to 16% by weight, ginkgo biloba extract containing as an active ingredient, health functional food composition for the improvement of thrombotic diseases. The method of claim 6,
The terpene lactone is a health functional food composition for the improvement of thrombotic diseases, characterized in that at least one selected from the group consisting of ginkgorides A, B, C, M, J and bilobaroid. The method of claim 6 or 7,
The health functional food composition further comprises one or more extracts selected from the group consisting of grape extract, blueberry extract, Audi extract, Seomoktae extract, blacktae extract and frosttae extract, health function for improving thrombus disease Food composition. The method of claim 6 or 7,
The health functional food composition is a health functional food composition for improving thrombus disease, characterized in that it has a platelet aggregation inhibitory activity. The method of claim 6 or 7,
The thrombotic disease is a health functional food for improving thrombotic disease, characterized in that it is selected from the group consisting of arterial thrombosis, venous thrombosis, portal vein thrombosis, pulmonary artery embolism, chronic venous ischemia, lower veins, deep vein thrombosis, angina pectoris, cerebral infarction, and brain hemorrhage. Composition. i) first step of washing and drying ginkgo biloba powder;
ii) a second step of extracting ginkgo biloba powder in step 1 with at least one solvent selected from the group consisting of water, lower alcohols of C1-4 and acetone, and concentrating the filtrate obtained by filtration;
iii) after adding hexane to the concentrate of step 2, removing the hexane layer and separating the remaining layer;
iv) After removing the hexane layer of step 3 and adding saturated ammonium sulfate to the remaining layer, butanone (ethyl methyl ketone) is added to separate the butanone layer, and the separated butanone layer is dried to dry the butanone layer. Four steps to obtain a fraction;
v) After adding ethanol to the butanone fraction of step 4, the filtrate obtained by filtration is concentrated and dried to obtain a ginkgo biloba extract;
vi) step 6 to further secure the ginkgo biloba extract through steps 1-5;
vii) adding an aqueous methanol solution to the additional ginkgo biloba extract obtained in step 6, and then concentrating under reduced pressure to evaporate methanol and secure a water layer;
viii) an eight step of concentrating and drying the ethyl acetate fraction obtained by adding ethyl acetate to the water layer of step 7 to obtain a ginkgo biloba fraction; And
ix) step 9 of mixing the ginkgo leaf fraction of step 8 with the ginkgo leaf extract of step 5;
Method of producing ginkgo biloba extract characterized in that it contains 18 to 23% by weight of flavonoids and 10 to 16% by weight of terpene lactone based on the total weight of ginkgo biloba extract.

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