KR20220036035A - Pharmaceutical composition comprising the extract of leaf of cannabis sativa l.as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing an extract of cannabis leaves (Leaf of Cannabis sativa L.) as an active ingredient, and more specifically, to immature young cannabis leaves. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation and platelet aggregation, containing the extract of as an active ingredient. The extract of immature cannabis leaves (Leaf of Cannabis sativa L.) as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis of the present invention acts as an initiator of thrombus formation along with inhibition of thrombogenesis-related enzymes and blood coagulation factors. It exhibits strong antithrombotic activity by inhibiting the aggregation of platelets, and at the same time does not show any hemolytic activity on human red blood cells, has excellent thermal stability, and has a blood coagulation factor inhibitory effect even under acidic conditions of pH 2 and in plasma. Since there is no loss of the effect of inhibiting enzymes related to blood clot formation, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation. In addition, the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. and has excellent effects in that it can be prepared in a form that can be taken at all times, making it a very useful invention in the pharmaceutical and food industries.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing hemp leaf extract as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF LEAF OF CANNABIS SATIVA L. THE SAME}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing an extract of cannabis leaves (Leaf of Cannabis sativa L.) as an active ingredient, and more specifically, to immature young cannabis leaves. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation and platelet aggregation, containing the extract of as an active ingredient.

As a component of the human body, blood has a variety of important functions, including transport of oxygen, nutrients, and waste, buffering action, maintenance of body temperature, regulation of osmotic pressure and maintenance of ionic balance, maintenance of moisture content, fluid regulation, maintenance and control of blood pressure, and biological defense. there is. Normal blood circulation facilitates blood circulation through complementary regulation of the blood coagulation response system and the thrombolysis response system in the body. Among these, the mechanism of the blood coagulation response system is that platelets adhere and aggregate to the blood vessel wall to form a platelet thrombus. It has been reported that the blood coagulation system is activated and a fibrin thrombus is formed centered on platelet aggregates.

The formation of a fibrin clot goes through a multi-step reaction of numerous blood coagulation factors, and thrombin, which is involved in fibrin coagulation, is activated, ultimately producing fibrin monomers from fibrinogen. The fibrin monomers are polymerized by calcium to form platelets and endothelial cells. They combine to form a fibrin polymer cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factor V, and factor VII to promote blood coagulation. Therefore, substances that inhibit thrombin activity can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation abnormalities. Meanwhile, in the endogenous thrombus formation pathway, sequential activation of factor XII, factor are becoming a target. To date, various anticoagulants, antiplatelet agents, and thrombolytics such as heparin, coumarin, aspirin, and urokinase have been used to prevent and treat thrombotic diseases. However, these are not only very expensive, but also cause hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions. Due to this, its use is limited.

Meanwhile, cannabis ( Cannabis sativa L.) is an annual plant of the hemp family. The stem bark is made into hemp and used for clothing, shoes, and crafts, the stem pith is used as paper and building materials, and the seeds have been used for food and medicine. Additionally, hemp seed oil is a very valuable plant resource used as oil and cosmetics. The seed coat, flower buds, and leaves of cannabis contain the hallucinogenic ingredient THC (Tetrahydrocanabinol), which has been misused as a hallucinogenic drug.

Hemp's origin is Central Asia, and it is currently cultivated in various regions around the world. In Oriental medicine, the root of hemp is called hemp root and is used to relieve stagnant blood, treat dystocia, and make stones come out. The stem bark of hemp is used to treat bruises, difficulty urinating, and intestinal pain, and hemp leaves are used to treat bruises, difficulty urinating, and intestinal pain. (Cannabis) has been used as an asthma, analgesic, anesthetic and diuretic. In addition, hemp flowers (hemp flower) have been used to treat paralysis and itching, and hemp flower spikes (hemp powder) have been used to treat dystocia, constipation, gout, and insomnia. Meanwhile, hemp seeds are called mazain in oriental medicine and are used for intractable constipation, hypodipsia, various pain diseases, menstrual irregularities, skin diseases, and dysentery. Recently, they are well known for their activity in suppressing neuroinflammation and protecting nerve cells.

Hemp seeds, which have been stripped of the hallucinogenic substance (THC) from the shell of hemp seeds (mazaine), are recently used as a health food. They have a nutty yet soft texture, taste similar to other nuts, and are high in protein, essential amino acids, and minerals. It is nutritionally excellent and has a high fat content of 25-35%, so it is pressed and extracted and used as cooking oil. In addition, hemp seed oil is rich in polyunsaturated fatty acids such as alpha-linolenic acid (ALA) and gamma-linoleic acid, which has the effect of reducing cholesterol, alleviating vascular inflammation, skin diseases, and atopy. is known to be excellent.

Most of the research related to cannabis is focused on hemp seeds. Studies related to hemp seeds include fatty acid analysis (Koh DH, 1990, Korean J Food Nutr 3: 201-206), antioxidant activity peptides of hemp seeds (Lu RR, et al., 2010. Food Chem 123: 1210-1218), It is known to have activities such as antihypertensive peptide (Girgih AT et al., 2014. J. Funct Foods 6: 384-394) and neuronal anti-inflammatory and protection (Zhou Y et al., 2018. Phytochem Lett 23: 57-61). In addition, it is known that consumption of hemp seeds improves cognitive function (Bae Gil-jun et al., 2015, Journal of Korean Medicine Rehabilitation 25: 1-15) and alleviates atopic dermatitis (Park Shin-hee, 2018, doctoral thesis at Chosun University). In addition, in relation to thrombosis, platelet aggregation inhibitory activity has been reported in rats when hemp seeds were supplied as a diet (Richard MN et al., 2007, Journal of thrombosis and haemostasis, 5: 424-425), but this is due to the hemp seed's It is known to be caused by polyunsaturated fatty acids such as linoleic acid (LA) and alpha-linolenic acid (ALA). Patents related to hemp seeds include Republic of Korea Patent No. 10-1844849 [Complex composition containing hemp seed oil and kenaf seed oil and method for manufacturing the same], No. 10-1828217 [Cordyceps sinensis using hemp seeds and mineral water as a medium] Culture and growth method], No. 10-1492976 [Noodles containing hemp seeds and their manufacturing method], No. 10-1886421 [Tofu manufacturing method using hemp seeds], No. 10-2030923 [Tofu using hemp seeds] Form of processed food and its manufacturing method], No. 10-1921331 [Yukgaejang kalguksu containing hemp seeds and its manufacturing method], No. 10-1820836 [Manufacturing method of granules using hemp seeds], No. 10-1811000 [ Cheongsam seed oil and its manufacturing method], No. 10-0829997 [Cheongsam seed oil and its manufacturing method], No. 10-0829999 [Cheongsam seed oil's quaternary amine salt manufacturing method and its manufacturing method Quaternary amine salt of Cheongsam seed oil], No. 10-1871343 [Manufacturing method of anti-hair loss soap], No. 10-2011349 [Mixed seed juice with improved antihypertensive activity], and Japanese patent JP-0044143 [PRODUCTION OF MISO CONTAINING] THRESHED HEMPSEED] is disclosed. In addition, Republic of Korea Patent Publication Nos. 10-2012-0111125 [Composition for treating kidney cancer and health functional food containing majaine extract], No. 10-2012-0111126 [Composition for treating pancreatic cancer and health containing majaine extract] Functional food], No. 10-2010-0008541 [Composition for promoting differentiation of human mesenchymal stem cells into adipocytes containing guaruin or majain extract] are disclosed, and No. 10-2019-0066702 [Hemp seed] Beverage and its manufacturing method], No. 10-2019-0068982 [Method for extracting protein and/or fat from hemp seeds], No. 10-2019-0087701 [Toothpaste composition containing hemp seed oil and yam for preventing cold teeth ], No. 10-2019-0066701 [Pretreatment method for shelling hemp seeds], No. 10-2017-0128028 [Functional noodles containing hemp seeds and manufacturing method of functional noodles], No. 10-2014-0081966 [Sec. Extraction method of Cheongsam seed oil using critical fluid and Cheongsam seed oil produced thereby], No. 10-2014-0081967 [Atopic skin improvement functional cosmetic composition containing Cheongsam seed oil], No. 10-2016-0014963 [Hemp Pack and its manufacturing method], No. 10-2013-0076658 [Herbal medicine rice prescription name and diabetic rice] are published, and international patents WO-9531176 [COSMETIC PRODUCT CONTAINING HEMPSEED OIL AND METHOD FOR MANUFACTURING THE SAME], WO-0180358 [ AGENT FOR PROMOTING HAIR GROWTH AND METHOD FOR PREPARING SAME] has been released.

In addition, domestic research on cannabis leaves is very limited, and its use is legally regulated as it can be misused as a hallucinogenic agent. On the other hand, in foreign countries, various medicinal products using hemp leaves are being developed, mainly through isolation of various cannabinoids (Bakro F. et al., 2020. J Sep Sci. doi: 10.1002/jssc.201900822; Nagy DU et al., 2019. Chem Biodivers. 16: e1800562) and are known to be effective in improving insomnia and treating epilepsy (Choi S. et al., 2020. J Clin Neurophysiol. 37: 39-49). Other known physiological activities include insecticidal effect (Ahmed M. et al, 2020, Sci Rep. 10: 522.) and peripheral nerve injury recovery effect (Aziz N. et al., 2019. Pak J Pharm Sci 32 (Supplementary): 785-792), blood cholesterol lowering effect (Guo T. et al., 2018. Food Funct. 9: 6608-6617), antibacterial activity against antibiotic-resistant bacteria MRSA (Chakraborty S. et al. , 2018. J Integr Med. 16: 350-357), tissue necrosis inducing activity (Shoyama Y et al., 2008, Plant Signal Behav. 3: 1111-1112), cancer cell proliferation inhibitory activity (Suzuki M. et al., 2017. Nat Prod Commun. 12: 759-761) has been recently reported.

In addition, as a patent related to hemp leaves, Korean Patent No. 10-1714345 discloses [cosmetic or dermatological composition containing an essential oil mixture and its use in the care of especially sensitive or sensitized skin], and the published patent No. 10-2018-0002839 [Hydrogenation of cannabis oil], No. 10-2017-0080608 [Cannabis extract and method of producing and using the same], No. 10-2019-0025485 [CANNABIS SATIVA ) from cannabichromenic acid synthase], in No. 10-2016-0141755 [protein-bound cannabinoid composition], in No. 10-2014-0037124 [canna for use in the treatment of neuropathic pain Nabinoids] are known, and in US patent US-20200060111, [METHOD OF PRODUCTION OF PHYTOCANNNABINOIDS FOR USE IN MEDICAL TREATMENTS], and in US-20190167749, [THE OLEO GEL COMPOSITION AND DELIVERY SYSTEM WITH ACTIVE COMPOUNDS FROM CANNABIS SATIVA AND MENTHA ARVENSIS FOR REDUCTION OF INFLAMMATION AND PAIN IN DEEP TISSUES] has been released.

Meanwhile, research on thrombosis related to cannabinoids in cannabis has been conducted for a very long time, and the effect of promoting platelet aggregation and resulting blood clot formation by cannabinoids in cannabis is well known (Randall MD. 2007. Br J Pharmacol. 152: 671-675: Kvasnicka T, 2010. Vnitr Lek. This thrombopromoting effect is understood to result in serious side effects such as myocardial infarction, increased carotid artery thrombosis, and increased ischemic stroke caused by incorrect use and overuse of cannabis cannabinoid hallucinogens (Faroqui R. et al., 2018. Radiol Case Rep. 13: 747-752; Moeller S. et al., 2017. Asian J Psychiatry 25: 127-130).

In addition, it has been reported that anandamide, an endocannabinoid produced from endothelium (single-layer squamous epithelial tissue lining the lumen of circulatory systems such as arteries, veins, capillaries, heart, and lymphatic vessels) in vivo, inhibits platelet aggregation (De Angelis et al. al., 2014, PLoS One, 29;9(9):e108282), which is different from externally administered cannabinoids such as hemp extract, and all other recent research results contradict the above results with anandamide, 2-arachidonoylglycerol, and virodhamine. It has been confirmed that major endocannabinoids in vivo, such as those that promote platelet aggregation, increase the risk of thrombosis (Brantl SA et al., 2014, Platelets. 25: 151-161; Brantl SA et al., 2014, Platelets. 25: 465-466; Baldassarri S. et al., 2008. J Thromb Haemost. 6: 1772-1779; Vnitr Lek. 2007. Br J Pharmacol. 675). Therefore, as in the case of cannabis smokers, cannabinoids in cannabis leaves are also known to increase the possibility of cardiovascular diseases such as thrombophlebitis by increasing platelet aggregation and blood clot formation (Lee C, Moll S. 2014. Circulation 130: 214-215).

KR 10-2017-0080608 A

De Angelis et al., 2014, PLoS One, 29;9(9):e108282

The present invention was devised to solve the problems of the prior art as described above. The problem to be solved by the present invention is the inhibition of blood coagulation and platelet inhibition containing an extract of immature cannabis leaves (Leaf of Cannabis sativa L.) as an active ingredient. The object is to provide pharmaceutical compositions and health functional foods for preventing or treating/improving thrombosis by inhibiting aggregation.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing leaf extract of immature cannabis ( Cannabis sativa L.) as an active ingredient.

It is preferable that the leaves have not been heat treated.

The extract is preferably an ethanol extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a leaf extract of immature cannabis ( Cannabis sativa L.).

It is preferable that the leaves have not been heat treated.

The extract is preferably an ethanol extract.

The extract of immature cannabis leaves (Leaf of Cannabis sativa L.) as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis of the present invention acts as an initiator of thrombus formation along with inhibition of thrombogenesis-related enzymes and blood coagulation factors. It exhibits strong antithrombotic activity by inhibiting the aggregation of platelets, and at the same time does not show any hemolytic activity on human red blood cells, has excellent thermal stability, and has a blood coagulation factor inhibitory effect even under acidic conditions of pH 2 and in plasma. Since there is no loss of the effect of inhibiting enzymes related to blood clot formation, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation. In addition, the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. and has excellent effects in that it can be prepared in a form that can be taken at all times, making it a very useful invention in the pharmaceutical and food industries.

Figure 1 shows the human platelet aggregation inhibitory activity of extracts from different parts of cannabis. 1: Solvent control (DMSO), 2: Aspirin (0.25 mg/ml), 3: Aspirin (0.12 mg/ml), 4: Immature cannabis leaf ethanol extract (0.25 mg/ml), 5: Mature cannabis stem ethanol extract ( 0.25mg/ml), 6: Ethanol extract of mature hemp root (0.25mg/ml), 7: Ethanol extract of hemp seed (0.25mg/ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of cannabis, the inventors of the present invention prepared extracts of cannabis leaves, stems, roots, and seeds (hemp seeds) at each stage of maturation using a certain method, and evaluated the anticoagulant activity and platelet aggregation inhibitory activity of immature cannabis. The leaf extract of hemp was recovered as an anti-thrombotic active ingredient, and it was confirmed that the extract had excellent heat stability and acid stability while showing no hemolytic activity on human red blood cells, making it possible to use the extract for the prevention or treatment of thrombosis. It was intended to be used as a pharmaceutical composition for improvement and as a health functional food.

Specifically, the present inventors developed a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis using cannabis, which is known in the private sector to be effective in various diseases such as blood vessels, circulatory system, digestive system, and metabolic system, Various solvent extracts were prepared from leaves, stems, roots, and seeds (hemp seeds) at different stages of maturity, and the antithrombotic activity of these extracts was measured by direct thrombin inhibition of human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time), and By evaluating the activated partial thromboplastin time (aPTT), it was confirmed that the extract of immature cannabis leaves (Leaf of Cannabis sativa L.) had excellent anticoagulant activity and strong platelet aggregation inhibitory activity. In addition, it was confirmed that the extract did not exhibit hemolytic activity against human red blood cells, confirming that it can be used practically.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing leaf extract of immature cannabis ( Cannabis sativa L.) as an active ingredient.

It is preferable that the leaves have not been heat treated.

The extract is preferably an ethanol extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a leaf extract of immature cannabis ( Cannabis sativa L.).

It is preferable that the leaves have not been heat treated.

The extract is preferably an ethanol extract.

Below, the manufacturing method and efficacy test of the immature cannabis leaf (Leaf of Cannabis sativa L.) extract of the present invention will be described in more detail.

The present invention includes the steps of preparing extracts through solvent extraction from various parts of hemp at different maturation times; A step of evaluating the antithrombotic activity of the extract; and investigating the stability of the immature hemp leaf extract.

The “immature cannabis leaf extract” included in the composition of the present invention can be obtained by extracting immature cannabis leaves with an organic solvent, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating it under reduced pressure. You can. Here, “immature” can be understood to indicate growth of the above-ground part of hemp less than 1 m in length.

Organic solvents used in the present invention include water (cold water, hot water), hexene, methylene chloride, acetone, alcohol, anhydrous or hydrous lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), and the above lower alcohols. A mixed solvent of ethanol and water can be used, and hot water or 95% ethanol extraction is preferred.

As a preferred embodiment, the immature cannabis leaf extract of the present invention may be an ethanol extract of young cannabis leaves with an aerial part less than 1 m in length. In addition, it is preferable that the leaves are not subjected to separate heat treatment. Here, the ethanol extract may be sequentially or separately fractionated with organic solvents of hexene, ethyl acetate, and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

In the present invention, various parts of cannabis at different maturation times were extracted with ethanol, the extract was adjusted to a concentration of 5 mg/ml, and thrombin time, prothrombin time, and APTI time were measured. As a result, in the immature leaf, mature stem, and mature root extracts, It was confirmed that it inhibited the formation of blood clots by exhibiting stronger inhibition than the inhibition of blood coagulation factors shown by aspirin at a concentration of 1.5 mg/ml. In addition, extracts of immature leaves, mature stems, and mature roots also showed prothrombin inhibition equivalent to aspirin at a concentration of 1.5 mg/ml. However, hemp seed extract showed almost negligible blood coagulation inhibitory activity compared to aspirin (1.5 mg/ml).

Meanwhile, the inhibition of platelet aggregation was confirmed by using ethanol extracts from various hemp parts at different stages of maturity at a concentration of 0.25 mg/ml. As a result, the hemp seed extract was almost the same as the no-added control and had almost no aggregation effect, but the mature stem extract had almost no aggregation effect. and mature root extract showed a platelet aggregation ability of 124% and 148%, respectively, compared to the non-added extract, showing a thrombus formation promoting activity. However, the immature cannabis leaf extract showed a strong platelet aggregation inhibitory activity with an aggregation rate of 56% compared to the no-added extract, which was similar to the platelet aggregation inhibitory activity of aspirin at normal concentration. These results indicate that extracts from different parts of cannabis have various activities related to blood clot formation, and in particular, immature cannabis leaf extract possesses antithrombotic activity through inhibition of blood coagulation factors and strong platelet aggregation inhibitory activity. It is suggested that it can replace antiplatelet drugs such as aspirin, which have a high risk of side effects.

The immature cannabis leaf extract of the present invention can be manufactured into powder through a conventional powdering process such as reduced pressure drying, freeze drying, or spray drying. They are not decomposed by various decomposition enzymes in plasma, and remain active even when heat treated at 100°C and at pH 2 in the human stomach.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. These diseases include, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis. , deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial occlusion, and venous thrombosis includes deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be formulated and used in various forms, and can be administered through various routes, including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the pharmaceutical composition, such as starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

As a preferred embodiment, oral liquid preparations may include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg per day. Alternatively, it can be administered every other day or divided into 1 to 3 doses per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., 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 a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

In the present invention, “administration” means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages that are effective in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

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

As the flavoring agent, natural flavoring agents such as thaumatin, rebaudioside A, or stevia such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

All experiments such as hemp cultivation, harvesting, extraction, and activity evaluation described in the examples below were conducted legally in accordance with the narcotics handler's permit, narcotic raw material handling permit, and hemp grower's permit.

Example 1: Preparation of ethanol extracts from various parts of cannabis according to growth period and analysis of their useful components

In June 2018, immature young cannabis leaves less than 1 m in length above the ground were harvested, and mature cannabis stems and roots were harvested in July, and ethanol extracts were prepared by purchasing domestic commercially available cannabis seeds (hemp seeds) harvested in 2018. Specifically, 10 times the amount of organic solvent was added to each sample, extraction was repeated twice at room temperature, the extracts were collected, filtered, and concentrated under reduced pressure to prepare an ethanol extract. The extraction yields of extracts from various parts of cannabis and their useful content analysis results are shown in Table 1.

[Table 1] Extraction efficiency of extracts from various parts of cannabis and analysis of useful components of the extracts

First, in the case of hemp seeds and stems, the yield was high at 9.4~9.8%, and the leaf extract was excellent at 6%. On the other hand, the root extract showed a very low yield of 1.4%. To analyze the useful components of the extracts, the total polyphenol, total flavonoid, total sugar, and reducing sugar contents were measured. At this time, the total polyphenol content was measured by adding 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution to 400 μl of the extraction sample solution, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. To determine the total flavonoid content, each sample was extracted with methanol stirring for 18 hours, 4ml of 90% diethylene glycol was added to 400μl of the filtered extraction solution, 40μl of 1 N NaOH was added again, and the absorbance was measured at 420nm after reacting for 1 hour at 37°C. Rutin was used as a standard reagent. Reducing sugars were quantified using the DNS method, and total sugars were quantified using the phenol-sulfuric acid method.

As shown in Table 1, the polyphenol content was highest in the leaf extract at 43.2 mg/g, while the seed extract was very low at 0.8 mg/g. In terms of total flavonoid content, the leaf extract showed the highest content of 29.1 mg/g, while the root and seed extract showed a relatively low content of 2.1 to 2.7 mg/g. Therefore, it was confirmed that the leaf extract contained superior polyphenol and flavonoid substances than the stem, root, and seed extracts. In the case of total sugar content, root extract (153.5 mg/g) > stem extract (120.0 mg/g), leaf extract (117.5 mg/g) > seed extract (12.0 mg/g), while reducing sugar content was in leaf extract ( 74.9mg/g) > stem extract (49.9mg/g), root extract (41.1mg/g) > seed extract (12.6mg/g), confirming that the cannabis leaf extract contains a significant amount of total sugar and reducing sugar. .

Example 2: Evaluation of blood coagulation inhibitory activity of cannabis extracts from various parts at different maturation times

The blood coagulation inhibitory activity of cannabis extracts from various parts of Example 1 was evaluated, and the results are shown in Table 2. At this time, the blood coagulation inhibitory activity of the cannabis extract was evaluated based on previously reported methods (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 APTI time were measured. As plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time, and AP time measurements were performed as follows.

Thrombin Time

At 37°C, 50μl of 0.5U thrombin (Sigma Co., USA), 50μl of 20mM CaCl ₂ , and 10μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, then 100μl of plasma was added. After that, the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 32.1 seconds. The thrombin inhibitory effect was expressed as the average value of experiments repeated three or more times, and the thrombin inhibitory activity was expressed as the coagulation time when adding the sample divided by the coagulation time of the solvent control.

prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, then 130 μl of PT reagent was added and the plasma was coagulated. The time until this was achieved was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 18.1 seconds. Prothrombin inhibitory activity was expressed as the coagulation time upon sample addition divided by the coagulation time of the solvent control.

activated Partial Thromboplastin Time (aPTT)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added and incubated again at 37°C for 3 minutes. It was incubated for a minute. Afterwards, 50μl CaCl ₂ (35mM) was added and the time until the plasma coagulated was measured. As a solvent control, DMSO was used instead of the sample, and in this case, the coagulation time was 55.1 seconds. The results of aPTT were expressed as the average value of three repeated experiments, and the blood coagulation factor inhibitory activity was expressed as the aPTT at the time of sample addition divided by the aPTT of the solvent control.

[Table 2] Blood coagulation inhibitory activity of extracts from various parts of cannabis by maturity period

As shown in Table 2, as a result of measuring thrombin time, prothrombin time, and APTI time of extracts prepared from various parts of cannabis at a concentration of 5 mg/ml, excellent blood coagulation factors (Coagulation) were found in the leaf, stem, and root extracts. factors) was observed, and the AP time was confirmed to be 1.84 to 1.90 times longer than that of the no-additive group. This was a superior activity than the 1.48-fold extended AP time shown by aspirin (1.5 mg/ml), which was used as a control for thrombus formation inhibition. Additionally, prothrombin inhibitory activity comparable to that of aspirin was confirmed in cannabis leaf, stem, and root extracts. However, in terms of thrombin inhibitory activity, leaf, stem, and root extracts showed weak activity. Meanwhile, in the case of the seed extract, the blood coagulation inhibitory activity was minimal and no significant thrombus formation inhibitory effect was observed. Therefore, it was determined that extracts of leaves, stems, and roots of cannabis can inhibit the formation of blood clots through inhibition of various blood coagulation factors and prothrombin.

Example 3: Platelet aggregation inhibitory activity of cannabis extracts from various parts at different maturation times

The human platelet aggregation inhibitory activity of extracts prepared from various parts of cannabis prepared in Example 1 was evaluated, and the results are shown in Table 3 and Figure 1. Platelets are small, disk-shaped cells that circulate in blood vessels together with various blood cells. Instead of having a nucleus, they have cytoplasmic granules that contain a high concentration of various substances related to protecting blood vessels from damage and platelet aggregation. When damage to the inner wall of blood vessels occurs, platelets aggregate. It is an important cell that initiates thrombus formation by secreting factors and combining with collagen exposed due to damage to endothelial cells to form a primary hemostatic plug. Therefore, inhibition of platelet aggregation is very important to prevent thrombus formation. It is active. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Concentrated human platelets were used, which were washed once with washing buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 1mM EDTA, pH 6.5). Afterwards, resuspend in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4) and run at 3,000 rpm for 10 minutes. After centrifugation, the platelets were resuspended in suspending buffer, and the platelet count was adjusted to ^4x109 /ml. Afterwards, 2.5 μl collagen was added to 1 ml suspension and reacted for 5 minutes, and platelet aggregation was measured at 37°C using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Platelet aggregation inhibitory activity of cannabis extracts from various parts at different maturation times

As shown in Table 3 and Figure 1, aspirin showed platelet aggregation of 49.7% at a concentration of 0.25 mg/ml and 82.2% at a concentration of 0.125 mg/ml, demonstrating the basis for its use as an antithrombotic agent in clinical practice due to its excellent platelet aggregation inhibitory activity. I was able to. Meanwhile, the seed extract had no effect on platelet aggregation at a concentration of 0.25 mg/ml, and the stem and root extracts showed aggregation rates of 124.8% and 148.4%, respectively, at a concentration of 0.25 mg/ml, which promoted platelet aggregation. However, the leaf extract showed a strong platelet aggregation inhibitory activity, showing an aggregation rate of 56.0% compared to the no-added control at a concentration of 0.25 mg/ml. Therefore, the above results suggest that the unheated ethanol extract of immature cannabis leaves inhibits prothrombin and blood coagulation factors related to the formation of blood clots, and strongly inhibits platelet aggregation and can be used as an antithrombotic agent.

Example 4: Human Red Blood Cell Hemolytic Activity of Immature Cannabis Leaf Ethanol Extract

Hemp leaves are used as a food in foreign countries (Aziz N. et al., 2019. Pak J Pharm Sci. 32 (Supplementary): 785-792) and are used as an adjuvant for the treatment of peripheral nerve damage, so their safety as a food is recognized. However, since the safety of cannabis extracts by site has not been objectively evaluated, human red blood cell hemolytic activity was evaluated to evaluate the potential for acute toxicity of these extracts.

At this time, hemolytic activity was evaluated according to an existing report (Ho-yong Son, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292), and simply, 100 μl of human red blood cells washed three times with PBS were placed in a 96-well microplate. 100 μl of sample solutions of various concentrations were added and reacted at 37°C for 30 minutes. Afterwards, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. It was measured in . DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 4] Human red blood cell hemolytic activity of extracts from various parts of cannabis by maturity period

As shown in Table 4, DMSO and water used as controls had no hemolytic activity, and triton In addition, in the case of amphotericin B, which is used as an anticancer and antifungal agent, it was confirmed that it haemolyzed more than 50% of red blood cells at a concentration of 0.025 mg/ml.

Meanwhile, cannabis leaf extract and stem and root extract did not cause red blood cell hemolysis at all up to a concentration of 1 mg/ml, confirming that there was no acute toxicity and no red blood cell hemolytic activity. On the other hand, the seed extract showed strong hemolytic activity of 90.8% at a concentration of 1 mg/ml and 72.1% red blood cell hemolysis even at a concentration of 0.25 mg/ml. These results suggest that the ethanol extract of immature cannabis leaves of the present invention can safely replace antithrombotic agents that have side effects such as aspirin in the future without separate hemolytic activity.

Example 5: Evaluation of plasma, acid and heat stability of immature cannabis leaf ethanol extract

The ethanol extract of immature cannabis leaves obtained in Example 1 was confirmed for its blood coagulation inhibitory and platelet aggregation inhibitory activities including plasma stability, heat stability, and acid stability. The extract did not show a decrease in blood coagulation inhibitory and platelet aggregation inhibitory activities even when heat-treated at 100°C for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, and treated in plasma for 1 hour. Therefore, it was confirmed that the ethanol-activated extract of immature cannabis leaves contains anti-thrombotic substances with acid resistance and heat resistance, confirming that it has high practical usability.

[R&D tasks]

Assignment number: 2020-0205

Organizing Ministry: Gyeongsangbuk-do

Research management agency: Andong University Industry-Academic Cooperation Foundation

Research project name: Verification of changes in effective ingredients and physiological activity of domestic hemp by growth stage and part

Contribution rate: 100%

Research period: 2020. 04. 01. - 2020. 12. 31.

Host organization: Andong University Industry-Academic Cooperation Foundation

Research institution: Department of Food and Nutrition, Andong University

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis containing leaf extract of immature cannabis ( Cannabis sativa L.) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the leaves are not heat treated. The pharmaceutical composition according to claim 1, wherein the extract is an ethanol extract. A health functional food for preventing or improving thrombosis containing the active ingredient according to any one of claims 1 to 3.