Pharmaceutical composition comprising the extract of hong-zam as an effective component for prevention or treatment of thrombosis and health functional food comprising the same

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of *Hongjam* (Steamed / lyophilized Mature Silkworm cultured for 5-instar 7-days) as an active ingredient. More specifically, the invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation containing, as an active ingredient, a hot water extract of *Hongjam* obtained by steaming and freeze-drying silkworm larvae (*silkworm, Bombyx mori L.*) of the *Orange Silkworm* and *White Silkworm* varieties grown for 5 instar 7 days. The hot water extract of red silkworms (Steamed / lyophilized Mature Silkworm cultured for 5-in-star 7-days) of yellow silkworms and white silkworms, used as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis according to the present invention, exhibits strong antithrombotic activity by inhibiting enzymes related to thrombosis formation and blood coagulation factors, has excellent thermal stability, and does not show loss of blood coagulation factor inhibitory effects and thrombosis-related enzyme inhibitory effects even under acidic conditions of pH 2 and in plasma, so it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through the improvement of blood circulation. In addition, the hot water extract of the red silkworm (Steamed / lyophilized Mature Silkworm cultured for 5-in-star 7-days) of the orange silkworm or white silkworm is used for food and does not exhibit toxicity. It has excellent effects in that it can be processed into various forms such as extract, powder, pills, and tablets, and prepared in a form that can be taken at any time, making it a very useful invention for the pharmaceutical and food industries.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of Hongjam (Steamed / lyophilized Mature Silkworm cultured for 5-instar 7-days) as an active ingredient, and more specifically, to silkworms of the Orange Silkworm and White Silkworm varieties grown for 7 days in the 5th instar. Bombyx mori L.) This relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation, containing as an active ingredient a hot water extract of red silkworm pupae obtained by steaming and freeze-drying the larvae. Background Technology

[0002] As a component of the human body, blood possesses various important functions, including the transport of oxygen, nutrients, and waste products, buffering, maintaining body temperature, regulating osmotic pressure and ion balance, maintaining constant fluid levels, regulating humors, maintaining and regulating blood pressure, and providing biological defense. Normal blood circulation is facilitated by the complementary regulation of the blood coagulation and thrombolytic systems within the body. Among these, the mechanism of the blood coagulation system is reported to involve platelets adhering to and aggregating on the blood vessel walls to form a platelet thrombus, after which the blood coagulation system is activated to form a fibrin thrombus centered around the platelet aggregate.

[0003] The formation of fibrin clots involves a multi-step reaction of numerous blood coagulation factors, through which thrombin, which is involved in fibrin coagulation, is activated. This ultimately leads to the generation of fibrin monomers from fibrinogen. These fibrin monomers are polymerized by calcium and bind to platelets and endothelial cells, forming a permanent blood clot by creating a fibrin polymer cross-linked by Factor XIII. Furthermore, thrombin plays a pivotal role in clot formation by activating platelets, Factor V, and Factor VII to promote blood coagulation reactions. Therefore, substances that inhibit the activity of thrombin can be used as highly useful preventive and therapeutic agents for various thrombotic diseases resulting from excessive blood coagulation abnormalities.

[0004] It is known that the endogenous thrombosis pathway involves the sequential activation of Factor XII, Factor XI, Factor IX, and Factor X, followed by the activation of prothrombin, which ultimately leads to the activation of thrombin; therefore, the specific inhibition of blood coagulation factors is also an important target for the development of treatments for thrombotic diseases. To date, various anticoagulants, antiplatelet agents, and thrombolytics such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases; however, their use is currently limited due to their very high cost, as well as hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

[0005] Meanwhile, silkworms ( Bombyx mori Silkworms are holometabolite insects that undergo four stages: egg, pupa, and moth. The term "silkworm" typically refers to the "larva of the silkworm moth belonging to the family Bombycidae." Silkworm larvae feed on mulberry leaves throughout their lives, grow rapidly, and can be mass-reared; therefore, they have long been used as silkworm insects, edible insects, and medicinal insects. Recently, as detailed genetic information has become known, they are being used in various genetic and physiological experiments.

[0006] Silkworm larvae and pupae are known to be nutritionally superior, being rich in protein, free amino acids, monounsaturated or polyunsaturated fatty acids, minerals, and fiber; they are already registered as food ingredients in Korea. Furthermore, the efficacy of silkworms is recorded in ancient Korean texts such as the *Donguibogam* and *Bencao Gangmu*, and they have been used in folk medicine for malnutrition, pulmonary tuberculosis, wind pathogens, semen enhancement, and stroke. Recently, with the report of an anti-diabetic active ingredient (deoxynojirimycin), they are gaining attention as functional food ingredients, and due to advancements in life science technology, they are also being developed as materials for cosmetics and medical applications.

[0007] To date, about 250 varieties of silkworms are known, including Jangchunjam, Bunongjam, Eunbaekjam, Dakwangjam, Daeseongjam, Segwangjam, Samgwangjam, Yangchujam, Yonggangjam, Bunongjam, Baegokjam, Juhwangjam, Yeonnokjam, and Golden Silk. New varieties are continuously being developed not only for the sericulture industry but also for tourism, experiential activities, cosmetic materials, and medicinal purposes.

[0008] Looking at the life cycle of the silkworm, it grows from an egg through five instars. While the duration of the larval period up to the end of the fifth instar varies depending on the variety and environment, it is generally known to be around 20 days. An instar is a term used to distinguish the intervals between molts, and a fifth-instar silkworm refers to a silkworm that has molted four times and emerged. The first-instar silkworm is called the "ant silkworm"; it is very small, initially less than 2 mm in size, but by the fifth instar, it grows to about 8 cm, becoming approximately 8,000 to 10,000 times larger than the ant silkworm. In particular, silkworms consume the most mulberry leaves and grow rapidly during the fifth instar. Typically, starting from the third day of the fifth instar, silk glands develop to build a cocoon, and after the seventh day of the fifth instar, the silkworm stops eating leaves and begins building a cocoon. At this stage, the silkworm reaches its largest size as a larva, a state known as "mature silkworm."

[0009] Sukjam, which refers to a silkworm just before it spins a cocoon, is a state in which the body is filled with silk protein, appearing clear and transparent. However, the silk protein inside the sukjam changes into a very hard structure when dried, making it impossible to chew, and if one tries to chew it forcefully, it is in an inedible state where teeth can be damaged. Therefore, previously, only silkworms from the 5th instar 3 days prior were used as edible silkworms. However, in order to produce edible sukjam, which is more than twice the size of the 5th instar 3-day-old and has superior nutritional value, the Rural Development Administration has made it possible to consume sukjam after years of research. The method involves rapidly steaming the silkworms to cook them, then freeze-drying them to produce a powder state that is easy to digest and absorb. The silkworms produced in this way were named "Hongjam."

[0010] Therefore, the name Hongjam refers to 'steamed, cooked, and dried silkworms' obtained by steaming 5th instar silkworms on their 7th day and then freeze-drying them. It is a name decided through a public contest by the Rural Development Administration in 2017 and carries the meaning of 'silkworms that benefit humans with broad and diverse functionalities.' According to existing reports, Hongjam is known to contain approximately 70% abundant protein, various amino acids, approximately 5% omega-3 fatty acids, various vitamins, as well as various functional components such as flavonoids and polyphenols.

[0011] Studies on fifth-instar silkworms and red silkworm pupae include the antidiabetic activity of edible silkworms (Ryu, KS, et al., 1997. Kor. J. Seric. Sci. 39. 79-85), the antidiabetic activity of silkworm cocoon extracts (Yoon, Ju-hwa et al., 2005. The Korean Society of Food Science and Nutrition 18: 140-145), the development of functional antidiabetic beverages using silkworm extracts (Choi, Jin-ho et al., 2003. Kor. J. Seric. Sci, 45: 96-102), the preparation and quality evaluation of Dacquoise with an optimal ratio of mixed red silkworm pupae and mulberry powder (Kim, Jong-sung, 2019. Master's thesis, Graduate School of Alternative Medicine, Kyonggi University), the general and nutritional components of freeze-dried silkworm powder (Kwon, Hae-yong et al., 2019. Journal of the Korean Society of Sericulture and Entomology 55: 33-39), and recent research on the various health-promoting functionalities of red silkworm pupae. The current status is known (Lee, Hyun-tae, 2019. Korean Journal of Sericulture and Entomology 55: 40-43). In particular, the health functional properties of red silkworm pupae have been confirmed to include preventive effects against Alzheimer's dementia, alcoholic fatty liver disease, hepatitis and liver inflammation, hyperlipidemia, and hangovers, as well as inhibition of skin darkening caused by ultraviolet rays (Lee, Hyun-tae, 2019. Kor. J. Seric. Sci. 55. 40-43). Furthermore, research has been conducted on *Baekokjam* (white cocoon), *Yeonnokjam* (light green cocoon), *Juhwangjam* (red cocoon), and *Goldensilk* (golden cocoon) among red silkworm pupae; in the case of *Baekokjam*, results of nutritional component analysis, such as fatty acid, amino acid, and mineral composition, have been reported (Ji SD et al, 2019. J. Asia-Pacific Entomology 22: 969-974). In particular, regarding blood circulation improvement activity, silkworm powder Bacillus subtilis and Aspergillus kawachi It has been reported that thrombolytic activity is observed when fermented with strains (Cha Jae-young et al., 2011. Journal of Life Sciences 21: 81-88), and platelet aggregation inhibitory peptides have been reported from *Baekgangjam* (White Silkworm Pupa) (Yi Kong et al., 2014. Peptides. 53:70-78). *Baekgangjam* refers to the 4th to 5th instar larvae of silkworm moths larvae infected with the white silkworm pathogen. Beauveria bassiana It refers to the rigorously dead larvae of silkworm white muscardine caused by natural or artificial infection with *Bals.* Vuill. (Moniliaceae).

[0012] Patents regarding fifth-instar silkworms (5th instar 3 days to 5th instar 7 days) containing red silkworms include Korean Registered Patent No. 10-2286139 [Composition for inducing differentiation of brown adipocytes in canine adipose-derived stem cells using red silkworm extract, method for inducing differentiation, and pet food additive for reducing body fat and preventing obesity containing the same], No. 10-1812499 [Processed food using silkworm powder and method of manufacturing the same], No. 10-0151731 [Hypoglycemic agent containing silkworm powder as an active ingredient and method of manufacturing the same], No. 10-1132337 [Processed flavored oil using silkworm powder and method of manufacturing the same], No. 10-0564202 [Functional health supplement containing silkworm powder extract and method of manufacturing the same], and No. 10-1406109 [Composition for the prevention and treatment of diabetes containing silkworm extract]. Disclosed in Patent No. 10-1246266 [Method for manufacturing fermented silkworm powder and composition for preventing or treating hyperlipidemia and fatty liver containing fermented silkworm powder], Patent No. 10-1359502 [Food composition for preventing and improving osteoporosis using silkworms and health supplement containing the same], Patent No. 10-2270000 [Composition for maturing dendritic cells containing silkworm extract and method for maturing dendritic cells using the same], etc., and Korean Published Patent No. 10-2019-0061544 [Cosmetic composition for antioxidant and whitening containing fermented silkworm powder as an active ingredient] and Published Patent No. 10-2020-0067490 [Method for manufacturing a functional food composition containing silkworm extract] are disclosed.

[0013] However, to date, there are no known reports or patents regarding the potent anticoagulant activity of hot water extracts of red silkworm pupae, particularly those of the orange silkworm and white silkworm pupae varieties. Prior art literature

[0014] Ji SD et al, 2019. J. Asia-Pacific Entomology 22: 969-974; Analysis of Nutritional Components of Red Silkworm Pupa, Lee Hyun-tae, 2019. Kor. J. Seric. Sci. 55. 40-43; Health Functional Properties of Red Silkworm Pupa, Cha Jae-young et al, 2011. Journal of the Korean Society of Life Sciences 21: 81-88; Thrombolytic Activity of Fermented Silkworm Powder, Yi Kong et al, 2014. Peptides. 53: 70-78; Report of Platelet Aggregation Inhibitory Peptide in White Silkworm Pupa The problem to be solved

[0015] The present invention was devised to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to provide a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through strong inhibition of blood coagulation containing steamed / lyophilized mature silkworm cultured for 5-in-star 7-days extract as an active ingredient. means of solving the problem

[0016] In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing a red silkworm extract as an active ingredient.

[0017] Preferably, the above red silkworm extract is a hot water extract of red silkworms from orange silkworms or white jade silkworms.

[0018] In addition, the present invention provides a blood coagulation inhibitor comprising a red silkworm extract as an active ingredient.

[0019] Preferably, the above red silkworm extract is a hot water extract of red silkworms from orange silkworms or white jade silkworms.

[0020] In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis comprising a red silkworm extract.

[0021] Preferably, the above red silkworm extract is a hot water extract of red silkworms from orange silkworms or white jade silkworms. Effects of the invention

[0022] The hot water extract of red silkworms (Steamed / lyophilized Mature Silkworm cultured for 5-in-star 7-days) of yellow silkworms and white silkworms, used as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis according to the present invention, exhibits strong antithrombotic activity by inhibiting enzymes related to thrombosis formation and blood coagulation factors, has excellent thermal stability, and does not show loss of blood coagulation factor inhibitory effects and thrombosis-related enzyme inhibitory effects even under acidic conditions of pH 2 and in plasma, so it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through the improvement of blood circulation. In addition, the hot water extract of the red silkworm (Steamed / lyophilized Mature Silkworm cultured for 5-in-star 7-days) of the orange silkworm or white silkworm is used for food and does not exhibit toxicity. It has excellent effects in that it can be processed into various forms such as extract, powder, pills, and tablets, and prepared in a form that can be taken at any time, making it a very useful invention for the pharmaceutical and food industries. Brief explanation of the drawing

[0023] Figure 1 is a photograph of the four types of red silkworm pupae powder of the present invention. Specific details for implementing the invention

[0024] The present invention will be described in detail below.

[0025] To verify the antithrombotic efficacy of red silkworm pupae, the inventors of the present invention prepared red silkworm pupae powder by steaming and freeze-drying 7-day-old, 5th-instar silkworms of four varieties (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) cultivated by a specific method. Ethanol and hot water extracts were prepared from this powder, and their anticoagulant activity was evaluated to recover the hot water extracts of red silkworm pupae from Juhwangjam and Baekokjam silkworms as antithrombotic active components. By confirming that the hot water extracts of red silkworm pupae from Juhwangjam and Baekokjam silkworms possess excellent thermal and acid stability and do not exhibit any hemolytic activity on human red blood cells, the inventors intended to utilize the hot water extracts of red silkworm pupae from Juhwangjam and Baekokjam silkworms as pharmaceutical compositions and health functional foods for the prevention, treatment, or improvement of thrombosis.

[0026] Specifically, in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using silkworms, which are known in folk medicine to be effective against various diseases such as diabetes, the vascular and circulatory systems, the digestive system, and the metabolic system, the inventors prepared red silkworms from 7-day-old 5th instar silkworms, the largest in size and most nutritious among the larvae, and prepared various solvent extracts from them. As a result of evaluating their antithrombotic activity by direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time), and activated partial thromboplastin time (aPTT) against human thrombin, it was confirmed that the hot water extracts of red silkworms from orange silkworms and white silkworms exhibited excellent thrombin inhibition and thrombus formation inhibition activity through inhibition of blood coagulation factors.

[0027] Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing steamed / lyophilized mature silkworm cultured for 5-in-1-days as an active ingredient.

[0028] The above red silkworm extract is obtained from orange silkworms and white jade silkworms grown for 7 days in the 5th instar. Bombyx mori L.) It is preferable that the hot water extract of red silkworm pupae obtained by steaming the larvae with steam and freeze-drying them.

[0029] In addition, the present invention provides a blood coagulation inhibitor comprising an extract of steamed / lyophilized mature silkworm cultured for 5-in-1-days as an active ingredient.

[0030] The above red silkworm extract is obtained from orange silkworms and white jade silkworms grown for 7 days in the 5th instar. Bombyx mori L.) It is preferable that the hot water extract of red silkworm pupae obtained by steaming the larvae with steam and freeze-drying them.

[0031] In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising an extract of steamed / lyophilized mature silkworm cultured for 5-in-1-days.

[0032] The above red silkworm extract is obtained from orange silkworms and white jade silkworms grown for 7 days in the 5th instar. Bombyx mori L.) It is preferable that the hot water extract of red silkworm pupae obtained by steaming the larvae with steam and freeze-drying them.

[0033] Below, the method for preparing the red silkworm extract of the present invention and efficacy experiments, etc., will be explained in more detail.

[0034] The present invention comprises the steps of: recovering four types of silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) cultured for 7 days in the 5th instar; preparing an extract by solvent extraction from powder of steamed / lyophilized mature silkworm cultured for 7 days in the 5th instar obtained by steaming and freeze-drying the four types of silkworm larvae; evaluating the antithrombotic activity of the extract; and investigating the safety and stability of the hot water extract of the steamed silkworm.

[0035] The "red silkworm extract" included in the composition of the present invention can be obtained by the steps of: harvesting four types of silkworm larvae reared for 7 days in the 5th instar, drying them, steaming them with steam at 100°C for 15 to 30 minutes, then freeze-drying them to produce a powder, extracting the powder with an organic solvent or hot water, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating the powder under reduced pressure.

[0036] The organic solvent used in the present invention may be water (cold water, hot water), hexene, methylene chloride, acetone, ethanol, anhydrous or hydrated lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc., and hot water extraction is preferred.

[0037] The above hot water extract can be further obtained by sequentially or separately fractionating it with organic solvents of hexene, ethyl acetate, and butanol to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

[0038] In the present invention, hot water extracts and ethanol extracts of red silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared for 7 days in the 5th instar were adjusted to a concentration of 5 mg / ml, and thrombin time, prothrombin time, and APT time were measured. As a result, regarding thrombin time, the hot water extract of Juhwangjam showed a coagulation delay extended 1.79 times compared to the control group, and the hot water extract of Baekokjam showed a coagulation delay extended 1.58 times. Regarding prothrombin time, although overall weak activity was observed, the ethanol extract of Baekokjam showed a coagulation delay extended 1.25 times. In the measurement of APT time due to inhibition of blood coagulation factors, the hot water extract of Juhwangjam showed a coagulation delay extended 1.87 times compared to the control group, and the hot water extract of Baekokjam showed a coagulation delay extended 1.75 times. Therefore, among the red silkworm extracts, the hot water extracts of the red silkworms of the orange silkworm and white silkworms exhibited excellent thrombin inhibition and blood coagulation factor inhibition, leading to the conclusion that they possess strong actual anticoagulant activity. The thrombus formation inhibitory activity of these hot water extracts of the red silkworms of the orange silkworm and white silkworms is comparable to the results of aspirin (1.5 mg / ml), which is used as an antithrombotic agent in clinical practice, showing that it extends thrombin time by 1.51 times, proprobin time by 1.34 times, and APT time by 1.38 times compared to the control group. Furthermore, it was confirmed that the hot water extracts of the red silkworms of the orange silkworm and white silkworms can be practically utilized as a preventive or therapeutic / improving agent for thrombosis, as they possess excellent thermal and acid stability while having no hemolytic activity against human red blood cells, allowing them to be consumed or drunk in various forms.

[0039] The red silkworm extract of the present invention can be prepared as a powder through a conventional pulverization process. These are not degraded by various degrading enzymes in plasma and maintain their activity even with heat treatment at 100°C and a pH of 2 in the human stomach.

[0040] The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. The diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, sensory abnormalities, personality changes, visual impairment, epileptic seizures, pulmonary thrombosis, deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial occlusion, and venous thrombosis such as deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

[0041] In addition, the active ingredient of the present invention can be used as a blood coagulation inhibitor, that is, as a pharmaceutical composition for inhibiting blood coagulation.

[0042] A pharmaceutical composition containing the active ingredient of the present invention can be formulated into various forms according to conventional methods to suit each intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injectable formulations like sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

[0043] These pharmaceutical compositions may additionally include 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical compositions of the present invention may additionally include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0044] As a preferred embodiment, solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid formulations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above pharmaceutical composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

[0045] As a preferred embodiment, oral liquid formulations may be exemplified as suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as humectants, sweeteners, flavorings, preservatives, etc.

[0046] As a preferred embodiment, formulations for parenteral administration may be exemplified as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

[0047] The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. 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 as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above-mentioned factors into consideration, and this can be easily determined by a person skilled in the art.

[0048] As 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 body weight, and generally, 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0049] The pharmaceutical composition of the present invention may be administered to a subject via various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0050] In the present invention, "administration" means providing a specific substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention may be oral or parenteral through any general route capable of reaching the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0051] In the present invention, "object" includes, but is not specifically limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably means mammals, more preferably humans.

[0052] In addition, the health functional food of the present invention can be used in various ways in foods and beverages, etc., that are effective in preventing or improving thrombosis. Foods containing the active ingredients of the present invention include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0053] The active ingredient of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition can be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

[0054] In addition to containing the above compound as an essential component in the indicated proportions, the health functional food of the present invention may contain food-grade acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional components.

[0055] Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0056] As the above flavoring agents, natural flavoring agents such as stevia, thaumatin, rebaudioside A, or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the above natural carbohydrates is generally about 1 to 20g, preferably about 5 to 12g, per 100ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. These ingredients may be used independently or in combination. The proportion of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0057] The present invention will be explained in more detail below through examples. The following examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[0058] [Example]

[0059] Example 1: Preparation of red silkworm extract prepared by steaming and drying four types of silkworm larvae (White Silkworm, Light Green Silkworm, Orange Silkworm, and Golden Silk varieties) reared for 7 days in the 5th instar.

[0060] Red silkworm powder of commercially available Baekokjam and Golden Silk varieties was purchased from a silkworm rearing farm in Sangju, Gyeongbuk. For Yeonnokjam and Juhwangjam, which are not currently on the market, red silkworm powder prepared by the rearing farm was used. First, four types of silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared for 7 days in the 5th instar were steamed at 100°C for 20 minutes, then rapidly cooled and freeze-dried, and then ground to prepare fine powder of approximately 50 mesh (Fig. 1). The results of measuring the color difference of the prepared red silkworm fine powder, as well as their pH and brix, are shown in Table 1.

[0061] [Table 1] pH, brix, and color difference of 4 types of red silkworm cocoon fine powder

[0062]

[0063]

[0064] As shown in Table 1, the pH of the red silkworm powder from all four silkworm varieties was 6.6; however, regarding brix, which indicates the content of water-soluble substances, there were significant differences in the order of Golden Silk (39.6) > Baekokjam (30.0) > Juhwangjam (26.0) > Yeonnokjam (22.0). This indicates that there are differences in the composition of the components depending on the silkworm variety. In addition, as a result of the color difference analysis of the red silkworm fine powder, the lightness was similar for the Baekokjam, Golden Silk, and Yeonnokjam varieties (51.2–52.8), but Juhwangjam showed a uniquely low lightness of 37.6. In the case of redness, the Baekokjam, Golden Silk, and Yeonnokjam varieties were similar (-2.03–-2.68), but Juhwangjam showed a relatively high redness of 0.10. In terms of yellowness, the Baekokjam and Golden Silk varieties were similar, ranging from 18.12 to 18.26, while the Juhwangjam and Yeonnokjam varieties were differentiated from the Baekokjam and Golden Silk varieties, ranging from 21.22 to 21.32. Therefore, regarding the overall color difference, Baekokjam and Golden Silk were almost identical, ranging from 43.07 to 43.52, making them difficult to distinguish with the naked eye; Yeonnokjam was characterized by a high yellowness of 45.86, while Juhwangjam was characterized by high redness and yellowness of 58.29. This indicates that there are differences in the pigment components contained depending on the silkworm variety.

[0065] Example 2: Preparation of red silkworm extract and analysis of extract components

[0066] After confirming that there were no silk protein particles in the fine powder of the red silkworm cocoon from Example 1, 20 times the amount of distilled water was added to the red silkworm cocoon powder sample to prepare a hot water extract, and the mixture was extracted at 100°C for 2 hours. To prepare an ethanol extract, 10 times the amount of ethanol (95%, Deoksan, Korea) was added to the red silkworm cocoon powder sample, and the mixture was extracted twice at room temperature. Subsequently, each extract was collected, filtered, and concentrated under reduced pressure to produce a powder. The extraction yield of the red silkworm cocoon extracts and the results of the analysis of their useful components are shown in Table 2. At this time, the total polyphenol content was determined by adding 50 μl of Folin-Ciocalteau and 100 μl of saturated Na2CO3 solution to 400 μl of the extract sample, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as the standard reagent. Total flavonoid content was determined by stirring each sample in ethanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extract, adding 40 μl of 1 N NaOH, reacting at 37°C for 1 hour, and measuring the absorbance at 420 nm. Rutin was used as the standard reagent. Total sugars were quantified using the phenol-sulfuric acid method, with sucrose used as the standard reagent.

[0067] [Table 2] Extraction Efficiency and Analysis of Useful Components of Red Silkworm Extract

[0068]

[0069] As shown in Table 2, for the four types of red silkworms, extraction efficiency ranged from 21.1% to 27.0% in hot water extraction and from 10.2% to 16.2% in ethanol extraction, confirming that hot water extraction is more efficient. In the case of hot water extraction, extraction efficiency was highest in the order of soft green silkworm > orange silkworm > golden silkworm > white jade silkworm, whereas in the case of ethanol extraction, extraction efficiency was highest in the order of white jade silkworm > soft green silkworm > golden silkworm > orange silkworm.

[0070] Meanwhile, analysis of the total polyphenol content of the extracts revealed that the hot water extract contained 5 to 10 times more polyphenols than the ethanol extract, confirming that hot water extraction is preferable for red silkworm pupae for beneficial physiological activity. On the other hand, the total flavonoid content was found to be similar in both the hot water and ethanol extracts, ranging from 2.5 to 4.4 mg / g. Analysis of total sugar content showed that the hot water extract was several times higher than the ethanol extract; in particular, the hot water extracts of the light green silkworm pupae and orange silkworm pupae varieties were found to be more than 10 times higher than the ethanol extracts. Based on these results, it was predicted that the hot water extracts of red silkworm pupae from the orange and light green varieties, which have very high polyphenol content, would exhibit various physiological activities. Actual analysis results confirmed that these extracts displayed stronger antioxidant activity and nitrite scavenging ability than other fractions. Therefore, the hot water extracts of red silkworm pupae from the orange and light green varieties are expected to contribute additionally to antithrombotic activity by exhibiting excellent antioxidant and anti-cancer activities.

[0071] Example 3: Evaluation of blood coagulation inhibitory activity of red silkworm extract

[0072] The blood coagulation inhibitory activity of the red silkworm extract prepared in Example 2 was evaluated, and the results are shown in Table 3. The blood coagulation inhibitory activity of the red silkworm extract samples was evaluated according to 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), and thrombin time, prothrombin time, and APT time were measured. Commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and the thrombin time, prothrombin time, and APT time measurements were performed as follows.

[0073] Thrombin Time

[0074] At 37°C, 50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl2, and 10 μl of sample extracts of various concentrations were mixed in the tube of an Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. Afterward, 100 μl of plasma was added, and the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 32.1 seconds was observed. The thrombin inhibitory effect was expressed as the average value of experiments repeated at least three times, and thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0075] Prothrombin time

[0076] 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tubes of an Amelung coagulometer KC-1A (Japan). After heating at 37°C for 3 minutes, 130 μl of PT reagent was added, and the time until the plasma coagulated was recorded as the average of three repeated experiments. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 18.1 seconds was observed. Prothrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0077] activated Partial Thromboplastin Time (aPTT)

[0078] 100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of an Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, after which 50 μl of aPTT reagent (Sigma, ALEXIN TM) was added and incubated again at 37°C for 3 minutes. Afterwards, 50 μl of CaCl2 (35 mM) was added, and the time until plasma coagulation was measured. DMSO was used as the solvent control instead of the sample, and in this case, a coagulation time of 55.1 seconds was observed. The aPTT result was expressed as the average of three repeated experiments, and blood coagulation factor inhibitory activity was expressed as the value obtained by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[0079] [Table 3] Inhibitory activity of red silkworm extract on blood coagulation

[0080]

[0081] As shown in Table 3, hot water and ethanol extracts of red silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared for 7 days in the 5th instar were adjusted to a concentration of 5 mg / ml, and thrombin time, prothrombin time, and APT time were measured. In the case of thrombin time, the hot water extract of Juhwangjam showed a coagulation delay 1.79 times longer than the control group, and the hot water extract of Baekokjam showed a coagulation delay 1.58 times longer. In the measurement of APT time due to inhibition of blood coagulation factors, the hot water extract of Juhwangjam showed a coagulation delay 1.87 times longer than the control group, and the hot water extract of Baekokjam showed a coagulation delay 1.75 times longer. On the other hand, in the measurement of prothrombin time, all 8 red silkworm samples showed generally weak activity, and the ethanol extract of Baekokjam showed a coagulation delay 1.25 times longer. Therefore, among the red silkworm extracts, the hot water extracts of the red silkworms of the orange silkworm and white silkworms exhibited excellent thrombin inhibition and blood coagulation factor inhibition, and it was determined that they would exhibit actual anticoagulant activity. The thrombus formation inhibitory activity of these hot water extracts of the red silkworms of the orange silkworm and white silkworms is very superior when compared to the results of aspirin (1.5 mg / ml), which is currently used as an antithrombotic agent in clinical practice, which extended thrombin time by 1.51 times, proprobin time by 1.34 times, and APT time by 1.38 times compared to the control group. It is judged that this could replace aspirin, which has severe side effects such as gastrointestinal disorders.

[0082] Example 4: Human erythrocyte hemolytic activity of red silkworm extract

[0083] To evaluate the potential for acute toxicity of the eight types of silkworm extracts prepared in Example 2, human erythrocyte hemolytic activity was evaluated. At this time, hemolytic activity was evaluated according to a previous report (Son Ho-yong, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292). Simply put, 100 μl of human erythrocytes washed three times with PBS were placed in a 96-well microplate, 100 μl of sample solutions of various concentrations were added, and the mixture was reacted at 37°C for 30 minutes. Afterward, the reaction mixture was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. The degree of hemoglobin leakage due to hemolysis was then measured at 414 nm. DMSO (2%) was used as the solvent control for the samples, and Triton X-100 (1 mg / ml) was used as the experimental control for erythrocyte hemolysis. Hemolytic activity was calculated using the following formula.

[0084]

[0085] [Table 4] Human erythrocyte hemolytic activity of red silkworm extract

[0086]

[0087] As shown in Table 4, DMSO and water used as controls showed no hemolytic activity, while Triton X-100 was confirmed to cause 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Additionally, amphotericin B, which is used as an anticancer and antifungal agent, was confirmed to cause more than 50% hemolysis of red blood cells at a concentration of 0.0032 mg / ml. Meanwhile, all eight types of prepared red silkworm extracts showed no hemolysis of red blood cells up to a concentration of 1 mg / ml, confirming that they lacked acute toxicity and hemolytic activity. These results suggest that the red silkworm extracts of the present invention can safely replace antithrombotic agents such as aspirin, which cause side effects, without exhibiting separate hemolytic activity.

[0088] Example 5: Evaluation of plasma, acid, and thermal stability of hot water extracts of red silkworms from orange silkworms and white silkworms

[0089] The red silkworm cocoon hot water extracts of yellow silkworms and white silkworms prepared in Example 2 are used as food, so it is judged that there are no separate safety issues. Accordingly, the plasma stability, thermal stability, and acid stability of the red silkworm cocoon hot water extracts of yellow silkworms and white silkworms regarding blood coagulation inhibition were confirmed. The red silkworm extracts did not show a decrease in blood coagulation inhibitory activity due to thrombin inhibition and coagulation factor inhibition 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 red silkworm cocoon hot water extracts of yellow silkworms and white silkworms contain antithrombotic active substances with acid resistance and heat resistance, confirming their high potential for practical use.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of thrombosis containing, as an active ingredient, a powder prepared by steaming silkworm larvae of the orange silkworm or white jade silkworm variety, reared for 7 days in the 5th instar at 100°C for 20 minutes, then rapidly cooling and freeze-drying them, grinding them to prepare a fine powder of 50 mesh, adding 20 times the amount of distilled water to the fine powder and extracting at 100°C for 2 hours, collecting the extract, filtering it, and then concentrating it under reduced pressure. Claim 2 delete Claim 3 A blood coagulation inhibitor comprising the active ingredient described in claim 1. Claim 4 A health functional food for the prevention or improvement of thrombosis containing the active ingredient described in Paragraph 1.

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