KR100576972B1 - Compositions comprising larvae or pupae of bee or extract thereof - Google Patents

Abstract

The present invention relates to an anticancer composition comprising bee larva itself or a water-soluble, alcohol or organic solvent extract thereof as an active ingredient.

The present invention relates to a thrombus dissolving composition comprising the bee larva itself or a water-soluble, alcohol or organic solvent extract thereof as an active ingredient.

Compositions of the present invention include pharmaceutical and food compositions useful for the prevention and treatment of various cancers, including breast cancer, bladder cancer, skin cancer, gastric cancer, liver cancer, leukemia and lymphoma.

The compositions of the present invention include pharmaceutical and food compositions useful for the prevention and treatment of various thrombus related diseases including coronary artery disease and cerebrovascular disease.

Description

Composition containing larvae or pupae of bee or extract etc.

1 is a diagram showing the effect of the composition of the present invention on topoisomerase II inhibitory effect.

FIG. 2 is a diagram illustrating a ladder pattern of bands obtained by extracting DNA from cervical cancer cell lines.

The present invention relates to a composition for anticancer or thrombosis dissolving bees larvae or extracts thereof.

DNA topoisomerase, an enzyme that selectively shows anticancer effects, has been identified by the recent development of biochemical analytical techniques. It controls the conformation of DNA, and is used in various cells such as DNA replication, RNA transcription, and gene recombination. It is an enzyme involved in the process. The enzymatic activity of topoisomerase modulates the DNA topology by cleaving DNA and then temporarily forming and sewing up the DNA-protein complex. Various types of topoisomerase inhibitors are known to inhibit the enzyme-induced sewing process, and these inhibitors have anti-cancer effects by selectively inhibiting the action of topoisomerase in cancer cells with high enzyme activity. The facts have been reported, indicating the clinical significance of this enzyme. Currently, various types of inhibitors of topoisomerase are used for cancer treatment, and efforts to develop higher-efficiency anticancer agents are of interest worldwide. Representative topoisomerase inhibitors that have been applied clinically as anticancer agents include camptothecin (CPT) and its derivatives topotecan, 9-amino-campotesin (9-amino-CPT), CPT-11, stauro Staurosporine, Bis-indolyl malemide, etoposide, amsacrine, CP-115.953, genistein, and the like.

In addition, thrombus is formed in the cerebrovascular system due to the bioreaction mechanism, and when thrombosis occurs, half body becomes incapacitated, and microvasculature of the brain is ruptured, leading to cerebral hemorrhage and bleeding into the space between the brain and the head bone. In addition to causing problems, clogging of blood vessels by blood clots causes heart failure or heart failure, and many studies have been conducted to treat thrombus. Thrombolytic agents such as streptokinase, urokinase and tPA (tissue-type plasminogen activator), which are used in the past, have a high risk of side effects for clinical use and are very expensive, and exclude urokinase. And oral administration is impossible. Therefore, in recent years, attention has been focused on preparations that can increase the thrombolytic ability in the blood by direct oral administration or in combination with vascular injection in addition to the thrombolytic agent which can be administered only by vascular injection.

The inventor of the present invention, while searching for an active ingredient of an anticancer agent and a thrombolytic agent that can overcome the disadvantages of the above-described cost or administration method, the bee larvae or their extracts are largely bred in bee farms in Korea. The present invention was found to have an inhibitory effect and a thrombolytic effect.

The present invention is to provide an anticancer composition containing bees larvae or extracts thereof as an active ingredient.

The present invention is to provide a composition for dissolving a thrombus containing bees larvae or extracts thereof as an active ingredient.

The present invention provides an anticancer composition containing the larvae of bees or extracts thereof.

The present invention provides a composition for dissolving thrombosis containing bee larvae or extracts thereof.

By larvae of bees in the present invention is meant everything in the stage prior to adulthood, in particular larvae or pupa or mixtures thereof.

The larvae of the bee of the present invention means not only the larva itself, but also the honeycomb from which the larva is released or a mixture of the larva and the honeycomb.

The larvae extract of bee of the present invention means a water-soluble, alcoholic or organic solvent extract. The alcohol means lower alcohols such as methanol and ethanol, and the organic solvent means ethyl acetate, hexane and the like.

Compositions of the present invention include pharmaceutical compositions useful for the prevention and treatment of various cancers, including breast cancer, bladder cancer, skin cancer, gastric cancer, liver cancer, leukemia and lymphoma.

The composition of the present invention includes food compositions useful for the prevention and improvement of various cancers, such as breast cancer, bladder cancer, skin cancer, gastric cancer, liver cancer, leukemia and lymphoma.

Compositions of the present invention include pharmaceutical compositions useful for the prevention and treatment of various thrombus related diseases including coronary artery disease and cerebrovascular disease.

The composition of the present invention includes a food composition useful for the prevention and improvement of various thrombus related diseases including coronary artery disease and cerebrovascular disease.

The bees used in the present invention are conventional bees (Dongyang species), beekeeping bees (Western species), bumble bees, chicks and bees, wasps, king bees, unidentified bees, etc., taxonomy (Korea Insect Collection, Korean Insectological Society, 1994) Rewards include all bees belonging to the bee family, bee family, calabash, wasp, twin bee, and bee family (including honeybee, bee family, back young bee, and bee family).

Extraction method of bee larvae extract used in the present invention is as follows.

The water-soluble extract is eluted with bee larvae in a mixed solution of Tris-HCl buffer solution at pH 7.4 and 0.1 M aqueous sodium chloride solution, and then centrifuged to collect the supernatant.

The larvae alcohol extract (or organic solvent extract) of the bee of the present invention is pulverized larvae (or honeycomb) and then eluted with alcohol (or organic solvent), filtered and concentrated the filtrate, then using a vacuum condenser To dry completely.

The composition of the present invention exhibits cytotoxic effects in both cervical cancer cell lines and gastric cancer cell lines.

The composition of the present invention shows a DNA topoisomerase II inhibitory effect.

The composition of the present invention exhibits an apoptosis effect in cervical cancer cell lines.

The composition of the present invention is a very excellent result of measuring antithrombotic activity by activated partial thromboplastim time (APTT) anticoagulant activity and TT (thrombin time) anticoagulant activity for measuring the anticoagulant activity for the endogenous blood coagulation system, It has an antithrombotic effect.

The composition of the present invention may further comprise at least one active ingredient exhibiting the same or similar function in addition to the bee larva itself or its extract.

The composition of the present invention may include one or more active ingredients exhibiting other functions in addition to the bee larva itself or extracts thereof.

The composition of the present invention, in addition to the above-described active ingredient for administration may be prepared by including one or more carriers that are acceptable in pharmaceutical or food.

Pharmaceutical or food acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered parenterally or orally according to the desired method, the dosage is depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, etc. Although the range varies, it is preferable that the adult daily dose is divided into one to several times the amount of 10 ~ 100mg / kg.

The compositions of the present invention can be used alone or for the use of surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of various cancers and thrombosis related diseases, including coronary artery disease and cerebrovascular disease. It can be used in combination with.

The larvae or extracts thereof of the bee of the present invention may be added to food or beverages for the purpose of anticancer and thrombolysis. In this case, the amount to be added may be added as 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight in the case of a health food composition, 1 to 30g, preferably based on 100ml in case of a health beverage composition Can be added at a rate of 3 to 10 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, synthetic flavors and natural flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, and stabilizers. It may contain a topical agent, preservatives, glycerin, alcohol, carbonation agent used in the carbonated beverage and the like. In addition, the compositions of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to 20 parts by weight per 100 parts by weight of the composition of the present invention.

Foods to which the larvae or extracts of the bee of the present invention can be added for development for health foods include various foods, beverages, gums, vitamin complexes, health supplements, and the like.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Effect of the Compositions of the Invention on Cytotoxicity

The water-soluble extract was added to 50 ml of 40mM tris-hydrochloric acid buffer solution and 0.1M aqueous sodium chloride solution at pH 7.4, and eluted at 4 ° C for 15 g of larvae frozen larvae. The supernatant was collected by centrifugation at 8,000 rpm for 30 minutes.

Methanol extract (or ethyl acetate extract) was pulverized 15 g of larvae frozen larvae, eluted in 50 ml of methanol (or ethyl acetate), and filtered with Whatman paper No. 6 (Whatman paper No. 6). The filtrate was concentrated using an evaporator and then completely dried using a Hanil vaccum concentration system.

The aqueous extract was based on the amount of protein in Bradford, and the methanol extract and ethyl acetate extract were dissolved in 0.5% ethanol in 1 mg / ml of the dried sample based on their respective dry weights. .

The cell line was composed of cervical cancer cell line (HeLa; cervix cancer cell line) and gastric cancer cell line (SNU-1; CRL 597.1), which were distributed at the Cancer Research Center of the College of Medicine, Seoul National University. 10% Fetal Bovine Serum (GIBCO) ), 5% CO ₂ using RPMI 1640 medium (GIBCO, New York, USA) containing 1 mmol / L L-glutamine, 100 units / ml penicillin G, and 100 μg / ml streptomycin sulfate (Sigma) And incubated at 37 ° C.

Mitomycin C (MMC, 0.1 mg / ml, USA Sigma) was used as a positive control.

Adriamycin (10 u M) was used as a comparative material.

In order to determine the effect of the composition of the present invention on the cytotoxicity in cervical cancer cell line (HeLa), gastric cancer cell line (SNU-1), XTT [sodium 3 '-[1- (phenylamino-carbonyl) -3,4-tetrazolium ] -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate kit solution (Boehringer Mannheim)] Assay assay for cell viability, the results are shown in Table 1.

In addition, the cytotoxic effects on cervical cancer cell lines are shown in Table 2 reduced to IC ₅₀ .

Cell survival rate of cervical cancer cell line and gastric cancer cell line of bee larvae extract (%) Cervical Cancer Cells (HeLa) Gastric Cancer Cells (SNU-1) Ethyl acetate fraction Water soluble fraction Mitomycin C (0.1mg / ml) 73 54 Adriamycin (10 u M) 89 77 King Beehive (0.1mg / ml) 90 108 Bumblebee Bee Pearl (0.1mg / ml) 94 76 Bumblebee Larvae (0.1mg / ml) 94 77 Bumblebee Worker Bee (0.1mg / ml) 94 81

Cytotoxic Effects of Bee Larva Extract on Cervical Cancer Cell Lines IC ₅₀ (mg / ml) Methanol fraction Mitomycin C (0.1mg / ml) 7.6 × 10 ^-2 Adriamycin (10 u M) 2.4 Beehive 1.1 Bumblebee Soap 8.1 Bumblebee larva 16.3 Bumblebee worker bee 0.5

As shown in Table 1 and Table 2, it can be seen that both the water-soluble, bee larvae, methanol and ethyl acetate extracts are excellent in cytotoxicity.

Example 2 Effect of the Compositions of the Present Invention on DNA Topoisomerase II Inhibitory Effects

In order to determine the effect of the composition of the present invention on the DNA topoisomerase II inhibitory effect, experiments were performed using the TopoGen Topoisomerase II Drug kit.

50 g of larvae of the bee were ground and eluted with 50 ml of buffer, and the eluate was adjusted to 1 mg / ml of protein and used as a sample.

1 μl supercoiled DNA (pRYG DNA), 300 mM Tris-HCl buffer, pH 7.6, 3 mM ATP, 15 mM mercaptoethanol, 8 mM MgCl ₂ , 60 μl NaCl, 20 μl distilled water, human topoisomerase II 2 units / Μl was added and reacted at 37 ° C for 30 minutes.

At this time, etoposide (10 mM) was used as a control.

2 μl of 10% SDS, protein kinase K, chloroform: isoamyl alcohol (CIA 24: 1) was added, stirred, and centrifuged to repeat the CIA operation once in the supernatant, and then the supernatant was 0.5 μg / ml ethidium bromide (ethidium). electrophoresis on 1% agarose gel containing bromide).

The results are shown in FIG.

As shown in Fig. 1, the supercoiled DNA treated with the bee larvae extract of the present invention does not release the two strands of the supercoiled DNA, so the band does not fall down. It can be seen that the level changes to etoposide level, which is a control that does not split into.

Example 3 Effect of the Compositions of the Invention on Apoptosis

The bee larvae buffer extract was inoculated at 10 µg / ml in cervical cancer cell lines for 45 hours in a CO ₂ incubator and then DNA was extracted. 10 μg of DNA was added to a 1% agarose gel (including 0.5 μg / ml ethidium bromide) and then electrophoresed to observe a ladder pattern of bands.

The results are shown in FIG.

As shown in FIG. 2, it was confirmed that cervical cancer cell DNA treated with the bee larvae extract of the present invention showed a DNA ladder pattern remarkably compared to cervical cancer cell DNAs not treated with the bee larvae extract.

Therefore, it can be seen that the bee larvae extract of the present invention exhibits anticancer effects specifically to cancer cells without showing anticancer effects on normal cells.

Example 4  : Effect of the Composition of the Present Invention on the Activity of Thrombolytic Compound

The water-soluble extract was put in 10ml 0.1M Tris-hydrochloric acid buffer solution (pH 7.5) per 10g insects and then pulverized with a mixer and left in a cold room for 1 day. A protein concentration of 1 mg / ml was prepared, and the activity of the thrombolytic substance was measured by a fibrinolytic activity assay.

Thrombin (10 NIH / mL) was added to 0.8% fibrinogen solution to make a fibrin plate, and 10 μl of the sample was dropped. After standing at 37 ° C. for 6-18 hours, the size of the lysis zone was measured, and the degree of thrombolytic activity was calculated by converting it into a standard enzyme unit of plasmin.

The results are shown in Table 3.

Thrombolytic Solubility of Bee Bee Larva Extract of the Present Invention Thrombus Solubility (U / mg) Water soluble fraction Control (saline) - Beehive 1.9 Bumblebee Soap 0.5 Bumblebee larva 1.0 Bumblebee worker bee 0.3

As shown in Table 3, among the bee larva water soluble extracts of the present invention, the bee larvae water soluble extract and the bumble bee larva water soluble extract are 1.9 U / mg and 1.0 U / mg, respectively.

Example 5  : Effect of the Composition of the Present Invention on Anticoagulant Activity

The water-soluble extract was used as a sample prepared in Example 4.

Methanol extract (or ethyl acetate extract) was added 100 ml of methanol (or ethyl acetate) per 10 g of insects, sonicated for 10 minutes and left for 1 day, and then filtered. The solvent of the filtrate was removed by concentration under reduced pressure, and the dry extract concentration was prepared to 5 mg / ㎖ was used as a sample.

1) Determination of Activated Partial Thromboplastine Time (APTT)

After the APTT reagent was warmed at 37 ° C., the plasma and the sample were mixed at 2: 1, and then incubated with APTT, followed by 0.02M CaCl ₂ .

2) measuring thrombin time (TT)

After 250 μl of fibrinogen was warmed up in advance, the thrombin and the sample were mixed 1: 1 and reacted at 37 ° C., and the coagulation time was measured.

Heparin was used as a comparative material.

The results are shown in Table 4.

Anticoagulant Time of Bee Bee Larva Extract of the Present Invention APTT (seconds) TT (seconds) Water soluble fraction Methanol fraction Ethyl acetate fraction Water soluble fraction Methanol fraction Ethyl acetate fraction Control group (solvent) 69 68 68 50 55 60 Heparin (10 μg / ml) 496 400> Beehive 294 42 61 54 65 80 Bumblebee Soap 77 54 47 65 58 86 Bumblebee larva 99 70 68 61 66 95 Bumblebee worker bee 137 67 62 52 63 165

As shown in Table 4, the coagulation time of the bee larvae extract and king beehive extract of the present invention has a longer coagulation time than the control. Therefore, it can be seen that the degree of thrombosis is high.

Bee larvae and extracts thereof of the present invention exhibit anticancer effects specifically to cancer cells.

In addition, bee larvae and extracts of the present invention exhibit a thrombolytic effect.

Therefore, the composition of the present invention is useful for the prevention and treatment of various cancers, such as breast cancer, bladder cancer, skin cancer, gastric cancer, liver cancer, leukemia and lymphoma.

In addition, the composition of the present invention is useful for the prevention and treatment of various blood clot-related diseases including coronary artery disease and cerebrovascular disease.

Claims (8)

An anticancer composition comprising a wasp, a bee larva or a bee belonging to a bee family, or an extract thereof as an active ingredient. The anticancer composition according to claim 1, wherein the extract is water soluble, C1 to C4 lower alcohol, ethyl acetate or hexane extract. The pharmaceutical composition according to claim 1 or 2, for preventing and treating cervical cancer or stomach cancer. delete A thrombi soluble composition comprising a wasp, a bee larva or a bee larva or an extract thereof as an active ingredient. The composition of claim 5, wherein the extract is water-soluble, C1 to C4 lower alcohol, ethyl acetate or hexane extract. The pharmaceutical composition according to claim 5 or 6, for the prevention and treatment of coronary artery disease or cerebrovascular disease. delete

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