KR101735239B1 - A pharmaceutical composition for preventing or treating cancer comprising Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang - Google Patents
A pharmaceutical composition for preventing or treating cancer comprising Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang Download PDFInfo
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- KR101735239B1 KR101735239B1 KR1020160016499A KR20160016499A KR101735239B1 KR 101735239 B1 KR101735239 B1 KR 101735239B1 KR 1020160016499 A KR1020160016499 A KR 1020160016499A KR 20160016499 A KR20160016499 A KR 20160016499A KR 101735239 B1 KR101735239 B1 KR 101735239B1
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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient, a mixed extract of Angelica gigas Nakai, Ganoderma lucidum, Ganoderma lucidum, Sekisui, Seshin, Prevention or improvement of food compositions.
The mixed extracts or fractions thereof of the present invention do not show toxicity to vascular endothelial cells but inhibit angiogenesis caused by vascular endothelial cells around the tumors, And thus can be usefully used for the prevention or treatment of cancer without any side effects such as toxicity to normal cells other than cancer cells.
Description
The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient, a mixed extract of Angelica gigas Nakai, Ganoderma lucidum, Ganoderma lucidum, Sekisui, Seshin, Prevention or improvement of food compositions.
Every cell in our body is fed oxygen and nutrients from the blood vessels and waste products are removed. Cancer cells also receive nutrients from their blood vessels, and cancer cells grow very rapidly, requiring a very large amount of nutrients, resulting in abnormal and excessive vascular proliferation. After the hypothesis that the inhibition of neovascularization could be a new therapeutic agent for solid tumors as cancer growth and metastasis are dependent on neovascularization (Folkman J., N Engl J Med., 1971 Nov 18; 285 (21): 1182-6), many studies have been conducted to develop a new concept of cancer treatment. That is, since new blood vessels are essential for the growth of cancer cells, the new blood vessel formation process is blocked by using an angiogenesis inhibiting factor, thereby inhibiting the growth of cancer and blocking the route of cancer metastasis.
The innermost part of the blood vessel is composed of endothelial cells, and blood vessels are formed based on the three-dimensional arrangement of these endothelial cells. The angiogenesis in the body is greatly reduced as compared with the fetal period, but the angiogenesis in the adult plays an important role in the progression and treatment of many diseases. In the case of cancer, diabetic retinopathy, psoriasis, rheumatoid arthritis, chronic inflammation, angiogenesis inhibition is required, whereas in the case of myocardial infarction, ulcer, cerebral infarction, and delayed wound healing, an increase in angiogenesis is required.
On the other hand, it is known that Angelica giganti is a mixed herbal medicine extract of Angelica gigas, Ganoderma lucidum, Ganoderma lucidum, Ganoderma lucidum, Sezin, Licorice, Contrast, Sour oil and Ginger and is mainly effective for cold circulation. In this regard, BMC Complement Altern. (Evid Based Complement Alternative Med. 2014; 2014: 549307), a disease in which ischemic attacks of the fingers or toe veins are caused by cold or psychological changes and skin tone changes, .
Angelica gigas is a dried perennial roots of Angelica gigas , a perennial paste that is very sweet and very warm. The efficacy of Angelica gigas has a blood-producing effect when blood is lacking. Angelica promotes blood flow in the coronary arteries and stimulates erythropoiesis.
Cinnamomi cassia Blume belongs to the genus Cambium and is a medicinal substance made from young branches of broiler tree. It has a unique fragrance. Its skin is thicker and its fever is spicy and warm. The stomach opens the pores of the skin in the early cold to sweat, relieves the pain of the shoulder and back, the pain of the limbs, promotes the circulation of the blood, and cures the lack of ovaries.
Paeonia lactiflora Pallas (Paeoniaxeae) belong to the family Ranunculaceae, and are resistant to cold and many fragrant varieties. The roots are used as medicinal materials. It is mainly used for pain, abdominal pain, dysmenorrhea, amenorrhea, blood clots, anemia, bruises.
Akebia I have quinata . polyphylla Nak . ) Refers to a stem that belongs to the genus Vinegranulaceae and which has removed the juniper. It has little odor, flavor, and properties. The throat is used for palsy pain when swelling of the bladder is caused by pyelonephritis, cystitis, urethritis due to wet heat, rash in the mouth due to deepening, heartburn, red urine.
Seasin ( Asarum sieboldii var. Seoulense Nakai ) is a medicinal herb that belongs to the genus Ruptured Vulgaris and dries the roots of bamboo shoots. It has a peculiar smell and slightly paralyzed tongue. Ssein is used for a variety of symptoms such as wet headache, limb paralysis, abdominal pain, headache, chills, fever, telegraph, seawater, asthma, sputum,
Licorice ( Glycyrrhiza uralensis Fischer (Leguminosae)) is a part of legumes and roots, peeled or peeled off, with a characteristic odor and taste. Licorice harmonizes the toxicity of all medicines and makes the medicinal effect appear, regulates the fever and morale of the book, makes good communication of all blood vessels, and strengthens the muscles and bones.
Contrast ( Zizyphus I have jujuba . inermis Rehder ) is a seagull, and ripe fruit is used as a medicinal material. Its characteristic odor is slight, its taste is sweet and its quality is fair. Contrast is used for liver protection, dysentery, abdominal pain, astonishment, chest pounding, hysterical dry cough, dry mouth, etc. and has the effect of reducing the toxicity of medicinal products.
Evodia There is rutaecarpa . bodinieri Huang ) belongs to the genus Udon and uses the fruit as a medicine, poisonous and has a spicy taste. Chronic eczema and dermatitis are caused by powder and attached to the affected area to remove inflammation and promote the regenerative power of new tissue, and to be used as dryness, antiparasitic, detoxification and diuretic.
Ginger (Zingiber officinale Rosc . ) Uses ginger and roots for edible and medicinal purposes. It is lumpy, yellowish and has a spicy and fragrant smell. Ginger treats chills, fever, headache, vomiting, seawater and sputum caused by a cold. It also works against abdominal pain, diarrhea, and complications caused by food poisoning.
As described above, the various pharmacological effects of Angelicae gigantis, Ganoderma lucidum, Ganoderma lucidum, Sukin, Seshin, Lycopene, Control, Fennel, and Ginger are known, but the anticancer effect of the mixed extract of Angelica gigasso There are no studies on this.
Under these circumstances, the present inventors have made intensive efforts to develop a novel anticancer agent, and as a result, they have found that a mixed extract of Angelica gigas, Lycopersicum, Lycopersiconazole, Lycopersicon esculentum, Licorice, Inhibiting angiogenesis and thus inhibiting the growth of cancer cells, thereby completing the present invention.
It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a mixed extract of Angelica gigas Nakai, Ganoderma lucidum, Peony root, Gramineae, Sezin, Licorice, Control, Fennel and Ginger or a fraction thereof.
Another object of the present invention is to provide a composition for inhibiting angiogenesis, which comprises the extract or fraction as an active ingredient.
It is still another object of the present invention to provide a food composition for preventing or ameliorating cancer comprising the extract or fraction as an active ingredient.
As one aspect for solving the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a mixed extract of Angelica gigasum, Angelica keiskei, Peanut, Thyme, Sezin, Licorice, Lt; / RTI >
The term " Angelica gigas " of the present invention means a plant referred to as scientific name " Angelica gigas &"Glue" refers to a plant called scientific name " Cinnamomi cassia Blume & quot ;; The term "peony" means the name " Paeonia lactiflora " Pallas &Quot; Paeoniaxeae " The word " thong " refers to the scientific name ' Akebia quinata var. polyphylla Nak . A plant referred to as'"Seishin" means the scientific name " Asarum sieboldii var. seoulense Plants called Nakai ; "Licorice" is a plant known under the scientific name ' Glycyrrhiza uralensis Fischer (Leguminosae)';"Contrast" refers to the scientific name " Zizyphus " I have jujuba . inermis Plants referred to as ' Rehder ';" Fresh water " refers to the scientific name " Evodia rutaecarpa var. bodinieri Plants referred to as ' Huang '; And "ginger" is a scientific name ' Zingiber officinale Rosc . Quot; and " plant "
In the present invention, commercially available products such as Angelica keiskei koidz., Ganoderma lucidum, Peony root, Thrush, Sezin, Licorice, Control, Fennel, and Ginger may be purchased and used, or collected or cultivated in nature.
The anticancer activity of the mixed extracts of Angelica gigas, Glycyrrhiza uralensis, Glycyrrhiza uralensis, Glycyrrhiza uralensis, Sezin, Licorice, Control, Fennel, and Ginger provided by the present invention is not known at all and has been firstly described by the present inventor. As an example of the above extract, the present inventor used a sweet potato starch syrup.
The term " Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang " of the present invention means a mixture of a mixture of Angelica keiskei koidz., Ganoderma lucidum, The composition of the present invention is a liquid extract obtained by removing a solid component from a mixture of ginseng, guacamole, peony root, thymus, sesquin, licorice, Can be mixed in a weight ratio of from 10: 2 to 4: 1 to 3: 1 to 7: 4 to 6: 1 to 3: 3 to 5 and more specifically from 3: 9: 3 to 9: 3 to 9: 3: 2 : 2 to 6: 5: 2: 4, but the present invention is not limited thereto.
According to an embodiment of the present invention, the Angelica gherkin, the ginger, the peony root, the throat, the sesame ginger, the licorice, the control, the sour oil and the ginger are shredded and shredded at a weight ratio of about 1 to 20 times, Extracts are obtained by the method of hot-water extraction at a temperature of about 50 ° C to 150 ° C, particularly about 90 ° C to 110 ° C, for about 1 hour to 4 hours, particularly for 1 hour to 3 hours, in about 9 to 11 times the volume of water , And the extract was filtered to remove the solid component, thereby obtaining a liquid extract of Angelica gigas Nakuru ginger tea.
In the present invention, the same meaning as "Danggui minjung tang kami room" or "DSGOST" The angiogenesis inhibitory activity and tumorigenic inhibitory activity of the above-mentioned Angelica gigas Streptococcus pneumoniae ginger were not known to date, and were first identified by the present inventors.
The term "extract " of the present invention means an extract obtained by extracting a mixture of Angelica keiskei keitii, Ganoderma lucidum, Peanut, Thyme, Sesame, Licorice, Control, Fennel and Ginger, a diluted solution or concentrate of the extract, , An adjusted product of the above extract or a purified product thereof, or a mixture thereof, and an extract of all the formulations which can be formed using the extract itself.
The method for extracting the mixture of the mixture of Angelica gigas Nakai, Lycopersicum glaze, Peony root, Ginkgo biloba, Sesame ginseng, Licorice, Control, Fennel and Ginger of the present invention is not particularly limited and may be carried out by a method commonly used in the art Can be extracted. Non-limiting examples of the extraction method include hydrothermal extraction, ultrasonic extraction, filtration, and reflux extraction. These may be performed alone or in combination with two or more methods.
The kind of the extraction solvent used for extracting the mixture in the present invention is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water, alcohol, and a mixed solvent thereof. These solvents may be used alone or in combination. When an alcohol is used as a solvent, an alcohol having 1 to 4 carbon atoms can be specifically used.
The term "fraction " of the present invention means a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.
The fractionation method for obtaining the fraction in the present invention is not particularly limited and may be carried out according to a method commonly used in the art. As a non-limiting example of the above-mentioned fractionation method, a predetermined solvent is treated with an extract obtained by extracting a mixture of Angelica gigas Nakai, Ganoderma lucidum, Ganoderma lucidum, Ganoderma lucidum, Sesqui, Licorice, Control, Fennel and Ginger of the present invention to obtain fractions from the extract Method.
The kind of the fraction solvent used for obtaining the fraction in the present invention is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fraction solvent include polar solvents such as water and alcohol; And non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of one or more. When an alcohol is used in the fraction solvent, an alcohol having 1 to 4 carbon atoms can be specifically used.
The extract or fraction of the present invention may be prepared in the form of a dry powder after extraction.
The term "cancer" of the present invention means a tumor that has abnormally grown by autonomous overgrowth of body tissue, or a tumor that forms a tumor.
In the present invention, the cancer is not particularly limited as long as symptoms can be alleviated, alleviated, ameliorated, or treated by the ginseng root of the present invention, for example, gastric cancer, colon cancer, breast cancer, lung cancer, Ovarian cancer, colon cancer, small intestine cancer, rectal cancer, anorectal cancer, fallopian tube carcinoma, endometrial cancer, cervical cancer, vaginal cancer, ovarian cancer, ovarian cancer, ovarian cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, Cancer of the prostate, cancer of the prostate, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer , Renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, or pituitary adenoma, and more specifically pancreatic cancer.
The term "prevention" of the present invention means any action that inhibits or delays cancer by administering a composition comprising the hydrolyzate ginger root of the Danggui ministry.
The term "treatment" of the present invention means all the actions for improving or alleviating the symptoms of cancer by administration of the composition containing the ginseng hot spring of Danggui ministry.
In a specific embodiment of the present invention, the mixture of Angelica gigas Nakai, Gamji, Peony root, Ginkgo biloba, Seshin, Licorice, Control, Fennel and Ginger at a weight ratio of 3: 3: 3: 3: 2: 2: 5: 2: The mixture was subjected to hot extraction with water at 100 ° C for 2 hours, followed by filtration, followed by concentration under reduced pressure and drying, thereby preparing powders of these mixed extracts (Danggui mining, sweet potato and ginger root) (Preparation Example 1).
In another embodiment, in order to confirm the effect of inhibiting angiogenesis in the above-mentioned Danggui Gangwon Ganjang ginger juice, vascular endothelial cells were treated with the above-mentioned Angelica gigassoi ganjanggang to inhibit vascular endothelial cell migration, tube formation and cell infiltration And inhibited the formation of angiogenesis in the ear or the back of the mouse (Figs. 2 to 5 and Figs. 9 to 10). Further, in order to evaluate the antitumor effect of the angiogenesis inhibitory effect of the angelic ginger in Danggui ministry, the growth of cancer tissue was inhibited as a result of administration of Angelica gigas Nakai soup stock to mice having pancreatic cancer (Figs. 12 to 13 ).
In another embodiment, the compositions of Angelica gigas Streptococcus pyogenes are prepared in a ratio of 9: 3: 3: 3: 2: 2: 5: 2: 4 3: 3: 3: 2: 5: 2: 4, 3: 3: 9: 3: 2: 2: 5: 2: 4 or 3: 3: 3: 3: 3: : 3: 3: 2: 2: 5: 2: 4 in weight ratio of 2: 4 to the inhibitory activity of angiogenesis inhibitor (Fig. 17). This suggests that the angiogougi gangbang produced by changing the weight ratio of each constituent can inhibit the formation of new blood vessels and can exhibit anticancer effects in the in vitro and in vivo animal models.
Therefore, the Danggui ministry of the present invention has the effect of inhibiting the growth of cancer by inhibiting the generation of new blood vessels, which is a means for supplying nutrients necessary for growth of cancer, so that it is useful for preventing or treating cancer .
In another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject other than a human, to provide.
The term "administering" of the present invention means introducing the pharmaceutical composition into a subject in an appropriate manner.
The term "individual" of the present invention means all animals such as mice, mice, livestock and the like, including humans who have developed or can develop cancer. By effectively administering the composition of the present invention to an individual, . As a specific example, it may be a mammal including a human.
The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined by the kind and severity of the subject, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the mixed extract may be administered as an active ingredient at a dose of 0.01 to 500 mg / kg per day, specifically 10 to 100 mg / kg, and the administration may be administered once a day or divided into several times have. In addition, the pharmaceutical composition of the present invention may contain 0.001 to 50% by weight of the above-described mixed extract with respect to the total weight of the composition.
The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.
The pharmaceutical composition for preventing or treating cancer of the present invention may contain, in addition to the above-described effective ingredient, a pharmaceutically acceptable carrier, excipient or diluent. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. Specifically, when formulating, it can be prepared by using diluents or excipients such as fillers, weights, binders, humectants, disintegrants, surfactants and the like commonly used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration, liquid paraffin, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be determined depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.
In another aspect of the present invention, the present invention provides a composition for inhibiting angiogenesis, which comprises the mixed extract or fraction as an active ingredient. At this time, The above-mentioned mixed extracts and fractions are as described above.
The term "angiogenesis" of the present invention means a physiological process in which new blood vessels are generated from existing blood vessels. In the present invention, the same as "angiogenesis" And can be mixed and used. In the case where the above-mentioned angiogenesis is excessive, various diseases may occur due to such angiopoiesis. In the case of such excessive angiogenesis, angioproliferative diseases may be induced, and such angioproliferative diseases may be caused by the above- The composition can be prevented or treated.
The vascular proliferative diseases are collectively referred to as diseases in which the production of new blood vessels is accelerated. For example, the vascular proliferative diseases include diabetic nephropathy caused by excessive production of new adipocytes, Obesity disease; Ocular diseases such as keratitis, corneal degeneration, macular degeneration, and diabetic retinopathy, which are caused by the formation of excessive blood vessels in the eye tissue of the cornea and the like; And spinal diseases such as rheumatoid arthritis and spondyloarthropathy which are caused by abnormally generated blood vessels in cartilage tissue. For example, in the present invention, it has been confirmed that the effect on neovascularization in the ear or the back skin of a mouse is inhibited to inhibit neovascularization in the ear skin of a mouse. As a result, It was found that the anti-angiogenesis-induced diseases were also prevented or treated.
In another aspect of the present invention, the present invention provides a food composition for preventing or ameliorating cancer comprising the above-mentioned mixed extract or fraction as an active ingredient. At this time, The definitions of the mixed extract, the fraction and the cancer are as described above.
The term " improvement "of the present invention means any action that at least reduces the degree of symptom associated with a condition to be treated by administration of a composition comprising a mixed extract or fraction of the present invention.
When the food composition of the present invention is used as a food additive, the composition can be added as it is, or can be used together with other food or food ingredients, and can be suitably used according to a conventional method.
Specifically, the food composition may be any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, Lt; / RTI >
It is also possible to use a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, The composition may contain a colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated beverage, and the like, as well as flesh for the production of natural fruit juice and fruit juice drinks and vegetable drinks . These may be used alone or in combination of two or more.
The above food composition may further contain food additives, and whether or not the food additive is suitable as a "food additive" includes, unless otherwise specified, the standard for the relevant item in accordance with the general provisions of the Food Additives Ordinance approved by the Food and Drug Administration, And criteria.
Examples of the products listed in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, coloring matter, licorice extract, crystalline cellulosic, high- , A sodium L-glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent, and the like.
At this time, the content of the mixed extract or the fraction thereof according to the present invention, which is added to the food including the beverage in the course of preparing the food composition, can be appropriately increased or decreased, and specifically 0.01 to 10 wt% %, But the present invention is not limited thereto.
The mixed extracts or fractions thereof of the present invention do not show toxicity to vascular endothelial cells but inhibit angiogenesis caused by vascular endothelial cells around the tumors, And thus can be usefully used for the prevention or treatment of cancer without any side effects such as toxicity to normal cells other than cancer cells.
FIG. 1 is a table showing a list of herbal medicine constituting the sweet potato sweet potato (DSGOST) of the present invention.
FIG. 2 is a graph showing the effect of the DSGOST of the present invention on the vascular endothelial cell HUVEC growth by the vascular endothelial growth factor (VEGF).
FIG. 3 shows the effect of the DSGOST of the present invention on migration of vascular endothelial HUVEC by VEGF, wherein A is an image showing the migration of HUVEC and B is a graph showing the number of migrated HUVEC.
FIG. 4 shows the effect of the DSGOST of the present invention on the vascular endothelial cell HUVEC capacity by VEGF, wherein A is an image showing a tube formed by HUVEC and B is a graph showing the number of tubes formed by HUVEC .
Figure 5 shows the effect of the DSGOST of the present invention on invasion of vascular endothelial cell HUVEC by VEGF, where A is an image showing HUVECs infiltrated with matrigel, B is an image showing infiltration of Matrigel HUVEC < / RTI >
FIG. 6 is an image showing the effect of the DSGOST of the present invention on signaling-related signaling angiogenesis in vascular endothelial cell HUVEC.
FIG. 7 shows the effect of the DSGOST of the present invention on the neovascularization-related NF-κB signaling mechanism in vascular endothelial cell HUVEC, wherein A is a luciferase assay performed to confirm the regulation of NF-κB gene And B is an image showing the degree of phosphorylation of the NF-κB signaling-related protein.
FIG. 8 is a graph showing the effect of DSGOST of the present invention on the expression of tumor angiogenesis-related genes in vascular endothelial cell HUVEC, wherein A shows the results of treatment with
FIG. 9 shows the effect of DSGOST of the present invention on neovascularization in the ear of a mouse, wherein A is an image obtained by staining a new blood vessel using Evans Blue, and B is an image obtained by measuring the amount of the stained Evans blue FIG.
10 shows the effect of the DSGOST of the present invention on the development of neovascularization in the dorsal skin of a mouse wherein A is an image obtained by staining a new blood vessel using evans blue and B is an image of the amount of the dyed Evans blue And the results are shown in FIG.
11 is a graph showing the effect of the DSGOST of the present invention on the viability of pancreatic cancer cells Pacn-28 in human.
Figure 12 shows the effect of the DSGOST of the present invention on the growth of cancer tissues during the experimental period in an animal model of pancreatic cancer. The pancreatic cancer animal model was prepared by injecting the pancreatic cancer cell line Panc-28-luc expressing fluorescence. The image A shows the fluorescence emitted by the pancreatic cancer cell line Panc-28-luc present in the mouse. It is proportional to size and intensity. B is a graph showing the result of measuring the intensity of the fluorescence.
FIG. 13 shows the effect of the DSGOST of the present invention on the size change of cancer tissue during the experiment period in an animal model of pancreas cancer, wherein A is an image showing the cancer tissue isolated from the mouse after the end of the experiment, FIG. 5 is a graph showing a change in size of the cancer measured. FIG.
FIG. 14 is a graph showing the effect of the DSGOST of the present invention on weight change of a mouse during an experimental period in an animal model of pancreatic cancer.
15 is an image showing the result of observing the pathological change of cancer tissue in an animal model of pancreatic cancer.
FIG. 16 is a graph showing the number of neovasculature formed in cancer tissue of an animal model of pancreatic cancer based on anti-CD-31 staining results. FIG.
17 is an image and a graph showing the influence of VEGF on the mobility of vascular endothelial cell HUVEC among the components constituting the DSGOST of the present invention, wherein the extract prepared by varying the mixing ratio of Angelica gigas Nakai, And B is a graph showing the number of migrated HUVECs.
Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
Manufacturing example One. Gangwon-do Produce
As shown in Fig. 1, there are various kinds of foods such as ginseng, gujee, peony, sesame, sesin, licorice, And Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST) were prepared using the ginger.
Specifically, 1 g of root of Angelica gigas Nakai, 1 g of cortex of roots, 1 g of peony root, 1 g of root of root, 0.67 g of sesamin, 0.67 g of licorice, 1.67 g of control, 0.67 g of fruit juice and 1.33 g of ginger root The mixture was pulverized and extracted at 100 DEG C with 10 times water for 2 hours. Thereafter, the extract was filtered, concentrated under reduced pressure, and dried to prepare a powder of a sweet potato starch gruel.
Example 1. For angiogenesis Gangwon-do Impact Analysis
Example 1-1. For the growth of vascular endothelial cells Gangwon-do Impact Analysis
In order to examine the effect of ganghwang ganjang on the growth of vascular endothelial cells, vascular endothelial cells were treated with vascular endothelial growth factor and.
Specifically, HUVEC, a vascular endothelial cell line cultured in 96-well plates, was injected with 50 ng / ml of vascular endothelial growth factor (VEGF) and 50 to 200 μg / The cells were treated with Denggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST) and cultured for 24 hours at 37 ° C and 5% CO 2 . Then, each cell was treated with MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reagent and further cultured for 2 hours. Then, the supernatant was removed, DMSO (dimethyl sulfoxide) was added. The absorbance was finally measured at a wavelength of 590 nm, and the absorbance value was compared with the control group in which no substance was treated.
As a result, as shown in Fig. 2, it was confirmed that the growth of vascular endothelial cells regardless of the concentration of Angelicae gigasuri of the present invention was not affected.
Example 1-2. For the migration of vascular endothelial cells Dangwi ministries Analysis of effect of steel
Scratching assay was carried out to examine the effect of Gangwon Ganjang, an egg plant, on the vascular endothelial cell mobility.
Specifically, HUVEC, a vascular endothelial cell line, was cultured on a 12-well plate, and cells were scraped off to make gaps between the cells. The cells were treated with 50 ng / ml of VEGF and 50 to 200 占 퐂 / ml of DSGOST and cultured for 9.5 hours, and then the number of migrated cells was measured.
As a result, as shown in Fig. 3, it was confirmed that the cell migration of HUVEC, a vascular endothelial cell line, can be remarkably inhibited by the Ganoderma sp.
Example 1-3. For angiogenesis of vascular endothelial cells Gangwon-do Impact Analysis
Tube formation assay was performed to examine the effect of ganghwang (ginseng root) on blood vessel formation of vascular endothelial cells.
Specifically, HUVEC, a vascular endothelial cell line, was cultured in an upper chamber coated with matrigel, treated with 50 ng / ml of VEGF and 50 to 200 μg / ml of DSGOST, and cultured for 6 hours After incubation, the number of tubes formed was measured.
As a result, as shown in Fig. 4, it was confirmed that the tube formation ability of HUVEC, a vascular endothelial cell line, was significantly inhibited by the Ganoderma root of the present invention.
Example 1-4. For invasion of vascular endothelial cells Danggui ministry area Impact Analysis
In order to examine the effect of Ganghwangsang, Gangwagi 's soup, on the infiltration of vascular endothelial cells, invasion essays were performed.
Specifically, HUVEC, a vascular endothelial cell line, was cultured in an upper chamber previously coated with matrigel, followed by treatment with 50ng / ml VEGF and 50-200ng / ml DSGOST for 7 days. Then, the infiltrated cells were stained with a crystal violet, and the number thereof was measured.
As a result, as shown in Fig. 5, it was confirmed that the angiogenic gingerang of the present invention was able to significantly inhibit cellular infiltration of HUVEC, a vascular endothelial cell line.
Example 1-5. The signaling mechanism of angiogenesis in vascular endothelial cells Danggu ministry Analysis of the effect of Kao milk ginger tea
In order to examine the effect of angelic ginger on the signaling mechanism of angiogenesis in vascular endothelial cells, a protein expression assay was performed.
Specifically, HUVEC, a vascular endothelial cell line, was treated with 50 ng / ml of VEGF and 100 占 퐂 / ml of DSGOST and cultured for 30 to 120 minutes. The cultured cells were washed to obtain proteins and electrophoresed. Then, p-VEGFR2 (Y1175), p-VEGFR2 (Y1214), VEGFR2, p-FAK (T397), p- P-ERK1 / 2, ERK1 / 2, p-AKT (S473), AKT, p-JNK, JNK, p-IKK? / ?, IKK ?, p- κB or α-tubulin antibody was used to compare the amount of protein expressed in the cells.
As a result, as shown in Fig. 6, it was confirmed that the angiogenic gingerang of the present invention inhibits the phosphorylation of VEGFR2, FAK, SRC, AKT and NF-κB which are involved in the signal related to angiogenesis in vascular endothelial cells .
Example 1-6. Vascular endothelial cell angiogenesis NF - κB Analysis of the effect of Ganggang tuna on the signal mechanism
In order to examine the effect of Gangwang-dong, Ohwoo Gijang-tang on the vascular endothelial cell neovascularization-related NF-κB signal transduction, we used a reporter gene (luciferase) assay and NF-κB signaling protein expression assays Respectively.
Specifically, for the reporter gene assay, a plasmid in which NF-κB-luc was bound to HUVEC, a vascular endothelial cell line, was transfected, and then 50 ng / ml of VEGF and 100 μg / ml of DSGOST Respectively. After 15 hours, the supernatant was collected and the expression of the NF-κB gene was observed using the Dual-Luciferase Reporter Assay. For the expression of NF-κB signaling protein, HUVEC, a vascular endothelial cell line, was treated with
As a result, as shown in Fig. 7, it was confirmed that the angiogenic gangbang of the present invention inhibits the expression of NF-κB, which is a gene involved in angiogenic endothelial cell angiogenesis, and inhibits the protein involved in the neovascularization signaling pathway Phosphorylation of the protein was inhibited.
Example 1-7. On the expression of angiogenic genes in vascular endothelial cells Angelica Analysis of the effect of Gyu-tang
Protein expression assays were carried out to examine the effect of angelic ginger on the expression of genes (MMP-9, Bcl-2, Cyclin D1 and Cox-2) involved in vascular endothelial cell neovascularization.
Specifically, HUVEC, a vascular endothelial cell line, was treated with 50 ng / ml of VEGF and 100 μg / ml of DSGOST and cultured for 4 to 24 hours. The cultured cells were washed to obtain proteins, and the amounts of proteins expressed in the cells were compared using MMP-9, Bcl-2, Cyclin D1, Cox-2 or α-tubulin antibody.
As a result, as shown in FIG. 8, it was confirmed that the angiogenic ginger tea of the present invention had a marked inhibition of vascular endothelial neovascularization-related MMP-9 protein expression.
Example 1-8. Invivo (in vivo ) On Angiogenesis in Animal Models Danggu ministry Analysis of the effect of Kao milk ginger tea
Evans blue assay was performed to examine the effect of ginseng root of Angelica gigas Nakai on the neovascularization in the ear or back skin of mice.
Specifically, mice were treated with 50 ng / ml of VEGF and 100 μg / ml of DSGOST in skin blood vessels of the mice or the like, and after 30 minutes, Evans Blue was injected. Mice were sacrificed and the ears or back tissues were isolated and photographed, and then Evans blue solution, which was immersed in formamide for 24 hours, and evolved from the ear or back tissue, was measured.
As a result, as shown in Fig. 9, it was confirmed that the angelic ginger root of the present invention inhibits the angiogenesis in the ear skin of the mouse.
In addition, as shown in Fig. 10, it was confirmed that the danggui gangwoo ganjang tang suppressed the formation of ischemic neovascularization in mice.
Example 2. In vitro Gangwon-do Analysis of antitumor activity
In order to examine the effect of ganghwang ganjang on the growth of human pancreatic cancer cells, pancreatic cancer cells were treated with.
Specifically, the pancreatic cancer cell line Panc-28 cultured on a 96-well plate was treated with various concentrations of 50 to 500 占 퐂 / ml of Angelica gigas Nakai soup stock, and then cultured for 24 hours at 37 ° C and 5% CO 2 . Then, each cell was treated with MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reagent and further incubated for 2 hours. Then, the supernatant was removed and 100 μl of DMSO Was added. The absorbance was finally measured at a wavelength of 590 nm, and the absorbance value was compared with the control group in which no substance was treated.
As a result, as shown in Fig. 11, it was confirmed that the growth of the pancreatic cancer cells was not affected regardless of the concentration of the ginseng root of the present invention.
Example 3. Invivo (in vivo In animal models Gangwon-do Analysis of antitumor activity
Example 3-1. About the growth of cancer tissue Gangwon-do Impact Analysis
The following experiment was carried out in order to confirm the anticancer activity in the animal model of pancreatic cancer.
A pancreatic cancer cell line, Panc-28-luc, was injected subcutaneously into a 5-week-old nu / Nu mouse (Oriental Science) at a dose of 1 x 10 6 . The mice were randomly grouped and then administered orally three times a day for 49 days at a concentration of 20 mg / kg of Angelica gigas L. var. For the control group, water was orally administered. Body weight and cancer size were measured three times a week. In order to observe the growth of cancer, the expression level of luciferin was injected once or twice a week, and the size of the cancer was measured by calipers.
As a result, it was confirmed that the present invention has the effect of suppressing the growth of pancreatic cancer during the experiment (Fig. 12), and suppresses the growth of cancer tissue more than the water-fed control (Fig. 13) .
In addition, it was confirmed that the ginseng root of Angelica gigas Nakai in the present invention did not affect the body weight of the mice during the experiment, and it was found that the material was safe without any side effects in vivo (FIG. 14).
Example 3-2. Necrosis and neovascularization-related protein expression in cancer tissues Party Analysis of Influence of Feeding Ginger Tea
In order to confirm whether the anticancer activity of ginseng root of Angelica gigas Nakai was confirmed by inhibiting the formation of angiogenesis, the effect of ginger root of Tanggui mungbean milk on the expression of cancer tissue and neovascularization related protein was analyzed Respectively.
Specifically, after completion of the experiment performed in Example 10-1, the mice were sacrificed to separate cancer tissues, and the cancer tissues were fixed with 4% formaldehyde. The fixed tissues were embedded in paraffin and stained with hematoxylin and eosin (H & E) for histological examination and others were treated with anti-Ki67, anti-CD31, anti-p-VEGFR2 ), Anti-MMP-9, and anti-cleaved caspase-3 antibody. In addition, to confirm inhibition of angiogenesis, the number of blood vessels stained with anti-CD31 was measured.
In the H & E staining results, pink indicates necrosis of cells causing inflammatory reaction. As a result of H & E staining of cancer tissues, it was confirmed that the intensity of staining in pink was decreased in the group administered with Danggui mince, And necrosis of the cells that cause cell death were decreased. In addition, the growth of cancer tissues was analyzed by staining with anti-Ki67 antibody showing brown color. As a result, the growth of cancer was decreased by confirming that the degree of brown staining was decreased in the group administered with Danggui mince, (Fig. 15).
As a result of staining with anti-CD31, anti-pVEGFR2 (Y1175) and anti-MMP-9 antibody showing brown color, it was confirmed that the degree of brown staining was decreased in the group treated with Angelica gigantosa It was confirmed that the expression of the protein was decreased.
On the other hand, the cells were stained with an
Example 3-3. For the generation of neovascularization in cancer tissues Danggui ministries feeding milk ginger Impact Analysis
In order to confirm whether the anticancer activity of ginseng root of Angelica gigas var. Sanguineus, which was confirmed in Example 3-1, was shown by suppressing the formation of angiogenesis, the cancer tissue identified in Example 10-2 was stained with anti-CD- . Specifically, the staining of the anti-CD31 antibody was brown, and blood-stained blood vessels stained with anti-CD-31 were measured by direct visual observation through an optical microscope.
As a result, it was confirmed that the blood vessel count of cancer tissues was decreased in the group administered with Danggui minced eggplant, Ganjanggang, compared with the control group (Fig. 16).
From the above results, it was confirmed that the present invention has excellent effects on the inhibition of cancer cell growth through inhibition of angiogenesis.
Example 4. Gangwon-do Analysis of vascular endothelial cell mobility by mixing ratio
The following experiment was carried out to confirm the optimal mixing ratio of the Angelica gigas var.
3: 3: 3: 2: 2: 5: 2: 4, 3: 3, 3: 3, 3: 3: 2: 6: 5: 2: 4: 3: 2: 4 were added to each other. Then, a scratching assay was carried out by using a method according to the above Example 1-2, using each of the Danggui dairy milking ginger baths having different mixing ratios.
As a result, as shown in Fig. 17, it was confirmed that the Angelica gigas var. Ganjang ganggang, which had different ratio of Angelicae gigantis, Ganoderma lucidum, Peonies or licorice, showed excellent cell migration inhibitory activity as compared with the negative control group not treated with it, Were able to significantly inhibit the migration of HUVEC, a vascular endothelial cell, to a similar extent to extracts containing 3: 3: 3: 3: 3: 2: 2: 5: 2: 4.
3: 3: 2: 2: 5: 2: 4, 4: 3, 3: 2, 3, 4, 3: 3: 3: 2: 5: 2: 4, 3: 3: 9: 3: 2: 2: 5: 2: 4 or 3: 3: 3: 3: 3: 3: 3: 2: 2: 5: 2: 4 in the weight ratio of 2: 4: 2: 4. This suggests that the angiogougi gangbang produced by changing the weight ratio of each constituent can inhibit the formation of new blood vessels and can exhibit anticancer effects in the in vitro and in vivo animal models.
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
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