WO2014168447A1

WO2014168447A1 - Pharmaceutical composition and functional health food for preventing or treating cancer containing natural extract as active ingredient

Info

Publication number: WO2014168447A1
Application number: PCT/KR2014/003138
Authority: WO
Inventors: 마진열; 김애영; 임남희; 임민주; 김태수; 정영필
Original assignee: 한국한의학연구원
Priority date: 2013-04-11
Filing date: 2014-04-11
Publication date: 2014-10-16
Also published as: KR101544470B1; KR20140123444A

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing a mixed medicine extract derived from natural plants as an active ingredient; to a functional health food for preventing or ameliorating cancer; to a method for preventing or treating cancer comprising a step of administering a composition containing a mixed medicine extract derived from natural plants and, more specifically, to a pharmaceutical composition for preventing or treating cancer containing an extract of mixed medicines composed of Cnidium officinale, Ledebouriella seseloides, Korean angelica, Paeonia lactiflora, Forsythia viridissima, Mentha arvensis, Ephedra sinica, Rheum palmatum, gypsum, Platycodon grandiflorum, Scutellaria baicalensis, Atractylodes japonica, Gardenia jasminoides, Schizonepeta tenuifolia, ginger, talc, licorice, Lonicera japonica, Epimedium koreanum, Polygala tenuifolia, and Inula helenium; to a functional health food for preventing or ameliorating cancer; to a method for preventing or treating cancer comprising a step of administering a composition containing a mixed medicine extract derived from natural plants. The mixed medicine extract has activities to inhibit metastasis, induce apoptosis, and inhibit cell growth, which are effective against cancer cells, and thus is pharmaceutically usable as a composition for preventing or treating cancer and is useful as a functional health food.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of cancer comprising natural extract as an active ingredient

The present invention administers a pharmaceutical composition for the prevention or treatment of cancer comprising a mixed medicinal herb extract derived from natural plants as an active ingredient, a health functional food for preventing or improving cancer, and a composition comprising a mixed medicinal herb extract derived from natural plants. It relates to a method for preventing or treating cancer, including the step of, more specifically, cheongung, windproof, Angelica, peony, pontoon, peppermint, ephedra, manganese, rhubarb, gypsum, Giltyeong, golden, baekchul, gardenia, mold, ginger, A pharmaceutical composition for the prevention or treatment of cancer, the health functional food for the prevention or improvement of cancer, and a mixed plant derived from a natural plant, comprising as an active ingredient an extract of a mixed medicine consisting of talc, licorice, gold and silver, yin and yang, raw paper and throat A method for preventing or treating cancer comprising administering a composition comprising an extract.

Cancer has the highest mortality rate worldwide and is the second most common cause of death in Western society after cardiovascular disease. In particular, lung cancer is increasing due to the aging population and the increase of smoking population and air pollution.In general, eating habits are westernized due to westernized eating habits, and rapid increase in environmental pollutants and increase in alcohol consumption. Prostate cancer is on the rise. In this situation, the creation of anti-cancer substances that can contribute to the promotion of human health, the improvement of healthy quality of life and the promotion of human health by enabling early prevention and treatment of cancer are urgently required.

Cancer metastasis refers to the migration of early tumors into blood vessels or lymphatic vessels to other organs with respect to the growth and proliferation of cancer cells (Cavallaro U, Christofori G. Cell adhesion in tumor invasion and metastasis: loss of the glue is not enough Biochem Biophys Acta 2001; 1552: 39-45), metastasis is a secondary process of metastatic cancer cells that proliferate at other sites by invasion through the blood vessels into the surrounding tissues after metastases are released from the site of origin. By a series of processes that form a tumor. This process begins with the breakdown of the extracellular matrix. The enzymes that break down the extracellular matrix include matrix metalloproteinase (MMP), serine protease (plasmin), and urokinase plasminogen activator (urokinase). plasminogen activator, uPA), a cysteine protease (cysteine protease) has (Liotta LA, Trygguason K, Garbisa S, Hart I, Foltz CM, Shafie S. Metastasis potent alcor relate with enzymatic degradation of basement membrane collagen Nature 1980; 284.: 67-68).

Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in inducing and metastasis of cancer cells by decomposing extracellular matrix and basement membrane. Thus, more than 20 species have been isolated and identified. MMP is an endoproteinase that uses zinc as a coenzyme, and is divided into collagenase, gelatinase, stromelysins, and Membrane-type MMP according to structure and characteristics. . In particular, MMP-2 (72 kDa type IV collagenase; gelatinase A) and MMP-9 (92 kDa type IV collagenase; gelatinase B), which are type IV collagenase, are enzymes that degrade type IV collagen, an important component of the basement membrane. , Most commonly associated with cancer migration and metastasis (Nabeshima, K. et al., Pathol. Int. , 52, pp255-64, 2002). In addition, MMP-9 is known to play an important role in the invasion and metastasis of breast cancer (Scorilas A. et al., Br. J. Cancer , 84, pp 1488-96, 2001). In addition, since the promoter region of MMP-9 has a transcription factor AP-1 and NF-κB binding region, cancer-causing stimulants such as cytokines and PMA (phorbol 12-mysistate 13-acetate) may It is known to regulate the expression of MMP-9 by regulating the activation of transcription factors such as NF-κB (Sato H., et al., Oncogene , 19, pp2904-2912, 2000; Lee SO, et al., Biochem Biophys.Res.Commun . , 354, pp165-171, 2007).

On the other hand, a method for treating cancer is to remove cancer tissue from the body at the cellular level. Current cancer treatments are divided into surgical, radiotherapy, and chemotherapy. Surgery is the removal of cancer from the body at the tissue level, which is most rational, but at the cellular level, it is difficult to completely remove the microscopic lesions that invade surrounding tissue or metastasize to the lymph gland. Therefore, surgery is very effective for early cancers or cancers with only a limited number of lesions (cancer stage 1 or 2), but for advanced stage 3 cancers, radiotherapy or chemotherapy is used. It is necessary to use together. Radiation therapy and chemotherapy are mainly used for advanced cancer or terminal cancer after stage 3, but in laryngeal cancer and cervical cancer, even one stage of cancer is cured only by radiotherapy, and radiotherapy is the first priority. In particular, in laryngeal cancer, in order to preserve the function of the vocal cords, the first stage may be radiotherapy only, and the second stage may be combined with partial surgery or chemotherapy. In addition, small cell carcinoma of lung cancer has a poor prognosis even when conventionally performed by surgical therapy, but now chemotherapy is a priority and often use radiation therapy in combination. This therapy has recently been able to increase the survival rate, albeit slightly. As described above, it is difficult to say that cancer treatment is safe except for early cancer, and the above surgical, radiotherapy, and chemotherapy all affect normal cells, causing serious side effects. Therefore, there is a need for an alternative cancer treatment method that is safer and has a high therapeutic effect. Recently, attention has been focused on researching functional foods and medicines using natural resources such as plants and microorganisms, which are guaranteed stability. Indeed, natural products are the main source of new drug development, and as much as 60 percent of the drugs used today are derived from natural products, as aspirin is derived from willow extract.

In particular, research on the regulation of human immune system of natural extracts is being actively conducted, because the abnormalities of the immune system breaks health and causes various diseases. Therefore, the development of immunomodulators for safe natural products that can minimize side effects can be a very important material for food, cosmetics and pharmaceutical development. For example, baekbaekpi, saponin of ginseng, shiitake mushrooms are immune enhancing agents, starfish, ganoderma lucidum mushroom, ginseng derived from ginseng, and ginsenoside derived from anticancer supplements, and licorice, licorice glycyrrhizin, sea cucumber In addition, research is being carried out on cell differentiating substances such as UDCA and ungdam components. The development of immunomodulators using such natural materials has attracted the interest and interest of many people because they can safely treat cancer or chronic inflammatory diseases without side effects.

Meanwhile, Cnidii Rhizoma is a dried perennial herbaceous perennial herb, Cnidium officinale MAKINO, which is taken from September to October to remove heat. It has a pain relief effect that helps blood circulation and alleviates pain, so it is often applied to headaches, and it is known to have a hematopoietic effect when applied to anemia in addition to activating the function of the liver.

Windproof (Ledebouriella seseloides WOLFF.) Is made using the roots and rhizomes of Sapshnikovia divaricata Schiskin. It has a peculiar smell, weakness, spicy, sweet and warm. It is effective for all kinds of symptoms such as external headache, chills, fever, systemic pain, sore throat, etc., and it is used for cold-limb joint pain, tetanus, muscle cramps, paralysis of paralysis, paralytic pain, itching, and ringworm. In addition, as a pharmacological action, antipyretics, anti-inflammatory, jingyeong, immune activation, anti-allergic, anti-ulcer, antibacterial, inhibiting skin improvement bacteria have been reported.

Angelica Gigantis Radix is the root of the umbeliferae plant, Angelicae, which is warm in nature, sweet in taste and very non-poisonous. It is known to be effective in improving blood circulation, blood pressure lowering effect, pain relief, spasm control, constipation improvement through promoting colon circulation, and removing blood blood, which alleviates all wind sickness, blood clots and pain.

Peony (Paeonia lactiflora Pall.) Is a perennial herb and peony perennial herb, distributed in Korea, Mongolia and East Siberia, and used for horticulture because of its beautiful flowers. Root is known to be effective in the treatment of pain, abdominal pain, amenorrhea, dysmenorrhea, hemostasis, anemia, bruises.

It is the fruit of Forsythia Viridissima Lindley or Forsythia suspensa Vahi, which belongs to Oleaceae. It has a peculiar smell, taste and bitterness. Yeonkyo lowers and detoxifies the fever, so the heart heat is lowered early in the fever and used for high fever and mental confusion. Boils, rashes, appendicitis, lung abscesses, lymphadenitis, and sore throat are known to have diuretic and anti-inflammatory effects. In addition, pharmacological action is known to have antibacterial action, anti-inflammatory action, blood pressure lowering, hemostatic action, liver treatment action, fever, clay, diuretic action.

Peppermint leaf is a leaf of Mentha arvensis var. Piperascens, a perennial herb that is a dicotyledon plant moth plant. It is cold, sour, dry, dry, and feverish. Therefore, it is widely used as a treatment for indigestion, chest bloating, bloating (cold), headache, toothache, sore throat, purpose, and sores.

Ephedra sinica is a medicinal herb made from Ephedra sinica Stepf or soft stems of the same plant. It has a tendency to numb the tongue slightly, tastes spicy, bitter, and warm. . Dissipate the wind, sweating, chills, fever, headache, asthma, seawater, species, paralysis of the upper body, skin paralysis, hyperemia, blood is used.

The forget-me-not (Erigeron canadensis) is a biennial plant of the dicotyledon plant Campanula Asteraceae. It is known to be effective in stomatitis, globules, stool irritation, swelling, pain, diarrhea, sebum, detoxification, fever, fatigue, and customs.

Rhubarb is an adverse odor that is made by using the root stems of Rheum palmatum L., Rheum tanguticum Maximowicz, and Rheum officinale Baillon, a perennial herb that belongs to the genus. If you chew it in, it feels like chewing on the fine sand, and it is steamed, bitter, and cold. Headache, congestion, sore throat, constipation, nosebleeds, bleeding, boils and is effective for diuretic, edema. Pharmacological action is known to promote colon movement, fever, body temperature, bile secretion, blood clotting time, antibacterial, diuretic, liver function protection.

Gypsum fibrosum is a very good medicine that lowers heat, tastes sweet and sweet and is very cold. It is also effective for headaches and toothaches caused by severe heat in the body. Recently, it is known that it is used for epidemic type B encephalitis, epidemic meningitis, and pneumonia. In addition, antipyretic, sedative, hypoglycemic, anti-inflammatory, mild diuretic effects have been reported.

Gilkyung is a medicine made by removing the roots or cuticles of Platycodon grandiflorum A. De Candolle, which has a slight smell, tastes bitter, and has a flat nature. It is used for sore throat, cold cough, phlegm, nasal congestion, asthma, bronchitis, pleurisy, headache, chills, tonsillitis, etc.As pharmacological actions include expectorant action, hypoglycemic action, cholesterol lowering action, and inhibitory action. have.

Gold is a medicinal herb made from the root of Scutellaria baicalensis GEORGE, a perennial herb that belongs to the Lamiaceae family.It is effective in pediatric acute respiratory infections, chronic bronchitis, acute dysentery, infectious hepatitis, nephritis, pyelonephritis, and hypertension. Pharmacological actions include anti-inflammatory, antipyretic, diuretic, lowering blood pressure, lowering blood sugar and hyperlipidemia, promoting bile secretion, and sedative effects.

Baekchul is a dried herb that removes rhizome or bark of Atractylodes japonica Koidzumi or Atractylodes macrocephala Koidzumi. It is slightly viscous and chewy and warm when chewed. Intestinal inhibitory and excitatory control, anti-ulcer and hepatic function protection, immune hyperactivity, vasodilation, diuretic and hypoglycemic activity have been reported.

Gardenia jasminoides is the fruit of the gardenia jasminoides, which is known for its effects on diarrhea, rhizome, clear heat, diuresis, bleeding and detoxification.

Schizonepeta rhizome is an annual herb that belongs to the Lamiaceae family. It has antipyretic effect and is used for sweating and antipyretic in the early stage of cold. It is used for sore throat, skin disease, stroke, etc., and it is applied to uterine bleeding, nosebleed, fecal bleeding, hemorrhage, urine bleeding when it is blackened. .

Ginger (Zingiberis Rhizoma) means fresh rhizome of Zingiber officinale Roscoe in Korea, China and Japan. Ginger treats chills, fever, headache, vomiting, seawater, and phlegm caused by an external cold, and it is effective for abdominal diarrhea and abdominal pain caused by food poisoning. Pharmacological action has been reported to promote gastric juice secretion, digestion, cardiac excitement, blood circulation, fungi.

Talc is a drug whose main effect is to stimulate water metabolism. Temper is sweet, light and cold. Talc has been reported to protect the skin mucosa, antibacterial action, diuresis, sedation (stop thirst), fever, detoxification, anti-inflammatory, urination pain, fever of heat stroke, canning, diarrhea.

Licorice (Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra L.) is a perennial herb of the dicotyledonous rosewood legume, with an unusual smell and taste. Licorice harmonizes the toxic effects of all drugs to make the drug appear well, manages the heat and morale of the book, and communicates all blood vessels, and strengthens muscles and bones. Pharmacological action is effective in detoxification, hepatitis, urticaria, dermatitis, eczema, etc. It is known to have antitussive, expectorant, muscle relaxation, diuretic, anti-inflammatory and inhibit peptic ulcer.

Gold and silver coins refer to the buds of the locust (Lonicera japonica Thunberg) or its varieties. It has a peculiar smell, sweet taste and cold nature. Gold and silver coins are used to reduce heat, when the chest is stuffy and thirsty. It is used for inflammation, and it is used to release boils, skin toxins, organ inflammation, and pus. In addition, it is used for necrosis of the skin tissue due to dysentery and heat poisoning, mastitis, etc., and has been applied to colitis, gastric ulcer, cystitis, sore throat, tonsillitis, bronchitis, conjunctivitis and swelling, mumps and musculitis. Pharmacological action, antibacterial action, anti-inflammatory action, antipyretic action, leukocyte phagocytosis increase, central nervous system excitability, serum cholesterol lowering, ulcer prevention effect has been reported.

Eum yang-gyeok means the perennial herbaceous plant (Epimedium koreanum Nakai) or other perennial roots (stems and leaves) of dicotyledonous plant (Prunus paniculata). It is odorless, tastes sweet and warm. Yin and Yang are used for erectile dysfunction, oil well, cold uterus, cold limb, skin paralysis, ophthalmology, forgetfulness, paraplegia, low back and knee weakness, hypertension, polio. Pharmacological actions include semen secretion promotion, blood pressure strengthening, coronary blood flow increase, hypoglycemia, cholesterol lowering, immune function enhancement, Jinhae, expectoration, Pyeongcheon, sedation, fungi, anti-inflammatory action, chicken femoral growth and protein polysaccharide activation Is reported.

Polygala tenuifolia is a perennial herb of the dicotyledonous mouse handicap grass family. It is used as an expectorant, tonic, or pill.

The elecampane refers to the root of the perennial herbaceous Aucklandia lappa Decne., Which has a peculiar smell, the taste is spicy, bitter and warm. Mokyang is used for abdominal pain caused by the abdomen, flat stomach symptoms, vomiting and diarrhea. It is effective for chronic inflammation of the digestive organs and stomach pain. Pharmacological action is to release bronchial and small intestine spasms, blood pressure lowering action, antibacterial action is known.

The inventors of the present invention have been studying safer and more effective cancer treatment methods that can replace conventional anticancer therapies that affect normal cells and cause serious side effects. HT1080 is a classic cancer cell line extract consisting of Peony, Peony, Mint Leaf, Ephedra, Forget-me-not, Rhubarb, Gypsum, Giltyeong, Golden, Whitening, Gardenia, Hungae, Ginger, Talc, Licorice, Gold Silver Coin, Yin Yang Kwak, Wonji and Mokji. Treatment with human fibroscarcoma (human fibrosarcoma) and B16F10 (murine melanoma; mouse melanoma) cell lines effectively inhibits the migration and invasion of cancer cells, as well as various cancer cell lines (HT1080 (human fibroscarcoma; human fibrosarcoma), Effective cell death after treatment with AGS (human gastric carcinoma), A431 (human epidermoid carcinoma) and B16F10 (murine melanoma; mouse melanoma) The present invention was completed by confirming that induction and inhibit tumor growth.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, which is safe for the human body and contains a mixed medicinal herb extract having an effective anticancer effect as an active ingredient.

Another object of the present invention to provide a health functional food for the prevention or improvement of cancer comprising the mixed medicinal herb extract as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating cancer, comprising administering to a subject in need thereof a composition comprising the mixed medicinal extract.

The present invention relates to a natural extract effective for the prevention or treatment of cancer, and has an effective metastasis inhibition, induction of cell death and cell growth inhibitory activity against cancer cells, as well as pharmaceutically usable as a composition for the prevention or treatment of cancer It can also be usefully used as a dietary supplement.

Figure 1 shows the results of analyzing the cell death induction effect of the mixed medicinal herb extract (KIOM-C) against various classical cancer cell lines (B16F10, HT1080, AGS and A431).

Figure 2 shows the results of analyzing the effect on the HT1080 cell cycle progression according to the treatment of the mixed medicinal extract (KIOM-C).

Figure 3 shows the results of analyzing the expression of the cell cycle-related protein changes according to the treatment of the mixed medicinal extract (KIOM-C).

4A to 4E show the results of analysis of apoptosis and autophagy induction according to the treatment of the mixed medicinal extract (KIOM-C).

5a to 5e show the results of analyzing the cell death induction mechanism by the treatment of the mixed medicinal extract (KIOM-C) by Western blot.

6a to 6c show the results of analyzing the effect of tumor cell death caused by the induction of intracellular oxidative stress following the treatment of the mixed medicinal herb extract (KIOM-C).

7a to 7e show the results of analyzing the effect on JNK activation according to the treatment of the mixed medicinal extract (KIOM-C).

Figure 8 shows the non-adherent colony forming ability of cancer cells (B16F10 (A) and HT1080 (B)) according to the treatment of the mixed medicinal extract (KIOM-C).

9A and 9B show wound healing assay results according to the treatment of the mixed medicinal herb extract (KIOM-C).

10A and 10B show the results of a transwell migration and invaation assay according to the treatment of the mixed medicinal herb extract (KIOM-C).

Figure 11 shows the effect of mixed medicinal herb extract (KIOM-C) on MMP-9 activity according to the treatment of mixed medicinal herb extract (KIOM-C) RT-PCR (A), Western blot (B and D) and gelatin The results are analyzed by Gelatin zymography (B and C).

Figure 12 shows the results of Western blot analysis of the effect of the mixed medicinal extract (KIOM-C) on NF-κB activation by PMA stimulation.

13A and 13B, the mixed medicinal herb extract (KIOM-C) was administered to mice injected with B16F10 lung cancer cell line, and the degree of lung metastasis was analyzed by visual observation analysis (FIG. 13A) and the number of colonies (FIG. 13B). One result is shown.

Figures 14a to 14d, after the administration of the mixed medicinal extract (KIOM-C) to the mice inoculated with HT1080, a classic cancer cell line, the tumor volume change for about 15 days (Fig. 14a) and tumors extracted 15 days later The results of the mass observation (FIG. 14b), tumor mass change (FIG. 14c) and interferon-gamma (IFN-γ) secretion concentration (FIG. 14d) are shown.

Fig. 15 shows the results of comparative analysis of cancer cell viability (cancer apoptosis effect) of the mixed medicinal herb extracts (KIOM-C) and the extracts of each sweetener component.

FIG. 16 shows the results of analysis of gelatin zymography on the change of MMP-9 activity caused by rhubarb extract and ephedra extract in the sweetening ingredients.

As one embodiment for achieving the above object, the present invention is a cheongung, windproof, donkey, peony, yeonkyo, peppermint, ephedra, forget-me-not, rhubarb, gypsum, gilyeong, golden, white leach, gardenia, mold, ginger, talc, licorice It provides a pharmaceutical composition for the prevention or treatment of cancer, including the extract of the mixed medicinal herb, gold and silver, yin and yang, paper and neck.

The composition is based on 1 part by weight of yin and yang, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight windproof, 1 part by weight to 3 parts by weight, peony 1 part by weight to 3 parts by weight, 1 part by weight of duct bridge To 3 parts by weight, peppermint 1 to 3 parts by weight, ephedra 1 to 3 parts by weight, 1 to 3 parts by weight of forget-me-not, 1 to 3 parts by weight of rhubarb, 0.5 to 3 parts by weight of gypsum, 0.5 to 3 parts by weight, 0.5 to 3 parts by weight of gold, 0.5 to 2 parts by weight, 0.5 to 2 parts by weight, 0.5 to 2 parts by weight of gardenia, 0.5 to 2 parts by weight, 1 part by weight of ginger 3 parts by weight, talc 1 part by weight to 5 parts by weight, licorice 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight of gold coins, 1 part by weight to 3 parts by weight, and 1 part by weight to 3 parts by weight of wood It is preferable to prepare and then extract the herbal mixture.

The term "extract" of the present invention refers to a formulation prepared by squeezing the herbal medicine with a suitable leach solution and evaporating the leach solution, but not limited thereto, but the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the extract It may be a dried product obtained by drying, these adjustment products, or a refined product. The mixed medicinal extract can be prepared using common extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably, hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used.

In the present invention, the extract may be prepared by extracting with an extracting solvent or by fractionating the fractional solvent to an extract prepared by extracting with an extracting solvent. The extraction solvent is not limited thereto, but water, an organic solvent or a mixed solvent thereof may be used, and the organic solvent may be a C 1-4 alcohol, a polar solvent such as ethyl acetate or acetone, hexane or dichloro Non-polar solvents of methane or mixed solvents thereof can be used. In addition, preferably water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof may be used, and more preferably ethanol may be used. In one embodiment of the present invention, an extract using ethanol was prepared as the solvent.

The extract of the mixed medicinal herb of the present invention is the above-mentioned ratios of Cheongung, Windproof, Angelica, Peony, Yeongyo, Peppermint, Ephedra, Forget-me-not, Rhubarb, Gypsum, Giltyeong, Golden, White, Red Gardenia, Penguin, Ginger, Active, Licorice, Gold and Silver Coin , The mixed medicine consisting of raw paper and wood was obtained by adding 2-10 times the amount of water and hot water extraction for 3 to 4 hours at a temperature of 70 ℃ to 125 ℃. The mixed medicinal herb extract was concentrated and lyophilized, and the lyophilized sample was dissolved in distilled water and used for the experiment.

As used herein, the term "cancer" refers to a lump grown abnormally by autonomous overgrowth of body tissue, and may be classified into benign tumor and malignant tumor. While benign tumors grow relatively slowly and do not metastasize, malignant tumors grow rapidly as they infiltrate surrounding tissues and spread or spread to various parts of the body, thereby threatening life. Malignant tumors can therefore be thought of as cancer.

The subject cancer which can be prevented or treated by the composition of the present invention is not limited to a particular cancer, preferably breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer , Skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer It may be prostate cancer, bronchial cancer or bone marrow cancer. More preferably, the cancer may be fibrosarcoma, skin cancer, lung cancer, breast cancer, gastric cancer, but is not limited thereto.

According to an embodiment of the present invention, a total of four cell lines of HT1080 (human fibroscarcoma), B16F10 (murine melanoma), A431 (human epidermoid carcinoma) and AGS (human gastric carcinoma) were used, and the mixed medicinal extract of the present invention was used. Fibrosarcoma through HT1080 cell death and growth inhibition, skin cancer through B16F10 cell death and growth inhibition, lung and breast cancer through A431 cell death and growth inhibition, gastric cancer through AGS cell death and growth inhibition or Proven that it can be treated.

For the purposes of the present invention, the cancer is a disease that is the object of prevention or treatment by the mixed medicinal herb extract of the present invention, the composition comprising the mixed medicinal herb extract to inhibit the metastasis of cancer cells, induce cell death or inhibit cell growth It can show anticancer effect by showing activity.

According to one embodiment of the present invention, the mixed medicinal herb extracts were treated in the classical ear cancer cell line and the effects on cell metastasis (cell migration and cell invasion activity), cell death and cell growth through in vitro and in vivo animal experiments. Confirmed.

First, as a result of treatment of mixed medicinal herb extracts on normal cells and various cancer cell lines in an in vitro study to see the efficacy of inhibiting the metastasis of the mixed medicinal herb extracts, there was no toxicity to normal cells, and the cell death ability of the classical medicinal cancers was significant. It was confirmed that it is suppressed (Fig. 1). In addition, analysis of the effects on the cell cycle progression and the expression of cell cycle-related proteins according to the treatment of the mixed medicinal herb extract, induces G1 cell cycle arrest, delay cell proliferation, effectively inhibit abnormal cell proliferation and cell death It was confirmed that the induced (Fig. 2 and 3). And said Analysis of factors related to apoptosis and autophagy by mixed medicinal herb extracts confirmed that the mixed medicinal herb extracts had a close relationship with related factors, demonstrating induction of cell death and autophagy. (FIGS. 4A-5E). In addition, it was confirmed that due to the intracellular oxidative stress induced by the treatment of the mixed medicinal extract, it shows a tumor cell killing effect (Figs. 6a to 6c). In addition, the mixed medicinal herb extract was confirmed to induce cell death by affecting the activation of JNK in the cell signaling system pathway (FIGS. 7A to 7E). In addition, non-adherent colony forming ability of cancer cells was inhibited by the mixed medicinal extract (FIG. 8), and cell migration and cell invasion were inhibited (FIGS. 9A to 10B). In addition, it was confirmed that the expression levels of factors related to cancer metastasis such as MMP-9 and NF-κB according to the treatment of the mixed medicinal extracts are closely related to each other (FIGS. 11 and 12).

Similarly, in vivo experimental results, it was confirmed that the intravenous injection of the mixed medicinal herb extract significantly reduced the lung metastatic potential of cancer cell lines (FIGS. 13A and 13B). When high concentrations of mixed medicinal extracts were treated with various cancer cell lines, cancer cell viability was significantly reduced, and when the mixed medicinal extracts were orally administered to nude mice, it was confirmed that the growth of cancer cell lines was significantly suppressed (FIGS. 14A to FIG. 14d).

Additionally, in order to compare and analyze the effects of the mixed medicinal herb extracts (KIOM-C) and the extracts of each of the sweeteners constituting the same, the cancer cell killing effect and metastasis after treatment of each extract to HT1080, respectively. As a result of confirming the inhibitory effect, it was confirmed that the anti-cancer effect such as cell death or metastasis inhibition did not show when the individual extracts of sweeteners were treated at low concentrations equal to the concentration ratios of the sweeteners contained in the mixed medicinal extracts (KIOM-C). 15 and 16. Through this, the mixed medicinal herb extract of the present invention was confirmed to have a synergistic effect of the mixing of the 22 medicinal herbs.

The term "prevention" as used in the present invention means any action that inhibits or delays the disease by administration of the composition containing the mixed drug extract. In addition, the term "treatment" used in the present invention means all the actions that improve or cure the symptoms of the disease by administration of the composition containing the mixed drug extract.

The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredient for administration. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Specifically, when formulated, it may be prepared by using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants which are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like, in the mixed medicinal extract. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, uthepsol, macrosol, tween 61, cacao butter, laurin butter, glycero gelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the condition and weight of the patient, the extent of the disease, Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art. The daily dosage of the mixed medicinal extract is preferably 1 mg / kg to 600 mg / kg, may be administered once to several times daily if necessary.

In another aspect, the present invention provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a composition comprising a mixed medicinal extract.

The cancer-related diseases may be various cancer diseases such as skin cancer, stomach cancer, lung cancer, but are not limited thereto.

As used herein, the term "individual" means all animals, including humans, who have already developed or may develop cancer, and the composition of the present invention is effective in effectively preventing and treating the disease by administering to the individual. have.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment in a medical treatment, the effective dose level being the type of subject and its severity, age , Sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

As another aspect, the present invention is a cheongungung, windproof, donkey, peony, yeonkyo, peppermint, ephedra, forget-me-not, rhubarb, gypsum, Gilgyeong, golden, baekchul, gardenia, hyungge, ginger, talc, licorice, gilding, yin and yang, It provides a health functional food for the prevention or improvement of cancer containing the extract of the mixed medicinal herb consisting of raw paper and throat.

The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. It may also contain natural fruit juices and pulp for the production of fruit juices and vegetable drinks. These components can be used independently or in combination. The dietary supplement may be any one of meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex. Can be.

In addition, the health functional food may further include food additives, and the suitability as a "food additive" is applied to the item according to the General Regulations of the Food Additives Code and General Test Law, etc., unless otherwise specified. Determined by the relevant standards and standards.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, cinnamon acid, natural additives such as color pigments, licorice extract, crystalline cellulose, high-quench pigments, guar gum, L Mixed preparations, such as a sodium glutamate preparation, a noodles addition alkali preparation, a preservative preparation, and a tar pigment preparation, etc. are mentioned.

At this time, the mixed medicinal extract according to the present invention added to the food in the process of manufacturing a health functional food can be appropriately added or subtracted as needed, preferably 1 to 15% by weight in 100% by weight food It is preferable to add as much as possible.

Through the embodiments will be described in more detail the configuration and effects of the present invention. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

실시예 1: 혼합 약재 추출물의 제조Example 1: Preparation of Mixed Medicinal Extracts

KIOM, a mixed herb consisting of Cheongung, Windproof, Angelica, Peony, Fellowship, Peppermint, Ephedra, Forget-me-not, Rhubarb, Gypsum, Giltyeong, Golden, White, Gardenia, Hungae, Ginger, Talc, Licorice, Gold and Silver Coins, Yin and Yang In order to manufacture -C, the herbal medicines were purchased from the Yeongcheon Herbal Medicine Market (Youngcheon, South Korea), and large-scale hot water extraction was performed.

KIOM-C is a mixed medicine, 1 part by weight of Cheongung, windproof, Angelica, Peony, Peony, Peppermint Leaf, ephedra, forget-me-not and rhubarb, based on 1 part by weight of yin and yang, and 0.5 parts by weight of gypsum, gilyeong and gold 1 part to 2 parts by weight, 1 part to 3 parts by weight, 1 part to 3 parts by weight, 1 part to 5 parts by weight of talc, 1 part to 5 parts by weight of licorice, gold and silver, and 1 part to 3 parts by weight of raw wood, 1 part by weight It is made by mixing the medicines in a ratio of 3 to 3 parts by weight, and 15 L of distilled water is added to 2456.5 g of KIOM-C mixed medicines, and the extractor (Cosmos-600 extractor, Gyeongseo Machinery Industry, Incheon, South Korea) is 115 ℃. Hot water was extracted for 3 hours to the temperature.

The extracted KIOM-C blended medicinal herb extract was filtered using a standard test sieve (sieve) (150 μm, Retsch, Han, Germany) and concentrated to dryness in a freeze dryer. The lyophilized KIOM-C mixed medicinal herb extract powder (50 mg) was dissolved in 1 ml of distilled water, filtered through a 0.22 μm disk filter, and stored at −20 ° C. until use.

비교예 1 내지 22: 각 단미재 추출물의 제조Comparative Examples 1 to 22: preparation of each sweetener extract

For comparative experiments on the anticancer effect of the mixed medicinal herb extract (KIOM-C) and the extract of each of the sweet rice extract prepared in the above example, the cheongung of each sweet rice constituting the mixed medicinal extract (KIOM-C) (Comparative Example 1) Windproof (Comparative Example 2), Angelica (Comparative Example 3), Peony (Comparative Example 4), Fellowship (Comparative Example 5), Park Ha-Yeop (Comparative Example 6), Ephedra (Comparative Example 7), Manganese (Comparative Example 8), Rhubarb (Comparative Example 9), Gypsum (Comparative Example 10), Gil-kyung (Comparative Example 11), Golden (Comparative Example 12), Baekchul (Comparative Example 13), Sanchija (Comparative Example 14), Hungae (Comparative Example 15), Ginger (Comparative Example 16), Talc (Comparative Example 17), Licorice (Comparative Example 18), Geumeunhwa (Comparative Example 19), Yin Yang Guk (Comparative Example 20), Paper (Comparative Example 21) and Mokhyang (Comparative Example 22) It was.

1 L of distilled water was added to 50 g of each sweetener, and hydrothermal extraction was performed for 3 hours at a temperature of 115 ° C. in an extractor (Cosmos-600 extractor, Gyeongseo Machinery Industry, Incheon, South Korea).

The hot water extracted sweetwood extract was filtered using a test sieve (150 μm, Retsch, Han, Germany), and concentrated to dryness in a freeze dryer. The freeze-dried sweetwood extract powder (50 mg) was dissolved in 1 ml of distilled water, filtered through a 0.45 μm syringe filter, and stored at 4 ° C. until use.

실시예 2: 혼합 약재 추출물(KIOM-C)의 암세포 증식 저해 활성 평가 및 작용기작 분석Example 2: Evaluation of cancer cell proliferation inhibitory activity and analysis of mechanism of action of mixed medicinal herb extract (KIOM-C)

A total of four cell lines of different origins and characteristics, HT1080 (human fibroscarcoma; human fibrosarcoma), AGS (human gastric carcinoma), A431 (human epidermoid carcinoma), and B16F10 (murine melanoma; Mouse melanoma) was used to evaluate the cancer cell proliferation inhibitory activity and the mechanism of action of the mixed medicinal extract (KIOM-C).

실시예 2-1: 암 세포주 배양Example 2-1: Cancer Cell Line Culture

HT1080 (human fibroscarcoma; human fibrosarcoma), AGS (human gastric carcinoma), A431 (human epidermoid carcinoma; human squamous cell carcinoma), and B16F10 (murine melanoma; mouse melanoma) cells are the American Type Culture Collection ) Was used. The various cancer cell lines were prepared using DMEM medium (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 ug / ml streptomycin. Incubation was carried out in an incubator where temperature (37 ° C.) and humidity were kept constant and supplied with 5% CO ₂ .

실시예 2-2: 암세포 증식 저해 활성 및 세포 독성 평가 Example 2-2 Evaluation of Cancer Cell Proliferation Inhibitory Activity and Cytotoxicity

In order to evaluate the cancer cell proliferation inhibitory activity of the mixed medicinal herb extract (KIOM-C) against various cancer cell lines, MTT assay was performed. At this time, using the normal hepatocyte (hepatocyte) as a control, the cytotoxicity of the mixed medicinal herb extract (KIOM-C) of the Example to the normal cells was evaluated. In addition, using an inverted microscope, the change in cell morphology of the cancer cell line by the mixed medicinal extract (KIOM-C) was also observed.

Specifically, in order to determine the effect on the cell viability of the mixed medicinal extract (KIOM-C) for various cancer cell lines, cultured as in Example 2-1, each cancer cell line (5 × 10 ³ cells / Well / 96-well plate) was incubated after treatment with the mixed medicinal herb extract (KIOM-C) at a concentration of 100 μg / ml to 1000 μg / ml prepared in Example 1. After 48 hours of treatment, 10 μl of MTT solution (5 mg / ml in PBS) was added to the cells and further incubated for 4 hours. Next, the formazan precipitate was dissolved in dimethyl sulfoxide (DMSO), and then absorbance was measured at 570 nm using an Infinite® M200 microplate reader (TECAN Group Ltd, Switzerland) to confirm cell viability. It was.

As a result of the experiment, when compared to the untreated control cells, the cytotoxicity was noticeably observed when the four kinds of cancer cell lines were treated with the mixed medicinal extract (KIOM-C) at a concentration of 500 μg / ml to 1000 μg / ml. It became. Concentration IC ₅₀ values representing 50% cell viability were derived at 720.34 μg / ml (B16F10 cell line), 408.22 μg / ml (HT1080 cell line), 406.59 μg / ml (AGS cell line) and 607.86 μg / ml (A431 cell line), respectively. In contrast, in the case of normal cells, hepatic cells did not show any cytotoxicity even when the mixed medicinal extract (KIOM-C) was treated at a concentration of 1000 μg / ml (FIG. 1A). This means that the mixed medicinal extract (KIOM-C) exhibits a concentration-dependent cancer cell proliferation inhibitory effect specifically for all cancer cell lines.

In addition, as a result of microscopic observation of the change in the cell morphology of the cancer cell line by the mixed medicinal extract (KIOM-C), the number of cells attached to the dish decreased in a concentration-dependent manner, the shape of the cell was rounded, did not adhere to the cytoplasm It was confirmed that induction of autophagic and apoptosis of cancer cells such as many vacuoles were observed (FIG. 1B).

Therefore, the mixed medicinal extract (KIOM-C) does not show cytotoxicity to normal hepatocytes, and has the effect of inhibiting concentration-dependent cancer cell proliferation and autophagic and apoptotic cancer cells in all cancer cell lines except normal hepatocytes. It means to have an effect.

실시예 2-3: 세포 주기 분석Example 2-3 Cell Cycle Analysis

In order to determine how cancer cell proliferation inhibitory effect of the mixed medicinal extract (KIOM-C) is related to cell cycle progression control, the following experiment was performed.

HT1080 cells were seeded to 5 × 10 ⁵ per 60 mm dish and then incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. After incubation, the mixed medicinal extract (KIOM-C) prepared in Example 1 was treated at a concentration of 1000 ㎍ / ㎖, and further cultured for 12 hours and 24 hours. After further incubation, the cells that were not attached to the dish and attached cells were collected and washed twice with PBS, and the cells were fixed by the slow addition of 70% ethanol. Fixed cells were stored at −20 ° C. for 24 hours. To remove ethanol, after centrifugation, cells were washed twice with PBS and 50 μg / ml propidium iodide containing RNase A (0.05 mg / ml), 0.1% Triton X-100 and 0.1 mM EDTA ( propidium iodide (PI) solution was added thereto, followed by shading staining at 4 ° C. for 30 minutes. After staining, the PI solution was removed, the stained cells were suspended in PBS, transferred to a polystyrene round-bottom tube, and then FACS caliber flow cytometer (BD Biosciences, San) using CellQuest software. Jose, CA) was used to analyze the cell cycle. Intracellular DNA changes were calculated using WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, Calif.) For each cell cycle.

The experimental results, as compared with the untreated control, when the ratio of the cells distributed in the G ₁ phase by mixing medicinal extract (KIOM-C) was increased by 57.14% (12 hours) and 55.53% (24 hours), the G ₁ phase Increasing the proportion of cells distributed led to a decrease in the proportion of cells distributed in the S and G ₂ / M phases. Apoptotic sub-G ₁ / G ₀ peaks significantly increased to 7.92% (12 hours) and 13.96% (24 hours) when treated with mixed medicinal herb extracts (KIOM-C) compared to the untreated control. (FIG. 2).

Therefore, the mixed medicinal herb extract (KIOM-C) of the present invention induces G ₁ cell cycle arrest (cell cycle arrest) to delay cell proliferation, which means that effectively induced cell death.

실시예 2-4: 세포 주기 관련 단백질 발현 분석Example 2-4 Cell Cycle-Related Protein Expression Assays

In order to determine how the mixed medicinal extract (KIOM-C) prepared in Example 1 affects the expression of the G ₁ phase regulatory proteins p21, p27 and cyclin D1, Western blot was performed.

Specifically, HT1080 cells were seeded to 5 × 10 ⁵ cells per 60 mm dish, and then cultured in 37 ° C., 5% CO ₂ incubator for 24 hours. After incubation, the mixed medicinal herb extract (KIOM-C) prepared in Example 1 was treated at a concentration of 500 µg / ml and 1000 µg / ml, and further cultured for 12 hours and 24 hours. After further incubation, the cells that were not attached to the dish and attached cells were collected and washed twice with PBS, and the whole cell lysate of the cells was washed with M-PER Mammalian Protein Extraction. Reagent) (Thermo Scientific, Rockford, Ill.). Cell lysates were quantified for protein concentration by BCA protein assay (Bicinchoninic Acid) assay, and the same amount of protein was obtained from 10% sodium dodecyl sulfate polyacrylamide gel (SDS). Each well was injected and electrophoresed. Proteins developed on gels were transferred to Immunobilon PVDF transfer membranes (Millipore, Bedford, Mass.). After transferring the expression protein to the membrane, immunoblotting was performed using anti- ^{p21 Waf1 / Cip1} , anti-p27 ^Kip1 and anti-cyclin D1. Later, the proteins were prepared using Power Opti-ECL Western blotting detection reagent (Animal Genetics, Inc., Korea) and ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA). Visualization was performed using. The expression levels of p21, p27 and cyclin D1, which are the G ₁ phase regulatory proteins, were derived by quantifying the visualized protein bands using software ImageJ (National Institutes of Health, USA).

As a result, compared with the untreated control group, in the group treated with the mixed medicinal extract (KIOM-C), the expression level of p21 and p27, cyclin-dependent kinase inhibitors, was upregulated, whereas cyclin It was confirmed that the expression level of D1 is down regulated (FIG. 3).

Therefore, the mixed medicinal herb extract (KIOM-C) of the present invention regulates cyclin-dependent kinase inhibitors such as cell cycle regulators p21 and p27, thereby inhibiting abnormal cell proliferation and cell death It can be derived.

실시예 2-5: 세포 사멸(apoptosis) 및 자가포식(autophagy) 작용 유도 분석Example 2-5: Induction Assay of Apoptosis and Autophagy Action

To confirm whether the mixed medicinal extract (KIOM-C) induces programmed cell death (PCD), the following experiment was performed.

실시예 2-5-1: YO-PRO-1의 흡수 탐지Example 2-5-1: Absorption Detection of YO-PRO-1

The HT1080 cell line was treated with the mixed medicinal extract (KIOM-C) prepared in Example 1 at concentrations of 500 μg / ml and 1000 μg / ml and incubated for 12 hours and 24 hours. At this time, YO-PRO-1 (1 uM, Molecular Probes, Eugene, OR), which is an apoptosis-specific dye, was added thereto, followed by shading staining at 4 ° C. for 30 minutes. After staining, YO-PRO-1 uptake was measured using a FACSSCalibur flow cytometer without cell washing or fixation, and analyzed using WinMDI 2.8 software.

As a result of the experiment, when the mixed medicinal extract (KIOM-C) was treated for 24 hours at 500 μg / ml and 1000 μg / ml, the cell death was 5.9% and 8.9%, respectively. In addition, when the mixed medicinal extract (KIOM-C) was treated for 48 hours at a concentration of 1000 ㎍ / ㎖, compared to the untreated control (3.6%), it was confirmed that the number of dead cells increased 7.5 times (26.95%). (FIG. 4A).

실시예 2-5-2: TUNEL assayExample 2-5-2: TUNEL assay

In order to evaluate the apoptosis-inducing activity of the mixed medicinal herb extract (KIOM-C) prepared in Example 1, TUNEL staining was performed using an in situ cell death detection kit (Roche diagnostics GmbH, Mannheim, Germany). .

Specifically, 1 × 10 ⁴ HT1080 cell lines suspended in 200 μl of PBS were cytospin and attached to a microscope slide, followed by fixation at 4 ° C. in formaldehyde at 20 ° C. for 1 hour. After fixing for 1 hour, blocking was blocked for 10 minutes at 20 ° C. with 3% hydrogen peroxide (H ₂ O ₂ ), and the solution was allowed to stand for 2 minutes by adding 0.1% TritonX-100, a permeabilization solution, in ice. . Next, TUNEL reaction mixture, which is a labeling solution containing terminal transferase and fluorescein-dUTP, was added to the cells at 37 ° C. for 1 hour. Shading staining. 2 × SSC (0.3 M NaCl, 30 mM sodium citrate) was added to stop the dyeing reaction, and a loading medium containing DAPI (Vector Laboratories, Burlingame, Calif.), Which stained the nucleus, was added, followed by a coverslip. It was sealed with a coverslip and collected under a microscope using a fluorescence microscope (Olympus TH4-200; Olympus Optical Co. LTD.).

As a result of the experiment, 15.53% and 57.33% of TUNEL positive cells were observed when the mixed medicinal extract (KIOM-C) was treated at concentrations of 500 μg / ml and 1000 μg / ml, respectively (FIG. 4B). ). Accordingly, depending on the concentration of the mixed medicinal extract (KIOM-C), by confirming that the TUNEL positive cells significantly increased, it was found that the mixed medicinal extract (KIOM-C) is closely related to cell death.

실시예 2-5-3: RFP-LC3 분포 형광 분석 및 LC3 단백 발현량 분석Example 2-5-3: RFP-LC3 distribution fluorescence analysis and LC3 protein expression analysis

In order to confirm the autophagy induction activity of the mixed medicinal extract (KIOM-C) prepared in Example 1, microtubule-associated protein1 light chain 3 (LC-3), which is an important molecular marker of autophagy Fluorescence analysis was performed using this fluorescently labeled RFP-LC3, and Western blot was performed to confirm the expression level of the LC3 protein. In addition, when Bafilomycin A1, a lysosomal inhibitor known to exacerbate cell death, is treated with the mixed medicinal herb extract (KIOM-C) of the present invention, it has no effect on autophagy-inducing activity. Influence was also confirmed.

Specifically, the HT1080 cell line (5 × 10 ⁴ ) grown in a 24-well culture plate containing coverslips was prepared by transiting an LC3 plasmid tagged with RFP to TransIT2020 (Mirus, Madison, WI). Transfection was performed using. The transfected cells were cultured as described above for 24 hours, and after the culture, the mixed medicinal extracts (KIOM-C) were treated with the concentration of 500 µg / ml and 1000 µg / ml for 24 hours. After 24 hours, a loading medium (Vector Laboratories, Burlingame, Calif.) Containing DAPI, which stains the nucleus, was added and then sealed with a coverslip to confocal microscope (FV10i-W; Olympus Optical Co. LTD) was examined and photographed the distribution of RFP-LC3. In addition, Western blots were performed using antibodies LC3 and anti-p62 / SQSTM1, the major markers of autophagy, as described in Examples 2-4 above.

As a result, when the mixed medicinal extract (KIOM-C) was treated at concentrations of 500 μg / ml and 1000 μg / ml, it was confirmed that the RFP-LC3 fluorescent dot was significantly increased as compared to the untreated control (FIG. 4C). ).

Bacillomycin A1 at a concentration of 10 nM, a lysosomal inhibitor known to exacerbate cell death, was applied to cells treated with a mixed medicinal extract (KIOM-C) at a concentration of 500 μg / ml for 24 hours. As a result of pretreatment for a time, RFP-LC3 fluorescence dot was increased more than the mixed medicinal extract (KIOM-C) alone compared to the untreated control group. After treatment with the mixed medicinal extract (KIOM-C), it was an important molecular marker of autophagy. As a result of Western blot analysis of LC-3 protein, it was confirmed that the amount of LC-3II protein was increased. In addition, when treated with mixed medicinal extract (KIOM-C) and Bafilomycin A1 (Bafilomycin A1) that inhibits the formation of autophagosome, autophagy than when treated with mixed medicinal extract (KIOM-C) alone It was confirmed that the expression levels of the markers p62 and LC3 proteins were significantly increased (FIG. 4D). Accordingly, autophagy was also involved in the killing of cancer cells by the mixed medicinal extract (KIOM-C), and LC-3II increased by the mixed medicinal extract (KIOM-C) was caused by autophagy.

실시예 2-5-4: 자가포식 액포(autophagic vacuole) 탐지Example 2-5-4: Autophagic Vacuole Detection

The HT1080 cell line was treated with the mixed medicinal extract (KIOM-C) prepared in Example 1 at a concentration of 500 μg / ml and 1000 μg / ml and incubated for 24 hours. At this time, 50 uM of MDC (monodansyl cadaverine; Sigma Chemical), an autophagy vacuole tracer, was added thereto, and stained at 37 ° C. for 40 minutes. After staining, the cells were washed twice with PBS, and then microscopically and photographed using a fluorescence microscope.

As a result, 33.10% and 57.63% of MDC positive cells were observed when the mixed medicinal extract (KIOM-C) was treated at concentrations of 500 μg / ml and 1000 μg / ml, respectively, compared to the untreated control (FIG. 4E). ). Accordingly, depending on the concentration of the mixed medicinal extract (KIOM-C), MDC-positive cells were found to increase significantly, indicating that the mixed medicinal extract (KIOM-C) is closely related to the formation of autophagy vacuoles. .

Thus, through the above results, the mixed medicinal extract (KIOM-C) means that cancer cells can be killed by inducing autophagy as well as apoptosis (apoptosis).

실시예 2-6: 종양세포사멸 유도 기작 분석Example 2-6 Analysis of Mechanism of Induction of Tumor Apoptosis

How does the mixed medicinal extract (KIOM-C) prepared in Example 1 affect the protein expression of various factors related to induction of apoptosis and autophagy, and inhibition of cancer cell proliferation and death-inducing activity Western blots were analyzed to confirm their association with the activation of this cellular signaling system.

실시예 2-6-1: 종양세포사멸 유도 관련 단백질의 발현 분석Example 2-6-1 Analysis of Expression of Tumor Apoptosis-related Proteins

In order to determine how the mixed medicinal extract (KIOM-C) prepared in Example 1 affects the protein expression of various factors related to induction of apoptosis and autophagy, various cancer cell lines (HT1080) , B16F10 and AGS) treated with the mixed medicinal extract (KIOM-C) at a concentration of 500 μg / ml or 1000 μg / ml and after 24 and 48 hours, the protein was recovered from each cell and then important for cell death. Western blot analysis revealed the expression of Beclin-1, which is important for the caspase-3 activity, PARP cleavage, LC3 and p62 proteins involved in autophagy, and autophagosome formation.

As a result, 24 hours after the treatment of the mixed medicinal extract (KIOM-C) on the HT1080 cell line, it was confirmed that LC3 protein, which is an autophagy marker, was converted from LC3 I of 16 kDa to 14 kDa LC3 II. When the mixed medicinal extract (KIOM-C) treatment, it was confirmed that the reduction efficiently. In addition, the expression level of Beclin-1 protein, which is important for the formation of autophagosomes during autophagy, was markedly increased, and caspase-3 activity and PARP cleavage were observed in the high concentration treatment group after 48 hours (FIG. 5A).

In addition, the B16F10 and AGS cell lines, like the HT1080 cell line, after 24 hours of treatment with the mixed medicinal extract (KIOM-C), confirmed that the autophagy marker LC3 protein was converted from 16 kDa LC3 I to 14 kDa LC3 II. It was. In addition, the expression level of Beclin-1 protein, which is important for the formation of autophagosomes during autophagy, was markedly increased and PARP cleavage was identified (FIG. 5B).

Through this, it was confirmed that a combination of apoptosis and autophagy by capase-3 activity by the mixed medicinal extract (KIOM-C).

실시예 2-6-2: 종양세포사멸 유도 관련 세포 신호 전달 체계의 분석Example 2-6-2: Analysis of Cell Signaling System Related to Tumor Apoptosis Induction

In order to determine whether cancer cell proliferation inhibition and death-inducing activity by the mixed medicinal herb extract (KIOM-C) prepared in Example 1 is related to activation of related cellular signaling systems, various cancer cell lines (HT1080, B16F10 and AGS) treated with the mixed medicinal extract (KIOM-C) at a concentration of 500 μg / ml or 1000 μg / ml and after 3 hours, 6 hours and 24 hours, the protein was recovered from each cell, and then cell death was controlled. Expression patterns of AMPK, ULK, JNK, c-jun, p53, p38 and ERK 1/2, which are factors related to signal transduction system activation, were confirmed by Wester blot analysis.

As a result, after 3 hours of treatment with KI1080-C on the HT1080 cell line, the phosphorylation of AMPK and ULK1 gradually increased with time, and the important JNK activity and p53 phosphorylation in inducing autophagy and cell death. When measured, by the mixed medicinal extract (KIOM-C), the phosphorylation was significantly increased over time. In addition, by the mixed medicinal extract (KIOM-C), the expression level of phosphorylated c-jun also increased continuously with time (Fig. 5c). In the case of p38, it was confirmed that the level of activated p38 is increased by about 2 times from the beginning (0 hours) after 24 hours. In contrast, there was no effect on the activation of ERK 1/2 (FIG. 5D).

In addition, the B16F10 and AGS cell lines, like the HT1080 cell line, when the activity of JNK, which is important for induction of autophagy and apoptosis, was measured after 3 hours of the mixed medicinal extract (KIOM-C) treatment, the mixed medicinal extract (KIOM) -C) showed an increase in phosphorylation with time. In contrast, there was no effect on the activation of p38 and ERK 1/2 (FIG. 5E).

Through this, it was confirmed that the mixed medicinal extract (KIOM-C) is involved in apoptosis and autophagy by regulating various signaling systems.

Therefore, through the above results, the mixed medicinal extract (KIOM-C) self-kills cancer cells by regulating the protein expression and cell signaling system of various factors related to induction of apoptosis and autophagy. (apoptosis) as well as autophagy (induced autophagy), it was confirmed that the cells can be killed.

실시예 2-7: 산화 스트레스에 의한 종양세포사멸 유도 분석Example 2-7 Induction of Tumor Cell Death by Oxidative Stress

In order to determine whether the mixed medicinal extract (KIOM-C) prepared in Example 1 causes intracellular oxidative stress (oxidative stress) to cause cell death, JNK mediated intracellular reactive oxygen species (reactive oxygen species; ROS) was assessed using flow cytometry. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were performed to confirm the expression level of Protein-Homologous Protein (CHOP), which is an important factor in the cell death process. At this time, SP600125, which is a NAC (N-acetyl-L-cysteine) or a JNK-specific inhibitor (JNK-specific inhibitor), which is known as an antioxidant, is treated simultaneously with the mixed medicinal extract (KIOM-C), and the mixed medicinal extract (KIOM- JNK activity by C) was directly related to ROS activity and CHOP expression.

실시예 2-7-1: 세포 내 활성산소종(reactive oxygen species; ROS) 레벨 측정Example 2-7-1: Measurement of Reactive Oxygen Species (ROS) Levels in Cells

Intracellular reactive oxygen species (ROS) levels were measured using DCF-DA, a peroxide-sensitive fluorescent probe. Specifically, the mixed medicinal extract (KIOM-C) prepared in Example 1 was treated with cells at a concentration of 500 ㎍ / ㎖ or 1000 ㎍ / ㎖ and incubated for 3 hours. At this time, NAC (1 mM) or J600-specific inhibitor SP600125 (5 uM) was pretreated for 1 hour. 5 M of DCF-DA was added to the cells incubated as above and incubated at 37 ° C. for 30 minutes. After incubation, cells were washed twice with PBS, detached and suspended in PBS to determine the generation of intracellular reactive oxygen species using a FACS caliber flow cytometer, and intracellular reactive oxygen species using WinMDI 2.8 software. The production rate of was calculated.

As a result, it was confirmed that intracellular ROS was significantly increased by 2.9 and 4.7 times when the mixed medicinal extract (KIOM-C) was treated at 500 μg / ml or 1000 μg / ml, respectively. . In addition, this activity was found to be lost in the generated intracellular reactive oxygen species when treated simultaneously with NAC or SP600125 (Fig. 6a). Accordingly, depending on the concentration of the mixed medicinal extract (KIOM-C), by confirming a significant increase in the active oxygen species in the cell, the mixed medicinal extract (KIOM-C) induces intracellular oxidative stress (oxidative stress) cells Caused death.

실시예 2-7-2: 역전사중합효소 연쇄 반응(RT-PCR)Example 2-7-2: Reverse Transcriptase Chain Reaction (RT-PCR)

After culturing the cells for 24 hours and 48 hours in a medium containing the mixed medicinal extract (KIOM-C) (500 ㎍ / ㎖ or 1,000 ㎍ / ㎖) prepared in Example 1, the RNA of the cell is extracted PureHelix RNA After separation using the kit (NanoHelix, Daejeon), cDNA was synthesized using the Helixcript 1'st strand cDNA synthesis kit (NanoHelix, Daejeon), and then PCR was performed. The hCHOP and GAPDH primer sequence information used is shown in Table 1 below.

Table 1

target	primer		SEQ ID NO:
target	direction	Sequence (5'-3 ')	SEQ ID NO:
hCHOP	Forward	GCTTGGCTGACTGAGGAGGAG	One
hCHOP	Reverse	CTGACTGGAATCTGGAGAGTGAGG		2
GAPDH	Forward	TCATGACCACAGTCCATGCC		3
GAPDH	Reverse	TCCACCACCCTGTTGCTGTA		4

As a result, the mRNA expression level of CHOP, which is an important factor of cell death process, was increased depending on the treatment concentration and time-dependently of the mixed medicinal extract (KIOM-C), and more specifically, about 20 times in the high concentration treatment group after 48 hours. The amount of CHOP mRNA expression could be increased (Fig. 6b). Accordingly, it was found that the mixed medicinal extract (KIOM-C) induces oxidative stress in cells and increases the mRNA expression of CHOP, which is an important factor in the cell death process, thereby causing cell death.

실시예 2-7-3: 웨스턴 블롯 분석(western blot analysis) Example 2-7-3: Western blot analysis

In order to determine how the increase in intracellular ROS by the mixed medicinal herb extract (KIOM-C) prepared in Example 1 affects the protein expression level of CHOP, SP600125 (5 uM), a JNK-specific inhibitor, was used for 1 hour. After pretreatment, the mixed medicinal extract (KIOM-C) was treated with cells at a concentration of 500 μg / ml or 1000 μg / ml and incubated for 24 hours. Whole cell lysates of cells incubated as described above were prepared using M-PER Mammalian Protein Extraction Reagent, according to the manufacturer's instructions. Cell lysates were quantified for protein concentration by BCA protein assay (Bicinchoninic Acid) assay, and after immunoblotting, the protein was subjected to a power-opti-chemiluminescent Weston blotting detection reagent ( Visualization was performed using Power Opti-ECL Western blotting detection reagent (Animal Genetics, Inc., Korea) and ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA). The degree of expression of CHOP was derived by quantifying the visualized protein bands using software ImageJ (National Institutes of Health, USA).

As a result of the experiment, the expression level of CHOP protein was significantly increased depending on the treatment concentration of the mixed medicinal extract (KIOM-C) compared to the untreated control group. In addition, the simultaneous treatment of the JNK-specific inhibitor SP600125, it was confirmed that the expression of the CHOP protein is inhibited when the generated active oxygen species are lost (Fig. 6c). Accordingly, it can be seen that the mixed medicinal extract (KIOM-C) induces oxidative stress in cells and increases the expression level of CHOP protein, which is an important factor in the cell death process, thereby inducing cell death.

Therefore, it can be seen from the above results that the increase of intracellular ROS by the mixed medicinal extract (KIOM-C) is closely related to the inhibition of cancer cell proliferation and death-inducing activity by the mixed medicinal extract (KIOM-C). .

실시예 2-8: 세포 사멸(apoptosis) 및 자가포식(autophagy) 작용 동시 유도Example 2-8: Simultaneous Induction of Apoptosis and Autophagy Actions

The medicinal herb extract (KIOM-C) prepared in Example 1, which mediates cell death by simultaneously inducing apoptosis and autophagy, activates JNK, which is very important in cell signaling system. In order to confirm the effect on, using the JNK-specific inhibitor and JNK-targeted siRNA, the following experiment was performed.

실시예 2-8-1: JNK-특이 저해제에 의한, 혼합 약재 추출물(KIOM-C)의 세포사멸 유도 저해Example 2-8-1: Inhibition of Apoptosis Induction of Mixed Medicinal Herb Extracts (KIOM-C) by JNK-Specific Inhibitors

The HT1080 cell line was pretreated with pharmaceutical inhibitors such as JNK-specific inhibitor SP600125, p38 inhibitor SB203580, ERK 1/2 inhibitor PD98059, and lysosome inhibitor Bafilomycin A1 for 1 hour. After 24 hours of treatment with the mixed medicinal extract (KIOM-C) at a concentration of 500 ㎍ / ㎖, after recovering the protein in each cell, including JNK, a factor involved in the activation of signal transduction systems that regulate cell death, The expression of Beclin-1 important for autophagosome formation, PARP cleavage and Bcl-2 important for cell death, and p62 protein involved in autophagy were confirmed by Wester blot analysis. In addition, after 48 hours of treatment with the pharmaceutical inhibitor and the mixed medicinal extract (KIOM-C) of the present invention, the observed cytotoxicity and cell morphological changes may be induced.

As a result, 24 hours after the treatment with the mixed medicinal extract (KIOM-C), when the important JNK activity was measured in induction of autophagy and apoptosis, the phosphorylation was significantly increased compared to the untreated control group. In addition, the expression level of Beclin-1 protein, which is important for autophagy formation during autophagy, was also significantly increased. p62 protein and BCl-2 were decreased in expression level when the mixed medicinal extract (KIOM-C) was treated, and PARP cleavage was confirmed. However, the expression pattern of the cell death-related factors whose expression level was controlled by the mixed medicinal extract (KIOM-C) was restored to a level similar to that of the untreated control group by the pretreatment of SP600125, a JNK-specific inhibitor (FIG. 7A). In addition, when SP600125 and the mixed medicinal extract (KIOM-C) was treated together, it was confirmed that cell death was not induced and cytoplasmic vacuole formation was also inhibited (FIG. 7B). Accordingly, it was found that SP600125, a JNK-specific inhibitor, blocks the regulation of cell death induction and expression of related factors by the mixed medicinal herb extract (KIOM-C) of the present invention.

실시예 2-8-2: JNK를 타겟으로 한 siRNA에 의한, 혼합 약재 추출물(KIOM-C)의 세포사멸 유도 저해Example 2-8-2: Inhibition of apoptosis induction of mixed medicinal herb extracts (KIOM-C) by siRNA targeted to JNK

First, HT1080 cells were cultured about 60% confluent in a 60 mm culture dish. Cells cultured as described above were transfected with JNK-specific siRNA (small interfering RNA) using TransIT2020 (Mirus, Madison, Wis.). At this time, scrambled siRNA (Scr siRNA) was used as a control. The transfected cells were cultured as described above for 72 hours, and after the culture, the mixed medicinal extract (KIOM-C) was treated for 24 hours at a concentration of 500 µg / ml. After this time, the protein was recovered from each cell and caspase-3 activity and PARP cleavage and autophagy, which are important for cell death, including JNK, a factor involved in the activation of signal transduction systems that regulate cell death. The expression patterns of LC3 proteins involved in) and the extent of Beclin-1 expression in autophagosome formation were confirmed by Western blot analysis. Sequence information of the used Scr siRNA and JNK siRNA is shown in Table 2 below.

TABLE 2

target	Sequence Information (5'-3 ')	SEQ ID NO:
Scr siRNA	CCUACGCCACCAAUUUCGU		5
JNK siRNA	AAAAAGAAUGUCCUACCUUCU		6

As a result, it was confirmed that the JNK protein expression is significantly reduced in the cells transformed with JNK-specific siRNA (Fig. 7c), each cell after 24 hours treatment with the mixed medicinal extract (KIOM-C), autophagy and cells In the control of Scr siRNA transfected, there was a marked increase in phosphorylation, and the changes in cell morphology were observed. Death induction patterns could be observed (FIGS. 7D and 7E). In addition, the autophagy marker LC3 protein was converted from 16 kDa LC3 I to 14 kDa LC3 II, and the expression level of Beclin-1 protein, which is important for autophagy, was markedly increased. In addition, caspase-3 activity and PARP cleavage were also confirmed (FIG. 7E). However, when transfected with JNK-specific siRNA and treated with the mixed medicinal extract (KIOM-C), the expression level and cell morphological changes of cell death-related factors were restored and did not induce cell death and formed cytoplasmic vacuoles. It was also confirmed that the inhibition (Fig. 7d and 7e). Accordingly, it can be seen that the JNK-specific siRNA blocks cell death induction and expression control of related factors by the mixed medicinal herb extract (KIOM-C) of the present invention.

Therefore, the above results, it can be seen that the use of inhibitors and siRNA of cell signaling system-related factors, can inhibit the induction of cell death. That is, this means that the mixed medicinal extract (KIOM-C) mediates cell death by inducing apoptosis and autophagy at the same time.

실시예 3: 비-부착성 콜로니 형성능 분석Example 3: Non-Adhesive Colony Formation Assay

When the cancer cells were treated with the mixed medicinal extract (KIOM-C) prepared in Example 1, the following experiment was performed to confirm whether non-adherent colony forming ability is inhibited.

Mixed medicinal extract (KIOM-C) prepared in Example 1 (25 μg / ml, 50 μg / ml, 100 μg / ml and 250 μg / ml), 3 ml containing 0.3% agar, and 10% FBS Suspension of cancer cells (B16F10 and HT1080) (1 × 10 ⁴ ) in the medium of and applied to the solidified bottom agar containing 0.6% agar and 10% FBS, followed by a phase-contrast with incubation for 3 weeks. The microscope observed colonies formed in soft agar and photographed them.

In addition, 200 cancer cells (B16F10 and HT1080) were suspended in 1 ml of 10% FBS / DMEM medium, plated in a 12-well plate, and cultured to adhere to the plate, followed by mixed medicinal extract (KIOM-C). (25 μg / ml, 50 μg / ml, 100 μg / ml and 250 μg / ml) were incubated for 10 days. After 10 days, the colonies formed were stained with 0.2% crystal violet / 20% methanol stain and observed under a microscope.

Experimental results showed that the mixed medicinal extract (KIOM-C) showed a significant decrease in non-adherent colony forming ability in a concentration-dependent manner compared to the untreated control, and non-adherent compared to non-adherent colony forming ability in control cells. Sex colony forming ability was found to be significantly reduced by about 60% to 95% (FIG. 8).

실시예 4: 세포 이동(cell migration) 능력 분석Example 4 Analysis of Cell Migration Ability

When treating the mixed drug extract (KIOM-C) to cancer cells, wound healing assays were performed as follows to determine whether cell migration ability was inhibited.

First, cancer cell lines (B16F10 and HT1080) were cultured in a 60 mm culture dish about 80% confluent. To exclude the healing by cell proliferation after wound formation, as described above, about 25% of mitomycin C (mitomycin C) in cancer cells (B16F10 and HT1080) proliferated in monolayers was about 25%. C; Sigma chemical Co.) was stopped for 30 minutes to stop cell proliferation, and then the single layer was about 2 mm wide. Then, after removing the suspended cell suspension, and filled with 10% FBS-DMEM culture medium containing the mixed medicinal extract (KIOM-C) (50 ㎍ / ㎖-250 ㎍ / ㎖) prepared in Example 1 , And cultured in the medium. Specifically, the cells were cultured for 24 hours and 48 hours for the B16F10 cell line and 40 hours for the HT1080 cell line. After incubation for the above time, the ability of cell migration at the site of injury was observed through a phase contrast microscope. The interval between the time points at which the wound was formed (0 hour) was set at 100% and the extent to which the interval was narrowed due to cell movement was calculated as time passed.

As a result, B16F10 cells of the untreated control group were cured about 25% of the damaged area after 24 hours and about 70% after 48 hours, whereas in the group treated with the mixed medicinal extract (KIOM-C) Healing was suppressed according to the concentration. In particular, when the mixed medicinal herb extract (KIOM-C) was treated at concentrations of 100 μg / ml and 250 μg / ml, after 48 hours, cell migration to the site of injury was concentration-dependent, compared to control cells. It was found to be significantly inhibited by 55% to 60% (FIG. 9A).

In addition, in the case of HT1080 cells of the untreated control group, about 70% was cured after 40 hours, whereas in the group treated with the mixed medicinal herb extract (KIOM-C), healing was inhibited depending on the concentration. The results were shown. In particular, when the mixed medicinal herb extract (KIOM-C) was treated at a concentration of 250 ㎍ / ml, healed about 25% after 40 hours, and inhibited cell migration about 3 times as compared to the control group. One thing was confirmed (FIG. 9B).

Therefore, the mixed medicinal herb extract (KIOM-C) of the present invention means that it effectively inhibited cell migration.

실시예 5: 시험관 내 세포 이동(cell migration) 및 세포 침윤능(cell invasion) 분석Example 5 In Vitro Cell Migration and Cell Invasion Assays

When treating the mixed drug extract (KIOM-C) to cancer cells (B16F10 and HT1080), in order to determine whether cell migration ability and invasion ability is inhibited, the following transwell migration / invasion assay (Transwell migration / invasion assay) ) Was performed.

Mobility analysis was performed by filling 600 μl of 10% FBS / DMEM medium in the lower chamber of a 10 mm diameter transwell chamber with an 8 μm pore size polycarbonate membrane (Corning costar, Cambridge, Mass.), the upper chamber and then fill in the mixed medicine extract (KIOM-C) and tumor cells (B16F10 and HT1080) (1 × 10 ⁵ gae / 100 ㎕) a, 100 ㎕ DMEM medium without serum containing the 24 hours at 37 ℃ to Incubate for 36 hours. Then, to identify the cells that have migrated or infiltrated into the filter, remove the cells remaining on the upper surface of the unmigrated filter and remove the filter with a 0.2% crystal violet / 20% methanol (w / v) solution. After staining with a microscope was observed. In this case, the HT1080 cell line was confirmed the efficacy of the mixed medicinal extract (KIOM-C) under PMA stimulation conditions.

Invasion assays were performed by coating the transwell chamber with a 20 μl of Matrigel: DMEM 1: 2 mixture (Matrigel, BD Biosciences, Bedford, Mass., USA) to form an intervening invasive barrier. It was performed after.

Experimental results showed that the mixed medicinal extract (KIOM-C) significantly reduced the cell's migration ability in a concentration-dependent manner compared to its untreated control, and also mixed the cell's invasion capacity (the ability to penetrate the Matrigel boundary). Medicinal extracts (KIOM-C) were found to be significantly reduced in treated cells. Specifically, in the B16F10 cell line treated with the mixed medicinal herb extract (KIOM-C), cell mobility and cell invasion were inhibited by approximately 55% compared to untreated control cells (FIG. 10A). In addition, in the case of HT1080 cell line, cell mobility and cell invasion ability were inhibited by about 50% by the mixed medicinal herb extract (KIOM-C) treatment compared to untreated control cells (FIG. 10B).

Therefore, it was confirmed that the mixed medicinal herb extract (KIOM-C) of the present invention effectively inhibits cell migration and cell invasion of cancer cell lines.

실시예 6: MMP-9의 발현 및 활성 감소 분석Example 6: Analysis of Decreased Expression and Activity of MMP-9

MMP-9 is known to play an essential role in cancer metastasis by breaking down the extracellular matrix (ECM). In the case of treating the mixed drug extract (KIOM-C) on cancer cells (B16F10 and HT1080), in order to determine whether the expression and activity of MMP-9 is reduced, RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) Western blotting and gelatin zymography were performed.

In this experiment, phorbol myristate acetate (PMA) was used as an inducer to increase the expression and activity of MMP-9. That is, cancer cells (B16F10 and HT1080) were incubated for 12 hours in serum-free DMEM containing mixed medicinal extract (KIOM-C) (100 μg / ml and 250 μg / ml), and then 5 nM of PMA was added thereto. Treatment was performed for 24 hours for the HT1080 cell line and an additional 48 hours for the B16F10 cell line to stimulate the cells, followed by the following experiment.

실시예 6-1: 역전사중합효소 연쇄 반응(RT-PCR)Example 6-1 Reverse Transcriptase Chain Reaction (RT-PCR)

After culturing in the medium containing the mixed medicinal extract (KIOM-C), the RNA of PMA-stimulated cells was isolated using PureHelix RNA extraction kit (NanoHelix, Daejeon), and then synthesized Helixcript 1'st strand cDNA. PCR was performed after cDNA was synthesized using the kit (NanoHelix, Daejeon). The hMMP-9 and GAPDH primer sequence information used is shown in Table 3 below.

TABLE 3

target	primer		SEQ ID NO:
target	direction	Sequence (5'-3 ')	SEQ ID NO:
hMMP-9	Forward	TCTTCCCTGGAGACTGAGAA		7
hMMP-9	Reverse	GGCAAGTCTTCCGAGTAGTTT		8

실시예 6-2: 웨스턴 블롯 분석(western blot analysis) Example 6-2 Western blot analysis

After culturing in the medium containing the mixed medicinal extract (KIOM-C), the total cell lysate and nuclear / cytosolic extract of PMA-stimulated cells according to the manufacturer's instructions M-PER Mammalian Protein Extraction Reagent and NE-PER Nuclear & Cytosolic Extraction Reagent (Pierce Biotechnology, Inc., Rockford, IL, USA Each). Cell lysates were quantified for protein concentration by BCA protein assay (Bicinchoninic Acid) assay, and after immunoblotting, the protein was subjected to a power-opti-chemiluminescent Weston blotting detection reagent ( Visualization was performed using Power Opti-ECL Western blotting detection reagent (Animal Genetics, Inc., Korea) and ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA). The expression level of MMP-9 was derived by quantifying the visualized protein bands using software ImageJ (National Institutes of Health, USA).

실시예 6-3: 젤라틴 자이모그래피(Gelatin zymography) Example 6-3 Gelatin zymography

The culture medium of the PMA-stimulated cells was electrophoresed in 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) containing 0.1% gelatin, and the gel was washed with buffer (50 mM Tris-HCl, pH 7.5, 100). After washing thoroughly with mM NaCl, 2.5% Triton X-100), it was soaked in active buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl ₂ , 0.02% NaN ₃ , 1 μM ZnCl ₂ ) And incubated at 37 ° C. The gel was then stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories, Hercules, Calif., USA) and 10% isopropanol / 10% acetic acid (v / v) Destained with solution. The gelatin degradation capacity of MMP-9 was detected at 92 kDa with a transparent band on a dark blue background.

As a result, when the mixed medicinal extract (KIOM-C) was treated, it was confirmed that compared with the untreated control group significantly reduced the expression and activity of MMP-9 by PMA stimulation depending on the concentration (Fig. 11). .

Therefore, it was confirmed that the expression and activity of MMP-9, which is mainly involved in tumor migration and invasion, by the mixed medicinal extract of the present invention (KIOM-C).

실시예 7: PMA 자극에 의한 NF-κB 활성에 대한 혼합 약재 추출물(KIOM-C)의 효능 분석Example 7: Analysis of Efficacy of Mixed Herbal Extract (KIOM-C) on NF-κB Activity by PMA Stimulation

NF-κB is a transcription factor known to regulate MMP-9 gene expression and enhance cancer cell migration and invasiveness. Whether the mixed medicinal extract (KIOM-C) prepared in Example 1 inhibits NF-κB activity To confirm, the degree of pIκB and IκB expression and the degree of nuclear transfer of the NF-κB p65 subunit were analyzed by Western blot as described in Example 6-2. At this time, in order to confirm the degree of nuclear migration of the NF-κB p65 subunit by Western blot, the cell lysate was performed by dividing the cytosol and the nuclear part. The expression levels of pIκB, IκB and the nuclear transfer NF-κB p65 subunits were quantified by standardizing α-tublin or TBP, which is an internal control.

Specifically, HT1080 cell line pretreated with 250 μg / ml of mixed medicinal extract (KIOM-C) was treated with PMA 5 nM to stimulate NF-κB by stimulating the cell line. After 15 minutes, 30 minutes, and 60 minutes of PMA treatment, each protein was harvested, and the expression levels of pIκB, IκB, and NF-κB p65 subunits transferred in the nucleus were confirmed. -C), pIκB was increased after PMA stimulation and IκB expression was decreased in the untreated control group, but there was no increase in pIκB due to PMA stimulation in the group treated with the mixed medicinal extract (KIOM-C). IκB expression did not decrease. Intranuclear migration of the NF-κB p65 subunit was also significantly reduced in the group treated with the mixed medicinal extract (KIOM-C) compared to the control. These results support that the mixed medicinal herb extract (KIOM-C) of the above example has an effect of inhibiting NF-κB activity (FIG. 12).

Therefore, it was confirmed that the mixed medicinal extract (KIOM-C) inhibits the expression and activity of MMP-9 by inhibiting the activity of NF-κB, resulting in the effect of inhibiting the migration and invasion of cancer cells.

실시예 8: 생체 내(Example 8: In vivo in vivoin vivo ) 실험) Experiment

In order to evaluate the anticancer activity and anticancer activity of the mixed medicinal herb extract (KIOM-C) in vivo, the following experiment was performed.

실시예 8-1: 자연 전이 마우스 종양 모델(spontaneous lung metastasis model)을 이용한 혼합 약재 추출물(KIOM-C)의 암 전이 억제 활성 평가Example 8-1 Evaluation of Cancer Metastasis Inhibitory Activity of Mixed Medicinal Herb Extracts (KIOM-C) Using Natural Metastatic Mouse Metastasis Model

In order to determine the efficacy of the mixed drug extract (KIOM-C) in vivo cancer metastasis was carried out the following experiment.

The tail veins of 15 C57BL / 6J mice were injected with B16F10 cell line (3 × 10 ⁵ cells / 0.2 ml PBS), each of which is a classical cancer cell line (day 0), and then randomly divided the mice into three groups ( N = 5) in each group, the first group (experimental group) mixed medicinal herb extract (KIOM-C) 170 mg / kg / day, the second group (experimental group) mixed medicinal extract (KIOM-C) 510 mg / kg / day, the third group (control) was orally administered saline solution for 17 days. After 17 days, the mice were sacrificed and the lungs of each mouse were fixed with Bowin's solution (Sigma), and the number of black colonies of B16F10 cells on the surface of the lungs was visually examined. The test results were calculated as the average number of colonies in each group.

Experimental results showed that the average number of colonies that metastasized to the lung was 542.3 ± 93.56 in the control mice, while 227.5 ± 20.10 and 510 mg / kg in the 170 mg / kg of the mixed medicinal extract (KIOM-C). The group administered with kg of mixed medicinal extract (KIOM-C) was observed to have metastasized 154.7 ± 52.81 tumor colonies (Figs. 13A and 13B). This is more than 58% and 71% metastasis was inhibited compared to the control group administered with saline, respectively, indicating that the metastatic ability of the classical cancer cells significantly decreased by the administration of the mixed medicinal extract (KIOM-C).

Therefore, the mixed medicinal herb extract (KIOM-C) of the present invention is a result supporting the excellent efficacy of inhibiting cancer cell metastasis in vivo.

실시예 8-2: 암 이식 모델(Tumor xenograft model)을 이용한 혼합 약재 추출물(KIOM-C)의 항암 활성 평가 Example 8-2: Anticancer Activity Evaluation of Mixed Medicinal Extracts (KIOM-C) Using Tumor xenograft Model

Human fibrosarcoma cell line (HT1080) 2 × 10 ⁶ pieces / a 200 ㎕ saline Balb / c of about 6 week-old female nude mice (Balb / c athymic nude mice) were inoculated in the thigh to avoid mixing medicinal extract (KIOM-C of ) Orally administered daily to confirm tumor growth for about 15 days. The dosage of the mixed medicinal extract (KIOM-C) was divided into three dose groups of 85 mg / kg, 170 mg / kg, and 340 mg / kg in consideration of the amount taken by a 60 kg adult and the yield of agar. As a negative control group, saline administration group was included. After inoculating the tumor subcutaneously, the mixed medicinal extract (KIOM-C) was orally administered daily for about 15 days, and the tumor volume was measured and calculated through the following calculation formula.

[tumor volume (mm ³ ) = length (length (long, mm) × width ² (short ² , mm ² ) × 0.52]

In addition, the secretion level of interferon-gamma (INF-γ), an important cytokine that kills cancer, was collected from the blood of each mouse in the experimental group, and the level of INF-γ in serum was produced. depending on the character indicated were determined using a ^{LEGEND MAX TM ELISA kit (BioLegent Inc.} San Diego, CA).

As a result, the negative control group began to observe tumor mass after 5 days of tumor inoculation, and showed rapid growth after about 10 days, whereas the group of the mixed medicinal extract (KIOM-C) showed significantly reduced tumor growth. It was confirmed (Fig. 14a). In addition, it was confirmed that the size of the tumor mass extracted after 15 days was also significantly smaller in the group to which the mixed medicinal extract (KIOM-C) was administered (FIG. 14B). After 15 days, tumors were collected from nude mice and the tumor weight was measured. The average weight of the tumors in the negative control group was 0.62 ± 0.18g, and the mixed medicinal extract 85 mg / kg administered group was 0.16 ± 0.06g, 170 mg / g. The kg administration group was 0.16 ± 0.09g and the 340 mg / kg administration group was 0.19 ± 0.11g (Fig. 14c). In addition, in the case of the group administered with the mixed medicinal extract (KIOM-C), it was confirmed that the INF-γ concentration significantly increased compared to the untreated control (FIG. 14D).

Therefore, it was confirmed that the mixed medicinal herb extract (KIOM-C) of the present invention significantly inhibits tumor growth even in in-vivo experiments, and there is no specific toxicity by repeated long-term administration.

실시예 9: 혼합약재 추출물 및 각 단미재의 효능 비교 분석Example 9 Comparative Analysis of Efficacy of Mixed Medicinal Extracts and Sweeteners

In order to analyze the tumor apoptosis effect and tumor metastasis inhibiting effect of the mixed medicinal extract (KIOM-C) and the sweet constituents comprising the same, the following experiment was performed.

실시예 9-1: 혼합약재 추출물 및 각 단미재의 종양세포사멸 효과 분석Example 9-1: Analysis of tumor cell death effect of the mixed medicinal extract and each sweetener

Mixed medicinal herb extract (KIOM-C) prepared in Example 1 and the sweet constituents (Kungung, Windproof, Angelica, Peony, Yeongyo, Peppermint, Ephedra, Manganese, Rhubarb, Gypsum, Giltyeong, Golden, Baekchul, Maize , Ginseng, ginger, talc, licorice, gilding, yin and yang, and paper and neck). 500 μg / ml of the mixed medicinal herb extract (KIOM-C) of the above example and the extracts of each sweetener of Comparative Example 1 to Comparative Example 22 (heavenly, windproof, donkey, peony, Yeongyo, Peppermint, Ephedra, Manganese, Rhubarb, Gypsum, Giltyeong, Golden, White Extract, Gardenia, Penguin, Ginger, Talc, Licorice, Geum, Eum, Yeonggwa, Wonji and Mokyang), and cultured for 48 hours and then cell viability was analyzed by MTT assay. Analyzed through. At this time, the extract of each sweetener was treated with the concentration of each sweetener component contained in 500 ㎍ / ㎖ of the mixed medicinal extract (KIOM-C).

As a result of the experiment, the group treated with the mixed medicinal herb extract (KIOM-C) prepared in Example 1 showed cancer cell survival rate (77.7% cancer cell death) of 22.3% compared to the group not treated, but each sweet rice Processed ash extracts (bowung, windbreak, donkey, peony, pontoon, peppermint, ephedra, forget-me-not, rhubarb, gypsum, gilsheng, gold, white peach, wild jasmine, hedgehog, ginger, talc, licorice, sterling silver, yin and yang, spring and wood) In the group, cancer cell survival inhibition (cancer apoptosis) efficacy was not confirmed (FIG. 15).

Therefore, the mixed medicinal herb extract (KIOM-C) of the present invention exhibits an effective anticancer activity (apoptosis) that can not be provided by each of the sweeteners alone by using a mixture of each of the sweeteners of low concentration that does not exhibit cancer cell death effects. It was confirmed.

실시예 9-2: 혼합약재 추출물 및 각 단미재의 종양 전이 억제 효과 분석Example 9-2: Analysis of Tumor Metastasis Inhibitory Effects of the Mixed Medicinal Extracts and Each Sweet Rice

Gelatin zymography of the effects of the ephedra (Comparative Example 7) extract and rhubarb extract (Comparative Example 9) of the MMP-2 / MMP-9 activity in each of the extracts prepared in Comparative Examples 1 to 22 ). At this time, the treatment concentrations of the ephedra and rhubarb of the ephedra and rhubarb contained in 500 ㎍ / ㎖ mixed medicinal extract (KIOM-C) of the Example used in the cancer cell killing effect experiment of Example 9-1 Each concentration (concentration that did not exhibit cancer cell death effect) was used.

In the case of rhubarb (Fig. 16, lane 1), as reported in the existing literature, the treatment of 300 ㎍ / ㎖ resulted in a significant decrease in the activity of MMP-9, but at the same time more than 80% Cytotoxicity was observed. On the other hand, when treated with rhubarb concentration (2 ㎍ / ㎖ to 15 ㎍ / ㎖) contained in 500 ㎍ / ㎖ mixed medicinal extract (KIOM-C) showed no toxicity, cancer cell death effect (cell Toxicity, FIG. 15) was not observed and did not affect MMP-9 activity (FIG. 16, lane 2). In the case of ephedra, cancer apoptosis effects (cytotoxicity, FIG. 15) were not observed even in the ephedra concentration (2 μg / ml to 15 μg / ml) contained in 500 μg / ml of the mixed medicinal extract (KIOM-C). There was no effect on MMP-9 activity (FIG. 16, lane 3).

Therefore, it was confirmed that the mixed medicinal herb extract (KIOM-C) of the above example has an effect of remarkably improving anti-transduction activity by using a mixture of low concentrations of safe sweeteners having no cancer cell killing effect (cytotoxicity).

Claims

Extracts of mixed medicinal herbs consisting of Cheongung, Windproof, Angelica, Peony, Fellowship, Mint Leaf, Ephedra, Forget-me-not, Rhubarb, Gypsum, Giltyeong, Golden, White Extract, Gardenia, Hungae, Ginger, Talc, Licorice, Gold Silver Coin, Yin Yang Kwak, Wonji and Mokji. Pharmaceutical composition for the prevention or treatment of cancer, including as an active ingredient.
According to claim 1, The composition is 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to about 3 parts by weight of peony 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight of mint leaves, 1 part by weight to 3 parts by weight of ephedra, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight of rhubarb, 0.5 parts by weight of gypsum 3 parts by weight, 0.5 parts by weight to 3 parts by weight, 0.5 parts by weight to 3 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight of mold , 1 part by weight to 3 parts by weight, 1 part by weight to 5 parts by weight of talc, 1 part by weight to 3 parts by weight of licorice, 1 part by weight to 3 parts by weight of silver coin, 1 part by weight to 3 parts by weight of raw paper, and 1 part by weight Characterized in that it comprises an extract of the mixed medicine comprising from 3 parts by weight to Pharmaceutical compositions.
The pharmaceutical composition of claim 1, wherein the extract is prepared by extracting the mixed medicine with water, C 1-4 alcohol, or a mixed solvent thereof.
The pharmaceutical composition according to claim 3, wherein the extraction is hot water extraction, ultrasonic extraction, room temperature extraction, cold needle extraction, reflux cooling extraction or steam extraction.
The pharmaceutical composition according to claim 1, wherein the composition has cancer cell metastasis suppression, cancer cell death, or cancer cell growth inhibiting activity.
According to claim 1, wherein the cancer is any one selected from the group consisting of fibrosarcoma, skin cancer, lung cancer, breast cancer or stomach cancer.
Extracts of mixed medicinal herbs consisting of Cheongung, Windproof, Angelica, Peony, Fellowship, Mint Leaf, Ephedra, Forget-me-not, Rhubarb, Gypsum, Giltyeong, Golden, White Extract, Gardenia, Hungae, Ginger, Talc, Licorice, Gold Silver Coin, Yin Yang Kwak, Wonji and Mokji. Health functional food for the prevention or improvement of cancer containing as an active ingredient.
According to claim 7, wherein the composition is 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to about 3 parts by weight of peony 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight of peppermint, 1 part by weight to 3 parts by weight of ephedra, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight, 1 part by weight to 3 parts by weight of rhubarb, 0.5 parts by weight of gypsum 3 parts by weight, 0.5 parts by weight to 3 parts by weight, 0.5 parts by weight to 3 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight, 0.5 parts by weight to 2 parts by weight of mold , 1 part by weight to 3 parts by weight, 1 part by weight to 5 parts by weight of talc, 1 part by weight to 3 parts by weight of licorice, 1 part by weight to 3 parts by weight of silver coin, 1 part by weight to 3 parts by weight of raw paper, and 1 part by weight Characterized in that it comprises an extract of the mixed medicine comprising from 3 parts by weight to Health functional food.
The health functional food according to claim 7, wherein the extract is prepared by extracting the mixed medicine with water, C 1-4 alcohol or a mixed solvent thereof.
The health functional food according to claim 9, wherein the extraction is hot water extraction, ultrasonic extraction, room temperature extraction, cold needle extraction, reflux cooling extraction or steam extraction.
The dietary supplement according to claim 7, wherein the composition has cancer cell metastasis suppression, cancer cell death induction, or cancer cell growth inhibitory activity.
The dietary supplement according to claim 7, which is any one selected from the group consisting of fibrosarcoma, skin cancer, lung cancer, breast cancer or stomach cancer.
A method of treating cancer, comprising administering to a subject in need thereof the composition of claim 1.