KR20140049218A - Composition comprising the extract of puerariae radix for anti-cancer activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a Puerariae radix extract for treating and preventing cancer. More specifically, the extract of the present invention not only shows a powerful anticancer activity for colon cancer like CT-26 cells, but also increases generation of MHC II, CD80, CD86, IL-12, CD4, and CD8 which are carcinomatous tissue growth inhibition and immunization modulating substances when co-administered with conventional anticancer medicines such as 5-FU and LV in an animal model experiment of cancer xenograft. Thereby, the extract can be used as a medicine and a functional health food having effects of anti-cancer drugs or anti-cancer adjuvants which is excellent for preventing, suppressing, and treating the cancer.

Description

Composition comprising the extract of Puerariae Radix for anti-cancer activity

The present invention relates to a composition for anticancer activity containing the root extract.

1 Dawson ™, et al., Annu . Rev. Med , 47 , pp.219-227, 1996

[Reference 2] Richard A. Goldsby, et al., KUBY Immunology . 2000

[Reference 3] Kim NH, Kim SN, Oh JS, Lee S, Kim YK. Anti-mitotic potential of 7-diethylamino-3 (2′-benzoxazolyl) -coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620 / 5-FU. Biochem Biophys Res Commun 2012; 418: 616-21; PMID: 22301191; DOI: 10.1016 / j.bbrc.2012.01.049.

C. Kanetaka K, Enjoji A, Furui J, Nagata Y, Fujioka H, Shiogama T, et al. Effects of Intermittent 5-Fluorouracil and Low-dose Cisplatin Therapy on Advanced and Recurrent Gastric Cancer. Anticancer Res 2012; 32: 3495-9; PMID: 22843936.

[5] Wettergren Y, Carlsson G, Odin E, Gustavsson B. Pretherapeutic uracil and dihydrouracil levels of colorectal cancer patients are associated with sex and toxic side effects during adjuvant 5-fluorouracil-based chemotherapy. Cancer 2012; 118: 2935-43; PMID: 22020693; DOI: 10.1002 / cncr.26595.

[Ref. 6] Herbal Medicine Reorganization Committee, Herbal Medicine, Dongmyeongsa, p.100-102, 2006

7 Alessandria G, Filippeschi S, Sinibaldi P, Mornet F, Passera P, Spreafico F, Cappa PM and Gullino PM: Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells. Cancer Res 47: 4243-4247, 1987

[Reference 8] Toxicological Test Standards of the Korea Food and Drug Administration and Drugs, Korea Food and Drug Administration Notice 2005-60, 2005.

Document 9 Holland EC (2004) Mouse models of human cancer. Wiley-Liss, New York.

Loganayaki N, Manian S. Antitumor activity of the methanolic extract of Ammannia baccifera L. against Dalton's ascites lymphoma induced ascitic and solid tumors in mice. J Ethnopharmacol. 2012 Jun 26; 142 (1): 305-9.

[Reference 11] Lee B, Lee DY, Yoo KH, Baek NI, Park JH, Chung IS. Calenduloside E 6'-methyl ester induces apoptosis in CT-26 mouse colon carcinoma cells and inhibits tumor growth in a CT-26 xenograft animal model. Oncol Lett. 2012 Jul; 4 (1): 22-28.

Cancer is widely classified into blood cancer and solid cancer, and it occurs in almost all parts of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer and skin cancer.

Leukocytes constitute a secondary or tertiary line of defense, breaking through the primary line of defense, and are responsible for foreign bodies entering the body.In the case of bacterial, viral infections, or inflammatory reactions, the regulation of macrophage and lymphocyte activity is necessary to determine the therapeutic effect of medicines. Play a pivotal role. Macrophage is a multifunctional cell that plays an important role in the immune system by producing various cytokines and oxygen monoxide (NO) in oxidative stress situations. In particular, iNOS expressed by stimulation such as lipopolysaccharide (LPS), cytokine, TNF-α in macrophages is known to produce a large amount of NO for a long time, promoting NFκB activity. Of reactive oxygen species (ROS) and nitric oxide (NO) such as superoxide anion (O ₂ −), hydrogen peroxide (H ₂ O ₂ ) by activated macrophages Production is an important cytotoxic and cytostatic mechanism in nonspecific immunity. Many studies have been conducted on which natural compounds influence the production of ROS and NO by macrophages. Macrophages are microbicidal cells that present antigen, present tumors, or kill microbial cells and play a central role in cell-mediated or humoral immunity. to control cells, is produced in the activated macrophage NO exhibits a non-specific host defense action agent macrophages, and bacteria, growth inhibitory activity of cancer cells (Dawson TM, et al., Annu. Rev. Med, 47, pp.219 -227, 1996).

Macrophages contain many lysosomes, which contain acid hydrolytic enzymes and peroxidase. It is also strongly attached to the glass surface and plastic surface and actively digests microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-γ. They produce cytokines such as complement components, interferons, interleukin-1 and tumor necrosis factors, and can be enhanced by a variety of cytokines produced from T-cells. T-cells, a type of leukocytes, account for about 70% of the bovine lymphocytes in the blood. T-cells differentiate in the thymus and have T-cell antigen receptors (TCRs). Peripheral blood T- cells were divided into CD4-positive T _H cells (helper T-cell) and CD8-positive T _C cells (cytotoxic T-cell), CD4 + T _H cells recognize the antigen binds to MHC II class (class) molecule And helps to stimulate B cells to enable the production of antibodies or help other T cells function. CD4 + T _H cells can be further classified as T _H 1 and T _H 2 based on the cytokines they produce. T _H 1 cells in mice (mouse) is Interleukin -2 (IL-2), interferon - T _H 2 cells, while the secretion of gamma (IFN-γ), etc., IL-4, IL-5, IL-6 , IL-9, IL-10, IL-13 and the like. However, the production of IL-2, IL-6, IL-10 and IL-13 in humans is not strictly classified. T _H 1 cells are involved in cellular immune responses and activate cytotoxic and inflammatory responses. T _H 2 cytokines produced from cells in promoting antibody formation, especially helping to produce IgE because of enhancing the proliferation and function of eosinophils, T _H 2 cytokines is commonly found in antibody production and an allergic reaction. It has been shown that T _H 1 and T _H 2 cytokines can inhibit hepatotoxicity and can alter the progression of the disease with anti-IL-4 and anti-IFN-γ antibodies in animal infectious diseases. In patients with rheumatoid arthritis In addition, IFN-γ has been shown to improve symptoms (Richard A. Goldsby, et al., KUBY Immunology . 2000).

Of the methods used to treat malignant tumors, chemotherapeutic agents other than surgery or radiation therapy are collectively referred to as anticancer drugs, and most of them exhibit anticancer activity by inhibiting the synthesis of nucleic acids.

Cancer treatments such as surgery, chemotherapy, and radiation have been studied until now, and 5-fluorouracil (5-FU) is a water-soluble fluorinated pyrimidine analog as an anti-tumor agent. (Kim NH, Kim SN, Oh JS, Lee S, Kim YK.Anti-mitotic potential of 7-diethylamino-3 (2′-benzoxazolyl) -coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620 / 5-FU.Biochem Biophys Res Commun 2012; 418: 616-21; PMID: 22301191; DOI: 10.1016 / j.bbrc.2012.01.049.). This includes liver cancer, stomach cancer, colon cancer, pancreatic cancer, breast cancer, etc. The same solid cancer alone or in combination with leucovorin (LV) is used for cancer treatment and has a plasma half-life of 10-20 minutes in systemic circulation (Kanetaka K, Enjoji A, Furui J, Nagata Y, Fujioka H, Shiogama T, et al. Effects of Intermittent 5-Fluorouracil and Low-dose Cisplatin Therapy on Advanced and Recurrent Gastric Cancer.Anticancer Res 2012; 32: 3495-9; PMID: 22843936.) May cause side effects such as bone marrow weakness, gastrointestinal reactions, leukopenia and thrombocytopenia (Wettergren Y, Carlsson G, Odin E, Gustavsson B. Pretherapeutic uracil and dihydrouracil levels of colorectal cancer patients are associated with sex and toxic side effects during adjuvant 5-fluorouracil-based chemotherapy. Cancer 2012; 118: 2935-43; PMID: 22020693; DOI: 10.1002 / cncr.26595.).

Although the efficacy of medicinal herbs has been mentioned in past oriental medicine books such as Dongbok-bong, the information in the literature is not associated with the current specific diseases or symptoms. In addition, it is not known which of the components of the herbal medicine has a direct effect. Among the herbal medicines listed in the consent agreement and Chinese medicine metabolism, the Chinese herbal medicines that are known to have an immune enhancing effect need to be verified by modern scientific research.

Puerariae Radix (葛根; Puerariae Radix) is a kudzu (Pueraria vines and beans (Leguminosae) It is the root of lobata (Willd.) Ohwi.), grows in the foothills of Korea, and is distributed in Japan and China (Bae Gi-hwan, Korean medicinal plant, Kyohaksa, p.260, 2000). The components of the root roots contain saponin and starch such as flavonoid compounds such as puerarin, daizein, and daidzin, and soyasaponin. It is known to be reorganized by the Pharmacognomy School Reorganization Committee, Pharmacognosy, Dongmyeongsa, p. 100-102, 2006.

In the oriental medicine, the temper of the drug is persimmon, kidney, and quantity, and it is returned to the stomach and stomach, and the regression of anatomy and the shot margin Efficacy in the appearance of vulgaris, Saengjingegal, and Ascendant Branches. External manifestations, marginal imperfections, febrile calculus, and heat stroke Iii), but it is being used for the treatment of non-hybrid diarrhea (Korea Herbal Medicine School Association, Herbology, Korean Pharmacy Society, pp.107-110, 1995). No disclosure has been made of the effect.

Therefore, the present inventors of the present invention not only show strong anticancer activity in colon cancer such as CT-26 cells, but also when co-administered with existing anticancer agents such as 5-FU and LV in cancer xenograft animal model experiments. The present invention was completed by confirming increased tissue growth inhibition and the production of immune modulators MHC II, CD80, CD86, IL-12, CD4 and CD8.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of cancer diseases containing the extract of Puerariae Radix as an active ingredient.

The present invention also provides an anticancer adjuvant composition for treating and preventing cancer diseases containing the extract of Puerariae Radix as an active ingredient.

The present invention provides a dietary supplement for improving and preventing cancer diseases containing the extract of Puerariae Radix as an active ingredient.

As defined herein, “cancer disease” includes colon cancer, prostate cancer, breast cancer, cervical cancer, colon cancer, leukemia, small intestine cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, gastric cancer, kidney cancer, uterine cancer, lung cancer, lymph gland cancer, thyroid cancer, Skin cancer, brain tumor, bladder cancer, ovarian cancer, or gallbladder cancer, preferably colon cancer, prostate cancer, breast cancer, cervical cancer, colon cancer, most preferably colon cancer.

"Extract" as defined herein includes water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably extracts soluble in water.

In another aspect, the present invention relates to a pharmaceutical composition for preventing and treating cancer diseases, including Puerariae Radix extract, and a method for treating the disease using the extract.

In another aspect, the present invention provides a pharmaceutical composition for the treatment and prevention of cancer diseases containing a extract of Puerariae Radix and an anticancer agent as an active ingredient.

Pharmaceutical dosage forms of the extracts of the present invention may be used alone or in pharmacologically active compounds, for example, existing anticancer agents well known in the art, namely 5-FU (Fluorouracil), LV (leucovorin), adriamycin , Cyclophosphamide, amsacrine, donomycin, taxol, and the like, preferably in combination with existing anticancer agents such as 5-FU (Fluorouracil) and LV (leucovorin). In addition, it can be used as an anticancer adjuvant for inhibiting the growth of cancer cells having multi-drug resistance (MDR) to the existing anticancer agents in a suitable combination.

Therefore, the present invention is a metabolism of the extract and other anticancer agents, preferably folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Cytarabine) used in conventional anticancer therapy Antagonists (antimetabolites); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; Platinum-containing anticancer agents such as cisplatin; more preferably at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, taxol, preferably Antimetabolites such as folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine) and pyrimidine derivatives (5-fluorouracil, Cytarabine); More preferably, a combination ratio of maximizing pyrimidine derivatives (5-fluorouracil, Cytarabine) and anticancer activity, for example, the weight combination ratio of the existing anticancer agent and the root extract is 1: 0.1 to 10 (w / w) It provides a pharmaceutical composition combined in the combination ratio of.

Therefore, the present invention is an antatabolites such as the root extract and folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, Cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer drugs such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine, vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Hormones such as corticosteroids and progesterone; A platinum-containing anticancer agent such as cisplatin; more preferably a combination with at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, taxol An anticancer agent for inhibiting growth of cancer cells as an active ingredient is provided.

The root extract of the present invention can be prepared by the following manufacturing process.

Dried roots may be prepared according to a conventional extraction method, C ₁ to C ₄ lower alcohols such as water, methanol, etc., 1 to 20 times, preferably 5 to 15 times the volume of dried dry roots, or these Mixed solvent, preferably water, 0.5 to 10 hours, preferably 2 to 5 hours, 70 ℃ to 100 ℃, 1 to 10 times, preferably 2 to 5 times repeated cold extraction, hot water extraction In the extraction method, such as ultrasonic extraction and reflux cooling extraction, preferably hot water extraction, the extract is filtered under reduced pressure, and then the filtrate is concentrated and lyophilized to obtain the root extract of the present invention.

The present invention provides a pharmaceutical composition for the treatment and prevention of cancer diseases containing the extract of the root (Puerariae Radix) obtained by the above production method as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for adjuvant anticancer agent for the prevention and treatment of cancer disease containing the extract of Puerariae Radix obtained by the above method as an active ingredient.

The pharmaceutical composition of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

The pharmaceutical composition of the present invention not only exhibits strong anticancer activity in colon cancer such as CT-26 cells, but also inhibits cancer tissue growth and immune regulation upon co-administration with existing anticancer agents such as 5-FU and LV in cancer xenograft animal model experiments. It can be used for the prevention and treatment of cancer diseases by confirming that it increases the production of the substances MHC II, CD80, CD86, IL-12, CD4 and CD8.

The pharmaceutical composition containing the extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical compositions containing extracts according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Carriers, excipients and diluents which may be formulated and used in the composition comprising the extract 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for preventing and improving cancer disease containing the root extract.

Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for anticancer effects. At this time, the amount of the extract in the food or beverage is generally added to the dietary supplement composition of the present invention to 0.01 to 50% by weight, preferably 0.01 to 15% by weight of the total food weight, the health beverage composition is 100 ml It can be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on the amount.

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health beverage composition of the present invention is not particularly limited in the other components except the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extract of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The extract of Puerariae Radix of the present invention not only shows potent anticancer activity in colon cancer such as CT-26 cells, but also grows cancerous tissue when co-administered with existing anticancer agents such as 5-FU and LV in animal xenograft animal model experiments. It has been shown to increase the production of inhibitors and immune modulators MHC II, CD80, CD86, IL-12, CD4 and CD8, which can be used as an anticancer or anticancer and health supplement for the prevention, inhibition and treatment of cancer diseases. have.

1 is a diagram showing the effect of the root extract on tumor volume, body weight and spleen weight in CT-26 tumor-xenograft mice (weight (A), tumor volume (B), and during the test period) Spleen weight (C) at the end of the test, Fluoruuracil, 5-FU; leucovorin, LV),
Figure 2 is a diagram showing the effect on the expression of immunomodulators of the root extract (weight (A), tumor volume (B), and spleen weight (C) after the end of the test, Fluoruuracil, 5-FU ; represents leucovorin, LV).

Below, The present invention will be described in detail by the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example  One. Changwon  Preparation of extract

After drying the roots (USE TECHNO CORPORATION, Korea) naturally, finely chopped, 3 liters of distilled water was added to 1 kg of dried roots, and repeated three times of heating extraction at 100 ° C. for 3 hours, followed by vacuum filtration to obtain a filtrate. The solution was concentrated under reduced pressure (EYELA, N-1000, Japan) and lyophilized to obtain 15.8 g of the root extract. (Hereinafter referred to as WK6-2S).

Reference Example  1. Cell Culture, Reagents and Reagents

The sample mouse carcinoma cell line (Mouse colon carcinoma cell line, CT-26 cells) used in this experiment was purchased from the company (Korean Cell Line Bank, KCLB; Seoul, Korea) to obtain 10% FBS and 1% penicillin / streptomycin. participated in DMEM and incubated under 5% CO _2, 37˚C conditions. Cell culture media and reagents were purchased from the company (Thermo Scientific Hyclone, Waltham, Mass., USA) and DMEM and FBS were purchased from the company (GibcoBRL, Grand Island, NY, USA). Lipopolysaccharide (LPS), dimethylsulfoxide (DMSO) and polymyxin B sulfate (PMB) were purchased from Sigma, St. Louis, MO, USA, and the kit (enzyme-linked immunosorbentassay (ELISA) kit for TNF-α) (R & D Systems, Minneapolis, MN, USA) The Easy BlueTotal RNA Extraction kit was purchased from the company (iNtRON Biotechnology, Inc, Seoul, Korea), PD98059, SB202190, SP600125 and PDTC (Pyrrolidine dithiocarbamate) Calbiochem, La Jolla, CA, USA). Enzymes (Polyclonal antibodies against phospho-JNK MAP kinase, JNK MAP kinase, phospho-ERK MAP kinase and ERK MAP kinase) were purchased from the company (Cell Signaling, Beverly, MA, USA) and antibodies (Polyclonal antibodies against phospho-p38 MAP) kinase, p-38 MAP kinase, NF-κB, IκBα, and HRP-conjugated anti-rabbit and anti-mouse IgGs) were purchased from Santa Cruz Biotechnology Inc, California, USA. Antibodies for flow cytometry (Antibodies, APC Rat anti mouse CD86, PE Hamster Anti-mouse CD80, PE Rat Anti-mouse IL-12 (p40 / p70), PE Rat Anti mouse IFN-γ, APC Rat Anti-mouse CD4 and PE-CyTm7 Rat Anti-mouse CD8a) is the company (BD Biosciences, San Jose, CA, USA) antibody (Anti-mouse F4 / 80 PECy7 and Fitc-Conjugated, Anti mouse MHC Class II) is the company (eBioscience, San Diego) , CA, USA, the reagent (Fluoruuracil, 5-FU) is a company (Ildong Pharmaceutical Co., Ltd., Seocho-Gu, Seoul, Korea) and the reagent (leucovorin, LV) is a company (Samhinpharm Co.Ltd., Mapo-Gu, Seoul, Korea).

Reference example  2. Statistical processing

The experimental results are expressed as the mean of three results (mean ± SD) of three individual experiments, and the statistical significance is tested by one way analysis of variance (ANOVA) and Student's t test, and the p-value is 0.05. The following cases were recognized as significant.

Experimental Example  One. Cancer  Anti-cancer experiments in animal models

In order to confirm the anticancer effect in the cancer xenograft animal model experiment of the sample of the example was tested by applying the method described in the literature (Holland EC (2004) Mouse models of human cancer. Wiley-Liss, New York; Loganayaki N, Manian S. Antitumor activity of the methanolic extract of Ammannia baccifera L. against Dalton's ascites lymphoma induced ascitic and solid tumors in mice.J Ethnopharmacol. 2012 Jun 26; 142 (1): 305-9; Lee B, Lee DY , Yoo KH, Baek NI, Park JH, Chung IS.Calenduloside E 6'-methyl ester induces apoptosis in CT-26 mouse colon carcinoma cells and inhibits tumor growth in a CT-26 xenograft animal model.Oncol Lett. 2012 Jul; 4 (1): 22-28

The effects on oriental growth, body weight and lymphocyte count in CT-26 xenograft mice were examined as follows.

Mice (Female Balb / c mice, 6 weeks, Central Laboratory Animal Inc., Seoul, Korea) were tested for temperature (22 ± 1 ° C), relative humidity (55 ± 1%) and 12 hours day-night Under conditions with a controlled cycle, they were raised as standard pellets and comply with the Institutional (Wonkwang University) Animal Care and Use Committee.

To establish an xenograft colon carcinoma animal model, cells (5 × 10 ⁵ CT-26 cells) in 200 μL PBS were injected into the right lobe of the mouse (BALB / cmice). Incubated for 7 days until tumors were seen and divided into 5 groups of 10 animals each. Individual groups were treated as follows: (a) Normal (no treatment and no cancer), (b) WK6-2S alone (50 mg / kg / dose), (c) 5-FU and LV Mixed dose group (36 mg / kg / dose; 18 mg / kg / dose 5-FU and 18 mg / kg / dose LV), (d) 5-FU and WK6-2S mixed dose group (68 mg / kg / dose; 18 mg / kg / dose 5-FU and 50 mg / kg / dose WK6-2S) and (e) 5-FU, LV and WK6-2S mixed dose groups (86 mg / kg / dose; 18 mg / kg / dose 5- FU, 18 mg / kg / dose 5-FU and 50 mg / kg / dose for WK6-2S) were dissolved in distillation and administered orally using garbage (3 hours ago). 5-FU and LV or 5-FU were treated once per week for three weeks and WK6-2S was treated three times per week for three weeks. Tumor volume was measured once a week with a caliper and calculated according to Equation 1 below.

Where length is the maximum tumor diameter and width is the minimum tumor diameter (Alessandria G, Filippeschi S, Sinibaldi P, Mornet F, Passera P, Spreafico F, Cappa PM and Gullino PM: Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells.Cancer Res 47: 4243-4247, 1987.). Body weight was measured twice per week.

At the end of the experiment, the next day of fasting overnight, blood was taken (dorsal vein) and used for lymphocyte measurements. Blood was analyzed by a reader (Fully Automated Blood Cell Counter LC-660 (HORIBA, Ltd .; Minami-ku, Kyoto, Japan)). It was.

As a result, there was no significant weight change in all groups (FIG. 1A), and in the blood analysis, the number of lymphocytes in the control group was lower than that in the normal group (FIG. 1B), and in the CT-26 tumor-xenograft mice 5- FU and LV mixture group showed a sharp decrease in lymphocytes (FIG. 1B). The group administered with WK6-2S in the 5-FU or 5-FU and LV mixtures showed a higher number of lymphocytes than the 5-FU alone or 5-FU and LV mixtures (FIG. 1B). CT-26 tumor-xenograft mice showed increased tumor size during the trial (FIG. 1C) and 5-FU and LV treatment groups showed smaller tumor sizes than untreated CT-26 tumor-xenograft mice (FIG. 1C). ). The WK6-2S treatment also showed a significantly reduced tumor size as compared to the tumor control group or 5-FU and LV administration (FIG. 1C). In particular, the mixed dose group of 5-FU and WK6-2S showed the most reduced cancer size (FIG. 1C).

Experimental Example 2. Flow cytometry experiment of immunomodulators

In order to confirm the effect on the immunomodulators of the sample of the above example, the experiment was performed by applying the flow cytometry method described in the literature (Chirasani SR, Leukel P, Gottfried E, Hochrein J, Stadler K, Neumann B, Oefner). PJ, Gronwald W, Bogdahn U, Hau P, Kreutz M, Grauer OM.Diclofenac inhibits lactate formation and efficiently counteracts local immune suppression in a murine glioma model.Int J Cancer. 2012. doi: 10.1002 / ijc.27712)

After grinding and filtration of the spleen, the cells are reached to reach 95% pure the cell population when measured on an analyzer (FACS Calibur, BD Biosciences; San Jose, Calif., USA) (cell isolation kit, MACS, Miltenyi Biotec, Germany). Purified cells were incubated with PMA (20 ng / mL), ionomycin (500 ng / mL) and brefeldin (1 mg / mL) at 37 ° C for 2 hours (2 x 10 ⁵ ). After collecting the cells on ice, the cells were centrifuged at 1,200 rpm for 5 minutes, ice-cold PBS was added and again centrifuged at 1200 rpm for 5 minutes. After suspending in 0.1 mL PBS, the cells were quenched at 4 ° C for 20 min at 0.25 μL individual cytochromes, 30 μL FACS supernatant and 0.5 μL individual antibodies (MHC II, CD80, CD86, IL-12, F4 / 80). , IFN-γ, CD4 and CD8). After washing with PBS, cells were suspended in 1 mL PBS and prepared under ice cooling with 2 × 10 ⁶ cells. An equal volume of 1% formaldehyde (formaldehyde in PBS) was added to the cells and left at 4 ° C. for 20 minutes. Cells were analyzed by flow cytometry. Three weeks later, splenocytes were prepared from tumor-induced mice and purified cells were analyzed by flow cytometry.

In vivo assay ( in vivo ) To determine the role of WK6-2S on the ability to produce immunomodulators (MHC II, CD80, CD86, IL-12, CD4 and CD8), cells from CT-26 tumor- xenograft mouse spleens were analyzed by flow cytometry. . Splenocytes were prepared from WK6-2S treated and untreated mice.

As shown in FIG. 2, the MHCII frequency of the normal group decreased when compared to untreated CT-26 tumor-xenograft mice. MHCII frequencies in the 5-FU and LV treatment groups were similar to cancer groups. Addition of WK6-2S to 5-FU alone or into the 5-FU and LV combination groups showed increased MHCII frequency when compared to untreated CT-26 tumor-xenograft mice.

In the frequency of CD80, 5-FU and LV treatment group showed lower reduction than cancer groups, and WK6-2S treatment group among 5-FU showed much higher frequency than cancer groups. .

In the frequency of CD86, 5-FU and LV treatment group showed lower reduction than cancer groups, and WK6-2S treatment group among 5-FU showed a much higher frequency than cancer groups. .

In terms of IL-12 frequency, the WK6-2S treatment group in 5-FU showed a much higher frequency.

The CD4 frequency was slightly decreased in cancer groups, but the WK6-2S treatment group in 5-FU showed a much higher frequency.

Experimental Example 3. Oral Toxicity Test

In this experiment, 7-week-old ICR mice (Oriental Bio, Japan) were used to fast the mice for 4 hours before administering the root extract, weigh them, and measure the toxicity standards of the Korea Food and Drug Administration (KFDA, Drugs, etc.). According to Toxicity Test Criteria, Korea Food & Drug Administration, 2005-60, 2005), the highest dose was set to 2,000 mg / kg, and diluted with purified distilled water to obtain 5 mg / kg, 50 mg / kg and 500 mg / kg of test substance. Prepared. The administration was made by oral administration, which is the main route of application in the clinic. The method of administration was as follows: The animal was fixed by the adriamycin fixation method and then injected directly into the stomach using a metal oral administration unit. Once daily, 10 ml / kg body weight was administered as a dose, and the feed was restrained for 2 hours after administration of the test substance to the test animals, and the feed and water were continuously supplied during the test period.

In accordance with the Toxicological Testing Standards of the Korea Food and Drug Administration and the guidelines of the International Organization for Economic Cooperation and Development (OECD) Guidelines for the Testing Chemicals revised draft guideline 420 (OECD, 2001) During the first 6 hours, with special attention, the test animal's skin, hair, eyes, mucous membrane, respiratory system, circulatory system, autonomic nervous system, central nervous system, physical activity, behavioral form and tremors of test animals, convulsions, We observed the presence of salivation, diarrhea, lethargy, sleep, and coma. General clinical symptoms were observed once a day for 14 days after administration, and the individual body weights of the test animals were measured before and after administration of the test substance and once a day during the test period.

ICR mice were anesthetized with ethyl ether on the 14th day after the injection, and all the organs were removed by bled lethal injection to observe gross lesions of individual test animals and weigh the organs.

As a result of acute toxicity test by single oral administration of the extract of the root extract which shows anticancer effect, no death was observed in the test animals used in this study and at the highest dose of 2,000 mg / kg administered under the conditions. Toxicity by the administration of the root extract was not induced in all the indicators observed according to the Toxicological Standards of the Korea Food and Drug Administration. In conclusion, it was confirmed that the root extract was safe without toxicity even at the recommended maximum dose of 2,000 mg / kg.

One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.

Formulation example  One. Sanje  Produce

Carrot Extract --------------------------- 20 mg

Lactose --------------------------------- 100 mg

Talc ---------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Carrot Extract --------------------------- 10 mg

Corn Starch --------------------------- 100 mg

Lactose --------------------------------- 100 mg

Magnesium Stearate -------------------- 2mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Carrot Extract --------------------------- 10 mg

Crystalline Cellulose ---------------------- 3 mg

Lactose ---------------------------- 14.8 mg

Magnesium Stearate ---------------- 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

Carrot Extract --------------------------- 10 mg

Mannitol ------------------------------- 180 mg

Sterile Distilled Water for Injection ------------------ 2974 mg

Na ₂ HPO ₄ 12H ₂ O ------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

Carrot Extract --------------------------- 20 mg

Isomerized sugar ------------------------------- 10 g

Mannitol ---------------------------------- 5 g

Purified Water --------------------------------- Proper

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

Brown root extract --------------------------- 1000 mg

Vitamin Mixture ---------------------------- Proper

Vitamin A Acetate --------------------- 70 μg

Vitamin E ------------------------------- 1.0 mg

Vitamin B ₁ ------------------------------ 0.13 mg

Vitamin B ₂ ------------------------------ 0.15 mg

Vitamin B ₆ ------------------------------- 0.5 mg

Vitamin B ₁₂ ------------------------------ 0.2 μg

Vitamin C -------------------------------- 10 mg

Biotin ---------------------------------- 10 μg

Nicotinamide ------------------------- 1.7 mg

Folic acid ------------------------------------ 50 μg

Calcium Pantothenate -------------------------- 0.5 mg

Mineral mixture ---------------------------- suitable

Ferrous Sulfate ----------------------------- 1.75 mg

Zinc Oxide ------------------------------ 0.82 mg

Magnesium Carbonate -------------------------- 25.3 mg

Potassium monophosphate ----------------------------- 15 mg

Dibasic calcium phosphate ----------------------------- 55 mg

Potassium Citrate ------------------------------ 90 mg

Calcium Carbonate ------------------------------- 100 mg

Magnesium Chloride -------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Carrot Extract --------------------------- 100 mg

Vitamin C -------------------------------- 15 g

Vitamin E (powder) ------------------------- 100 g

Iron Lactate ------------------------------- 19.75 g

Zinc Oxide ------------------------------- 3.5 g

Nicotinamide ------------------------- 3.5 g

Vitamin A ------------------------------- 0.2 g

Vitamin B ₁ ------------------------------ 0.25 g

Vitamin B ₂ ------------------------------- 0.3 g

Water -------------------------------------- Quantitative

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (8)

A pharmaceutical composition for the treatment and prevention of cancer diseases containing the extract of Puerariae Radix as an active ingredient. A pharmaceutical composition for the treatment and prevention of cancer diseases, comprising as an active ingredient a combination of the extract of Puerariae Radix and an existing anticancer agent. 3. The method of claim 2,
The existing anticancer agent is a pharmaceutical composition of at least one or more anticancer agents selected from adriamycin (adriamycin), cyclophosphamide (cyclophosphamide), 5-FU, amsacrine, or taxol. 3. The method according to claim 1 or 2,
The extract is a pharmaceutical composition that is an extract available in a solvent selected from water, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof. 3. The method according to claim 1 or 2,
The cancer is preferably selected from the group consisting of colon cancer, prostate cancer, breast cancer, cervical cancer, colon cancer, leukemia, small bowel cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, stomach cancer, kidney cancer, uterine cancer, lung cancer, , Ovarian cancer, or gallbladder cancer. An anticancer agent composition for preventing and treating cancer diseases containing the extract of Puerariae Radix as an active ingredient. Puerariae Radix Health functional food for cancer disease prevention and improvement containing the extract as an active ingredient. 8. The dietary supplement of claim 7, wherein the dietary supplement is in the form of a powder, granule, tablet, capsule or beverage.

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