KR102085774B1 - Pharmaceutical composition for prevention or treatment of colon cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer, which contains an extract of a mixture of Injinho, Cordyceps sinensis, and sterling silver as an active ingredient. Specifically, the extract of the mixture of Injinho, Cordyceps sinensis and Geumhwahwa of the present invention has a superior colon cancer cell proliferation inhibitory effect, and has an excellent antioxidant activity as compared to the extract of Chinese herbal medicine, antioxidant, induction of apoptosis, cancer suppressor protein Inhibition of cell proliferation through increased expression of phosphorus p21 and activation of p53 and inhibition of tumor formation through antioxidant activity in a colon cancer mouse model can be usefully used as a composition for preventing, treating or improving colon cancer.

Description

Pharmaceutical composition for prevention or treatment of colon cancer}

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer, which contains an extract of a mixture of Injinho, Cordyceps sinensis, and sterling silver as an active ingredient.

Colorectal cancer is a refractory disease that causes about 1 million cases worldwide and 530,000 deaths annually. It is estimated that about 100,000 people will be diagnosed with colorectal cancer in the United States in 2010 and about 50,000 will die. Recently, due to westernized eating habits, the incidence rate in Asia is increasing, and in Korea, the incidence rate is rapidly rising to 3rd place with 12.7% of all cancers. Colorectal cancer is associated with genetic predisposition and dietary factors, lifestyles such as smoking or drinking or physical activity, and metabolic diseases such as obesity and insulin resistance. The major causes of colorectal cancer in relation to dietary factors include excessive animal fats, sugars, alcohol consumption and lack of fiber, antioxidant vitamins, vegetables and fruits.

Apoptosis is active death caused by the regulation and expression of several genes and proteins in response to a programmed signal inside the cell. Apoptosis plays an important role in the processes of various morphological changes in the early developmental stages of life and in the functional self-organization of the immune or nervous system, regulating cell numbers to maintain homeostasis, removing damaged cells, and acting as a defense against infection. do. However, such abnormal suppression of apoptosis may cause cancer, and inducing apoptosis of cancer cells becomes a major mechanism of action in which most anticancer drugs exhibit cancer cell proliferation inhibitory effect.

Reactive oxygen species (ROS) or reactive oxygen species are chemically reactive molecules, including oxygen atoms, and are highly reactive due to unpaired electrons. Free radicals are naturally released by the normal metabolism of oxygen and play an important role in cell signaling and homeostasis. Cells have genes that produce free radicals and antioxidant genes that effectively remove them in order to regulate the homeostasis of free radicals, and have sophisticated mechanisms to regulate them. On the other hand, cancer proceeds in three stages: early progression, proliferation, and metastasis. The active oxygen causes DNA damage or abnormal gene expression in the early stage of cancer, and abnormal gene expression and intercellular signaling changes during the proliferation stage. Induces and participates in cancer progression by regulating more gene expression in the metastasis stage. Therefore, removing free radicals may also be a target of anticancer drug development.

Thus, the inventors of the present invention, while developing a colorectal cancer treatment using a safe natural product used for a long time, the extract of Injinho, Cordyceps sinensis and gold and silver coin mixture according to the present invention is excellent in inhibiting the growth of colorectal cancer cells compared to each herbal extract alone It has excellent antioxidant activity, inhibits cell proliferation through antioxidant, induction of apoptosis, increased expression of p21, a cancer suppressor protein, and activation of p53, and tumor formation through antioxidant activity in colorectal cancer mouse model. This invention was completed by confirming that it suppresses.

Republic of Korea Patent No. 10-1834485

 Inpyo Choi, Hanyang Med Rev, 33, 118-122, 2013.

An object of the present invention is to provide a composition for the prevention, treatment or improvement of colorectal cancer containing the extract of the mixture of Injinho, Cordyceps sinensis and gold and silver coin as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of colorectal cancer containing the extract of the mixture of Injinho, Cordyceps sinensis and gold and silver coins as an active ingredient.

In addition, the present invention provides a health functional food for the prevention or improvement of colorectal cancer containing the extract of the mixture of Injinho, Cordyceps sinensis and gold and silver coin as an active ingredient.

The extract of the mixture of Injinho, Cordyceps sinensis and Gold and Silver Coin of the present invention has superior colorectal cancer cell proliferation inhibitory effect and superior antioxidant activity as compared to the extract of herbal medicine alone, and has an antioxidant, apoptosis induction, and cancer suppressor protein of p21. By inhibiting cell proliferation through increased expression and activation of p53, and inhibiting tumor formation through antioxidant activity in a colon cancer mouse model, it can be usefully used as a composition for preventing, treating or improving colon cancer.

1 is a graph comparing the effects of inhibiting colon cancer cell proliferation of the extract of Injinho, Geummihwa and Cordyceps sinensis, two kinds of mixtures and three kinds of mixtures.
Figure 2 is a graph showing the effect of inhibiting colon cancer cell proliferation of the extract (FDY003) of Injinho, Geummihwa and Cordyceps sinensis.
Figure 3 is a graph of the results of measuring the DPPH radical scavenging ability of the extract (FDY003) and gallic acid (FDY003) of Injinho, Geummihwa and Cordyceps sinensis.
4 is a graph showing the results of DPPH radical scavenging ability of colorectal cancer cells (A) and extracellular cells (B) of the extracts (FDY003) of Injinho, Geummihwa and Cordyceps sinensis mixtures.
5 is a result graph showing the lipid peroxidation inhibitory effect of colon cancer cells extract (FDY003) of Injinho, Geummihwa and Cordyceps sinensis.
FIG. 6 shows the effects of increased Bax expression (A), caspase-3 activation (B), p21 expression (C) and p53 activation induction (D) effects of the extract of Injinho, Geummi and Cordyceps sinensis (FDY003) on colorectal cancer cells The results of analysis by Western blot are shown (A to D: graph quantifying the band intensities of each factor; E: representative blot image).
Figure 7 is a graph of the results of measuring the tumor size of colorectal cancer mice.
8 is a graph showing the result of measuring the tumor size of colorectal cancer mice.
9 is a graph showing the results of measuring lipid peroxidation levels in colorectal cancer mice.
10 is a graph showing the results of measuring DPPH radical scavenging ability of serum of colorectal cancer mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating colorectal cancer, which contains an extract of a mixture of Injinho, Cordyceps sinensis, and gold and silver coins as an active ingredient.

The injinho dried the above ground part of Artemisia capillaris Thungerg belonging to the chrysanthemum, can be used injinjin (茵 蔯 蒿) collected in the spring or Injinho (茵 蔯 蒿) taken in the fall.

The cordyceps can be used for all types of cordyceps known in the art, including the body of the fruiting body and the larvae, Cordyceps sinensis Sacc belonging to parasites of bat moths and insects. Specifically, the Cordyceps Sinensis ( C. sinensis ), Militaris Cordyceps ( C. misinensis ), Red Cordyceps ( C. militaris ), Cicada Cordyceps ( C. sobolifera ), Beetle Cordyceps ( C. sphecocephala ), Yellow Bunch C. bassiana or Paecilomyces japonica may be used.

The gold and silver coins can be used to dry the buds or flowers that have just started to bloom of Lonicera japonica Thunberg belonging to the genus Phloxaceae.

The extract may be prepared by a manufacturing method comprising the following steps:

1) preparing an extract by adding an extraction solvent to Injinho, Cordyceps sinensis and Geumhwahwa mixture;

2) filtering the extract of step 1); And

3) drying the filtered filtrate of step 2) under reduced pressure.

The extractant may be water, alcohol or a mixture thereof. The alcohol may be C ₁ to C ₄ lower alcohol, specifically, the lower alcohol may be ethanol or methanol. The extraction solvent may be added in an amount of 1 to 50 ml per 1 g of the total weight of the herbal medicine used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 1 to 50 hours, 10 to 40 hours or 15 to 30 hours. The extraction may be repeated 3 to 5 times. According to an embodiment of the present invention, the repeated extraction is performed using 70% ethanol in step 1), 80% ethanol is added to the concentrate of step 3), followed by filtration and concentration, and 90% of the concentrate. Filtration and concentration after the addition of% ethanol, and may be made of the step of filtration and concentration after adding purified water to the concentrate.

On the other hand, the reduced pressure concentration of step 3) may be a vacuum pressure reducer or a vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, specifically, may be freeze drying.

The extract is preferably extracted by mixing injinho, Cordyceps sinensis and gold and silver coins in a weight ratio of 0.1 to 20: 0.1 to 20: 1, more preferably can be extracted by mixing in a weight ratio of 0.5 to 4: 0.5 to 4: 1 Most preferably, it can extract by mixing in the weight ratio of 1-2: 1: 1-2: 1. In addition, the extract may be prepared by mixing the medicinal extracts, or by mixing the respective medicinal extracts.

The extract is root, supernatant, licorice, health, Guernsey, dried, missing, Gyeji, Goyanggang, Gosam, Gyo, Gujeolcho, Dansam, Angelica, Dangsam, Contrast, Guin, Tofu, Ephedra, Mansam, Magmun-dong, Neck barrel, Myrrh, Peppermint, halved, Baekduong, Bokbunja, Rich, Loquat leaf, Casualties, Sancho, Situation mushroom, Ginger, Gypsum, Seonhwa, Sessin, Wheat, Sokdan, Sukjihwang, Siho, Ogapi, Ogong, Osuyu, Keel, Emulsion, Yubaekpi, Broiler , Nutmeg, ginseng, jasmine herb, jasmine leaf, peony, earthly, fruit room, celestial organ, cheongho, incense stick, taklan, pogongyoung, haechocho, lower lobe, nasturtium, passerby, hyuncho, hedgehog, hodo, red ginseng, safflower, safflower , May be further mixed with one or more selected from the group consisting of hungry.

In a specific embodiment of the present invention, the present inventors prepared the extract (FDY003) of the mixture of Injinho, Cordyceps sinensis and gold and silver coin, and the FDY003 has a superior colon cancer cell proliferation inhibitory effect compared to each herbal extract alone (see Fig. 1). ), Has excellent antioxidant activity, inhibits cell proliferation through antioxidant, induction of apoptosis, increased expression of p21, a cancer suppressor protein, and activation of p53 in colorectal cancer cells (see FIGS. 2 to 6), and in a mouse model of colorectal cancer Tumor production was inhibited through antioxidant activity (see FIGS. 7 to 10). Therefore, FDY003 can be usefully used for the prevention or treatment of colorectal cancer.

The pharmaceutical composition according to the present invention may include 10 to 95% by weight of the extract of Injinho, Cordyceps sinensis, and gold and silver mixture, which are active ingredients, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredient.

The pharmaceutical compositions of the present invention may comprise carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivery of the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added as necessary.

When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used may be added.

The composition of the present invention may be formulated as an oral or parenteral preparation. Oral formulations may include solid and liquid formulations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, for example magnesium styrate, talc and the like. On the other hand, the liquid formulation may be a suspension, a liquid solution, an emulsion or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives and the like.

The parenteral preparations may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams, and the like. The injection may include a sterile aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. In this case, as the non-aqueous solvent or the suspension solvent, vegetable oil such as propylene glycol, polyethylene glycol, olive oil, or injectable ester such as ethyl oleate may be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, the drug being used simultaneously, and the like. However, for the desired effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration can be from one to several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous.

In addition, the present invention provides a health functional food for the prevention or improvement of colorectal cancer containing the extract of the mixture of Injinho, Cordyceps sinensis and gold and silver coin as an active ingredient.

The injinho dried the above ground part of Artemisia capillaris Thungerg belonging to the chrysanthemum, can be used injinjin (茵 蔯 蒿) collected in the spring or Injinho (茵 蔯 蒿) taken in the fall.

The cordyceps can be used for all kinds of cordyceps known in the art as described above, including the body of the fruiting body and the larva, which Cordyceps sinensis Sacc is a parasite of bat moths and insects grew. .

The gold and silver coins can be used to dry the buds or flowers that have just started to bloom of Lonicera japonica Thunberg belonging to the genus Phloxaceae.

The extract may be prepared by the preparation method as described above.

The extract is preferably extracted by mixing injinho, Cordyceps sinensis and gold and silver coins in a weight ratio of 0.1 to 20: 0.1 to 20: 1, more preferably can be extracted by mixing in a weight ratio of 0.5 to 4: 0.5 to 4: 1 Most preferably, it can extract by mixing in the weight ratio of 1-2: 1: 1-2: 1. In addition, the extract may be prepared by mixing the medicinal extracts, or by mixing the respective medicinal extracts.

The extract is root, supernatant, licorice, health, Guernsey, dried, missing, Gyeji, Goyanggang, Gosam, Gyo, Gujeolcho, Dansam, Angelica, Dangsam, Contrast, Guin, Tofu, Ephedra, Mansam, Magmun-dong, Neck barrel, Myrrh, Peppermint, halved, Baekduong, Bokbunja, Rich, Loquat leaf, Casualties, Sancho, Situation mushroom, Ginger, Gypsum, Seonhwa, Sessin, Wheat, Sokdan, Sukjihwang, Siho, Ogapi, Ogong, Osuyu, Keel, Emulsion, Yubaekpi, Broiler , Nutmeg, ginseng, jasmine herb, jasmine leaf, peony, earthly, fruit room, celestial organ, cheongho, incense stick, taklan, pogongyoung, haechocho, lower lobe, nasturtium, passerby, hyuncho, hedgehog, hodo, red ginseng, safflower, safflower , May be further mixed with one or more selected from the group consisting of hungry.

In a specific embodiment of the present invention, the present inventors prepared the extract (FDY003) of the mixture of Injinho, Cordyceps sinensis and gold and silver coin, and the FDY003 has a superior colon cancer cell proliferation inhibitory effect compared to each herbal extract alone (see Fig. 1). ), Has excellent antioxidant activity, inhibits cell proliferation through antioxidant, induction of apoptosis, increased expression of p21, a cancer suppressor protein, and activation of p53 in colorectal cancer cells (see FIGS. 2 to 6), and in a mouse model of colorectal cancer Tumor production was inhibited through antioxidant activity (see FIGS. 7 to 10). Therefore, FDY003 can be usefully used for the prevention or improvement of colorectal cancer.

The composition of the present invention may be added as it is to food, or used with other foods or food ingredients. At this time, the amount of the active ingredient added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of dietary supplement are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The dietary supplement may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include taumartin, stevia extract, and the like. On the other hand, examples of the synthetic sweeteners include saccharin, aspartame, and the like. In addition, the natural carbohydrate may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.

In addition to the above-mentioned additional ingredients, the health functional food of the present invention is a nutrient, vitamin, electrolyte, flavoring agent, coloring agent, pectane and salts thereof, alginic acid and salts thereof, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, and preservative. , Glycerin, alcohol, and the like may be further included. These components can be used independently or in combination. The proportion of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following Examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1 Preparation of Extract of Herbal Medicine Mixture (FDY003)

Buds of dried Artemisia capillaris Thungerg (Injinho), Cordyceps sinensis Sacc, and dried Lonicera japonica Thunberg (gold) were purchased at Gyeongdong Market. 6.25 g of Injinho Lake, 6.25 g of Cordyceps sinensis and 4.16 g of Gold Coated Silver were placed in 500 ml of 70% ethanol and extracted at 85 ° C. for 3 hours. The extract was filtered through a filter paper having a pore size of 1 μm, and the filtrate was concentrated under reduced pressure. 200 ml of 80% ethanol was added to the concentrate, followed by filtration with a filter paper having a pore size of 1 μm, followed by concentration under reduced pressure, and 200 ml of 90% ethanol was added to the concentrate, followed by stirring. After filtration and concentration under reduced pressure. 100 ml of purified water was further added to the concentrate, filtered through a filter paper having a pore size of 1 μm, and once again filtered through a filter paper having a pore size of 0.6 μm. The filtrate was lyophilized to obtain an extract of the final mixture (FDY003).

Comparative Example 1. Preparation of Injinho, Cordyceps sinensis and Gold and Silver Coin Extracts

Injinho, Cordyceps sinensis and Gold and Silver Coin were prepared each 50g and ground. Each extract was prepared in the same manner as described in Example 1 using the powder of each medicine.

Comparative Example 2. Preparation of Extracts of Two Herbal Medicine Mixtures

After pulverizing Injinho, Cordyceps sinensis and Gold and Silver Coin, respectively, 20g of Gold Silver Coin and 30g of Injinho were mixed, 25g of Injinho and 25g of Cordyceps were mixed, and 20g of Goldfinaceae and 30g of Cordyceps was mixed. Extracts of each of the two herbal mixtures were prepared in the same manner as described in Example 1 using the respective herbal mixture powders.

Example 2. in vitro  Preparation of Extract Samples for Experiment

The extract samples obtained in Example 1, Comparative Example 1 and Comparative Example 2 were each dissolved in distilled water at 10 mg / ml, and then filtered through a 0.45 μm polyethersulfone (PES) filter to prepare a highly concentrated stock solution. It was.

Experimental Example 1. Comparison of the Inhibitory Effect of Injinho, Geumunhwa and Cordyceps Sinensis, Extracts from Two Mixtures, and FDY003 on Colorectal Cancer

To investigate whether FDY003 shows an excellent anticancer effect against colorectal cancer, compared to the extracts of Injinho, Geummihwa and Cordyceps sinensis alone extracts and the two mixtures of the above herbal medicines, the extracts of Examples 1, Comparative Example 1 and Comparative Example 2 Proliferation inhibitory effect of human colon cancer cell line (Colo205 cells, Korea Cell Line Bank, Korea) by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltertrazolium) assay.

Specifically, Colo205 cells were cultured in 96-well plates at 1 × 10 ⁶ / well, and each drug prepared in Example 2 was RPMI (Roswell Park) so that the final concentration was 1, 5, 10 or 15 μg / ml. MemoCial Institute) was diluted in 1640 medium and treated with cells. After incubation for 24 hours, MTT solution was added to each well to a final concentration of 5 mg / ml and incubated at 37 ° C. for 4 hours. Thereafter, the medium was removed and DMSO (dimethyl sulfoxide) was added to dissolve formazan, and absorbance was measured using an ELLISA reader. The cell survival rate of each drug treatment group was calculated as a ratio of the absorbance of the control group treated with only the medium containing no drug.

As a result, the extract of Cordyceps sinensis extract showed the highest inhibitory effect on colon cancer cell proliferation, but the extract of Injinho or Geummi-hwa in the extract of Cordyceps sinensis decreased. However, FDY003, an extract of a mixture of Cordyceps sinensis, Injinho, and Gold and Silver, showed the highest inhibitory effect on colon cancer cell proliferation (FIG. 1). This suggests that when the extracts of the three kinds of Chinese herbal medicines are mixed, they show an unexpected effect of inhibiting colon cancer cell proliferation compared to the extracts of the mixtures of the two kinds of Chinese herbal medicines.

Experimental Example 2. Confirmation of the inhibitory effect of colon cancer cell proliferation of FDY003

As confirmed in Experimental Example 1, MTT analysis was performed to confirm the effect of inhibiting colorectal cancer cell proliferation at a broader concentration range with respect to FDY003 having the best effect of inhibiting colorectal cancer cell proliferation.

Specifically, except that Colo205 cells were treated with FDY003 prepared in Example 2 to a final concentration of 0.1, 0.5, 1, 5, 10 or 50 ㎍ / ㎖ in the same manner as described in Experiment 1 MTT analysis was performed.

As a result, FDY003 showed a colorectal cancer growth inhibitory effect at a concentration-dependent concentration of 0.1 μg / ml or more (FIG. 2).

Experimental Example 3. Confirmation of radical scavenging ability of FDY003

In order to investigate whether the anticancer effect of FDY003 against colorectal cancers identified in Experimental Examples 1 and 2 was due to the antioxidant effect of FDY003, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability of FDY003 was measured.

Specifically, 100 μL of each extract and 100 μl of 500 μg / ml DPPH dissolved in ethanol were mixed and reacted at 37 ° C. in the dark for 30 minutes, and then absorbance was measured at 540 nm using an ELISA reader.

As a result, FDY003 showed DPPH radical scavenging ability dependent on concentration up to 1000 ㎍ / ml, and the radical scavenging ability of FDY003 1000 ㎍ / ml was similar to that of 10 μg / ml gallic acid, a positive control ( 3).

Experimental Example 4. Confirmation of radical scavenging ability of colon cells in FDY003

In order to confirm whether DPDY radical scavenging ability of FDY003 is also observed in colorectal cancer cells, CoDY205 cells were treated with FDY003 prepared in Example 2 by concentration, and then cell and cell cultures were obtained to measure DPPH radical scavenging ability in and out of cells. .

Specifically, Colo205 cells were cultured at 1 × 10 ⁶ / dish in a 100 mm dish, and the FDY003 was diluted in RPMI 1640 medium so as to have a final concentration of 0.5, 1, or 5 μg / ml and treated to the cells. After incubation for 24 hours, cells and cell cultures were obtained, and DPPH radical scavenging ability in and out of cells in accordance with the manufacturer's instructions with an enzyme-linked immunosorbent assay (DPPH ELISA) kit (Cat. # EU3110, Fine test, China). Was measured.

As a result, FDY03 showed significant intracellular DPPH radical scavenging activity at 0.5 and 1 μg / ml concentrations as compared to the control group, and showed extracellular DPPH radical scavenging activity at 0.5 to 5 μg / ml concentrations as compared to the control group (FIG. 4A). And 4B).

Experimental Example 5. Inhibition of lipid peroxidation effect in colon cancer cells of FDY003

In order to investigate the antioxidant effect of FDY003, CoLO205 cells were treated with FDY003 prepared in Example 2 by concentration, and then cells were obtained to measure the degree of lipid peroxidation in cells.

Specifically, Colo205 cells were cultured at 1 × 10 ⁶ / dish in a 100 mm dish, and the FDY003 was diluted in RPMI 1640 medium so as to have a final concentration of 0.5, 1, or 5 μg / ml and treated to the cells. After incubation for 24 hours, the cells were obtained, and the degree of lipid peroxidation was measured by TBARS (thiobarbituric acid reactive substances) ELISA kit (Cat. # MBS480427, Mybiosource, USA) according to the manufacturer's instructions.

As a result, FDY003 showed a significant, concentration-dependent lipid peroxidation inhibitory effect compared to the control at a concentration of 0.5 to 5 ㎍ / ㎖ (Fig. 5).

Experimental Example 6. Confirmation of the effect of FDY003 on colorectal cancer cell death

In order to investigate whether the anticancer effect of FDY003 against colorectal cancers identified in Experimental Examples 1 and 2 was due to the apoptosis-inducing effect of FDY003, CoLO205 cells were treated with FDY003 prepared in Example 2 by concentration, and then the cells were treated. The expression of Bax protein and the degree of activation by cleavage of caspase-3 were analyzed by Western blot.

Specifically, Colo205 cells were incubated at 1 × 10 ⁶ / dish in a 100 mm dish, and FDY003 was diluted in RPMI 1640 medium to a final concentration of 0.5, 1, or 5 μg / ml and treated with cells. After incubation for 24 hours, the cells were collected, centrifuged, the supernatant was removed, and the cells were disrupted with RIPA lysis buffer containing a protease inhibitor and a phosphatase inhibitor. The cell lysate was centrifuged to recover the supernatant, the protein was quantified and electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the separated protein was transferred to the membrane. Membrane was used as primary antibody (anti-Bax, Cat. #. Ab53154, Abcam, USA; anti-caspase-3, Cat. #. Ab13585, Abcam; anti-cleaved caspase-3, Cat. #. Ab49822, Abcam; or After reacting with anti-beta-actin (β-actin), Cat. #. Ab8227, Abcam), it was reacted with a secondary antibody bound to horseradish peroxidase (HRP). Thereafter, the HRP conjugate was detected with an enhanced chemiluminescence (ECL) buffer to analyze the blot image.

As a result, FDY003 increased the expression of Bax at a concentration of 0.5 to 5 μg / ml and increased the expression of cleaved caspase-3 relative to total caspase-3, inducing the activation of caspase-3 (FIGS. 6A, 6B and 6E). ).

Experimental Example 7 confirmed the effect of FDY003 on p53 activation and p21 protein expression in colorectal cancer cells

In order to investigate whether the anticancer effect of FDY003 against colorectal cancers identified in Experimental Examples 1 and 2 was due to the increased expression of p21, a tumor suppressor protein of FDY003, and an activation inducing effect of p53, Colo205 cells were prepared in Example 2 After FDY003 treatment by concentration, cells were obtained and analyzed by Western blot for expression level of p21 protein and activation by phosphorylation of p53 protein.

Specifically, Colo205 cells were cultured and recovered in the same manner as described in Experimental Example 6, and anti-p21 (Cat. #. Ab109520, Abcam) and anti-p53 (Cat. #. Ab1101, Abcam) were used as primary antibodies. Or Western blot analysis was performed in the same manner as described in Experiment 6 except that an anti-beta-actin antibody was used and a corresponding secondary HRP conjugated antibody was used.

As a result, FDY003 increased the expression of p21 in a concentration-dependent manner at a concentration of 0.5 to 5 μg / ml, and increased the expression of phosphorylated p53 relative to the total p53 to induce p53 activation (FIGS. 6C to 6E).

Example 3 Construction of Colon Cancer Mouse Model

3-1. Preparation of Colo205 Cell Lines

Frozen colon cancer cell lines (Colo205 cells) were RPMI 1640 medium (Cat. #. 22400) containing 10% of heat-inactivated fetal bovine serum (Cat. #. 16000-044, Gibco, USA). -089, Gibco) was thawed and incubated in an incubator at 5% carbon dioxide and 37 ° C. Cells suspended in culture medium are obtained, transferred to tubes, adherent cells remaining in the flasks are washed with phosphate-buffered saline (PBS), 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid, Cat. #. 25200) -056, Gibco) was added to isolate, and then added to the tube with suspended cells. The tube was centrifuged at 1,000 rpm for 5 minutes, the supernatant was discarded and resuspended in fresh medium, and the number of cells was counted to prepare a concentration of 5.0 × 10 ⁷ cells / ml.

3-2. Construction of Colon Cancer Mouse Model by Colo205 Cell Line Inoculation

A 5 week old female nude mouse (Athymic nude mouse, Saronbio, South Korea) was obtained and purified for 7 days. After disinfecting the back of the mouse with 70% alcohol, Colo205 cells prepared in Example 3-1 were administered subcutaneously by 0.1 ml per horse using a 26 gauge needle syringe. When the tumor size reached about 100-150 mm ³ , group separation was performed so that the measured tumor size was distributed as evenly as possible.

Experimental Example 8. Confirmation of anticancer effect of FDY003 in colorectal cancer mouse model

In order to confirm the anticancer effect of FDY003 against colorectal cancer at the cellular level identified in the above experimental examples in an animal model, a colon cancer mouse model was used.

8-1. Medication Schedule

5 ml of sterile physiological saline solution (Korea Pharmaceutical Industry Co., Ltd., Korea) or 20 mg / kg / 5 ml of positive control group, irinotecan, was administered to the colon cancer mouse prepared in Example 3 twice a week for 4 weeks. In the drug administration group, FDY003 0.2 mg / kg / 5 ml was administered twice a week for 4 weeks. Intravenous administration was performed by inserting the mouse through a tail vein using a 31 gauge (gauge) insulin syringe after correction.

8-2. Find out the effect of FDY003 on mouse weight

During the entire experiment, mouse body weights were measured once a week after group segregation and drug initiation, and finally measured on autopsy day.

Mouse weight during the experiment Date (days) Average weight of mice by group ^a (g) Control Irinotecan administration group FDY003 administration group 0 17.00 ± 1.10 16.66 ± 1.24 16.40 ± 0.96 7 18.41 ± 1.46 17.10 ± 1.14 ^* 17.61 ± 1.04 14 17.98 ± 1.45 16.93 ± 1.29 17.18 ± 1.11 21 16.51 ± 1.66 17.54 ± 1.74 16.62 ± 1.34 28 16.63 ± 1.63 17.42 ± 2.01 16.29 ± 1.58

( ^a mean ± standard deviation, ^* p <0.05 compared to control)

As a result, the irinotecan-administered group, which was a positive control group, lost weight significantly compared to the control group at 7 days after drug administration, but the FDY003-administered group showed no significant weight difference compared to the control group for 4 weeks (Table 1).

8-3. Confirmation of tumor growth inhibitory effect by administration of FDY003

During the entire experiment, tumor size was measured twice a week after group separation and on the start of drug administration. Calipers were used to measure the long and short axis of the tumor and tumor size was calculated using the following formula:

Tumor size ^{= (a × b) 2/} 2 (a: major axis; b: minor axis).

As a result, in the case of the FDY003 administration group, the tumor size was significantly smaller than the control group from 14 days after the start of administration (Figs. 7 and 8).

8-4. Confirmation of Antioxidant Effect by FDY003 Administration

In order to investigate whether the effect of inhibiting the growth of colorectal cancer tumors of FDY003 confirmed in Experimental Example 8-3 was due to the antioxidant effect of FDY003, blood was collected on the autopsy day of the mouse and the degree of lipid peroxidation and DPPH radical scavenging ability in serum were measured.

8-4-1. Mouse Blood Sampling and Serum Isolation

Blood was collected from the posterior vena cava on the day of necropsy of the mice, and then the blood was injected into a vacuum vessel tube to which a clot activator was added and allowed to coagulate by standing at room temperature for about 15 to 20 minutes. Serum was isolated by centrifuging the coagulated blood for 10 minutes at 5,000 rpm. Serum samples were stored in a deep freezer set at −70 ° C. or lower until analysis.

8-4-2. Inhibition of Lipid Peroxidation in Serum by FDY003 Administration

Lipid peroxidation was measured using the Lipid peroxidation kit (Cat. #. K739, Biovision, USA) with the mouse serum obtained in Experimental Example 8-4-1.

As a result, serum lipid peroxidation was significantly inhibited in the FDY003 administration group compared to the control group (FIG. 9).

8-4-3. DPPH Radical Scavenging Activity of Mouse Serum FDY003

DPPH radical scavenging ability of the mouse serum obtained in Experimental Example 8-4-1 was measured. 80 μl of 0.2 mM DPPH solution dissolved in ethanol was added to 20 μl of each mouse serum or DMSO and reacted at room temperature for 10 minutes, and the absorbance was measured at 492 nm with an ELISA reader. The color of each sample was corrected after measuring its own absorbance, and the DPPH radical scavenging ability of each sample was calculated by the following equation using DMSO as a control:

% Radical scavenging ability = (1-A / B) x 100 (A: absorbance of the sample; B: absorbance of the control).

As a result, DPPH radical scavenging activity was significantly increased in the serum of FDY003-treated mice compared to the control group (FIG. 9).

Experimental Examples 8-1 to 8-4 confirmed that FDY003 did not affect the body weight in the colorectal cancer mouse model, and inhibited tumor production through the antioxidant effect.

Example 4. Preparation of Herbal Extracts

Root (root) of Pueraria lobata Ohwi, tuber of Euphorbia kansui Liou ex Wang, root and root stem of Glycyrrhiza uralensis Fischer, and root stem of dried ginger ( Zingiber officinale Roscoe) , Dried sap from dried roots of dried Rehmannia glutinosa (Gaertner) Liboschitz ex Steudel, dried rhubarb ( Rhus verniciflua Stokes) and dried seeds of Cassia tora Linne ( Cassia tora Linne) Maltose), young eggplant of broiler ( Cinnamomum cassia Presl), root stem of Alpinia officinarum Hance, root of red ginseng ( Sophora flavescens Solander ex Aiton), and seed of rice ( Oryza sativa Linne) with malt powder ( Gyo ), Chrysanthemum zawadskii Herbich var. Latilobum (Maxim.) Kitamura outpost, Root of Salvia miltiorrhiza Bunge, Root of Angelica gigas Nakai, Angelica gigas Nakai codonopsis pilosula Nannfeldt var. modesta L The roots of T Shen, the ripe fruit of the jujube ( Zizyphus jujuba Miller var. Inermis Rehder), the ripe seeds of the peach tree ( Prunus persica Batsch), the seeds of Eucommia ulmoides Oliver. Stem bark, stem of Ephedra sinica Stapf, ephedra, root of Codonopsis pilosula Nannfeldt, bulge of root of Liriope platyphylla Wang et Tang, stem of Akebia quinata Decaisne , Rubber resin (myrrh) obtained from Commiphora myrrha Engler, mint ( Mentha arvensis Linne var. piperascens Malinvaud ex Holmes, tuber of Pinellia ternata Breitenbach, roots of Pulsatilla koreana Nakai, unripe fruit of Rubus coreanus Miquel, Aconitum carmichaeli Root of the Debeaux (rich), leaves of the loquat ( Eriobotrya japonica Lindley), loquat ( Cnidium monieri (L), fruit of the Cussion), Zanthoxylum piperitum De Candolle Ripe Roots of Fruits ( Seaweed ), Phellinus linteus , Fresh Roots of Ginger ( Zingiber officinale Roscoe), Gypsum Fibrosum, Flowers of Lithuania ( Inula japonica Thunberg) the roots of Asiasarum heterotropoides F. Maekawa var. mandshuricum F. roots and rhizomes (slender), wheat (Triticum aestivum var. vulgare) grew seeds (wheat), cloth speed (Dipsacus asperoides CY Cheng et TM Ai ) of Maekawa) ( Fasting), Rehmannia glutinosa Liboschitz ex Steudel Root processing (Sukjihwang), Root of Bupleurum falcatum Linne, Root bark and stem of Acanthopanax sessiliflorum Seeman (Ogapi), Body of Scolopendra subspinipes mutilans Linne Koch, Fruit of Evodia rutaecarpa Bentham, Root (Wild) of Keel (Fossilia Ossis Mastodi), Root (Wild) of Achyranthes japonica Nakai, Bark (milk white skin), Broiler ( Cinnamomum cassia ) removed from bark of Ulmus macrocarpa Hance Stems of Presl, ripe seeds of nutmeg ( Myristica fragrans Houttuyn), roots of Panax ginseng CA Meyer, roots and root stems of Glycyrrhiza uralensis Fischer ( Perilla frutescens Britton var. Leaves and tip of the acuta Kudo, the roots of the peony ( Paeonia lactiflora Pallas), the fruit or seed (earth) of the fern ( Hovenia dulcis Thunb.), of the Poncirus trifoliata Rafinesque Unripe fruits (plows), root stems of the Cnidium officinale Makino, ground parts of the Artemisia annua Linne (Zhengho), resinous stems of the Aquilaria agallocha Roxburgh, incense, easily Lycopus lucidus Turczaininov before the flowering (Tranlan), dandelion ( Taraxacum platycarpum H. Dahlstedt) outpost (pogongyeong), nectar ( Prunella vulgaris Linne var. Lilacina Nakai), lotus ( Nelumbo nucifera Gaertner) ) of the leaf (foliage), hanryeoncho (aboveground (Geranium nepalense) of the ripe seeds (passer), cranesbill (Geranium thunbergii Siebold et Zuccarini) of the outpost, apricot trees (Prunus armeniaca Linne var. ansu Maximowicz ) of Eclipta prostrata Linne), mold opening Isaac flowers of (Schizonepeta tenuifolia Briquet), Donna non-braised root of the seeds (walnuts), ginseng (Panax ginseng CA Meyer) of (Juglans regia Linne) (ginseng), flowers (safflower), and the fruit of safflower (Carthamus tinctorius Linne) (honghwaja), Astragalus (Astragalus membranaceus The roots of Bunge, and the ground part of the Siegesbeckia pubescens Makino, were purchased from Kyungdong Market. Each extract was milled to prepare each extract in the same manner as described in Example 1, and extract samples for in vitro experiments were prepared in the same manner as described in Example 2.

Experimental Example 9. Confirmation of colon cancer cell proliferation inhibitory effect of herbal extracts

In addition to FDY003 having an anticancer effect against colorectal cancers identified in Experimental Examples 1 to 8, in order to discover a herbal medicine material having an anticancer effect against colorectal cancer, the effect of inhibiting colon cancer cell proliferation of the herbal medicine extracts prepared in Example 4 was MTT It was investigated by analysis.

Specifically, MTT analysis was performed in the same manner as described in Experiment 1 except that Colo205 cells were treated with the herbal extracts prepared in Example 4 to have a final concentration of 10 μg / ml.

Inhibitory Effects of Herbal Extracts on Colorectal Cancer Cell Proliferation Medicinal name Cell survival rate (%) Medicinal name Cell survival rate (%) Medicinal name Cell survival rate (%) Rooting 89.35 Half 87.01 Ginseng 89.05 subtrahend 90.91 Baekduong 86.37 Persimmon 90.04 licorice 89.48 Bokbunja 89.69 Lobule 90.47 health 88.63 Wealthy 87.09 Peony 89.07 Guernsey 89.77 Non-leafed 90.17 Earth 92.57 Dry 86.19 Casualties 90.92 Basement 88.04 A defector 91.87 Sancho 90.48 Cheongung 88.92 Cock 90.33 Situation Mushroom 88.04 Cheongho 90.57 High volume steel 89.10 ginger 89.94 Aroma 83.39 Gosam 92.36 gypsum 99.53 Talc 90.88 Gyo 88.92 Conquest 84.10 Poongyoung 90.31 Gujeolcho 95.33 Sesin 90.91 Hagocho 93.47 Salvia 87.11 Wheat 90.76 Lower lobe 90.51 Donkey 91.20 Fast 90.29 Nasturtium 90.96 Dangsam 89.25 Hungjiwang 91.37 Passerby 89.39 contrast 90.83 Shiho 87.94 The beginning 91.58 Doin 90.40 Ogapi 90.91 Brother-in-law 96.58 Tofu 89.06 Goku 93.08 Hodo 89.47 Ephedra 89.43 Osuyu 89.76 Red ginseng 89.18 Ginseng 89.38 keel 93.18 Safflower 88.03 Macmundong 91.28 Dew 93.14 Safflower 88.57 Neck 90.65 Milky skin 96.17 Astragalus 90.96 Myrrh 93.10 Broiler 90.85 Happiness 90.98 mint 87.72 nutmeg 93.18

As a result, the 71 herbal extracts showed a maximum inhibitory effect of colon cancer cell proliferation of about 17% at 10 ㎍ / ㎖ (Table 2).

In conclusion, through the above examples and experiments, Injinho, Cordyceps sinensis and the extract of the gold and silver coin mixture (FDY003) has excellent antioxidant activity, antioxidant, apoptosis induction in colon cancer cells, increased expression of p21, a cancer suppressor protein and p53 Inhibition of cell proliferation through the activation of and inhibited tumor production through the antioxidant activity in the colorectal cancer mouse model. Therefore, FDY003 can be usefully used for the prevention, treatment or improvement of colorectal cancer.

Claims (8)

A pharmaceutical composition for preventing or treating colorectal cancer, comprising as an active ingredient an extract of a mixture of Injinho, Cordyceps sinensis and Gold and Silver coins in a weight ratio of 1 to 2: 1 to 2: 1.
The pharmaceutical composition of claim 1, wherein the extract is extracted with water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof.
The pharmaceutical composition of claim 2, wherein the lower alcohol is ethanol or methanol.
According to claim 1, wherein the extract is brown root, water persimmon, licorice, health, dried Guangji, dried lacquer, gyeja, gyeji, Goyanggang, red ginseng, gyo, gujeolcho, sweet ginseng, Angelica, Tang ginseng, control, cabbage, tofu, ephedra, ephedra, mansam, Megmun-dong, keg, myrrh, peppermint, half, baekduong, bokbunja, rich, non-leafed, casualties, sancho, situation mushroom, ginger, gypsum, bokhwa, sesin, wheat, fast, sukji, shiho, ogapi, goku, osuyu, keel, Esoteric, Milk White Peel, Broiler, Nutmeg, Ginseng, Jagamcho, Jagobyeo, Peony, Earth, Jisil, Cheongung, Cheongho, Incense, Taekran, Pogongyeong, Hagocho, Lower lobe, Nasturtium, Pedestrian, Hyuncho, Hyungae, Hodo, Red ginseng , Safflower, safflower, astragalus, extracted from any one or more selected from the group consisting of, pharmaceutical composition.
Health functional food for the prevention or improvement of colorectal cancer containing the extract of the mixture of Injinho, Cordyceps sinensis, and gold and silver coin in a weight ratio of 1 to 2: 1 to 2: 1 as an active ingredient.
The health functional food according to claim 5, wherein the extract is extracted with water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof.
The dietary supplement of claim 6, wherein the lower alcohol is ethanol or methanol.
The extract of claim 5, wherein the extract is brown root, water persimmon, licorice, health, dried Guangji, dried fruit, gyeolja, gyeji, Goyanggang, red ginseng, gyo, gujeolcho, sweet ginseng, Angelica, Dangsam, control, cabbage, tofu, ephedra, ephedra, ginseng, Megmundong, keg, myrrh, peppermint, halved, baekduong, bokbunja, rich, non-leafed, casualty, sancho, situation mushroom, ginger, gypsum, bokhwa, sesin, wheat, fast, sukji, shiho, ogapi, goku, osuyu, keel, Esoteric, Milk White Peel, Broiler, Nutmeg, Ginseng, Jagamcho, Jasaeop, Peony, Earth, Fruits, Cheongung, Cheongho, Incense, Taekran, Pogongyeong, Hagocho, Lower lobe, Nasturtium, Pedestrian, Hyuncho, Hyungae, Hodo, Red ginseng , Safflower, safflower, Astragalus, extracted from any one or more selected from the group consisting of health functional food.

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