KR102289965B1 - A pharmaceutical composition for preventing or treating thyroid cancer comprising extract of ursi fel or fraction thereof - Google Patents

Abstract

One embodiment of the present invention relates to a use for improving, preventing or treating thyroid cancer, comprising an extract or a fraction thereof as an active ingredient. It can be utilized for the above purpose by promoting and inhibiting angiogenesis.

Description

A pharmaceutical composition for the prevention or treatment of thyroid cancer, comprising the extract or a fraction thereof as an active ingredient {A PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING THYROID CANCER COMPRISING EXTRACT OF URSI FEL OR FRACTION THEREOF}

The present invention relates to a use for improving, preventing, or treating thyroid cancer, comprising an extract of Woongdamae or a fraction thereof as an active ingredient.

Thyroid cancer, one of the most common endocrine tumors, is a malignant tumor with increasing incidence worldwide. Malignant thyroid cancer can be classified into four main types, including anaplastic thyroid cancer (ATC). Undifferentiated thyroid cancer is one of the most lethal malignancies with aggressive differentiation and invasion into surrounding tissues such as organs. Current clinical treatment for undifferentiated thyroid cancer, either alone or in combination with surgery, radiation therapy, or chemotherapy, has a low recovery rate and a high recurrence rate, so it is not effective in treatment and patient survival. Therefore, there is an urgent need for the development of novel therapeutic methods. Among the studies to find such new anticancer therapies, treatment through the use of natural resources has recently attracted attention as a new anticancer agent with excellent stability in the body that can reduce the side effects caused by chemotherapy and radiation therapy.

On the other hand, the bile of Ursus arctos (Brown bear bile, UF) is a representative herbal medicine for cooling and fever. Therefore, it has been reported that the sagebrush can be effective in detoxifying fever, relieving spasms, removing liver, and improving eyesight. In particular, it was commonly used in oriental medicine to reduce fever, inflammation, swelling, detoxification and pain. It is also known to help treat liver disease and other liver diseases including viral hepatitis, liver fibrosis and liver cancer.

Ursodeoxycholic acid (UDCA), a main component of bile acids collected from Woongdam, has been used for liver treatment and is known to have angiogenesis inhibitory effects (Republic of Korea Patent No. 10-0156227).

However, the anticancer action mechanism of the UDCA has not been fully elucidated, and in particular, there has been no report on the content that it is effective in preventing or treating thyroid cancer.

Therefore, the present inventors have been studying to solve the above problems, the substance containing the extract or a fraction thereof as an active ingredient inhibits the growth and proliferation of thyroid cancer cells, increases apoptosis, and inhibits angiogenesis , and found that it is effective in the prevention and treatment of thyroid cancer, thereby completing the present invention.

The technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating thyroid cancer comprising an extract of Woongdam or a fraction thereof as an active ingredient.

In addition, another technical problem to be achieved by the present invention is to provide a health functional food composition for preventing or improving thyroid cancer, which includes an extract of Woongdam or a fraction thereof as an active ingredient.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above technical object, an embodiment of the present invention provides a pharmaceutical composition for preventing or treating thyroid cancer comprising an extract or a fraction thereof as an active ingredient.

In an exemplary embodiment of the present invention, the extract of _{ferns may be those extracted from water, C 1-6} alcohol, or a mixed solvent thereof.

In an embodiment of the present invention, the Ungdam extract may be obtained by extracting for 1 month to 5 months under a temperature of 10 to 50 ℃.

In an embodiment of the present invention, the fraction may be fractionated using any one or more solvents selected from the group consisting of hexane (Hex), methylene chloride (MC) and ethyl acetate (EtOAc).

In an embodiment of the present invention, the extract of Woongdamum may be contained in an amount of 25 to 100 μg/mL based on the total composition.

In an embodiment of the present invention, the pharmaceutical composition may promote apoptosis of cancer cells.

In an embodiment of the present invention, the pharmaceutical composition may increase the expression of one or more proteins selected from the group consisting of Bax, cytochrome c and caspase-3 in cancer cells.

In an embodiment of the present invention, the pharmaceutical composition may inhibit angiogenesis of cancer cells.

In order to achieve the above technical object, another embodiment of the present invention provides a health functional food composition for preventing or improving thyroid cancer, which includes an extract or a fraction thereof as an active ingredient.

According to an embodiment of the present invention, the extract or a fraction thereof of the present invention may specifically inhibit growth, increase apoptosis, and induce nuclear fragmentation in thyroid cancer cells. Biochemical and morphological changes may occur during the apoptosis process.

In addition, the extract or a fraction thereof may increase the expression of pro-apoptotic proteins including Bax, caspase-3 and cytochrome C in thyroid cancer cells. That is, the extract or a fraction thereof can inhibit cell growth and proliferation, which can induce apoptosis by regulating Bax expression, mitochondrial dysfunction, and caspase activation in undifferentiated thyroid cancer cells.

In addition, when thyroid cancer cells are treated with the extract or a fraction thereof, the expression of TGF-βVEGF, N-cadherin and SIRT-1 is suppressed, and the extract or a fraction thereof has the ability to inhibit tumor proliferation and invasion of undifferentiated thyroid cancer cells. can represent In addition, it may exhibit an effect of inhibiting angiogenesis.

Furthermore, treatment of the extract or a fraction thereof on thyroid cancer cells (FRO) may exhibit the effect of increasing Akt phosphorylation. That is, it is possible to inhibit the growth, proliferation, survival and invasion of cancer cells by regulation of the Akt signal transduction pathway.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a graph showing the effect of ungdam extract (UF) and UDCA on cell growth of thyroid cancer cell viability according to different times and concentrations through MTT analysis.
Figure 2 is the result of confirming the effect of ungdam extract (UF) and UDCA on apoptosis through Hoechst staining.
Figure 3a shows the result of confirming the effect on pro-apoptotic protein expression through western blot after treatment with Ungdam extract (UF) and UDCA. Figure 3b is a graph showing the Bax / β-actin ratio when treated with ungam extract (UF) and UDCA.
Figure 4a shows the result of confirming the effect on the expression of angiogenesis regulation-related protein through western blot after treatment with ungam damme extract (UF). Figure 4b is a graph showing the ratio of TGF-β, VEGF, N-cadherin, and SIRT-1 / β-actin when treated with an ungam extract (UF).
FIG. 5a shows the results of confirming the effect on Akt phosphorylation through western blot after treatment with ungdam extract (UF). Figure 5b is a graph showing the p-Akt/Akt ratio in the treatment of male fern extract (UF) and UDCA.
6 is an HPLC test result of ungdam extract (UF) and UDCA.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in several different forms, and thus is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is said to be “connected (connected, contacted, coupled)” with another part, it is not only “directly connected” but also “indirectly connected” with another member interposed therebetween. "Including cases where In addition, when a part "includes" a certain component, this means that other components may be further provided without excluding other components unless otherwise stated.

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

The first aspect of the present application is

It provides a pharmaceutical composition for preventing or treating thyroid cancer, comprising the extract or a fraction thereof as an active ingredient.

Hereinafter, the pharmaceutical composition according to the first aspect of the present application will be described in detail.

First of all, in one embodiment of the present application, the pharmaceutical composition may be one comprising the extract or a fraction thereof as an active ingredient.

As used herein, the term “ungdam” refers to the gallbladder of a bear belonging to the brown bear (Ursus arctos Linne) or other bears (Ursidae), and is generally 10-20 cm in length and 5-8 cm in width. The ungdam may use a dried bear's gall, and a commercially available one may be used. It is known that the main components of bile acids collected from wild dam include UDCA, alkali metal salts, cholesterol, and bilirubin. The bile acids collected from the dam include different amounts of UDCA depending on the type of bear. According to an embodiment of the present application, the umbilical cord is from the brown bear (Ursus arctos Linne) and contains about 19% of UDCA (Hagey et al., J Lipid Res, 1993; 34: 1911-1917).

As used herein, the term “extract” is extracted from sagebrush using an appropriate solvent, and may include, for example, an immersion extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract.

The extract may be water, C _1-6 alcohol, or a mixed solvent thereof. Preferably, the alcohol may be methanol, ethanol or propanol, and more preferably a mixed solvent of water and ethanol. can The extraction solvent may be extracted by adding 2 to 40 times, specifically, 2 to 10 times the amount of dried ungdam.

In one embodiment of the present application, as the extraction method of the bogdam, conventional methods in the art such as immersion extraction, filtration, hot water extraction, cold extraction, reflux cooling extraction, steam distillation, ultrasonic extraction, elution method and compression method may be used. can

In one embodiment of the present application, the Ungdam extract may be obtained by immersion extraction at a temperature of 10 to 50 ℃ for 1 to 5 months. The extraction temperature may be preferably 10 to 30 °C, more preferably 10 to 20 °C. In addition, the extraction time may be preferably 2 months to 4 months, more preferably 3 months to 4 months. The ungdam may be subjected to low-temperature aging during the immersion extraction process, thereby causing chemical and physical transformation of the active ingredient.

In addition, the extraction process may be repeated 2 to 3 times.

In one embodiment of the present application, it may be a fraction of the Ungdam extract. For the fractionation, various methods of fractionation such as solvent fractionation of the extract, ultrafiltration fractionation, and chromatographic fractionation may be used. The fraction is subjected to a conventional fractionation process using a polar solvent and a non-polar solvent in the extract to obtain a polar solvent fraction and a non-polar solvent fraction, respectively. The polar solvent may be any one or more solvents selected from the group consisting of n-butanol and water, and the non-polar solvent is selected from the group consisting of hexane (Hex), methylene chloride (MC) and ethyl acetate (EtOAC). Any one or more solvents may be used. Preferably, the fraction of the Ungdam extract may be fractionated using any one or more solvents selected from the group consisting of hexane (Hex), methylene chloride (MC) and ethyl acetate (EtOAc).

In addition, in one embodiment of the present application, the extract or fraction may be further purified using a conventional purification method. There is no limitation on the method for preparing the extract of Woongdamae, and any known method may be used.

In one embodiment of the present application, the extract or fraction thereof of the present application may be concentrated or dried under reduced pressure after the extraction and fractionation. The reduced pressure concentration may be using a vacuum vacuum concentrator or a vacuum rotary evaporator. In addition, drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying.

As used herein, the term “thyroid cancer” refers to cancer occurring in the thyroid gland, papillary carcinoma, follicular carcinoma, Hurthle cell neoplasm (follicular adenocarcinoma), anaplastic carcinoma (undifferentiated) thyroid cancer), and medullary thyroid carcinoma. In addition, thyroid cancer has a high rate of metastasis due to its high cell mobility and invasiveness.

The pharmaceutical composition of the present invention is effective for preventing or treating thyroid cancer. As used herein, the term "prevention" refers to any action that suppresses or delays the onset of thyroid cancer by administration of the composition of the present invention, and "treatment" refers to any action in which the symptoms due to thyroid cancer are improved or beneficially changed by the composition of the present invention. means Specifically, the extract or a fraction thereof, specifically promotes apoptosis in thyroid cancer cells, and inhibits angiogenesis, so it is effective in preventing or treating thyroid cancer with high cell mobility and invasiveness.

In addition, in one embodiment of the present application, the extract or a fraction thereof is not particularly limited, but may preferably be contained in an amount of 25 to 100 μg/mL based on the total composition, more preferably 25 to 50 μg/mL. When the extract or its fraction is less than 25 μg/mL, it may have a disadvantage that it is difficult to sufficiently express the preventive or therapeutic effect of thyroid cancer to be achieved in the present invention, and the extract or its fraction is 100 μg/mL If it exceeds, there may be a disadvantage of limiting the content of the remaining substances for manufacturing a pharmaceutical application product.

In one embodiment of the present application, the pharmaceutical composition may promote apoptosis (apoptosis) of cancer cells.

In a method of treating cancer, apoptosis control therapy is used to block the pathological growth of uncontrolled cells such as cancer cells. At the same time, the cytotoxic enzymes that are released by destroying the cell membrane of pathological cells at the same time cause cytotoxicity even to surrounding normal cells, which inevitably accompanies excessive inflammatory expression, which has a disadvantage in that the side effects are large. However, apoptosis-induced apoptosis control therapy induces the spontaneous death of pathological cells or strongly inhibits the growth of these cells. there is.

Apoptosis is genetically regulated programmed cell death and means active apoptosis, and the apoptosis process requires energy and exhibits characteristic cell morphology. That is, when an apoptosis signal is transmitted, apoptosis is determined within the cell, and it proceeds to the execution stage. In the execution stage, the cells show characteristic biochemical and morphological changes. ), it separates from adjacent cells, and the cell membrane changes to bubble hair (blebbing), furthermore, the nucleus is condensed and the DNA in the nucleus is cut into small oligonucleotide fragments to form an apoptotic body. Cell death occurs through a series of processes in which the apoptotic body thus formed is phagocytosed by macrophages.

In one embodiment of the present application, the induction of cancer cell death may be one of the most important chemotherapeutic approaches with minimal off-target effec in which cancer cells are “self-destructed”. The intrinsic pathway is characterized by permeabilization of the mitochondria, and as stimulation activates the intrinsic pathway, it leads to the opening of the mitochondrial permeability transition pore, and the release of pro-apoptotic proteins such as cytochrome C to the mitochondrial membrane potential It can cause the loss of caspases and activate multiple caspases such as caspase-3. In addition, by regulating the expression of pro-apoptotic proteins (eg, Bax), it is possible to regulate the mitochondrial permeability transition.

In one embodiment of the present application, the pharmaceutical composition or a fraction thereof may increase apoptosis specifically in thyroid cancer cells and induce nuclear fragmentation. Specifically, biochemical and morphological changes may occur during apoptosis. In addition, the pharmaceutical composition or a fraction thereof may increase the expression of apoptotic proteins including Bax, caspase-3 and cytochrome C in thyroid cancer cells.

In one embodiment of the present application, the pharmaceutical composition or a fraction thereof may inhibit angiogenesis of cancer cells.

Specifically, in one embodiment of the present application, the extract of Woongdamum can inhibit the expression of TGF-βVEGF (Vascular endothelial growth factor), N-cadherin, and SIRT-1 in thyroid cancer cells, and the extract of Woongdam's ATC tumor It may have inhibitory ability against proliferation and invasion. In addition, the extract of fermented horseradish may inhibit angiogenesis and metastasis in ATC. Since thyroid cancer has high mobility and invasiveness, the treatment mechanism of the present invention can effectively achieve the therapeutic goal.

Angiogenesis is a process in which new blood vessels are formed from existing blood vessels, and is one of the processes necessary for the division and growth of cancer cells as well as the development and development stages and division and growth of normal cells and tissues. On the other hand, the development and progression of ATC may include complex and overlapping interactions between hormones, ligands such as epidermal growth factor (EGF) that regulate proliferation and differentiation of thyroid cells, and growth factors such as TGF-β. Target TGF-β is effective in inhibiting primary tumor proliferation in ATC cells. On the other hand, VEGF, essential for cell growth and metastasis, is a major cytokine involved in tumor angiogenesis. Most tumor types, including ATC, are known to overexpress VEGF, which is directly related to the area of angiogenesis. In addition, N-cadherin, an adhesion molecule, promotes tumor cell survival, migration and invasion, is expressed in endothelial cells, and is associated with increased cancer invasiveness. Class III histone deacetylases, SIRT-1, capable of deacetylating lysine residues in nuclear proteins, regulate vascular endothelial homeostasis by regulating angiogenesis and vascular function.

The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or flavoring agent may be used.

In addition, the pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Additionally, the formulation of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or required, suitable binders, lubricants, disintegrants and color-developers may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Furthermore, acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, Ethanol and one or more of these components may be mixed and used, and if necessary, other conventional additives such as antioxidants, buffers, and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

Furthermore, it can be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration. .

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by internalizing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The second aspect of the present application is

It provides a health functional food composition for preventing or improving thyroid cancer, comprising an extract of Woongdam or a fraction thereof as an active ingredient.

Although detailed descriptions of parts overlapping with the first aspect of the present application are omitted, the contents described with respect to the first aspect of the present application may be equally applied even if the description thereof is omitted in the second aspect.

As used herein, the term “improvement” means that symptoms caused by thyroid cancer are improved or the probability of occurrence is advantageously lowered.

Hereinafter, the health functional food composition according to the second aspect of the present application will be described in detail.

First, in one embodiment of the present application, the health functional food is a food prepared by adding or encapsulating, powdering, suspension, etc., to food materials such as beverages, teas, spices, gums, confectionery, etc. When consumed, it means that certain health effects are obtained, but unlike general drugs, there may be advantages in that there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material. The health functional food of the present invention obtained in this way can be very useful because it can be consumed on a daily basis. The added amount of the extract or fractions of fermented soybeans in such a health functional food varies depending on the type of health functional food and cannot be defined uniformly, but may be added within a range that does not impair the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in an amount of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

ingredient

Ungdam (Ursi Fel, UF) belonged to a brown bear (Ursus arctos Linne) and was purchased from a herbal medicine company (Jeondam Eun, Yangju, Korea).

UDCA (ursodeoxycholic acid), docetaxel (DC) and MTT were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-Bax and anti-caspase-3 antibodies (Abs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Akt was purchased from Cell Signaling Technologies (Boston, MA, USA).

Horeseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies were provided by Bio-Rad (Hercules, CA, USA). RIPA buffer was purchased from Thermo Scientific Co. (Rockford, IL, USA), 1× protease inhibitor cocktail kit (tissue 2 perfect) was purchased from Quartett (Berlin, Germany), and Xpert phosphatase inhibitor was GenDEPOT (Barker, Texas, USA). was purchased from A nitrocellulose (NC) membrane and Clarity enhanced chemiluminescence (ECL) western blot substrate were purchased from Bio-Rad.

Example 1 - Preparation of UF extract

After immersing the purchased Wogdam (Ursi Fel, UF) in water:ethanol (=1:1, v/v), it was left at 20° C. for 3 months to obtain an extract. The extracts were collected and concentrated under reduced pressure to obtain a powder through freeze-drying. The powder was dissolved in purified water and ethanol (=1:1, v/v) to prepare samples of 10 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL.

Although quantitative data is not shown here, according to the results of conventional studies, in general, the extract contains about 19% by weight of UDCA, and considering that the molecular weight of UDCA is 392.58 g/mol, 50 μg of It is thought to contain about 0.02 μmol of UDCA.

Experimental Example 1 - Comparative experiment on the effect of UF extract and UDCA on cell growth

1-1. Experiment preparation

To confirm the effect of UF extract and UDCA on cell growth, FRO cells were treated with different concentrations of UF extract and UDCA for 24, 48 and 72 hours, and their viability was measured by MTT assay.

The FRO cells were provided by Dr. Kim ES of Ulsan University Hospital as a human thyroid cancer cell line. The FRO cells were maintained at 37° C. in a humidified carbon dioxide atmosphere with _{5% CO 2} in DMEM (Dulbecco's Modified Eagle's Medium, Corning Incorporated, Corning, NY, USA) supplemented with 10% fetal bovine serum (Corning Incorporated). did. The cells were cultured in 30 mm dishes for 24 hours.

First, cells (1x104/well) were seeded in 96-well plates and incubated overnight, followed by UF extracts (0, 10, 25, 50 and 100 μg) and UDCA (0, 10, 25, 50 and 100 μM). 24 hours, 48 hours and 72 hours treatment. After each incubation, the medium was replaced with 100 μL of MTT solution (5 mg/mL) and incubated for 4 hours. Next, the reaction was stopped by removing the MTT solution and adding DMSO to each well, followed by incubation at room temperature for 15 minutes. The solubilized purple formazan crystals were transferred to a 96-well plate (100 μL/well). The colorimetric response was analyzed by measuring the optical density at 570 nm using a microplate reader (UVM, Cambridge, UK). Finally, cell viability was calculated for control cells that were not treated with UF extract and UDCA.

1-2. Experiment result

Figure 1A is a UF extract, Figure 1B shows the MTT analysis results of UDCA.

As shown in Figure 1A, cell viability was significantly inhibited by the UF extract for 24 hours (p<0.05), 48 hours (p<0.05) and 72 hours (p<0.001), and cell growth inhibition of 50% ( IC50) was 50.92 μg/mL at 48 hours and 49.45 μg/mL at 72 hours. On the other hand, IC50 by UDCA treatment was approximately 58.92 μM/mL at 48 hours and 52.49 μM/mL at 72 hours, and the inhibition of cell viability by UDCA treatment was lower than that of the UF extract. In particular, the UF extract treatment showed the best results at a concentration of 50 μg/mL for 72 hours, and it was found that optimal cell growth inhibition was achieved under the above conditions. In addition, in the 72-hour experiment, it was confirmed that the 50 μg/mL UF extract contained less UDCA than the dose of 100 μM/mL UDCA, showing effective inhibition of cell viability.

Experimental Example 2 - Comparative experiment on the effect of UF extract and UDCA on apoptosis

2-1. Experiment preparation

To investigate the effect of UF extract and UDCA on apoptosis, FRO cells were treated with different concentrations of UF extract and UDCA for 48 hours. After the treatment, it was stained with Hoechst.

The thyroid cancer cell line FRO cells prepared in Experimental Example 1-1 were used in the same manner in Experimental Example 2-1.

The cells were seeded and cultured overnight on Lab-Tek chamber slides (Nalge Nunc International) in an incubator at 37° C., 5% CO _{2 .} Cells were treated with UF extract (0, 25 and 50 μg) and UDCA (50 and 100 μM) for 48 h and washed 3 times with 1x PBS. Next, it was fixed with cold 4% paraformaldehyde and incubated with Hoechst 33342 dye (Thermo Fisher Scientific) for 15 minutes at room temperature. Thereafter, the stained cells were washed again 3 times with 1x PBS, and observed using a fluorescence microscope (Leica, Wetzlar, Germany).

2-2. Experiment result

The experimental results are shown in FIG. 2 . It was found that 25 μg/mL and 50 μg/mL of UF extraction treatment and 50 μM/mL and 100 μM/mL of UDCA treatment resulted in a higher degree of cell death compared to non-treated cells. 2 shows the results of Hoechst staining. As shown in FIG. 2 , it was confirmed that, when treated with UF extract, a dose-dependent increase in nuclear fragmentation was more efficient in cells than when treated with UDCA.

Experimental Example 3 - Pro-apoptotic protein expression measurement experiment

3-1. Experiment preparation

Expression of pro-apoptotic proteins including Bax, caspase-3 and cytochrome c was measured in FRO cells by Western blot.

The thyroid cancer cell line FRO cells prepared in Experimental Example 1-1 were used in the same manner in Experimental Example 3-1.

The cells were treated with UF extract (0, 25 and 50 μg/mL) and UDCA (50 and 100 μM/mL) and incubated for 48 hours. Total protein was lysed for isolation via RIPA lysis and extraction buffer (Thermo Fisher Scientific) containing 1x protease inhibitor cocktail (tissue 2 perfect) and Xpert phosphatase inhibitor (GenDEPOT, Barker, TX, USA). Cell debris was removed by centrifugation at 16,000×g for 20 min at 4°C. The same amount of protein was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad, Hercules, CA, USA). An electrical transfer system was used to transfer the protein from the gel to the NC membrane. Non-specific binding was blocked with 5% skim milk in TBST buffer (5 mm Tris-HCl, 136 mm NaCl and 0.1 % Tween-20 at pH 7.6) for 1 hour at room temperature. The membranes were then incubated with primary antibody overnight at 4 °C, washed 3 times with 1x TBS-T, and then 1 h with source-matched anti-rabbit or anti-mouse secondary mAbs in 5% skim milk at room temperature. incubated during Membranes were again washed three times with 1x TBS-T and developed using ECL Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, PA, USA) before analysis using the ChemiDoc MP imaging system (Bio-Rad Laboratories). Western blot bands for Bax, cytochrome c, and caspase-3 were confirmed, and the intensity of the Western blot band for β-actin was used as an internal control to quantify the intensity of the Western blot band for Bax.

3-2. Experiment result

The experimental results are shown in FIG. 3 . As shown in Figure 3a, UF extract or UDCA treatment was confirmed to increase the expression of Bax, caspase-3 and cytochrome c. Although the UF extract at 50 μg/mL had a lower content of UDCA than that of UDCA at 100 μM/mL, it showed better expression of Bax, caspase-3 and cytochrome c when treated with cells. Also, as shown in FIG. 3b, a significant increase in the expression ratio of Bax/β actin was observed in cells treated with 50 μg/mL of UF extract (p<0.05).

Experimental Example 4 - Measurement of protein expression related to angiogenesis regulation

4-1. Experiment preparation

To investigate the effect of UF extract on angiogenesis in anaplastic thyroid carcinoma (ATC), the expression of angiogenesis regulators, TGF-βVEGF, N-cadherin and SIRT-1 was measured in FRO cells by western blot. .

The thyroid cancer cell line FRO cells prepared in Experimental Example 1-1 were used in the same manner in Experimental Example 4-1. Except for analyzing TGF-β, VEGF, N-cadherin and SIRT-1 instead of Bax, cytochrome c and caspase-3, the above experiment was performed in the same manner as in the Western blot experiment performed in 3-1.

Western blot bands for TGF-β, VEGF, N-cadherin, and SIRT-1 were confirmed, and the intensity of the western blot band of β-actin was used as an internal control to show TGF-β, VEGF, N-cadherin and SIRT-1. The intensity of the Western blot band was quantified.

4-2. Experiment result

The experimental results are shown in FIG. 4 . As shown in Figure 4, when the UF extract was treated, it was confirmed that the expression of TGF-βVEGF, N-cadherin and SIRT-1 was reduced compared to the untreated cells. In particular, a significant decrease in N-cadherin expression was observed in 50 μg/mL of UF extract (p<0.05). In addition, the expression of TGF-β, VEGF, N-cadherin and SIRT-1 was decreased in a concentration-dependent manner at 50 μg/mL (p <0.05) than at 25 μg/mL (p <0.05) in the UF extract.

On the other hand, it was found that the decrease in N-cadherin and VEGF expression was excellent compared to docetaxel.

Experimental Example 5 - Akt phosphorylation level confirmation experiment

5-1. Experiment preparation

Western blot analysis was used to measure the phosphorylation level of Akt, a marker related to cell cycle regulation.

The thyroid cancer cell line FRO cells prepared in Experimental Example 1-1 were used in the same manner in Experimental Example 5-1. Except that Akt was analyzed instead of Bax, cytochrome c, and caspase-3, the above experiment was performed in the same manner as in the Western blot experiment performed in 3-1.

The band intensity of Akt in the Western blot analysis was calculated by the phosphorylation ratio from the total morphological expression.

5-2. Experiment result

The experimental results are shown in FIG. 5 . As shown in Figure 5, when UF extracts were treated with 25 μg/mL (p<0.05), 50 μg/mL (p <0.001) and UDCA 100 μM/mL (p<0.05), Akt phosphorylation decreased in untreated cells. was found to be significantly higher than In addition, it was confirmed that when UDCA was treated at a content of 100 μM/mL, it had a similar activity as when treated with a UF extract of 50 μM/mL.

Experimental Example 6 - HPLC analysis

6-1. Experiment preparation

High Performance Liquid Chromatography (HPLC) was performed using the UF extract and UDCA to confirm the components of the UF extract including UDCA. The HPLC instrument was equipped with a Waters Delta 600 (Waters, Milford, MA, USA) pump, a PDA detector and a CAPCELL PAK C18 UG80 column (SHISHEIDO Co., Ltd. Japan). Chromatographic separation was tested using a gradient solvent system, and the chromatography included acetonitrile containing 0.1% formic acid (B) and water (A). Gradient elution was as follows; 0 % B at 0 min, 3 % B at 5 min, 3 % B at 15 min, 10 % B at 25 min, 15 % B at 35 min, 15 % B at 45 min, 30 % B at 55 min, 65 50 % B at min, 70 % B at 75 min, and 0 % B at 80 min. Column elution was monitored at UV 280 nm and all solvents were degassed using 0.2 μm cellulose acetate filtration. Chromatography was performed at room temperature at a flow rate of 1.0 mL/min, and 25 μL was analyzed.

Pump Waters Delta 600 (Waters, Milford, MA, USA) pump Detector PDA: photodiode array detector Column CAPCELL PAK C18 UG80 Column (SHISHEIDO Co., Ltd. Japan) flow rate 1.0 mL/min mobile phase Acetonitrile with 0.1% formic acid (B) and water (A) Injection volume 25 μL

6-2. Experiment result

The experimental results are shown in FIG. 6 . In HPLC analysis of UF extracts, UDCA was confirmed by comparison with retention times of standard reference substances. UDCA and UF extracts were confirmed to have a retention time (retention times) of 72.2 minutes through comparison with the retention times (retention times) of the standard reference material.

In addition, it was confirmed that the UF extract contained many compounds in addition to the active ingredient UDCA.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (9)

A pharmaceutical composition for preventing or treating thyroid cancer, comprising an extract of Woongdam's water and ethanol mixed solvent or a fraction thereof as an active ingredient.
delete According to claim 1,
The water and ethanol mixed solvent extract of Ungdam will be obtained by extraction for 1 month to 5 months under a temperature of 10 to 50 ℃, a pharmaceutical composition.
According to claim 1,
The fraction is fractionated using any one or more solvents selected from the group consisting of hexane (Hex), methylene chloride (MC) and ethyl acetate (EtOAc), the pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the water and ethanol mixed solvent extract of Ungdam is contained in an amount of 25 to 100 μg/mL based on the total composition.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition promotes apoptosis of cancer cells.
The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition increases the expression of one or more proteins selected from the group consisting of Bax, cytochrome c and caspase-3 in cancer cells.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits angiogenesis of cancer cells.
A health functional food composition for preventing or improving thyroid cancer, comprising an extract of Woongdam's water and ethanol mixed solvent or a fraction thereof as an active ingredient.

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