KR20230168808A - A composition for preventing or inhibiting cancer metastasis comprising an extract of Copthdis Rhizoma - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition for preventing or inhibiting cancer metastasis containing a yellow lotus extract as an active ingredient, wherein the yellow lotus extract inhibits the metastasis and invasion of cancer cells resistant to anticancer drugs and affects cell cycle regulation. Because it has the effect of suppressing the proliferation of cancer cells, it can be usefully used in related medicine or health food fields.

Description

A composition for preventing or inhibiting cancer metastasis comprising an extract of Copthdis Rhizoma}

The present invention relates to pharmaceutical compositions and food compositions containing yellow lotus extract as an active ingredient for overcoming anticancer drug resistance.

Cancer is a disease whose mortality rate continues to increase every year due to changes in the environment, aging population, and changes in lifestyle. It is one of the top five causes of death worldwide and the number one cause of death in Korea. Cancer is treated with surgery, radiation therapy, and drug therapy. In the case of drug therapy, starting with first-generation chemotherapy, second-generation targeted anti-cancer drugs were developed and are currently the most widely used, but they are facing a major problem called anti-cancer drug resistance.

Anticancer drug resistance is caused by denaturation of target proteins of anticancer drugs, activation of prosurvival signaling pathways, increase in DNA repair system, failure to induce apoptosis signaling, overexpression of drug efflux pump proteins, etc. It occurs for a variety of reasons, and it is known that approximately 90% of cancer patients who fail chemotherapy are related to anticancer drug resistance. Anticancer drug resistance is a major obstacle to cancer treatment. However, there is no suitable method to overcome anticancer drug resistance, so the development of technology to overcome anticancer drug resistance is urgently needed.

Meanwhile, to overcome anticancer drug resistance, immunotherapy, next generation drugs that complement the problems of existing drugs, combination therapy with existing drugs, new anticancer substances derived from natural products, and new drug delivery systems are used. Although various researches such as development are being conducted, it has not yet been fully put into practical use in clinical settings.

Republic of Korea Patent Publication No. 10-2019-0106315 (2019.09.18) Republic of Korea Patent Publication No. 10-2237651 (2020.05.25)

The purpose of the present invention is to provide a pharmaceutical composition and a food composition for the prevention or treatment of anticancer drug resistance, which contain an anticancer extract as an active ingredient.

The present invention contains yellow lotus extract as an active ingredient, a column flow rate of 1 mL/min, and jatrorizin ( Jatrorrhizine), Coptisine detected at 28.5 ± 1 minutes, Palmatine detected at 32.4 ± 1 minutes, and Berberine detected at 34.0 ± 1 minutes, the peak of palmatine Provided is a pharmaceutical composition for preventing or inhibiting cancer metastasis, wherein the intensity of the peak of berberine is greater than that of the peak.

The pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention may be a pharmaceutical composition for preventing or treating cancer metastasis that has an anticancer effect through inhibition of cancer metastasis.

In the pharmaceutical composition for preventing or suppressing cancer metastasis according to an example of the present invention, the intensity of the peak of coptisine and palmatine is greater than the intensity of the peak of jatrolyzine, and it may be a food composition for preventing or improving cancer metastasis. .

The pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention further includes a first fraction detected at a retention time of 27.5 ± 1 minutes, wherein the intensity of the peak of the first fraction is 0.25 of the intensity of the peak of berberine. It may range from 0.5 to 0.5.

In the pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention, the cancer may be colon cancer, colorectal cancer, bladder cancer, ovarian cancer, stomach cancer, lung cancer, prostate cancer, or pancreatic cancer.

In the pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention, the Yellow Lotus extract is obtained by mixing the Yellow Lotus rhizome powder with a co-solvent in which ethanol and water are mixed in a volume ratio of 20 to 50:80 to 50. Extracting at room temperature; Concentrating the supernatant after removing the precipitate from the extracted solution; and drying the concentrated solution; It may include a first extract prepared from.

In the pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention, the Yellow Lotus extract is obtained by mixing the Yellow Lotus rhizome powder with a co-solvent in which ethyl acetate and hexane are mixed in a volume ratio of 60 to 90:40 to 10. Extracting at room temperature; Concentrating the supernatant after removing the precipitate from the extracted solution; and drying the concentrated solution; It may further include a second extract prepared from.

The pharmaceutical composition for preventing or inhibiting cancer metastasis according to an example of the present invention may further include an anticancer agent.

In the pharmaceutical composition for inhibiting cancer metastasis according to an example of the present invention, the anticancer agent is Oxaliplatin, Irinotecan, Doxorubicin, Cisplatin, 5-fluorouracil (5-FU) , 5-fluorouracil), UFT (Tegafur-uracil), Capecitabine, Dactinomycin, and Docetaxel.

In the pharmaceutical composition for inhibiting cancer metastasis according to an example of the present invention, the anticancer agent is 5-fluorouracil (5-FU, 5-fluorouracil), and yellow lotus extract and 5-fluorouracil (5-FU) , 5-fluorouracil) may have a solid weight ratio of 1:0.08 to 0.3.

In addition, the present invention contains yellow lotus extract as an active ingredient, a column flow rate of 1 mL/min, and a jatrogen extract detected at a retention time of 23.50 ± 1 min in HPLC under mobile phase conditions containing distilled water and acetonitrile in a 5:5 weight ratio. Jatrorrhizine, Coptisine detected at 28.46 ± 1 minutes, Palmatine detected at 32.40 ± 1 minutes, and Berberine detected at 34 ± 1 minutes. A food composition for preventing or improving cancer metastasis is provided, wherein the intensity of the peak of berberine is greater than that of the peak.

In the food composition for preventing or improving cancer metastasis according to an example of the present invention, the food composition is an oral dosage form selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups. It may be a food composition.

The pharmaceutical composition for inhibiting cancer metastasis containing the yellow lotus extract as an active ingredient according to the present invention is significantly excellent in preventing and inhibiting cancer metastasis. More specifically, compared to existing anticancer drugs that mainly have a pharmacological effect on killing cancer cells, the composition of the present invention regulates the expression of resistance-causing genes in cancer cells resistant to anticancer drugs, inhibits the growth and division of cancer cells, and prevents metastasis. The suppressing effect is excellent.

Figure 1 shows the results of HPCL analysis of four indicator components (Berberine, Palmatine, Coptisine, and Jatrorrhizine) of yellow lotus extract.
Figure 2 shows the results of confirming resistance to the anticancer drug 5-FU in HCT116/R cells and specifying the concentration of the anticancer drug 5-FU (A). This is the result of confirming the gene and protein expression levels of Thymidylate synthase (TS), an anticancer drug resistance mediator, in HCT116/R cells (B, C, D).
Figure 3 shows the results of confirming the harmless concentration of yellow lotus extract for anticancer drug-resistant cells.
Figure 4 shows the results of confirming the mobility inhibition effect in HCT116/R cells through wound healing analysis.
Figure 5 shows the results confirming the invasiveness inhibitory effect through cell invasion analysis in HCT116/R cells.
Figure 6 shows results showing changes in expression of proteins characteristic of EMT in HCT116/R cells.
Figure 7 shows the results of confirming gene expression of E-cadherin and Vimentin in HCT116/R cells by PCR.
Figure 8 shows the results of confirming the expression changes of TGF-β and its subordinate proteins involved in EMT.
Figure 9 shows the results of confirming whether yellow lotus extract inhibits mobility by inhibiting TGF-β using a TGF-β inhibitor.
Figure 10 shows the results of flow cytometry confirming cell cycle changes.
Figure 11 shows the results of confirming the expression levels of proteins involved in the cell cycle.
Figure 12 shows the results confirming the changes in expression of TS genes and proteins that appear when the yellow lotus extract and 5-FU are combined.

Hereinafter, with reference to the attached tables and drawings, a pharmaceutical composition and a food composition containing the yellow lotus extract of the present invention as an active ingredient and having an anticancer drug resistance suppressing effect will be described in detail.

When drawings are described, they are provided as examples so that the idea of the present invention can be sufficiently conveyed to those skilled in the art. Accordingly, the present invention is not limited to the presented drawings and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

At this time, if there is no other definition in the technical and scientific terms used, they have the meaning commonly understood by those skilled in the art to which this invention pertains, and the gist of the present invention is summarized in the following description and accompanying drawings. Descriptions of known functions and configurations that may be unnecessarily obscure are omitted.

Additionally, as used herein, the singular forms “a,” “an,” and “the” are intended to also include the plural forms, unless the context clearly dictates otherwise.

In addition, units used without special mention in the specification of the present invention are based on weight, and as an example, units of % or ratio mean weight % or weight ratio.

In addition, in the specification of the present invention, the expression “comprises” is an open description that has the same meaning as expressions such as “comprises,” “contains,” “has,” or “features,” and is not additionally listed. does not exclude elements, materials or processes that are not Also, “substantially… The expression “consists of” means that other elements, materials or processes not listed together with the specified elements, materials or processes do not have an unacceptably significant effect on at least one basic and novel technical idea of the invention. It means something that can exist in quantity. Additionally, the expression “consists of” means that only the elements, materials, or processes described are present.

Terms used in the specification of the present invention, “ingredient”, “composition”, “composition of compounds”, “compound”, “drug”, “pharmaceutically active agent”, “active agent”, “treatment”, “therapy”, “treatment” or "drug" refers to a compound or composition of compound(s) or substances that induces a desired pharmaceutical and/or physiological effect by local and/or systemic action when administered to a subject (human or animal). Used interchangeably.

As used herein, the term “treatment” or “therapy” as well as its different forms include prophylactic (eg, prophylactic treatment), curative or palliative treatment. As used herein, the term “treating” includes alleviating or reducing at least one detrimental or negative effect or symptom of a condition, disease or disorder.

The terms “prevention,” “improvement,” and “treatment” of the present invention should be interpreted in the broadest sense, and “prevention” refers to patients who may be exposed to or susceptible to a disease but have not yet experienced or revealed symptoms of the disease. This means preventing one or more of the clinical symptoms of the disease from progressing. “Treatment” means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.

In the present invention, “sample” or “sample” refers to an object for analysis and is used with the same meaning throughout the specification.

Hereinafter, the present invention will be described in more detail.

The present invention contains yellow lotus extract as an active ingredient, and jatrorizin ( Jatrorrhizine), Coptisine detected at 28.5 ± 1 minutes, Palmatine detected at 32.4 ± 1 minutes, and Berberine detected at 34.0 ± 1 minutes, the peak of palmatine Provided is a pharmaceutical composition for preventing or inhibiting cancer metastasis, wherein the intensity of the peak of berberine is greater than that of the peak.

Coptidis Rhizoma is a perennial herbaceous plant of the Ranunculaceae family and is cultivated in China, Korea, Japan, etc. It stops dysentery, diarrhea, pulmonary tuberculosis, hematemesis, nosebleeds, and fecal bleeding that are prevalent in the summer, and is also effective in diabetes, whooping cough, and sore throat. It has a remarkable sterilizing effect and is widely used for skin infections.

In the present invention, the extract is an extract obtained by extracting yellow lotus. The yellow lotus extract may refer to a result such as a liquid component obtained by immersing yellow lotus powder in a solvent and then extracting it at room temperature or at a heated state for a certain period of time, and a solid content obtained by removing the solvent from the liquid component, but is not limited thereto. Instead, it may include all of the extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof. The solvent may be one or more solvents selected from the group consisting of water, C1 to C4 alcohols, hexane, and mixtures thereof, but is not limited thereto.

Additionally, the extract may be a concept including a fraction of the extract.

The term "cancer" in the present invention refers to a condition in which problems occur in the normal division, differentiation, and death control functions of cells, causing abnormal hyperproliferation, infiltrating surrounding tissues and organs, forming lumps, and destroying or deforming existing structures. it means.

The cancer may be colon cancer, colorectal cancer, bladder cancer, ovarian cancer, stomach cancer, lung cancer, prostate cancer, or pancreatic cancer, but is not limited thereto.

In the pharmaceutical composition for preventing or inhibiting cancer metastasis of the present invention, the composition may have an anticancer effect through inhibition of cancer metastasis, but is not limited thereto.

Cancer metastasis refers to the spread of cancer cells to other tissues away from the original site of origin. Metastasis may be the progression of epithelial-mesenchymal transition (EMT).

The intensity of the peaks of coptisine and palmatine may be greater than the intensity of the peak of jatrorizine.

In the pharmaceutical composition for preventing or inhibiting cancer metastasis of the present invention, it further comprises a first fraction detected at a retention time of 27.5 ± 1 minutes, wherein the intensity of the peak of the first fraction is 0.25 to 0.5 of the intensity of the peak of berberine. It may have a range. It is preferable that the intensity of the peak of the first fraction and the intensity of the peak of berberine have the above-mentioned relationship, so that a more improved cancer metastasis inhibition effect can be achieved. More specifically, the intensity of the peak of the first fraction may range from 0.35 to 0.5 of the intensity of the peak of berberine, which is preferable because it can have a more significant cancer metastasis inhibition effect.

The yellow lotus extract includes mixing yellow lotus rhizome powder with a co-solvent in which ethanol and water are mixed in a volume ratio of 20 to 50:80 to 50, and then extracting it at room temperature; Concentrating the supernatant after removing the precipitate from the extracted solution; and drying the concentrated solution; It may include a first extract prepared from.

In addition, the yellow lotus extract includes the steps of mixing yellow lotus rhizome powder with a co-solvent in which ethyl acetate and hexane are mixed in a volume ratio of 60 to 90:40 to 10, and then extracting it at room temperature; Concentrating the supernatant after removing the precipitate from the extracted solution; and drying the concentrated solution; It may further include a second extract prepared from. As the first extract further includes the second extract, the intensity of the peak of the first fraction may range from 0.35 to 0.5 of the intensity of the peak of berberine, and thus may have a more significant cancer metastasis inhibition effect. It is desirable in that respect.

The yellow lotus extract may further contain an anticancer agent. The anticancer agent is an anticancer agent used in anticancer chemotherapy and refers to a compound designed to destroy cancer cells for cancer treatment.

The anticancer drugs specifically include Oxaliplatin, Irinotecan, Doxorubicin, Cisplatin, 5-FU, 5-fluorouracil, UFT (Tegafur-uracil), and Caffeine. It may be one or more selected from the group consisting of Capecitabine, Dactinomycin, and Docetaxel, but is not limited thereto.

In addition, the anticancer agent may specifically be 5-fluorouracil (5-FU, 5-fluorouracil), and the solid weight ratio of yellow lotus extract and 5-fluorouracil (5-FU, 5-fluorouracil) is 1: It may be 0.08 to 0.3, specifically 1:0.1 to 0.25, but is not limited thereto. When the yellow lotus extract and 5-fluorouracil are administered in a mixture of the above weight ratio, it is preferable because it has an excellent effect of controlling the expression of resistance-causing genes in cells resistant to anticancer drugs and reducing the mobility of cancer cells to inhibit metastasis.

The present invention provides a food composition for preventing or improving cancer metastasis, wherein the food composition contains yellow lotus extract as an active ingredient, a column flow rate of 1 mL/min, and mobile phase conditions including distilled water and acetonitrile in a weight ratio of 5:5. In HPLC, Jatrorrhizine was detected at a retention time of 23.50±1 minutes, Coptisine was detected at 28.46±1 minutes, Palmatine was detected at 32.40±1 minutes, and 34±1 minutes. It contains detected berberine, and is characterized in that the intensity of the peak of berberine is greater than that of the peak of palmatine.

The term “improvement” includes any action that causes the symptoms of a disease to improve or become beneficial.

The food composition may be an oral dosage form selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups, but is not limited thereto.

Additionally, the present invention can provide a pharmaceutical composition for suppressing anticancer drug resistance.

In addition, the present invention can provide a pharmaceutical composition for adjuvant anticancer drugs.

Hereinafter, the contents of the present invention will be described in more detail through examples. The examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example 1>

For Coptidis Rhizoma, powdered Coptidis Rhizoma purchased from an herbal medicine distributor (Jeong-Seong Inc., Daejeon, Korea) was used. 1L of co-solvent, which is a mixture of ethanol and water in a volume ratio of 30:70, was added to 100g of yellow lotus powder and extracted with stirring at room temperature (25°C) for 72 hours. After stirring, centrifugation was performed at 5000 rpm for 30 min to obtain the supernatant, which was filtered through 50 mm 300 mesh filter paper (Advantac, Tokyo Roshi Kaisah, Tokyo, Japan). The filtered liquid (fillterate) was concentrated in a circular evaporator and then freeze-dried to prepare the first yellow lotus extract.

<Example 2>

Except for extracting using a co-solvent of ethyl acetate and hexane in a volume ratio of 70:30 as the extraction solvent in Example 1, the remaining process was performed in the same manner to prepare a second yellow lotus extract. A third yellow lotus extract was prepared by mixing the first yellow lotus extract and the second yellow lotus extract prepared in Example 1 at a weight ratio of 50:50.

<Experimental Example 1> Identification of components of yellow lotus extract

As a step to identify the active ingredient of the yellow lotus extract, 25 mg of each yellow lotus extract of Example 1 was dissolved in 25 mL of 70% methanol and then filtered through a 0.2 μm nylon filter. Then, 10 μL of the filtrate was injected into an HPLC analysis device using a C18 column to perform fingerprint analysis, and the results are shown in Figure 1.

At this time, the column flow rate was 1 mL/min, and the mobile phase conditions were (A) distilled water and (B) acetonitrile as follows: 0 to 15 min, 10 to 25 % B (isocratic elution); 15 to 25 min, 25 to 30 % B (linear gradient elution); 25 to 40 min, 30 to 45% B (linear gradient elution); 40 to 45 min, 45 to 45% B (linear gradient elution); 45 to 50 min, 45 to 10% B (linear gradient elution); and 50 to 60 min (isocratic elution). This was detected with a UV detector. As a result, Jatrorrhizine, Coptisine, Palmatine, and Berberine, which are indicator substances of yellow lotus, were retained at a total of 4 main peaks at a UV wavelength of 265 nm with a coefficient of variation of 0.04%. It was confirmed that the retention time was 23.50 minutes (Jatrorrhizine), 28.46 minutes (Coptisine), 32.40 minutes (Palmatine), and 33.94 minutes (Berberine).

Referring to Figure 1, the peak intensity of coptisine and palmatine was found to be greater than that of jatrolyzine. Meanwhile, the first fraction was detected at a retention time of 27.5 ± 1 minutes, and the intensity of the peak of the first fraction was found to have a value of 0.23 of the intensity of the peak of berberine.

In addition, the yellow lotus extract of Example 2 was injected into the HPLC analysis device in the same manner as described above and fingerprint analysis was performed. As a result, the peak intensities of coptisine and palmatine were found to be greater than those of jatrorizin. In addition, the intensity of the peak of the first fraction detected at a retention time of 27.5 ± 1 minutes was found to have a value of 0.42 of the intensity of the peak of berberine.

<Experimental Example 2> Evaluation of anticancer drug metastasis inhibition activity

2-1. Preparation of cells resistant to anticancer drugs and identification of resistance factors

2x10³ of the normal human colon cancer cell line, HCT116/WT, and the anticancer drug-resistant cell line, HCT116/R, were dispensed into each 96-well plate, cultured for 24 hours, and representative anticancer drugs for colon cancer treatment, 5-FU and cisplatin, were added at each concentration (5). , 25, and 50 μM) were added. After culturing for an additional 48 hours, 1/10 volume of WST-8 reagent (DoGen, Seoul, Korea) was added to each well according to the WST cell viability measurement technique, and after 2 hours, the change in absorbance was measured at 450 nm. The results are shown in Figure 2A. It was confirmed that HCT116/R cells were resistant to 5-FU, and 25 μM of 5-FU, which had a cell viability of more than 80%, was specified as the concentration for subsequent experiments.

In addition, to compare gene expression of representative anticancer drug resistance mediators, P-gp (P-glycoprotein), GST (glutathione S-transferase), and TS (thymidylate synthase), HCT116/WT and HCT116/R cells were cultured for 60 pi. Each plate was distributed at 3x10 ⁴ and cultured for 48 hours, and then cells were collected using Trypsin. Total RNA was isolated using TRIZOL reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized using PrimeScript RT reagent kit (Takara, Dalian, China) using 1 μg of total RNA. The primer sequences used are as follows.

Human p-gp forward, 5' AAG GCC TAA TGC CGA ACA CA 3' (SEQ ID NO: 1) and reverse, 5' TCC AGG CTC AGT CCC TGA AG 3' (SEQ ID NO: 2), human GST forward, 5' TTC CTG TGG CAT AAT GTG AT 3' (SEQ ID NO: 3) and reverse, 5' CTG ATT CAA AGG CAA ATC TC 3' (SEQ ID NO: 4), human TS forward, 5' ACC GAG CTC CCG AGA CTT TTT GGA CAG CCT 3' (SEQ ID NO: 5) and reverse, 5' ACC AAG CTT AAG AAT CCT GAG CTT TGG GAA 3' (SEQ ID NO: 6), human GAPDH forward, 5' CAT GGC CTT CCG TGT TCC T 3' (SEQ ID NO: 7) and reverse , 5' CCT GCT TCA CCA CCT TCT TGA 3' (SEQ ID NO: 8).

cDNA was amplified using a SimpliAmp Thermal Cycler (Applied Biosystems, Forster City, CA, USA).

The results are shown in Figure 2B. As shown in the results, it was confirmed that the expression level of TS in HCT116/R cells was significantly increased compared to other genes, and it was confirmed that TS was the cause of anticancer drug resistance in HCT116/R cells.

To check the amount of TS protein expression in HCT116/R cells, cells were cultured for 48 hours under the same conditions, 200 uL of protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) was added to obtain protein, and an equal amount of protein was extracted with sodium dodecyl sulfate. (SDS)- Separated by polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was probed with specific primary and secondary antibodies. Complexes of proteins and antibodies were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). The primary antibodies used were Anti-thymidylate synthase (TS, Cell Signaling Technology, Danvers, MA, USA) and alpha-tubulin (Thermo-Fisher Scientific, Waltham, MA, USA), and the secondary antibodies were secondary horseradish peroxidase (HRP). )-conjugated antibodies (GeneTex, Inc., Irvine, CA, USA) were used. The protein expression confirmation results of TS are shown in Figures 2C and D. As shown in the results, it was confirmed that the protein expression of TS was the same as the gene expression trend, and it was confirmed that HCT116/R cells were appropriate cells for conducting anticancer drug resistance experiments.

2-2. wound healing assay

In order to determine the appropriate treatment concentration for Examples 1 and 2, a cytotoxicity evaluation was performed to determine the concentration. The results are shown in Figure 3.

It was confirmed that the concentration of 20ug/mL in Examples 1 and 2 was not cytotoxic, and experiments were conducted at this concentration.

To measure the metastasis inhibition activity of the anticancer drugs of Examples 1 and 2, a wound healing assay was performed to determine whether motility, one of the characteristics of cancer metastasis, is inhibited. HCT116/R cells were cultured by distributing 3x105 cells in a 60-pi culture dish. When the dish reached 90% density, the center was lined with a plastic pipette tip to remove cells within about 1 mm in width, destroying cell continuity. After washing once with culture medium, new culture medium was added, Examples 1 and 2 were added, culture was continued for 24 hours, and then the extent to which cells had moved to the area where persistence was lost was checked under a microscope. The results are shown in Figure 4. The characteristic of metastasis in HCT116/R cells is increased mobility. In HCT116/R cells treated with Example 1, mobility was suppressed by about 33%, and in the group treated with Example 2, mobility was suppressed by about 48%. It was confirmed that it was done.

2-3. 3D invasion assay

A 3D invasion assay was performed to confirm whether invasiveness, another characteristic of cancer metastasis, is suppressed.

A 24-well transwell with a pore size of 8.0㎛ was used. The outer membrane of the upper chamber was coated with 300 μg/mL Matrigel (BD Bioscience, San Jose, CA, USA). HCT116/R cells were prepared at 0.5-1.0 x 106 cells/ml and carefully sprinkled into the coated upper chamber. 500uL of medium was added to the bottom well, Example 1 was added at a concentration of 20ug/mL, and cultured for 24 hours. The results of quantifying the cells that migrated through the chamber 24 hours later are shown in Figure 5.

As seen in the results, it was confirmed that the group to which yellow lotus extract was added had an effect of suppressing the invasiveness of cancer cells.

2-4. Confirmation of metastasis-related protein expression changes

Western blot was performed to confirm expression changes in E-cadherin, vimentin, Snail, and ZEB2, which are well known as Epithelial-Mesenchymal Transition (EMT) markers in cancer.

HCT116/R cells were distributed at 3x10 ⁵ in a 60-pi culture dish, cultured for 12 hours, and then Example 1 (20 μg/mL) was added. After culturing for an additional 48 hours, cells were collected using Trypsin. Proteins were obtained by adding 200uL of protein extraction solution (iNtRON Biotechnology, Seongnam, Korea), and equal amounts of proteins were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was probed with specific primary and secondary antibodies. E-cadherin, vimentin, Snail, and ZEB (Santa Cruz Biotechnology, Dallas, TX, USA) were used as primary antibodies, and secondary horseradish peroxidase (HRP)-conjugated antibodies (GeneTex, Inc., Irvine, CA) were used as primary antibodies. , USA) was used. Complexes of proteins and antibodies were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Western blot was performed to confirm protein expression changes. The results are shown in Figure 6. In the group treated with Example 1, it was confirmed that E-cadherin, a tumor suppressor, increased, and vimentin, Snail, and ZEB2 decreased.

In addition, the experiment was conducted under the same conditions as above, and 1 mL of RNA extraction solution (TRIzol, Molecular Research Center, Cincinnati, OH, USA) was dispensed to each of the obtained cells to lyse the cells. RNA was then isolated, and cDNA was extracted. By synthesizing and performing PCR, changes in mRNA expression levels of E-cadherin and vimentin were confirmed. The results are shown in Figure 7. The primer sequences used are as follows. Human E-cadherin forward, 5' AAG AAG CTG GCT GAC ATG TAC GGA 3' (SEQ ID NO: 9) and reverse, 5' CCA CCA GCA ACG TGA TTT CTG CAT 3' (SEQ ID NO: 10), human Vimentin forward, 5' AGA ACC TGC AGG AGG CAG AAG AAT 3' (SEQ ID NO: 11) and reverse 5' TTC CAT TTC ACG CAT CTG GCG TT 3' (SEQ ID NO: 12), human GAPDH forward, 5' CAT GGC CTT CCG TGT TCC T 3' (SEQ ID NO: 7) and reverse, 5' CCT GCT TCA CCA CCT TCT TGA 3' (SEQ ID NO: 8).

2-5. Confirmation of inhibition of TGF-b, a top metastasis-related protein

Western blot was performed to confirm changes in the expression level of TGF-beta (Transforming growth factor-beta), which exists upstream in epithelial-mesenchymal transition (EMT) signaling in cancer.

HCT116/R cells were distributed at 3x10 ⁵ in a 60-pi culture dish, cultured for 12 hours, and then Example 1 (20 μg/mL) was added. After culturing for an additional 48 hours, cells were collected using Trypsin. Protein was obtained by adding 200 uL of protein extraction solution (iNtRON Biotechnology, Seongnam, Korea), and Western blot was performed to confirm protein expression changes. At this time, the primary antibodies used were TGF-beta, p-AKT, AKT, p-p38, p38 (Santa Cruz Biotechnology, Dallas, TX, USA), and alpha-tubulin (Thermo-Fisher Scientific, Waltham, MA, USA). was used. Secondary horseradish peroxidase (HRP)-conjugated antibodies (GeneTex, Inc., Irvine, CA, USA) were used as secondary antibodies. The results are shown in Figure 8. In the group treated in Example 1, it was confirmed that the expression of TGF-beta and its subordinate p-AKT and p-p38 expressions were decreased.

In addition, a wound healing assay was performed using the TGF-beta inhibitor SB431542 (Sigma-Aldrich, St. Louis, MO, USA) to confirm whether the yellow lotus extract truly inhibits mobility by inhibiting TGF-beta. The results are shown in Figure 9. Example 1 is TGF- It was confirmed that the group treated with the inhibitor SB431542 showed similar values to those treated alone. Therefore, it can be assumed that yellow lotus extract reduces mobility by inhibiting TGF-beta.

<Experimental Example 3> Testing the efficacy of cancer metastasis inhibition by combined treatment with anticancer drugs

3-1. Check cell cycle changes

After culturing HCT116/R cells under the same conditions as above, they were treated with Example 1 at a concentration of 20ug/mL, and 25uM of 5-FU, an anticancer drug, was combined. For cell cycle analysis after 48 hours of culture, cells were collected, washed with cold PBS, and fixed with 80% ethanol for 1 hour at 4°C. Cells were then stained with 50 μg/ml PI in the presence of 30 μg/ml DNase-free RNase A for 30 min at room temperature. Then, the stained cells were resuspended in 500 μl of PBS. The relative DNA content at each stage of the cell cycle was analyzed using flow cytometry (FACSCalibeur, BD Biosciences). The results are shown in Figure 10. It was confirmed that the combined treatment of yellow lotus extract and anticancer drugs had a strong effect on the S phase of DNA replication as cells accumulated in the G0/G1 phase, showing the effect of suppressing cancer metastasis.

In addition, cells were cultured and collected under the same conditions as above, and the expression of proteins involved in the cell cycle was confirmed using Western blot technique. Primary antibodies are cyclin-dependent kinase (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), cyclin A, cyclin B1, cyclin D1, and cyclin E. (Santa Cruz Biotechnology, Dallas, TX, USA) and beta-actin (Thermo-FisherScientific, Waltham, MA, USA) were used, and secondary horseradish peroxidase (HRP)-conjugated antibodies (GeneTex, Inc., Irvine) were used as secondary antibodies. , CA, USA) was used. The results are shown in Figure 11. It was confirmed that among the proteins related to the cell cycle, CDK4, CDK2, CDK1, cyclinB1, and cyclinD1 were regulated in the group treated with yellow lotus extract and the anticancer drug 5-FU. It has been shown that yellow lotus extract and anticancer drugs inhibit cancer cell proliferation and metastasis by regulating the expression of cyclin and CDK proteins, which play an important role in the progression from G1 phase to S phase.

3-2. Confirmation of inhibition of anticancer drug resistance

It was tested whether the combined treatment of 5-FU could exhibit synergistic efficacy in suppressing cancer metastasis. HCT116/R cells with anticancer drug resistance were treated with Examples 1 and 2 (20 μg/mL) and 5-FU (25 μM) singly or in combination, and changes in the expression levels of TS genes and proteins were confirmed. After treatment with Examples 1 and 2 and the anticancer drug 5-FU, cells were collected by culturing for 48 hours. Gene expression of TS was measured using PCR technique, and changes in protein expression were confirmed using Western blot. The results are shown in Figure 12. The primer sequences used are as follows. Human TS forward, 5' ACC GAG CTC CCG AGA CTT TTT GGA CAG CCT 3' (SEQ ID NO: 5) and reverse, 5' ACC AAG CTT AAG AAT CCT GAG CTT TGG GAA 3' (SEQ ID NO: 6), human GAPDH forward, 5' CAT GGC CTT CCG TGT TCC T 3' (SEQ ID NO: 7) and reverse, 5' CCT GCT TCA CCA CCT TCT TGA 3' (SEQ ID NO: 8).

The primary antibodies used in Western blot were Anti-thymidylate synthase (TS, Cell Signaling Technology, Danvers, MA, USA) and beta-actin (Thermo-FisherScientific, Waltham, MA, USA). Secondary horseradish peroxidase (HRP)-conjugated antibodies (GeneTex, Inc., Irvine, CA, USA) were used as secondary antibodies.

When Example 1 and anticancer drugs were treated together, it was confirmed that the expression level of the TS gene, which was increased by the anticancer drug 5-FU, was reduced by about 20%. In the group treated in combination with Example 2, the expression level of TS, a resistance gene, was confirmed to be reduced by about 20%. It was confirmed that this was reduced by about 37%.

As seen from the above results, it was confirmed that the yellow lotus extract of the present invention has a significantly excellent effect in suppressing and preventing cancer metastasis when treated alone or in combination with cancer cells resistant to anticancer drugs.

As the specific parts of the present invention have been described in detail above, those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Contains yellow lotus extract as an active ingredient,
In HPLC with a column flow rate of 1 mL/min and mobile phase conditions containing distilled water and acetonitrile at a weight ratio of 5:5, Jatrorrhizine was detected at a retention time of 23.5 ± 1 min, and Coptisine was detected at 28.5 ± 1 min. (Coptisine), Palmatine detected at 32.4±1 minute, and Berberine detected at 34.0±1 minute,
A pharmaceutical composition for preventing or inhibiting cancer metastasis, characterized in that the intensity of the peak of berberine is greater than that of the peak of palmatine. According to paragraph 1,
The composition is a pharmaceutical composition for preventing or treating cancer metastasis, which has an anti-cancer effect through inhibition of cancer metastasis. According to paragraph 1,
A pharmaceutical composition for preventing or inhibiting cancer metastasis, wherein the peak intensity of coptisine and palmatine is greater than that of jatrorizine. According to paragraph 1,
A pharmaceutical composition for preventing or inhibiting cancer metastasis, further comprising a first fraction detected at a retention time of 27.5 ± 1 minutes, wherein the intensity of the peak of the first fraction is in the range of 0.25 to 0.5 of the intensity of the peak of berberine. According to paragraph 1,
A pharmaceutical composition for preventing or inhibiting cancer metastasis, wherein the cancer is colon cancer, colorectal cancer, bladder cancer, ovarian cancer, stomach cancer, lung cancer, prostate cancer, or pancreatic cancer. According to paragraph 1,
The yellow lotus extract,
Mixing yellow lotus rhizome powder with a co-solvent containing ethanol and water in a volume ratio of 20-50:80-50 and then extracting at room temperature;
Concentrating the supernatant after removing the precipitate from the extracted solution; and
A pharmaceutical composition for preventing or inhibiting cancer metastasis, comprising a first extract prepared from the step of drying the concentrated solution. According to clause 6,
The yellow lotus extract,
Mixing yellow lotus rhizome powder with a co-solvent containing ethyl acetate and hexane in a volume ratio of 60-90:40-10 and then extracting at room temperature;
Concentrating the supernatant after removing the precipitate from the extracted solution; and
A pharmaceutical composition for preventing or inhibiting cancer metastasis, further comprising a second extract prepared from the step of drying the concentrated solution. According to clause 7,
A pharmaceutical composition for preventing or inhibiting cancer metastasis further comprising an anticancer agent. According to clause 8,
The anticancer drugs include Oxaliplatin, Irinotecan, Doxorubicin, Cisplatin, 5-FU, 5-fluorouracil, UFT (Tegafur-uracil), and Capecitabine ( A pharmaceutical composition for preventing or inhibiting cancer metastasis, which is at least one selected from the group consisting of Capecitabine, Dactinomycin, and Docetaxel. According to clause 8,
The anticancer agent is 5-fluorouracil (5-FU, 5-fluorouacil), and the solid weight ratio of yellow lotus extract and 5-fluorouracil (5-FU, 5-fluorouracil) is 1: 0.08 to 0.3. Pharmaceutical composition for preventing or suppressing lice. Contains yellow lotus extract as an active ingredient,
Jatrorrhizine was detected at a retention time of 23.50 ± 1 min in HPLC with a column flow rate of 1 mL/min and a mobile phase containing distilled water and acetonitrile at a weight ratio of 5:5, and coptisine was detected at 28.46 ± 1 min. (Coptisine), Palmatine detected at 32.40±1 minute, and Berberine detected at 34±1 minute,
A food composition for preventing or improving cancer metastasis, characterized in that the intensity of the peak of berberine is greater than that of the peak of palmatine. According to clause 11,
The food composition is a food composition for preventing or improving cancer metastasis, which is an oral dosage form selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups.