COMPOSITIONS FOR PREVENTING OR TREATING CANCER COMPRISING EXTRACTS OF ASARUM MACULATUM NAKAI

Abstract

Provided are compositions for preventing or treating cancer including Asarum maculatum Nakai extracts, or a fraction thereof. According to an aspect, an Asarum maculatum Nakai extract has an excellent anticancer efficacy against various cancers including diffuse-type gastric cancer, and a cancer treatment agent having an excellent effect may be developed by using the extract as an active ingredient.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising an Asarum maculatum Nakai extract or a fraction thereof as an active ingredient.

2. The pharmaceutical composition of claim 1, further comprising a Rubus tozawae Nakai ex J. Y. Yang [Rubus longisepalus var. tozawai (Nakai) T. B. Lee] extract, or a fraction thereof.

3. The pharmaceutical composition of claim 1, wherein the extract is obtained by extraction using at least one solvent selected from water, methanol, ethanol, propanol, butanol, glycerin, butylene glycol, propylene glycol, methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane.

4. The pharmaceutical composition of claim 1, wherein the extract is obtained by extraction using at least one method selected from reduced pressure high temperature extraction, boiling extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction, steam extraction, and fractional extraction.

5. The pharmaceutical composition of claim 1, wherein the cancer is gastric cancer, liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, perianal cancer, colon cancer, breast cancer, cervical cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, adenocarcinoma of endocrine glands, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

6. The pharmaceutical composition of claim 1, wherein the cancer is diffuse-type gastric cancer.

7. A method of treating or preventing cancer, comprising administering to a subject the pharmaceutical composition comprising an Asarum maculatum Nakai extract or a fraction thereof.

8. The method of claim 7, wherein the composition further comprises a Rubus tozawae Nakai ex J. Y. Yang [Rubus longisepalus var. tozawai (Nakai) T. B. Lee] extract, or a fraction thereof.

9. The method of claim 7, wherein the extract is obtained by extraction using at least one solvent selected from water, methanol, ethanol, propanol, butanol, glycerin, butylene glycol, propylene glycol, methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane.

10. The method of claim 7, wherein the extract is obtained by extraction using at least one method selected from reduced pressure high temperature extraction, boiling extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction, steam extraction, and fractional extraction.

11. The method of claim 7, wherein the cancer is gastric cancer, liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, perianal cancer, colon cancer, breast cancer, cervical cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, adenocarcinoma of endocrine glands, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

12. The method of claim 7, wherein the cancer is diffuse-type gastric cancer.

13. A food or feed composition for preventing or improving cancer, comprising an Asarum maculatum Nakai extract or a fraction thereof as an active ingredient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0065] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0066] FIG. 1 shows diagrams measuring expression levels of markers related to epithelial-mesenchymal transition for screening for diffuse-type gastric cancer cell lines,

[0067] FIG. 2 shows a diagram showing results of a primary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0068] FIG. 3 shows a diagram showing results of a secondary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0069] FIG. 4 shows a diagram showing results of a tertiary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0070] FIG. 5 shows diagrams confirming cell growth inhibitory ability of Asarum maculatum Nakai extracts for each solvent on diffuse-type gastric cancer cell lines,

[0071] FIG. 6 shows a diagram confirming cell viability inhibitory ability of Asarum maculatum Nakai extracts for each solvent on a normal cell line,

[0072] FIG. 7 shows diagrams confirming cell growth inhibitory ability of Asarum maculatum Nakai butanol extracts of each concentration on the diffuse-type gastric cancer cell lines,

[0073] FIG. 8 shows a diagram confirming cell viability inhibitory ability of Asarum maculatum Nakai butanol extracts of each concentration on normal cell lines,

[0074] FIG. 9 shows a diagram confirming cell growth inhibitory ability of Asarum maculatum Nakai butanol extracts of each concentration on organoid cohorts derived from patients with diffuse-type gastric cancer,

[0075] FIG. 10 shows a diagram confirming cell viability inhibitory ability of Asarum maculatum Nakai butanol extracts of each concentration on normal organoids,

[0076] FIG. 11 shows diagrams confirming cell growth inhibitory ability of an Asarum maculatum Nakai butanol extract, and extracts of Asarum heterotropoides var. seoulense (Nakai) Kitag for each solvent, on the diffuse-type gastric cancer cell lines, and

[0077] FIG. 12 is a diagram confirming cell growth inhibitory ability of an Asarum maculatum Nakai butanol extract on various cancers.

DETAILED DESCRIPTION

[0078] Reference will now be made in detail to embodiments, embodiments of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of at least one of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0079] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.

Example 1: Establishment of Diffuse-Type Gastric Cancer Cell Models

[0080] The following experiment was performed in order to establish diffuse-type gastric cancer cell models for screening for effective substances for treating diffuse-type gastric cancer.

[0081] Specifically, in order to select diffuse-type gastric cancer cell lines, epithelial mesenchymal transition levels of 11 gastric cancer cell lines (Hs746T, MKN1, SNU1, SNU-668, MKN28, MKN74, NCI-N87, SNU-601, SNU-719, SNU-1967) were measured. To this end, after culturing the cells, mRNA expression levels of 8 markers (CDH1, Claudin-7, GRHL2, EpCAM, Vimentin, ZEB1, SLUG, Fibronectin), which may confirm the degree of epithelial-mesenchymal transition, were confirmed (FIG. 1).

[0082] As a result, among cell lines with low expression levels of CDH1, Claudin-7, GRHL2, and EpCAM and high expression levels of Vimentin, ZEB1, SLUG, and Fibronectin, MKN1 and SNU668 were selected as diffuse-type gastric cancer cell lines. In the following examples, substances for treating diffuse-type gastric cancer were screened by using the cell lines.

Example 2: Screening for Active Substances for Treating Diffuse-Type Gastric Cancer

[0083] In order to screen for effective substances for treating diffuse-type gastric cancer from various natural products, the following experiments were performed.

[0084] A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was performed to evaluate cell growth inhibitory ability according to treatment of samples of natural extracts. Specifically, each cell line was seeded in a 96-well plate by 5000 each and cultured for one day, then, the experimental groups were treated with samples of each concentration, incubated for 72 hours, and 50 μL of MTT (2 mg/mL) dye was added. Thereafter, additional incubation was performed for 4 hours, the medium was removed, and dimethyl sulfoxide was added to each well to dissolve the dye for 20 minutes, and then absorbance was measured at a wavelength of 490 nm to analyze the degree of cell viability.

[0085] First, for a primary screening, after 63 kinds of natural substance extract samples of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated to MKN1, a diffuse-type gastric cancer cell line selected in Example 1, cell growth inhibitory ability was confirmed by an MTT assay, and samples showing 50% or more cell growth inhibitory activity when treated at a concentration of 100 μg/ml were primarily selected. As a result, it was confirmed that 19 samples out of a total of 63 samples exhibited 50% or more of cell growth inhibitory activity against MKN1, and the 19 samples were primarily selected (FIG. 2).

[0086] Next, for a secondary screening, after 19 kinds of primarily selected natural substance extract samples of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated to SNU688, a diffuse-type gastric cancer cell line selected in Example 1, cell growth inhibitory ability was confirmed by an MTT assay, and samples showing 50% or more cell growth inhibitory activity when treated at a concentration of 100 μg/ml were secondarily selected. As a result, it was confirmed that 14 samples out of a total of 19 samples exhibited 50% or more of cell growth inhibitory activity against SNU688, and the 14 samples were secondarily selected (FIG. 2).

[0087] Next, for a tertiary screening based on cytotoxicity on normal cells, after 14 kinds of secondarily selected natural substance extract samples of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated to HEK293, a normal cell line, cell viability inhibitory ability on normal cells was confirmed by an MTT assay, and samples showing 45% to 50% or less cell viability inhibitory activity when treated at a concentration of 100 μg/ml were tertiary selected. As a result, it was confirmed that 7 samples out of a total of 14 samples exhibited less than about 45% to about 50% of normal cell viability inhibitory activity on HEK293, and the 7 samples with low toxicity to normal cell lines were tertiary selected. (FIG. 4).

[0088] In addition, by comprehensively considering the inhibitory activity on MKN1 and SNU668, which are diffuse-type gastric cancer cell lines, and the cytotoxicity results for a normal cell line, a Rubus tozawae Nakai ex J. Y. Yang [Rubus longisepalus var. tozawai (Nakai) T. B. Lee] extract and an Asarum maculatum Nakai extract were finally selected as candidate active substances for treating diffuse-type gastric cancer.

Example 3: Evaluation of Anticancer Efficacy of Asarum maculatum Nakai Extract

[0089] In order to evaluate anticancer efficacy of an Asarum maculatum Nakai extract selected as a candidate active substance for treating diffuse-type gastric cancer in Example 2, the following experiment was performed.

3.1 Preparation of Asarum maculatum Nakai Extract

[0090] In order to prepare an extract of Asarum maculatum Nakai by using various solvents, the following experiment was performed.

[0091] Specifically, the whole plant of Asarum maculatum Nakai was collected in the Hantaek Botanical Garden in August 2020 and dried with warm air (30° C.) for 7 days to obtain 350 g of dried plant. Thereafter, the dried plant was crushed and put into an extraction container, 3.5 L of 70% ethanol/water was added, the sample was stirred by shaking at room temperature for 7 days for an extraction, the process of gravity filtration by using a Whatman filter paper having a film thickness of 0.34 mm and a glass funnel was repeated twice (‘extraction-filtration’ twice) on the mixture to obtain a filtered extract. The filtered extract was transferred to a round flask, placed in a vacuum concentrator, and the solvent was completely evaporated at 35° C. under reduced pressure to obtain 45.9 g of an Asarum maculatum Nakai ethanol extract (yield 13.1%).

[0092] Next, 45.9 g of the ethanol extract was suspended in 300 mL of water, 450 mL of n-hexane was added, and the mixture was stirred at room temperature and fractionated 3 times for 2 hours each, and then the mixture was placed in a vacuum concentrator, and the solvent was completely evaporated at 35° C. under reduced pressure and concentrated to obtain 6.7 g of n-hexane fraction extract.

[0093] After the fractionation, 450 mL of water-saturated n-butanol was added to the remaining water, the mixture was stirred by shaking at room temperature, and fractionation was carried out 3 times for 2 hours each, and then the mixture was placed in a vacuum concentrator, the solvent was completely evaporated at 35° C. under reduced pressure and concentrated to obtain 16.9 g of n-butanol fraction extract, and the water remaining after the fractionation was freeze-dried to obtain 21.7 g of a water fraction extract.

3.2 Evaluation of Treatment Efficacy for Diffuse-Type Gastric cancer and cytotoxicity for each extraction solvent

[0094] The following experiments were performed to evaluate a treatment efficacy for diffuse-type gastric cancer and cytotoxicity on normal cells of the Asarum maculatum Nakai extract prepared in Example 3.1 for each solvent.

[0095] First, in order to evaluate anticancer efficacy against diffuse-type gastric cancer, Asarum maculatum Nakai extracts of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) for each solvent were treated on MKN1 and SNU668, which are the diffuse-type gastric cancer cell lines selected in Example 1, and then the cell growth inhibitory ability was confirmed by an MTT assay. As a result, the hexane and butanol extracts showed an anticancer efficacy against the two types of diffuse-type gastric cancer cell lines, and in particular, it was confirmed that the butanol extract showed an excellent anticancer efficacy (FIG. 5).

[0096] Next, in order to evaluate cytotoxicity of the hexane and butanol extracts, which were confirmed to have an excellent anticancer efficacy above, Asarum maculatum Nakai extracts of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) for each solvent were treated on HEK293, a normal cell line, and then cell growth inhibitory abilities thereof were confirmed by an MTT assay. As a result, it was confirmed that the butanol extract showed significantly lower cytotoxicity on normal cells compared to the hexane extract (FIG. 6).

[0097] Based on the above results, it may be seen that an Asarum maculatum Nakai butanol extract, which has an excellent cell death effect on diffuse-type gastric cancer and low cytotoxicity to normal cells, has significantly superior anticancer efficacy among extracts for each solvent.

3.3 Evaluation of Treatment Efficacy for Diffuse-Type Gastric Cancer and Cytotoxicity of Asarum maculatum Nakai Butanol Extract—Cell Models

[0098] The following experiments were performed to evaluate a treatment efficacy for diffuse-type gastric cancer and cytotoxicity of an Asarum maculatum Nakai butanol extract confirmed to have an excellent efficacy in Example 3.2 in cell models.

[0099] First, in order to evaluate anticancer efficacy for diffuse-type gastric cancer, Asarum maculatum Nakai butanol extracts of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated on MKN1 and SNU668, which are the diffuse-type gastric cancer cell lines selected in Example 1, and then the cell growth inhibitory ability was confirmed by an MTT assay. As a result, it was confirmed that an Asarum maculatum Nakai extract is capable of strongly inhibiting growth of diffuse-type gastric cancer cells even at a low concentration of 50 μg/ml (FIG. 7).

[0100] Next, in order to evaluate cytotoxicity to normal cell lines, Asarum maculatum Nakai butanol extracts of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated to HEK293, normal kidney-derived cells, and HFE-145, normal gastric epithelium-derived cells, and then, cell growth inhibitory ability thereof was confirmed by an MTT assay. As a result, the Asarum maculatum Nakai extract did not significantly inhibit cell viability, even at a high concentration of about 100 μg/ml, and it was confirmed that its toxicity to normal cells is low (FIG. 8).

[0101] Based on the above results, it may be seen that an Asarum maculatum Nakai butanol extract has excellent anticancer efficacy for diffuse-type gastric cancer and low cytotoxicity to normal cells.

3.4 Evaluation of Treatment Efficacy for Diffuse-Type Gastric Cancer and Cytotoxicity of Asarum maculatum Nakai Butanol Extract—Organoid Models

[0102] The following experiment was performed in order to evaluate treatment efficacy for diffuse-type gastric cancer and cytotoxicity of the Asarum maculatum Nakai butanol extract, which was confirmed to have an excellent efficacy in Example 3.2, in organoid models.

[0103] First, in order to prepare diffuse-type gastric cancer organoids and normal gastric organoids, gastric tissues of patients with diffuse-type gastric cancer and normal persons obtained through surgical resection were washed with Dulbecco's phosphate-buffered saline (DPBS) (without Ca.sup.2+ or Mg.sup.2+) and then cut into small pieces, and the cut pieces were reacted with a stripping buffer [composed of Hank's balanced salt solution (HBSS), ethylenediaminetetraacetic acid (5 mmol/L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) (25 mmol/L), and 10% fetal calf serum (FCS)] for 10 minutes at 37° C. Afterwards, the stripping buffer was replaced with a fresh stripping buffer, and the tissue pieces were further reacted with the fresh stripping buffer for 5 minutes. After the tissue was washed with HBSS, a culture buffer [Roswell Park Memorial Institute (RPMI) medium+collagenase (1.5 mg/mL)+hyaluronidase (20 μg/mL)] was added and reacted at 37° C. for 30 minutes. Thereafter, the sample was diluted with 20 mL of DPBS and filtered with a 70 μm-size filter to obtain cancer cells. The obtained cells were precipitated by centrifugation, diluted with Matrigel, seeded, and then cultured by adding gastric cancer organoid medium to prepare diffuse-type gastric cancer organoids.

[0104] Next, in order to evaluate anticancer efficacy against diffuse-type gastric cancer organoids, Asarum maculatum Nakai butanol extracts of each concentrations (0.5 μg/ml, 5 μg/ml, 50 μg/ml, and 100 μg/ml) were treated on an organoid library prepared from cancer tissues of 17 diffuse-type gastric cancer patients, and cell growth inhibitory ability thereof was confirmed by an MTT assay. As a result, it was confirmed that the Asarum maculatum Nakai extracts showed an excellent inhibitory ability on all diffuse-type gastric cancer organoids of 17 patients, even at a low concentration of 5 μg/ml, and the extracts were capable of killing most of the cancer cells at a concentration of 50 μg/ml or higher (FIG. 9).

[0105] Next, in order to evaluate cytotoxicity against normal gastric organoids, Asarum maculatum Nakai butanol extracts of each concentrations (0.5 μg/ml, 5 μg/ml, 50 μg/ml, and 100 μg/ml) were treated on an organoid library prepared from stomach tissues of normal people, and cell viablility inhibitory ability thereof was confirmed by an MTT assay. As a result, it was confirmed that the Asarum maculatum Nakai extract showed cell viability of about 80% at a concentration of 50 μg/ml, and the toxicity to normal tissues was significantly lower than that to cancer tissues (FIG. 10).

[0106] Based on the above results, it may be seen that an Asarum maculatum Nakai butanol extract has excellent anticancer efficacy for diffuse-type gastric cancer and low cytotoxicity to normal tissues.

3.5 Comparison of Anticancer Efficacies of Asarum maculatum Nakai Extract and Extracts of Other Plants of the Genus Asarum

[0107] In order to compare anticancer efficacy of an extract of Asarum maculatum Nakai with that of an extract of Asarum heterotropoides var. seoulense (Nakai) Kitag., the following experiment was performed.

[0108] First, the roots and rhizomes of Asarum heterotropoides var. seoulense (Nakai) Kitag. plants were collected in Daegu, Gyeongbuk in November 2015 and dried to obtain 75 g of dried plants. Thereafter, the dried plant was crushed and put into an extraction container, 750 mL of 100% ethanol was added, the sample was reflux-extracted for 2 hours, and the process of gravity filtration of the mixture by using a Whatman filter paper having a film thickness of 0.34 mm and a glass funnel was repeated twice ('extraction-filtration' twice) to obtain a filtered extract. The filtered extract was transferred to a round flask, placed in a vacuum concentrator, and the solvent was completely evaporated at 35° C. under reduced pressure to obtain 11.7 g of an Asarum heterotropoides var. seoulense (Nakai) Kitag. ethanol extract (hereinafter referred to as ‘sesin’) (yield 15.6%). In addition, the sesin butanol extract and the water extract were obtained in the same manner as in the preparation method of an Asarum maculatum Nakai extract.

[0109] Next, the Asarum maculatum Nakai butanol extracts and sesin extracts of different concentrations (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated on the diffuse-type gastric cancer cell lines MKN1 and SNU668, and the cell growth inhibitory abilities were evaluated by an MTT assay.

[0110] As a result, it was confirmed that compared with the sesin extracts of various solvents, the Asarum maculatum Nakai butanol extract had a significantly superior growth inhibitory ability for diffuse-type gastric cancer (FIG. 11).

[0111] Therefore, based on the above results, it may be seen that an Asarum maculatum Nakai has superior anticancer efficacy for diffuse-type gastric cancer than other plants of the genus Asarum.

3.6 Evaluation of Anticancer Efficacy of Asarum maculatum Nakai Extract on Various Cancers

[0112] In order to confirm whether an Asarum maculatum Nakai extract exhibits anticancer efficacy against other cancers other than diffuse-type gastric cancer, the following experiment was performed.

[0113] Specifically, Asarum maculatum Nakai butanol extracts of each concentration (50 μg/ml, 100 μg/ml, and 200 μg/ml) were treated to intestinal-type gastric cancer cell lines (MKN28 and MKN74), a cervical cancer cell line (HeLa), a liver cancer cell line (Hep3B), and a colorectal cancer cell line (SW480), and then cell growth inhibitory abilities were confirmed through an MTT assay.

[0114] As a result, Asarum maculatum Nakai extracts showed excellent inhibitory activities in several cancers derived from other organs. Comparing the cancer growth inhibitory abilities of an Asarum maculatum Nakai extract on intestinal-type gastric cancer and the aforementioned diffuse-type gastric cancer, it was confirmed that the extract showed excellent anticancer efficacy specifically for the diffuse type rather than the intestinal type (FIG. 12).

[0115] Based on the above results, it may be seen that an Asarum maculatum Nakai extract has anticancer efficacy against various cancers, and in particular, has superior anticancer efficacy against diffuse-type gastric cancer.

[0116] The above description of the present disclosure is for illustrative purposes, and those skilled in the art to which the present disclosure belongs will be able to understand that the examples and embodiments can be easily modified without changing the technical idea or essential features of the disclosure. Therefore, it should be understood that the above examples are not limitative, but illustrative in all aspects.

[0117] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.