Compositions for preventing or treating cancer comprising extracts of Rubus longisepalus var. tozawai (Nakai) T.B.Lee

Abstract

Provided are compositions for preventing or treating cancer, the compositions including Rubus longisepalus var. tozawai (Nakai) T.B.Lee extracts, or a fraction thereof as an active ingredient.

According to an aspect, a Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract has an excellent anticancer efficacy against various cancers including diffuse-type gastric cancer, and thus, 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, the pharmaceutical composition comprising a Rubus tozawae Nakai ex J.Y.Yang [ Rubus longisepalus var. tozawai (Nakai) T.B.Lee] extract or a fraction thereof as an active ingredient.

2. The pharmaceutical composition of claim 1, further comprising Asarum maculatum Nakai extract or a fraction thereof.

3. The pharmaceutical composition of claim 1, wherein the extract is extracted by 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 extracted by 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, the method comprising administering a pharmaceutical composition comprising a Rubus tozawae Nakai ex J.Y.Yang [ Rubus longisepalus var. tozawai (Nakai) T.B.Lee] extract or a fraction thereof to a subject.

8. The method of treating or preventing cancer of claim 7, wherein the pharmaceutical composition further comprises Asarum maculatum Nakai extract or a fraction thereof.

9. The method of treating or preventing cancer of claim 7, wherein the extract is extracted by 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 treating or preventing cancer of claim 7, wherein the extract is extracted by 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 treating or preventing cancer 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 treating or preventing cancer of claim 7, wherein the cancer is diffuse-type gastric cancer.

13. A food or feed composition for preventing or improving cancer, the food or feed composition comprising a Rubus tozawae Nakai ex J.Y.Yang [ Rubus longisepalus var. tozawai (Nakai) T.B.Lee] extract or a fraction thereof as an active ingredient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0064] 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:

[0065] FIG. 1 shows diagrams for measuring expression levels of markers related to epithelial-mesenchymal transition for screening for diffuse-type gastric cancer cell lines,

[0066] FIG. 2 shows a diagram showing results of a primary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0067] FIG. 3 shows a diagram showing results of a secondary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0068] FIG. 4 shows a diagram showing results of a tertiary screening for selecting candidate active substances for treating diffuse-type gastric cancer,

[0069] FIG. 5 shows diagrams confirming cell growth inhibitory ability of Rubus longisepalus var. tozawai (Nakai) T.B.Lee extracts for each solvent on diffuse-type gastric cancer cell lines,

[0070] FIG. 6 shows diagrams confirming cell growth inhibitory ability of Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration on the diffuse-type gastric cancer cell lines,

[0071] FIG. 7 shows a diagram confirming cell viability inhibitory ability of Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration on normal cell lines,

[0072] FIG. 8 shows diagrams confirming cell growth inhibitory ability of Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration on diffuse-type gastric cancer organoids,

[0073] FIG. 9 shows a diagram confirming cell viability inhibitory ability of Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration on normal organoids, and

[0074] FIG. 10 shows a diagram confirming cell growth inhibitory abilities of hexane extracts of Rubus longisepalus var. tozawai (Nakai) T.B.Lee, Rubus coreanus Miq., or Rubus longisepalus Nakai against various cancers.

DETAILED DESCRIPTION

[0075] 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.

[0076] 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

[0077] 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.

[0078] 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).

[0079] 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

[0080] In order to screen for effective substances for treating diffuse-type gastric cancer from various natural products, the following experiments were performed.

[0081] 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 .Math.L 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.

[0082] First, for a primary screening, after 63 kinds of natural substance extract samples of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/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 .Math.g/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).

[0083] Next, for a secondary screening, after 19 kinds of primarily selected natural substance extract samples of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/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 .Math.g/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).

[0084] 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 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/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 .Math.g/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).

[0085] 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 Rubus Longisepalus Var. Tozawai (Nakai) T.B.Lee Extract

[0086] In order to evaluate anticancer efficacy of a Rubus longisepalus var. tozawai (Nakai) T.B.Lee 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 Rubus longisepalus Var. Tozawai (Nakai) T.B.Lee Extract

[0087] In order to prepare an extract of Rubus longisepalus var. tozawai (Nakai) T.B.Lee by using various solvents, the following experiment was performed.

[0088] Specifically, whole plants of Rubus longisepalus var. tozawai were collected from Geoje Island in August 2020, washed with water sufficiently, and dried with warm air (30° C.) for 7 days to obtain 7.4 kg of dried plant. Thereafter, the dried plant was placed in a crusher and sliced to a size of 20 mm, and put into an extraction container, 74 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, put in a low pressure evaporator, and concentrated by evaporating the solvent completely at 35° C. under reduced pressure to obtain 400 g of an ethanol extract of Rubus longisepalus var. tozawai (Nakai) T.B.Lee (yield 5.4 %).

[0089] Next, 350 g of the Rubus longisepalus var. tozawai (Nakai) T.B.Lee ethanol extract was suspended in 4 L of water, 4 L of n-hexane was added, and the mixture was stirred by shaking at room temperature and fractionated 3 times for 2 hours each, to obtain 14 g of n-hexane fraction extract. After the fractionation, 4 L 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, to obtain 260 g of n-butanol fraction extract, and the water remaining after the fractionation was freeze-dried to obtain a water fraction extract.

3.2 Evaluation of Treatment Efficacy for Diffuse-Type Gastric Cancer for Each Extraction Solvent

[0090] The following experiments were performed to evaluate a treatment efficacy for diffuse-type gastric cancer and cytotoxicity on cells of the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract prepared in Example 3.1 for each solvent.

[0091] First, in order to evaluate anticancer efficacy against diffuse-type gastric cancer, Rubus longisepalus var. tozawai (Nakai) T.B.Lee extracts of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/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, it was confirmed that the hexane extract exhibited excellent anticancer efficacy against the two types of diffuse-type gastric cancer cell lines (FIG. 5).

[0092] Based on the above results, it may be seen that an hexane extract of Rubus longisepalus var. tozawai (Nakai) T.B.Lee, which has an excellent cell death effect for diffuse-type gastric cancer, has significantly superior anticancer efficacy among extracts for each solvent.

3.3 Evaluation of Treatment Efficacy for Diffuse-Type Gastric Cancer and Cytotoxicity of Rubus Longisepalus Var. Tozawai (Nakai) T.B.Lee Hexane Extract - Cell Models

[0093] The following experiments were performed to evaluate a treatment efficacy for diffuse-type gastric cancer and cytotoxicity of a Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extract, which is confirmed to have an excellent efficacy in Example 3.2, in cell models.

[0094] First, in order to evaluate anticancer efficacy against diffuse-type gastric cancer, Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/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, it was confirmed that a Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extract is capable of strongly inhibiting growth of diffuse-type gastric cancer cells even at a low concentration of 50 .Math.g/ml (FIG. 6).

[0095] Next, in order to evaluate cytotoxicity to normal cell lines, Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/ml)were treated to HEK293, normal kidney cells, and HFE-145, normal gastric epithelium cells, and then, cell growth inhibitory ability thereof was confirmed by an MTT assay. As a result, the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract did not significantly inhibit cell viability, even at a high concentration of about 100 .Math.g/ml, and it was confirmed that its toxicity to normal cells is low (FIG. 7).

[0096] Based on the above results, it may be seen that a Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane 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 Rubus Longisepalus Var. Tozawai (Nakai) T.B.Lee Hexane Extract - Organoid Models

[0097] The following experiments were performed to evaluate a treatment efficacy for diffuse-type gastric cancer and cytotoxicity of a Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extract, which is confirmed to have an excellent efficacy in Example 3.2, in organoid models.

[0098] First, in order to evaluate anticancer efficacy against diffuse-type gastric cancer organoids, Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extracts of each concentrations (0.5 .Math.g/ml, 5 .Math.g/ ml, 50 .Math.g/ml, and 100 .Math.g/ml)were treated on an organoid library prepared from cancer tissues of 4 diffuse-type gastric cancer patients by the method described in Example 3.4, and cell growth inhibitory ability thereof was confirmed by an MTT assay. As a result, it was confirmed that the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extracts showed an excellent inhibitory ability on all the diffuse-type gastric cancer organoids of 4 patients, even at a level of 50 .Math.g/ml, and the extracts were capable of killing most of the cancer cells at a concentration of 100 .Math.g/ml or higher (FIG. 8).

[0099] Next, in order to evaluate cytotoxicity to a normal gastric organoid, Rubus longisepalus var. tozawai (Nakai) T.B.Lee extracts of each concentration (0.5 .Math.g/ml, 5 .Math.g/ml, 50 .Math.g/ml, and 100 .Math.g/ml)were treated to a organoid library prepared from a gastric tissue of a normal person, and then, cell viability inhibitory activity was confirmed by an MTT assay. As a result, it was confirmed that the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract showed cell viability of about 60 % at a high concentration of 100 .Math.g/ml, and the toxicity to normal tissues was confirmed to be low (FIG. 9).

[0100] Based on the above results, it may be seen that a Rubus longisepalus var. tozawai (Nakai) T.B.Lee hexane extract has excellent anticancer efficacy for diffuse-type gastric cancer and low cytotoxicity to normal cells.

3.5 Evaluation of Anticancer Efficacy of Extracts of Rubus Longisepalus Var. Tozawai (Nakai) T.B.Lee and Other Plants Belonging to Genus Rubus on Various Cancers

[0101] In order to compare and evaluate the anticancer efficacy of a Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract with extracts of other plants belonging to the genus Rubus, such as Rubus coreanus, and Rubus longisepalus Nakai, the following experiment was performed.

[0102] Specifically, whole plants of Rubus longisepalus Nakai were collected from Geoje Island in August 2020, washed with water sufficiently, and dried with warm air (30° C. for 7 days to obtain 4.36 kg of dried plant. Thereafter, the dried plant was placed in a crusher and sliced to a size of 20 mm, and put into an extraction container, 43.6 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, put in a low pressure evaporator, and concentrated by evaporating the solvent completely at 35° C. under reduced pressure to obtain 347.3 g of an ethanol extract of Rubus longisepalus Nakai (yield 8.0 %). In addition, the hexane extract was obtained by the same method as the preparation method of the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract.

[0103] Next, for the Rubus coreanus, fruits of Rubus coreanus Miq. plants were purchased from ‘Dusone Herb inc.’ in 2016, crushed, and put in an extraction container, after adding 600 mL of ethanol, reflux extraction was performed for 2 hours, and 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, put in a low pressure evaporator, and concentrated by evaporating the solvent completely at 35° C. under reduced pressure to obtain 9.8 g of a Rubus coreanus ethanol extract (Hereinafter referred to as ‘ Rubus coreanus’) (yield 16.3 %). In addition, the hexane extract was obtained by the same method as the preparation method of the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract.

[0104] Next, extracts of Rubus longisepalus var. tozawai (Nakai) T.B.Lee, Rubus coreanus, and Rubus longisepalus Nakai of each concentration (50 .Math.g/ml, 100 .Math.g/ml, and 200 .Math.g/ml)were treated on diffuse-type gastric cancer cell lines (MKN1 and SNU668), an intestinal-type gastric cancer cell line (MKN74), a prostate cancer cell line (DU145), and a colorectal cancer cell line (SNUC4), and then, cell viability inhibitory activity was confirmed by an MTT assay.

[0105] As a result, in the case of the diffuse-type gastric cancer, it was confirmed that the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract showed a significantly superior cancer cell suppression ability compared to the Rubus coreanus extract and the Rubus longisepalus Nakai extract at low concentrations of 25 .Math.g/ml and 50 .Math.g/ml, and the Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract also exhibited an anticancer efficacy against intestinal-type gastric cancer, prostate cancer, and colorectal cancer (FIG. 10). In particular, when comparing only the cases of diffuse-type gastric cancer and intestinal-type gastric cancer, it was confirmed that the cancer-suppressing ability was particularly strong in diffuse-type gastric cancer compared to intestinal-type gastric cancer.

[0106] Based on the above results, it may be seen that a Rubus longisepalus var. tozawai (Nakai) T.B.Lee extract has superior anticancer efficacy against various cancers compared to other plants belonging to the genus Rubus, and in particular, it may be seen that the anticancer efficacy against diffuse-type gastric cancer is more excellent.

[0107] 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.

[0108] 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.