KR20220052574A - Composition for treating cervical cancer comprising Citrus unshiu Markovich peel extract - Google Patents

Abstract

The present invention relates to a composition for treating, preventing or alleviating cervical cancer containing a Cirtus unshiu extract as an active component, wherein the Cirtus unshiu extract has an effect of inhibiting the proliferation and metastasis of cancer cells, inducing G2/M phase arrest, inducing apoptosis, inhibiting the formation of tumorsphere, and inhibiting the expression of a cancer stem marker, thereby being able to be usefully used in related medicines or health food fields.

Description

Composition for treating cervical cancer comprising Citrus unshiu Markovich peel extract

The present invention relates to a composition for treating cervical cancer, etc. comprising a dermis extract as an active ingredient.

Cervical cancer is the second most common cancer among women worldwide. High-risk human papillomavirus (HPV) 16 and HPV 18 cause more than 70% of cervical cancers. These cancers can be prevented with vaccines and can be treated if diagnosed early, but patients with advanced cervical cancer are still difficult to treat due to recurrence, metastasis, and resistance to radiation and chemotherapy. Therefore, it is necessary to discover promising anticancer drugs for the prevention and treatment of cervical cancer.

Cancer cells can proliferate abnormally by avoiding apoptosis and cell cycle arrest. HPV infection induces expression of oncogenes E6 and E7. E6 binds to p53 and interferes with its function through degradation, resulting in resistance to apoptosis. E7 inhibits another tumor suppressor, retinoblastoma 1 (RB1), releasing the E2F transcription factor, which induces the expression of cell cycle regulators that stimulate cell proliferation. Therefore, restoration of apoptosis and cell cycle arrest may be important strategies for the treatment of cervical cancer.

On the other hand, it has been reported that the recurrence of cervical cancer and resistance to radiation/chemotherapy are due to the presence of cancer stem cells (CSCs). CSC induces genetic heterogeneity in cervical cancer, lowering the effectiveness of chemotherapy and promoting metastasis to other tissues. Several potential markers of cervical CSC have been identified and they upregulate cancer stem-like functions, including self-renewal, tumorigenicity, and radiation/chemotherapy resistance. For this reason, targeting CSCs can contribute to better treatment outcomes for cervical cancer, and in-depth studies are required.

Republic of Korea Patent Publication No. 10-2016-0070912

The present inventors have completed the present invention by experimentally confirming that the dermis extract is treated with HeLa cells, etc., and suppression of cancer cell proliferation and migration, tumor cell formation, and cancer stem cell marker expression.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for treating or preventing cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for treating or preventing cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

In one embodiment of the present invention, the extract may be a non-aqueous fraction.

In one embodiment of the present invention, the extract may include hesperidin and hesperetin.

In one embodiment of the present invention, the composition can inhibit the proliferation and metastasis of cancer cells.

In one embodiment of the present invention, the composition may induce G2/M phase arrest in cancer cells.

In one embodiment of the present invention, the composition may induce apoptosis of cancer cells in a reactive oxygen species (ROS)-independent manner.

In one embodiment of the present invention, the composition can inhibit the formation of tumor cells.

In one embodiment of the present invention, the composition can inhibit the expression of one or more cancer stem cell markers selected from the group consisting of Sox2, Nanog, Oct4, ALDH1A1, integrin a6 and CD133.

The present invention relates to a composition for treating, preventing or improving cervical cancer comprising a dermis extract as an active ingredient, wherein the dermal extract inhibits proliferation and metastasis of cancer cells, induces G2/M phase arrest, induces apoptosis, and tumorsphere formation Since it has effects such as suppression of cancer stem marker expression, it can be usefully used in related medicines or health food fields.

1a and 1b show the HPLC analysis results of the present invention CECU (chloroform extract of C. unshiu Markovich peel).
Figure 2a confirms the effect of CECU on the growth of HeLa, SiHa, CaSki and 267B1 cells.
Figure 2b confirms the effect of CECU on the colony forming ability of HeLa cells.
Figure 2c shows the effect of CECU on the migration of HeLa cells through the wound healing assay.
Figure 3a shows the cell cycle distribution of HeLa cells after CECU treatment was confirmed through flow cytometry.
Figure 3b shows the change in the apoptosis rate of HeLa cells after CECU treatment was confirmed through flow cytometry.
Figure 3c shows the change in the nuclear morphology of HeLa cells after CECU treatment was confirmed through a fluorescence microscope.
FIG. 4a is a view illustrating specific protein levels through Western blot to confirm the apoptotic pathway of HeLa cells after CECU treatment.
Figure 4b confirms the effect of CECU on ROS generation in HeLa cells.
Figure 5a confirms the growth inhibitory effect when the HeLa cancer stem-like cells treated with CECU, hesperetin or hesperidin while increasing the dose.
5b and 5c confirm the effect of CECU on the tumorigenicity of HeLa cancer stem-like cells.
Figure 5d confirms the effect of CECU on the expression of cancer stem cell biomarkers in HeLa cancer stem-like cells.

The present inventors have completed the present invention by experimentally confirming that the dermis extract is treated with HeLa cells, etc., and suppression of cancer cell proliferation and migration, tumor cell formation, and cancer stem cell marker expression.

Accordingly, the present invention provides a pharmaceutical composition for treating or preventing cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

In the present invention, the dermis means the peel of Citrus unshiu Markovich, and may preferably be dried, but is not limited thereto. It has been reported that the essential oil component of the dermis promotes digestion, expectoration, anti-ulcer, anti-gastric secretion, cardiac arrhythmia, blood pressure increase, anti-allergic effect, etc., but the contents of treatment of cervical cancer, etc. have not been disclosed.

In the present invention, the extract is an extract obtained by extracting the dermis. The dermis extract may mean a result such as a liquid component obtained by immersing the dermis in a solvent and then extracting the dermis for a certain period of time at room temperature or in a warm state, and a solid obtained by removing the solvent from the liquid component, but is not limited thereto It may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or all of these crude products or purified products. The solvent may be one or more solvents selected from the group consisting of water, C ₁ to C ₄ alcohols, hexane and mixtures thereof, and preferably ethanol, but is not limited thereto.

In addition, the extract may be a concept including a fraction of the extract. In the present invention, the extract may be fractionated using chloroform, and preferably, the extract may be fractionated by distributing the extract in a solution having a ratio of chloroform and distilled water of 1:1, but is not limited thereto.

In the present invention, cervical cancer refers to a malignant tumor occurring in the cervix.

In the present invention, prevention means any action that suppresses symptoms or delays the onset of cervical cancer by administration of the pharmaceutical composition according to the present invention.

In the present invention, treatment means any action in which cervical cancer symptoms are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention includes the dermis extract as an active ingredient, and may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., ophthalmic eye drops, pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as eye drops, injections, or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, mucosal, intravenous, or subcutaneous) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, the degree of disease, the drug Although it varies depending on the form, administration route and time, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like.

In the present invention, the extract may be a water-insoluble fraction.

In the present invention, the extract may include hesperidin and hesperetin.

In the present invention, the hesperidin is a flavanone glycoside among flavonoid pigments.

In the present invention, the hesperetin is a 4'-methoxy derivative of eriodictyol, a flavanone.

In the present invention, the composition can inhibit the proliferation and metastasis of cancer cells.

In the present invention, the composition may induce G2/M phase arrest in cancer cells. Inducing G2/M stage arrest in the cancer cells of the present invention means increasing the G2/M stage cancer cell population and decreasing the G0/G1 stage cancer cell cell population.

In the present invention, the composition may induce apoptosis of cancer cells in a reactive oxygen species (ROS)-independent manner.

In the present invention, the composition may inhibit the formation of tumor spheres. Since the tumor spheres can be formed by tumor stem cells (Tumor Initiating Cell), inhibiting the formation of tumor spheres may mean inhibiting the proliferation of tumor stem cells.

In the present invention, the composition can inhibit the expression of one or more cancer stem cell markers selected from the group consisting of Sox2, Nanog, Oct4, ALDH1A1, integrin a6 and CD133.

In another aspect, the present invention provides a food composition for preventing or improving cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.

The description of the pharmaceutical composition may be equally applied to the food composition as long as there is no conflict.

The food composition is a concept including health functional food.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of symptoms.

In the food composition of the present invention, the dermis extract may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the dermis extract may be suitably determined according to the purpose of its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than or equal to the above range.

The health functional food composition of the present invention is not particularly limited in other ingredients other than containing the above ingredients as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like may be contained. These components may be used independently or in combination. The proportion of these additives may also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example 1: Materials and Methods>

Example 1-1. Reagents and Antibodies

Dulbecco's modified Eagle's medium (DMEM) was purchased from Corning Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS), DMEM/nutrient mixture F-12 (F12), B-27 serum-free supplement, L-glutamine, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and penicillin/streptomycin It was purchased from Gibco (Grand Island, NY, USA). Heparin, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), hesperidin, hesperetin, gallic acid, and crystal violet were obtained from Sigma-Aldrich (St. Louis, MO, USA). Purchased. Penicillin-streptomycin-amphotericin B was purchased from Lonza (Walkersville, MD, USA). p53 (53 kDa; cat. no. 2524), phospho-AKT (Ser473, 60 kDa; cat. no. 4060), AKT (60 kDa; cat. no. 9272), phospho-ERK1/2 (Thr202/Tyr204, 42,44 kDa; cat. no. 9101), ERK1/2 (42,44 kDa; cat. no. 9102), Bcl-2 (28 kDa; cat. no. 2872), Bcl-XL (30 kDa; cat . no. 2764), Bax (20 kDa; cat. no. 2772), cleaved caspase-3 (17,19 kDa; cat. no. 9661), cleaved caspase-9 (37 kDa; cat. no. 9501), cleaved caspase-8 (18,41,43 kDa; cat. no. 9496), PARP (89,116 kDa; cat. no. 9542), DR5 (40,48 kDa; cat. no. 8074), Fas (40-50 kDa; cat. no. 4233), cyclin B1 (55 kDa; cat. no. 12231), cyclin D1 (36 kDa; cat. no. 55506), CD133 (133 kDa; cat. no. 64326), Sox2 (35 kDa; cat. no. 3579), Oct4 (45 kDa; cat. no. 2750), Nanog (42 kDa; cat. no. 3580), integrin α6 (125,150 kDa; cat. no. 3750), ALDH1A1 (55 kDa) Antibodies against cat. no. 12035), rabbit IgG (cat. no. 7074), and mouse IgG (cat. no. 7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against p21 (21 kDa; cat. no. sc-397) was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against β-actin (42 kDa; cat. no. ab6276) and Bad (22 kDa; cat. no. ab62465) were purchased from Abcam (Cambridge, UK).

Example 1-2. Preparation of chloroform extract of C. unshiu Markovich peel (CECU)

Dried C. unshiu Markovich peels were purchased from the Yeongcheon Herbal Medicine Wholesale Market (Yeongcheon, Korea). CECU was extracted with 100% ethanol for 24 h and concentrated in vacuo. The extract was distributed between chloroform and distilled water in a 1:1 ratio to obtain a chloroform fraction. CECU was prepared at a concentration of 100 mg/mL using DMSO.

Examples 1-3. cell culture

HeLa, CaSki and SiHa human cervical cancer cell lines were purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were grown in DMEM containing 10% FBS and 1% penicillin-streptomycin-amphotericin B and maintained at 37° C. in a humidified 5% CO ₂ incubator (Thermo Scientific, Vantaa, Finland). HeLa-derived cancer stem-like cells were cultured in DMEM/F12 supplemented with 1x B-27, 5 µg/mL heparin, 2 mM L-glutamine, 20 ng/mL bFGF, 20 ng/mL EGF and 1% penicillin/streptomycin. became

Examples 1-3. Total polyphenol content

The total polyphenol content of CECU was estimated using Folin-Ciocalteu reagent (Sigma-Aldrich). CECU (100 mL) and Folin-Ciocalteu reagent (60 mL) were mixed for 5 minutes, and then 600 mL of 2% Na ₂ CO ₃ was added. After incubation for 2 hours in dark conditions, absorbance was measured at 750 nm using a multimode microplate reader (BioTek, Inc., Winooski, VT, USA). A calibration curve for gallic acid was prepared and linearity was obtained in the range of 0-0.9 mg/mL. The total phenol content of CECU was expressed as gallic acid equivalents in milligrams (mg GAE/g extract) using a standard curve.

Examples 1-4. HPLC analysis

HPLC was performed to confirm the content of hesperidin and heperetin in CECU. The reference compound was diluted with MeOH to a concentration of 0.5 mg/mL and CECU was prepared to a concentration of 20 mg/mL. HPLC (Shimadzu, LC-2030C, Kyoto, Japan) was performed using a gradient method using a RP C-18 (Shimadzu; 250 mm × 4.6 mm, 5 μm) column and water/acetonitrile solvent.

Examples 1-5. cell growth assay

HeLa, SiHa, CaSki and 267B1 cells (2 x 10 ³ cells/well) were seeded in 96-well culture plates. After 24 h incubation, cells were treated with various concentrations of CECU. After incubation for 72 hours, 50 mL of MTT solution (2 mg/mL) was added to each well. Cells were then incubated for 3 h and lysed in 100 µL of DMSO per well. Absorbance was measured at 540 nm wavelength using a microplate reader (BioTek). IC ₅₀ values of the obtained data were analyzed using a curve fitting program GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).

Example 1-6. Colony Formation Assay

HeLa cells (3 x 10 ² cells/well) were inoculated into 6-well culture plates and treated with CECU. Cells were grown for 13 days and colonies formed were fixed with 3.7% formaldehyde for 10 minutes. After washing with PBS, colonies were stained with 1 mL of 0.5% crystal violet for 20 minutes. Then, the stained colonies were washed with PBS and the number of colonies was counted.

Example 1-7. wound healing analysis

HeLa cells (15 x 10 ⁴ cells/well) were seeded in 24-well culture plates. After incubation for 24 h, cells were scratched using a 10 μL pipette tip, washed with medium and treated with CECU. Medium containing 10% FBS was used in the assay to stimulate cell migration. After 72 hours of incubation, images were obtained with a 40-fold optical microscope (Olympus, Tokyo, Japan) and the migrated cells were counted.

Examples 1-8. cell cycle analysis

HeLa cells (3 x 10 ⁵ cells/dish) were seeded in a 60 mm culture dish and treated with CECU for 24 hours. The cells were then collected, washed with PBS and fixed with cold 70% ethanol. After overnight storage at -20 °C, ethanol was removed and the cells were washed with PBS. In addition, 200 μL of propidium iodide (PI) solution (Muse Cell Cycle Assay Reagent; Merck Millipore, Darmstadt, Germany) was added and reacted in the dark for 30 minutes. Stained cells were analyzed by flow cytometry (Muse; Merck Millipore).

Examples 1-9. Apoptosis analysis

HeLa cells (5 x 10 ⁵ cells/dish) were placed in a 60 mm culture dish and treated with CECU for 24 hours. Cells were then harvested, washed with PBS, and stained with Annexin V-FITC and PI according to the manufacturer's instructions (Invitrogen, Waltham, Massachusetts, USA). Stained cells were analyzed by flow cytometry (Cyto FLEX; Beckman Coulter, USA).

Examples 1-10. ROS analysis

HeLa cells (1 x 10 ⁵ cells/well) were inoculated into a 96-black well culture plate and treated with CECU for 30 minutes. Then, the cells were incubated with 10 μM of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) for 20 minutes and washed with PBS. The fluorescence intensity of DCF was detected using a multimode microplate reader (BioTek) at excitation and emission wavelengths of 495 and 529 nm, respectively.

Examples 1-11. DAPI staining

HeLa cells (5 x 10 ⁴ cells/well) were seeded in 24-well culture plates and treated with CECU for 24 hours. The cells were then fixed with 3.7% formaldehyde for 10 minutes. Nuclei were stained with 4 μg/mL of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) for 30 minutes and washed with PBS. The nuclear morphology of the cells was captured using a fluorescence microscope (Korea Lab Tech, Seong Nam, Korea).

Examples 1-12. ATP-monitored luminescence assay

The growth of HeLa cancer stem-like cells was quantitatively evaluated using the ATPlite Luminescence Assay System (PerkinElmer, Waltham, MA, USA). Cells (3 x 10 ³ cells/well) were seeded into 96-white well culture plates using serum-free medium with EGF and bFGF and treated with CECU, hesperidin and hesperetin for 7 days. After that, 50 mL of the substrate solution was added to each well, and the culture plate was shaken for 5 minutes and incubated in the dark for 10 minutes. Luminescence was detected using a multimode microplate reader (BioTek).

Examples 1-13. Oncosphere formation assay

HeLa cancer stem-like cells (5 x 10 ² cells/well) were seeded into 96-well culture plates using serum-free medium with EGF and bFGF and treated with CECU. After culturing for 7 days, the size and number of tumor spheres were counted under a 200x optical microscope (Olympus).

Examples 1-14. Western blot analysis

Cells were lysed on ice using RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Roche Diagnostics). Extract protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). An equal amount of cell lysate (40 μg/lane) was separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the isolated protein was purified on a polyvinylidene difluoride (PVDF) membrane (EMD) according to standard electroblotting procedures. Millipore). Blots were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% skim milk for 1 h at room temperature. and against p53 (dilution 1:2,000), p21 (dilution 1:500), phospho-AKT (dilution 1:2,000), AKT (dilution 1:2,000), phospho-ERK1/2 (dilution 1:2,000), ERK1/ 2 (dilution 1:2,000), Bcl-2 (dilution 1:2,000), Bcl-XL (dilution 1:2,000), Bad (dilution 1:2,000), Bax (dilution 1:2,000), cleaved caspase-3 (dilution) 1:2,000), cleaved caspase-9 (dilution 1:2,000), cleaved caspase-8 (dilution 1:2,000), PARP (dilution 1:2,000), DR5 (dilution 1:2,000), Fas (dilution 1:2,000) , cyclin B1 (dilution 1:2,000), cyclin D1 (dilution 1:2,000), CD133 (dilution 1:2,000), Sox2 (dilution 1:2,000), Oct4 (dilution 1:2,000), Nanog (dilution 1:2,000) , integrin a6 (dilution 1:2,000), ALDH1A1 (dilution 1:2,000), and b-actin (dilution 1:10,000) were immunolabeled overnight at 4°C with primary antibodies.

After washing 3 times with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit (dilution 1:3,000) or anti-mouse (dilution 1:3,000) secondary antibody at room temperature for 1 hour. Immunolabeling was detected using an enhanced chemiluminescence (ECL) kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions.

Examples 1-15. statistical analysis

Results were expressed as mean ± standard deviation (SD). Differences between groups were analyzed using the SPSS statistical package (SPSS 9.0; SPSS Inc.) and analysis of variance (ANOVA). Post hoc analysis was performed using Tukey's test. A p-value of <0.05 was considered to indicate a statistically significant difference.

<Example 2: Configuration analysis of CECU>

Polyphenols have shown a variety of biological activities to prevent and treat human diseases, including cancer. As a result of measuring the total phenol content using the Folin-Ciocalteu method to evaluate the phytochemical composition of CECU, it was confirmed that CECU contained 8.3 mg GAE/g of polyphenol.

In order to determine the content of hesperidin and its aglycone, hesperetin, in CECU, the reference compound and CECU were analyzed by HPLC, and the detection wavelength of the compound was 288 nm. The HPLC chromatogram of CECU detected hesperidin and hesperetin at retention times of 10.82 minutes and 15.53 minutes, respectively ( FIGS. 1A and 1B ). The estimated hesperidin and hesperetin contents in CECU were 0.739% and 1.641%, respectively.

<Example 3: Confirmation of the effect of CECU on the growth and migration of HeLa cells>

To investigate whether CECU affects the growth of cervical cancer cells, three different cervical cancer cell lines (HeLa, SiHa, and CaSki) were treated with CECU (0–500 mg/mL) for 72 h and then cells by MTT assay. Growth was assessed. As a result, as shown in FIG. 2a , CECU treatment inhibited the growth of HeLa, SiHa and CaSki cells in a dose-dependent manner with IC ₅₀ values of 58.95, 73.41 and 69.63 mg/mL, respectively. In particular, CECU showed the highest inhibitory effect on the growth of HeLa cells.

We further evaluated the effect of CECU on the growth of 267B1 human normal prostate epithelial cells. CECU inhibited the growth of 267B1 cells with an IC ₅₀ value of 114.7 mg/mL, indicating that CECU more sensitively inhibited the growth of cervical cancer cells compared to normal cells (Fig. 2a).

Based on the above results, the anticancer effect of CECU on HeLa cells at a concentration ranging from 10-80 mg/mL was further evaluated. Next, the effect of CECU on colony formation of HeLa cells was evaluated. CECU treatment inhibited clonogenic growth of HeLa cells in a dose-dependent manner (Fig. 2b). In particular, the colony-forming ability of cells was significantly reduced at 40 mg/mL of CECU.

To confirm the effect of CECU on the migrating ability of HeLa cells, a monolayer wound healing assay was performed. After 72 hours of incubation, complete wound closure by HeLa cell migration was observed. Treatment with CECU (10, 20 and 40 mg/mL) significantly reduced the migration of HeLa cells compared to the untreated control (Fig. 2c). These results suggest that CECU effectively inhibits the growth and migration of cervical cancer cells.

<Example 4: Confirmation of effect of CECU on HeLa cell cell cycle and apoptosis>

To determine whether CECU inhibits the growth of HeLa cells by regulating the cell cycle, the effect of CECU on cell cycle distribution was investigated by flow cytometry. Compared with untreated control cells, CECU treatment increased the G2/M stage cell population and decreased the G0/G1 stage cell population (Fig. 3a). These results indicate that CECU induced G2/M phase arrest in HeLa cells.

To further investigate whether CECU-induced growth inhibition was associated with apoptosis induction, CECU-treated HeLa cells were stained with Annexin V-FITC and PI followed by flow cytometry. As a result, after treatment with CECU, early and late cell apoptosis rates were increased compared to untreated control cells (FIG. 3b). As a result, CECU exhibited a dose-dependent apoptosis-inducing effect in HeLa cells.

DAPI staining was performed to confirm that CECU induces apoptosis-related morphological changes in HeLa cells. As a result, as shown in Fig. 3c, CECU treatment caused nuclear condensation and fission. These results demonstrate that CECU inhibits the growth of HeLa cells through cell cycle arrest and apoptosis induction.

<Example 5. Confirmation of the effect of CECU on apoptosis-related pathways in HeLa cells>

The PI3K/AKT and Ras/MEK/ERK pathways contribute to the survival and growth of cervical cancer cells. To elucidate the molecular mechanism by which CECU inhibits the growth of HeLa cells, we first investigated whether CECU regulates the activation of AKT and ERK, key effectors of these signaling pathways. As a result, it was confirmed that CECU treatment significantly down-regulated AKT and ERK1/2 phosphorylation without affecting the total protein level of HeLa cells (FIG. 4a).

Activation of the tumor suppressor protein p53 arrests the cell cycle at the G2/M phase. Cell cycle arrest by p53 is mainly mediated by transcriptional activation of p21/WAF1. Therefore, the effect of CECU on the expression of p53 and p21 was investigated. As a result, it was confirmed that CECU treatment significantly increased the expression of p53 and p21 in HeLa cells (Fig. 4a). Furthermore, CECU reduced the expression levels of cyclin B1 and cyclin D1, which are related to the regulation of G2/M phase transition ( FIG. 4a ).

Cellular apoptosis can be induced through either the death receptor mediated extrinsic pathway or the mitochondrial mediated intrinsic pathway. Therefore, we evaluated whether CECU affects these apoptotic pathways in HeLa cells. As a result, it was confirmed that treatment with CECU clearly increased the expression levels of death receptors, DR5 and Fas, as well as the downstream apoptosis effector, an active form of caspase-8 (FIG. 4a). In addition, among the Bcl-2 family members involved in the intrinsic apoptotic pathway, the expression of anti-apoptotic proteins such as Bcl-XL and Bcl-2 was downregulated by CECU treatment, whereas pro-apoptotic proteins including Bad and Bax were downregulated by CECU treatment. ) protein levels were up-regulated (Fig. 4a). As a result, the expression of caspase-9, a downstream apoptotic effector, was activated. By regulating extrinsic and intrinsic apoptotic pathways, CECU triggered the activation of an important effector of apoptotic caspase-3 and its substrate, PARP.

Reactive oxygen species (ROS) play an important role in inducing apoptosis. Therefore, we further evaluated whether the apoptosis-inducing effect of CECU was mediated by ROS in HeLa cells. As a result, as shown in Fig. 4b, CECU treatment did not cause a significant change in ROS generation, which means that the apoptosis-inducing effect of CECU is independent of ROS.

Taken together, these results suggest that HeLa cell growth inhibition by CECU may be associated with inactivation of AKT and ERK signaling, upregulation of p53 and p21, downregulation of cyclin B1 and cyclin D1, and activation of ROS-independent apoptotic pathways. suggest

<Example 6. Effect of CECU on cancer stem-like characteristics of HeLa cells>

To evaluate the potential of CECU for suppressing cervical cancer stem cells (CSC), we investigated the effect of CECU on the cancer stem-like properties of HeLa cells, and the CSC population of HeLa cells together with EGF and bFGF. It was grown via spheroid culture using serum-free medium. As a result of the investigation, as shown in FIG. 5a , CECU treatment inhibited the growth of HeLa cancer stem-like cells in a dose-dependent manner.

We also evaluated whether hesperidin and hesperetin are possible active ingredients contributing to the anticancer activity of CECU against cervical CSC. Both compounds inhibited the growth of HeLa cancer stem-like cells in a dose-dependent manner (Fig. 5a). In particular, hesperetin showed a better growth inhibitory effect than hesperidin in these cells.

In addition, the clonogenic growth of HeLa cancer stem-like cells was significantly inhibited by CECU treatment (Figs. 5b and c), and the size and number of tumor cells were reduced.

Finally, we investigated whether CECU regulates the expression of key stem-related markers in cervical CSCs. As a result, CECU treatment significantly reduced the expression levels of stem function regulators including Sox2, Nanog, Oct4, ALDH1A1, integrin a6 and CD133 in HeLa cancer stem-like cells (Fig. 5d). These results suggest that CECU has therapeutic potential to eliminate cervical CSC.

Claims (16)

A pharmaceutical composition for treating or preventing cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the extract is a non-aqueous fraction.
The pharmaceutical composition according to claim 1, wherein the extract comprises hesperidin and hesperetin.
The pharmaceutical composition of claim 1, wherein the composition inhibits the proliferation and metastasis of cancer cells.
The pharmaceutical composition of claim 1, wherein the composition induces G2/M phase arrest in cancer cells.
The pharmaceutical composition of claim 1, wherein the composition induces apoptosis of cancer cells in a reactive oxygen species (ROS)-independent manner.
The pharmaceutical composition according to claim 1, wherein the composition inhibits tumorisphere formation.
The pharmaceutical composition of claim 1, wherein the composition inhibits the expression of one or more cancer stem cell markers selected from the group consisting of Sox2, Nanog, Oct4, ALDH1A1, integrin a6 and CD133.
A food composition for preventing or improving cervical cancer comprising an extract of Citrus unshiu Markovich peel as an active ingredient.
The food composition according to claim 9, wherein the extract is a non-aqueous fraction.
The food composition according to claim 9, wherein the extract comprises hesperidin and hesperetin.
The food composition according to claim 9, wherein the composition inhibits the proliferation and metastasis of cancer cells.
The food composition according to claim 9, wherein the composition induces G2/M phase arrest in cancer cells.
The food composition of claim 9, wherein the composition induces apoptosis of cancer cells in a reactive oxygen species (ROS)-independent manner.
The food composition according to claim 9, wherein the composition inhibits tumor sphere formation.
The food composition of claim 9, wherein the composition inhibits the expression of one or more cancer stem cell markers selected from the group consisting of Sox2, Nanog, Oct4, ALDH1A1, integrin a6 and CD133.

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