KR20200140098A - Composition for preventing, improving or treating cancer comprising extract of Parthenocissus tricuspidata from Pinus densiflora as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating cancer containing an extract of Parthenocissus tricuspidata from Pinus densiflora as an active ingredient. The extract of Parthenocissus tricuspidata from Pinus densiflora of the present invention has an effect of reducing a survival rate of cancer cells and inducing apoptosis, and is derived from plants to have excellent stability, and thus may be easily used as a health functional food composition and a pharmaceutical composition for the prevention, amelioration or treatment of cancer.

Description

Composition for preventing, improving or treating cancer comprising extract of Parthenocissus tricuspidata from Pinus densiflora as effective component, containing pine ivy extract as an effective component}

The present invention relates to a composition for preventing, improving or treating cancer containing pine ivy extract as an active ingredient.

Cancer is one of the incurable diseases that humanity must solve, and a huge amount of capital is being invested in the development to cure it worldwide. In Korea, it is the number one disease cause of death, and about 100,000 people per year It is diagnosed, and more than 60,000 people are dead. Cancer-causing factors such as these include smoking, ultraviolet rays, chemicals, food, and other environmental factors, but the causes of the various causes make it difficult to develop a treatment, and the effects of the treatment are also different depending on the site where it occurs. . Substances currently used as therapeutic agents are highly toxic, and there is an urgent need to develop effective anticancer drugs with low toxicity.

Ivy ( Parthenocissus tricuspidata ) is a deciduous broad-leaved vine plant belonging to the buckthorn grape family, a dicotyledonous plant, and it is also called now Sangchundeung (地錦常春藤) and grows attached to stone walls, rocks, or tree trunks. If the stem of the ivy grows sufficiently, it grows more than 10m, and tendrils face the leaves and split, and have rounded adsorbents at the ends, so they do not fall off easily when attached to a wall or rock wall. Leaves are alternate, wide egg-shaped, 10~20cm wide, sharp at the tip of the leaves, split into 3, there are no hairs on the front, fine hairs on the veins on the back, irregular serrates on the edge, and the petiole is longer than the leaf. The leaves are set in autumn, but the stems are still alive even in winter. Commonly, vine plants are characterized by winding other trees in a clockwise or counterclockwise direction, and ivy climbs straight up while riding down the bark of a tree and descending the fine roots of a sucker with adhesive at each node. Since ivy climbing on rocks or walls is toxic, it is recommended to use ivy climbing on other trees such as pine or oak when using it for medicinal purposes, and mainly stems, but roots, leaves, and fruits can all be used.

On the other hand, Korean Patent Application Publication No. 2010-0043521 discloses a composition for promoting hair growth containing ivy extract, and Korean Patent No. 1751799 discloses a blood vessel containing ivy extract or a compound isolated therefrom. A composition for preventing or treating diseases' is disclosed, but there is no description of a composition for preventing, improving or treating cancer containing the pine ivy extract of the present invention as an active ingredient.

The present invention was derived from the above requirements, and the present inventors prepared an extract using ethanol for the stems of ivy collected from pine trees and treated them to A549 lung cancer cells, resulting in a decrease in cell viability of cancer cells. And apoptosis is induced, the expression levels of proteins (p-mTOR and p-Akt) and anti-apoptotic proteins (Bcl-2) related to cancer cell proliferation are reduced, and cancer suppressing proteins The present invention was completed by confirming that the expression levels of (p53) and apoptotic protein (Bax) were increased.

In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving cancer containing pine ivy extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing pine ivy extract as an active ingredient.

Since the pine ivy extract of the present invention is a material derived from nature and has a low possibility of side effects and has no cytotoxicity, it can be usefully used for prevention, improvement and treatment of cancer.

Figure 1 is a result of measuring the cell viability after treatment of pine ivy extract (PTE) by concentration (25, 50, 75, 100, 125, 150 μg/ml) in normal cells (Fibroblast) and lung cancer cells (A549) A is a graph that measures the cell viability after treatment with PTE on Fibroblast cells and culture for 24 hours, and B and C is a graph for measuring the cell viability after treatment with PTE on A549 cells and incubation for 24 hours and 48 hours, respectively. It is a graph. N; PTE-free control, NS: not significant
FIG. 2 is a photograph illustrating a morphological change of cells after treatment with pine ivy extract (PTE) by concentration (50, 75, 100 μg/ml) on A549 lung cancer cells with a microscope.
3 is a flow cytometer after staining A549 cells treated with PTE with Annexin V and 7-AAD (7-Aminoactinomycin D) to confirm the apoptosis effect of pine ivy extract (PTE) on A549 lung cancer cells. This is the measurement result. Live; Annexin V(-)&7-AAD(-), Early Apop; Annexin V(+)&7-AAD(-), Late Apop/Dead; Annexin V(+)&7-AAD(+), Dead; Annexin V(-)&7-AAD(+).
Figure 4 is the ^expression of p-mTOR ^Ser2448 , p-AMPK ^Thr172 , p-Akt ^Ser473 , p53, PARP, cleaved-PARP, Bax, Bcl-2, pro caspase-3 and β-actin proteins in PTE-treated A549 cells. This is a Western blot analysis result confirming the level.

In order to achieve the object of the present invention, the present invention provides a health functional food composition for preventing or improving cancer containing pine ivy extract as an active ingredient.

In the health functional food composition for preventing or improving cancer of the present invention, the pine ivy extract may be extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably using ethanol. It may be extracted, but is not limited thereto.

In the health functional food composition for preventing or improving cancer of the present invention, the pine ivy extract decreases the survival rate of A549 lung cancer cells and induces apoptosis of lung cancer cells, and cancer cell proliferation protein (p-mTOR And p-Akt) and anti-apoptotic protein (Bcl-2), and increase the expression levels of cancer suppressor protein (p53) and apoptosis protein (Bax).

In the health functional food composition according to an embodiment of the present invention, the pine ivy extract is added to 100 g of the powder after crushing the stems of ivy vines collected from the pine, and then adding 800 ml of 95% ethanol to room temperature for 72 hours. It may be prepared by extracting under reflux to recover only the supernatant and then filtered, but is not limited thereto.

The term "cancer" as used herein refers to general cancer diseases including solid tumors and blood born tumors, preferably lung cancer, gastric cancer, colon cancer, breast cancer, non-small cell lung cancer, and bone cancer , Pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, 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 ureteral carcinoma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma, pituitary adenoma or fibrosarcoma cancer, preferably May be lung cancer, but is not limited thereto.

The health functional food composition for preventing or improving cancer may be prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup, effervescent tablets, and beverages, but is not limited thereto. The health functional food composition is not particularly limited as long as it can be ingested to increase anticancer activity.

When the pine ivy extract of the present invention is used as a food additive, the pine ivy extract may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). In general, when preparing food or beverage, the pine ivy extract of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health control, it can be used in an amount within a range without any problems in terms of safety.

There is no particular limitation on the type of food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and all health foods in the usual sense are included. The beverage includes carbonated beverages, functional ionic beverages, juices (eg, apples, pears, grapes, aloe, tangerines, peaches, carrots, tomato juice, etc.), sikhye, and the like.

Functional foods of the present invention include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (eg, taumatin, stevia extract, etc.) and a synthetic flavoring agent (eg, saccharin, aspartame, etc.). In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may further contain a carbonation agent used in beverages.

In addition to the above health functional food composition, various nutrients, vitamins, inhibitors, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, It may further contain a carbonating agent used in carbonated beverages. Although the ratio of the ingredients to be added is not very important, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer containing a pine ivy extract as an active ingredient.

In the pharmaceutical composition of the present invention, the pine ivy extract is as described above.

In addition, in the pharmaceutical composition of the present invention, the cancer is as described above.

In the pharmaceutical composition for preventing or treating cancer of the present invention, the pine ivy extract reduces the survival rate of A549 lung cancer cells and induces apoptosis of lung cancer cells, and cancer cell proliferation proteins (p-mTOR and p -Akt) and anti-apoptotic protein (Bcl-2) expression levels are reduced, and cancer suppression protein (p53) and apoptosis protein (Bax) expression levels are increased.

The pharmaceutical composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage form of the composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable set.

The pharmaceutical composition according to the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. However, it is not limited thereto. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, and sucrose. ) Or lactose (Lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, Witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be expected and may be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1. Materials and extraction

After pulverizing the stems of pine ivy (Jiri-san Herbal Products Farming Association), 800 ml of 95% ethanol was added to 100 g of the pulverized powder, followed by reflux extraction at room temperature for 72 hours, and the extract was 5 or 6 After filtration with ㎛ filter paper (Toyo Roshi Kaisha, Japan) and concentrated under reduced pressure using a vacuum concentrator (N-1200A, Tokyo Rikakikai Co., Japan), the concentrated extract was stored at -86 °C. The concentrated extract was made into 200 mg/ml stock using DMSO (dimethyl sulfoxide, Samchun, Korea) and stored at -20°C for use. The ethanol extract of pine ivy prepared by the above method was named PTE.

2. Cell culture

Lung cancer cells (A549) and human fibroblasts (Fibroblast) were sold and used by ATCC (American Type Culture Collection, USA). A549 cells were used in RPMI 1640 medium (HyClone Laboratories Inc.) containing 10% FBS (HyClone Laboratories Inc., USA) and 1% antibiotics (HyClone Laboratories Inc.), and Fibroblast cells were 10% FBS and 1% antibiotics. Was cultured in an incubator (MCO-15AC, SANYO Electric Biomedical Co., Japan) in a 5% CO ₂ condition at 37° C. using DMEM (HyClone Laboratories Inc.) medium containing. Each 48 hours of culture, the cells were suspended using Trypsin-EDTA (HyClone Laboratories Inc.), and the cells were dispensed at 1×10 ⁶ cells/ml and subcultured.

3. MTT Assay

A549 cells and Fibroblast cells were dispensed into a 12-well culture vessel at 1×10 ⁴ cells/ml and cultured for 24 hours, and then the prepared pine ivy extract (25, 50, 75, 100, 125, 150 Μg/ml) and incubated for 24 hours or 48 hours at 37°C and 5% CO ₂ . The cultured cells were treated with 40 µl of the MTT solution, and incubated for 50 minutes at 37°C and 5% CO ₂ . Thereafter, the medium of each well was removed, washed with 1 ml of phosphate buffered saline (PBS), treated with 150 µl of DMSO, and transferred to a 96-well culture vessel by 100 µl, and an absorbance meter (Bio-Rad Laboratories Inc., Japan). ) Was measured at 595 nm, and all measurements were performed at least three times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich Co. (USA, USA) and made into 5 mg/ml stock.

4. Observation of morphology of A549 cells

A549 cells were dispensed into a 6-well culture vessel at 1×10 ⁶ cells/ml, cultured for 24 hours, and treated with pine ivy extract (50, 75, 100 μg/ml), and 37°C, 5% CO Incubated for 24 hours under the conditions of ₂ . After the culture was completed, the change in the morphology of A549 cells was observed with an inverted microscope (Zeiss M, Carl Zeiss, Germany).

5. Annexin Cell death by V staining ( apoptosis ) Measure

In order to quantitatively analyze apoptosis, the Muse ^TM Annexin V & Dead Cell kit (MCH100105, Merck Millipore Co., USA) was used. A549 cells were suspended at 1×10 ⁵ cells/ml using 100 μl of RPMI 1640 medium, and 100 μl of Muse ^™ Annexin V&Dead Cell Reagent was mixed and reacted in a 37° C., CO ₂ incubator for 20 minutes. After the reaction is over, Muse ^TM It was analyzed using Cell Analyzer (PB4455ENEU, Merck Millipore Co.).

6. Western Blot (Western blot) analysis

A549 cells were dispensed into a 6-well culture vessel at 1×10 ⁶ cells/ml, cultured for 24 hours, and treated with pine ivy extract (50, 75, 100 μg/ml), and 37°C, 5% CO Incubated for 6 hours under the condition of ₂ . After incubation, 150 µl of RIPA lysis buffer (lysis buffer; 25 mM Tris-Cl (pH 7.4), 1% NP40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF) was added to each well to add protein. After extraction, the supernatant was taken by centrifugation at 14,000 rpm and 4° C. for 20 minutes. The extracted protein was quantified by measuring the absorbance at 595 nm using an absorbance meter. The quantified protein was loaded onto an 8% or 12% acrylamide gel, subjected to electrophoresis, and then transferred to a nitro-cellulose (NC) membrane. After the transfer is complete, a blocking process is performed at room temperature for 1 hour and 30 minutes using BSA (Bovine serum albumin), the primary antibody is reacted overnight at 4°C, the secondary antibody is reacted for 1 hour 30 minutes, and then Blue The test results were confirmed by sensitizing the X-ray film. β-actin was used as a housekeeping gene that always showed a certain degree of expression in cells.

7. Statistical processing

Using the SPSS (22.0, IBM Corp., Armonk , USA) statistical program was distributed analysis of the experimental design, were tested by the independent sample t -test (independent sample t -test) . Each data was tested with the results obtained through repeated experiments at least 3 times, and it was judged to be statistically significant when the p value was less than 0.05.

Example 1. Confirmation of the effect of pine ivy extract on the proliferation of normal cells (Fibroblast) and A549 lung cancer cells

As a result of performing MTT assay to confirm the effect of pine ivy ethanol extract (PTE) on the proliferation of Fibroblast cells and A549 cells, in Fibroblast cells, both the PTE-free control group and the PTE-treated group showed a cell survival rate of 90% or more. It was confirmed that it was maintained (FIG. 1A), and in A549 cells, it was confirmed that the cell viability was significantly decreased in the PTE-treated group compared to the untreated control group, depending on the PTE-treated concentration and incubation time (FIG. 1B and 1C).

Through this, it was found that the ethanol extract of pine ivy of the present invention has the effect of inhibiting the proliferation of lung cancer cells without affecting normal cells.

Example 2. Observation of Morphological Changes of A549 Lung Cancer Cells Following Treatment of Pine Ivy Extract

As a result of observing the shape of the cells by treating the A549 cells by concentration of PTE, in the untreated control group, cells of a certain shape were attached to the culture plate, but as the concentration of PTE increased, the shape of the cells changed irregularly. , It was confirmed that the cell density decreased by falling without attaching to the culture plate (FIG. 2). Through this, it was found that PTE treatment inhibited cell proliferation in A549 lung cancer cells, exhibited cytotoxic effects, and induced morphological changes in cells.

Example 3. Apoptosis of A549 Lung Cancer Cells by Pine Ivy Extract ( apoptosis ) Check the effect

To confirm whether the proliferation inhibitory effect of A549 lung cancer cells is due to apoptosis by PTE treatment, A549 cells treated with PTE were stained with Annexin V/7-AAD (7-Aminoactinomycin D), and then flow cytometry (Muse ^TM Cell Analyzer), and the cell death rate was expressed by summing the Late Apop/Dead value (%) and the Early Apop value (%). Annexin V is a substance that binds favorably to phosphatidylserine (PS) and is widely used as an early marker of cells in which apoptosis has occurred, and 7-AAD permeates the damaged cell membrane. Therefore, the staining of Annexin V and 7-AAD You can check whether death is triggered.

As a result, about 9.6% of the PTE-free control group, about 46.7% of the PTE 50 μg/ml treatment group, about 59.2% of the PTE 75 μg/ml treatment group, and about 74.8% of the PTE 100 μg/ml treatment group. It was confirmed that apoptosis was induced in the cells (Fig. 3). Through this, it was found that the inhibition of the proliferation of A549 lung cancer cells by PTE treatment was caused by apoptosis.

Example 4. Confirmation of apoptosis-related protein expression through the intrinsic pathway by pine ivy extract

As a result of Western blot analysis of the expression levels of proteins related to apoptosis by treating PTE in A549 lung cancer cells by concentration, p-mTOR ^Ser2448 , a protein involved in cell proliferation in a concentration-dependent manner of PTE treatment And p-Akt ^Ser473 and anti-apoptotic protein, Bcl-2, are reduced in expression levels, and p-AMPK ^Thr172 , a protein related to maintaining intracellular energy balance, p53, and apoptotic protein, a cancer suppressor protein. It was confirmed that the expression level of Bax (apoptotic protein) was increased (FIG. 4).

In addition, caspase that exists in the form of proenzyme and is activated through proteolysis and poly ADP ribose polymerase (PARP) protein that is cleaved by activated caspase are capable of recognizing and repairing damaged cells. Induces apoptosis by disappearing. Referring to FIG. 4, it was confirmed that the expression of pro caspase-3 was decreased in a concentration-dependent manner by PTE treatment, and the expression of the cleaved PARP (clevead-PARP) protein was increased.

Through this, the pine ivy extract (PTE) of the present invention regulates the expression of p-mTOR, p-AMPK, p-Akt, p53, cleaved-PARP, Bax, Bcl-2 and pro caspase-3 proteins in lung cancer cells. By doing so, it was found that apoptosis was induced by an endogenous pathway, thereby showing an excellent anticancer effect.

Claims (7)

Health functional food composition for the prevention or improvement of cancer containing pine ivy extract as an active ingredient. The health functional food composition for preventing or improving cancer according to claim 1, wherein the pine ivy extract is extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The method of claim 1, wherein the cancer is lung cancer, liver cancer, stomach cancer, colon cancer, colon cancer, skin cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, kidney cancer, fibrosarcoma, melanoma, and blood cancer. Health functional food composition for preventing or improving any one or more selected from the group. The health functional food composition for preventing or improving cancer according to claim 1, wherein the pine ivy extract is an ethanol extract of pine ivy, and the cancer is lung cancer. A pharmaceutical composition for the prevention or treatment of cancer containing pine ivy extract as an active ingredient. The pharmaceutical composition for preventing or treating cancer according to claim 5, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the pine ivy extract. The pharmaceutical composition for preventing or treating cancer according to claim 5, wherein the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups and aerosols.

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