KR101485317B1 - Food and pharmaceutical composition for preventing or improving cancer comprising extracts of Cinnamomum camphora Sieb. as effective component - Google Patents

Abstract

The present invention relates to a food composition and a pharmaceutical composition for preventing or ameliorating cancer, which comprises a camphor extract as an active ingredient. The present invention relates to a camphor extract of the present invention, which reduces the survival rate of cancer cells, induces apoptosis, It can be usefully used as a food or pharmaceutical composition for preventing, ameliorating or treating cancer.

Description

TECHNICAL FIELD The present invention relates to a food composition and a pharmaceutical composition for prevention or improvement of cancer, which contains camphor extract as an active ingredient. as effective component}

The present invention is camphor (Cinnamomum camphora Sieb . The present invention relates to a composition and a pharmaceutical composition for preventing or ameliorating cancer, which contains an extract as an active ingredient. The present invention relates to a cambium extract having an effect of reducing the survival rate of cancer cells, inducing cell suicide, , It can be usefully used as a food or pharmaceutical composition for preventing, ameliorating or treating cancer.

Camphor (Cinnamomum camphora Sieb . ) Is a tree that grows only in Jeju Island in Korea and is called evergreen tree or oak tree. It is an evergreen broad-leaved tree that leaves no leaves in winter. It belongs to the Camphoraceae family. It has a height of 20 m and a diameter of 2 m. It has no pests, has good growth and has a long life span. The fragrance ingredients contained in camphor are essential oil components such as camphor, safflower, and cinere, and contain about 1% of camphor wood, leaf, and fruit. Essential oil is obtained by distilling tree trunks and steam distillation. The essential oil thus obtained is called "camphor" and is used widely as a stimulant or a cardiotonic agent for the treatment of nervous breakdown, epilepsy, cystitis, and pyelonephritis in the private sector. Especially in Japan, camphor is considered very valuable and treated like national ginseng as a national monopoly, and camphor tree has been used as a cancer remedy in the private sector in Jeju Island. However, there are not many reports on the physiological activities of these components of camphor tree.

Korean Patent No. 0623972 discloses a composition for the prevention and treatment of inflammatory diseases containing camphor tree extract and Korean Patent No. 0706134 discloses a composition for prevention and treatment of diabetes comprising camphor leaf extract or fractions thereof as an active ingredient However, no food composition or pharmaceutical composition for preventing or ameliorating cancer containing camphor extract as an active ingredient has been disclosed in the present invention.

The present invention has been made in view of the above-mentioned needs. It is an object of the present invention to provide a method of treating cancer stem cells by treating a 95% ethanol extract of camphor with an effect of reducing the survival rate of HT1080 fibrosarcoma cancer cells, inducing apoptosis, By confirming, the present invention has been completed.

In order to solve the above-mentioned problems, the present invention provides a food composition for preventing or improving cancer, which contains camphor extract as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, which contains camphor extract as an active ingredient.

Through the present invention, the camphor extract can be effectively used as a food or pharmaceutical composition for preventing, ameliorating or treating cancer by confirming that camphor tree extract has an effect of reducing the survival rate of cancer cells, inducing apoptosis, and inhibiting cancer cell metastasis.

1 is a camphor tree (Cinnamomum camphora Sieb . ) 95% ethanol extract on the proliferation of HT1080 cells.
Figure 2 shows the apoptosis effect of the 95% ethanol extract of camphor tree.
Figure 3 shows confirmation of the p53 signal pathway using Western blot analysis.
FIG. 4 shows the cell mobility inhibitory effect of the 95% ethanol extract of camphor tree by Wound healing assay.

According to an aspect of the invention, the invention is camphor (Cinnamomum camphora Sieb. ) Extract as an active ingredient.

The camphor tree extract of the present invention may be extracted as water or an organic solvent or a mixed solvent thereof. The organic solvent is any conventional solvents which can be used, preferably water, C _{1 - 4} alcohol: may be a polar solvent or a mixture of these solvents, such as (for example methanol, ethanol, etc.). Preferably, the camphor extract may be an ethanol extract, and most preferably a 95% ethanol extract, but is not limited thereto.

The term "cancer ", as used herein, refers to a general cancer disease including solid tumors and blood born tumors, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non- Cancer of the pancreas, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small bowel cancer, rectum cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, Cancer of the urethra, cancer of the prostate, cancer of the prostate, chronic or acute leukemia, lymphocytic lymphoma (Hodgkin's disease), esophageal cancer, small bowel cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, (CNS), primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, or fibrosarcoma, and can be used to treat a wide variety of conditions, including, but not limited to, bladder cancer, kidney or ureteral cancer, kidney cell carcinoma, But are not limited to, fibrosarcoma.

In addition, the camphor extract of the present invention can be added to food or beverage for the purpose of preventing or improving cancer. At this time, the amount of the extract in the food or beverage may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, have.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tautatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention can also be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aging agents (cheese, chocolate, etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extracts of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These ingredients may be used independently or in combination, and the proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, which contains camphor extract as an active ingredient.

In the pharmaceutical composition according to one embodiment of the present invention, the extract is as described in the above food composition.

In the pharmaceutical composition according to one embodiment of the present invention, the cancer is as described in the above food composition.

The pharmaceutical composition for preventing or treating cancer according to the present invention may contain 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the above extract relative to the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

Or mixtures thereof. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

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

Materials and methods

Preparation of camphor ethanol extract

800 g of ethanol was added to 100 g of ground camphor powder, which was completely immersed in each extraction solvent and then extracted with refluxing at room temperature. The camphor extract prepared by the above extraction method was concentrated under reduced pressure using a vacuum concentrator, and the obtained extract was stored at -80 ° C. The camphor extract was dissolved in DMSO prior to use in the experiment and stored frozen.

Cell culture

HT1080 cells (fibrosarcoma cell line) were purchased from ATCC (America Type Culture Collection, Gaithersburg, MD, USA) and were inoculated with 10% fetal bovine serum (Gibco BRL, Grand Island, (WelGENE Inc., Seoul, Korea) containing 5% CO ₂ and 37 ° C in a DMEM medium (100 U / ml penicillin). Cells were allowed to float with the use of trypsin-EDTA (WelGene Inc., Seoul, Korea) every 24 hours, and cells were subcultured at 1 × 10 ⁶ cells / ml.

MTT  Measurement of cell viability by analysis

HT1080 cells were plated at a density of 1 × 10 ⁶ cells / ml in a 12-well plate, cultured for 24 hours, and treated with a 95% ethanol extract of camphor. Twenty microliters of MTT solution (mg / ml) was added and cultured in a CO ₂ incubator for 1 hour. After the medium containing the MTT solution was removed, 150 μl of DMSO was added to each well to dissolve all the generated formazan in the wells. Then, 100 μl of each of the generated formazan was transferred to a 96-well plate, followed by ELISA microplate reader (BIO- RAD medel 680, BIO- RAD Laboratories Inc., Tokyo, Japan) at 595 nm. All measurements were performed three times, and mean values and standard errors were analyzed using the Microsoft Excel program.

Hoechst  33342 Using staining Chromatin  dyeing( chromatin staining )

HT1080 cells were cultured on a cover glass, treated with 10 μM of Hoechst 33342 staining reagent (32 μg / μl), reacted for 30 minutes, and incubated with 3.5% formaldehyde Was added to each well and cultured for 15 minutes. Then, the cells were washed twice with PBS, and one or two drops of 50% glycerol were dropped on a slide glass. Using a tweezers, the cells cultured on the cover glass were directed to the bottom. Then, a fluorescence microscope (Olympus Optical, Tokyo, Japan) , Apoptotic cells were identified in HT1080 cells and photographed with a digital camera (Olympus SP-6600, Japan).

Western Blotting  analysis

The 95% ethanol extract of camphor tree was treated and cultured in CO ₂ incubator for 24 hours. 150 μl of RIPA lysis buffer (50 mM Tris-HCl (pH 8.0), 150 nM NaCl, 1% NP 40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl) was added to each well for protein extraction After the reaction, the supernatant was taken after centrifugation at 14,000 rpm, 4 ° C for 20 minutes. The absorbance at 595 nm was then measured by ELISA-reader to determine the protein concentration of each sample. 5 × Laemmi sample buffer (loading dye; 250 mM Tris-Cl (pH 6.8), 40% glycerol, 4% β-mercaptoethanol, 0.08% Bromophenol Blue, 8% SDS) and HT1080 cells To prepare a sample. The primary antibody was purchased from Cell Signaling Technology (Beverly, MA, USA) and the secondary antibody was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and diluted to the respective concentrations.

Example  1. A 95% camphor tree ethanol extract HT1080  Effect on cell proliferation

To investigate the effect of 95% ethanol extract of camphor tree on HT1080 cell proliferation, the survival rate of cancer cells was measured by MTT assay after treatment with 95% ethanol extract of camphor tree. As a result, it was confirmed that the survival rate of cells decreased as the concentration of 95% ethanol extract of camphor increased (Fig. 1).

Example  2. Cellular suicide of 95% ethanol extract of camphor tree apoptosis ) effect

The inhibition of cancer cell growth by 95% ethanol extract of camphor tree was induced by apoptosis induced by chromatin staining with Hoechst 33342. As a result, more apoptotic bodies were observed when treated with 95% ethanol extract of camphor tree (Fig. 2).

Example  3. Western Blot ( Western blotting ) Analysis p53  Identification of signal path

Western blotting was used to investigate the induction of apoptosis by 95% ethanol extract of camphor tree. As the concentration of 95% ethanol extract of camphor increased, the expression of p53 was increased and the expression of anti-apoptotic protein, Bcl-2, was decreased. In addition, pro-caspase-3 decreased and PARP cleavage was increased. In conclusion, it was confirmed that HT1080 cells treated with 95% ethanol extract of camphor showed apoptosis through p53 (FIG. 3).

Example  4. Analysis of wound healing ( Wound healing assay Effect of 95% Ethanol Extract of Camphor Tree on Cell Mobility

Wound healing assay was used to observe the effect of 95% ethanol extract of camphor tree on cell mobility. As a result of the experiment, it was confirmed that the cell mobility was inhibited by increasing the concentration of the 95% ethanol extract of camphor (Fig. 4). This indicates that the 95% ethanol extract of camphor tree not only induces apoptosis of cancer cells but potentially inhibits cancer cell metastasis.

Claims (7)

A food composition for preventing or improving fibrosarcoma cancer containing Cinnamomum camphora Sieb. Extract as an active ingredient. The food composition according to claim 1, wherein the extract is an ethanol extract. delete A pharmaceutical composition for preventing or treating fibrosarcoma cancer containing Cinnamomum camphora Sieb. Extract as an active ingredient. 5. The pharmaceutical composition according to claim 4, wherein the extract is an ethanol extract. delete The pharmaceutical composition according to claim 4, which induces apoptosis of cancer cells and inhibits metastasis of cancer cells.

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