KR20210065576A - Composition for anticancer or cancer supplement comprising extracts of raw pericarp derived from immature fruit of Camellia japonica - Google Patents

Abstract

The present invention relates to an anticancer pharmaceutical composition or a food composition for anticancer supplementation comprising a raw fruit peel extract of an immature fruit of Camellia japonica. The raw fruit peel extract of the immature fruit of Camellia japonica has excellent effects of inhibiting the growth of cancer cells and inducing apoptosis, unlike a dried fruit peel extract, thereby being able to be usefully used as an anticancer composition.

Description

Composition for anticancer or cancer supplement comprising extracts of raw pericarp derived from immature fruit of Camellia japonica

The present invention relates to an anticancer or anticancer auxiliary composition comprising, as an active ingredient, a raw peel extract derived from the immature fruit of Camellia japonica, specifically, a water extract or 100% of the raw fruit peel of an immature fruit of a camellia. It relates to a pharmaceutical composition for anticancer or a food composition for adjuvant anticancer comprising an alcohol extract as an active ingredient.

Despite advances in modern medicine, cancer is still one of the most difficult human diseases to treat. Cancer is a tissue composed of abnormal cells that arise from one or more tissues and spread to other tissues. Currently, more than 100 known types of cancer are known ("Defining Cancer". National Cancer Institute. 17 September 2007. Retrieved 28 March 2018). It is known that 90-95% of the main causes of cancer are genetic mutations caused by environmental factors. Most of these factors are smoking, obesity, radiation, UV rays, stress, and environmental pollution (Anand P, Kunnumakkara AB, Sundaram). C, Harikumar KB, Tharakan ST, Lai OS, Sung B, Aggarwal BB (September 2008). "Cancer is a preventable disease that requires major lifestyle changes". Pharmaceutical Research. 25 (9): 2097-116).

Current cancer treatment methods include chemotherapy using drugs such as antimetabolites, alkylating agents, topoisomerase inhibitors, monoclonal antibodies, or radiation therapy at the same time. also use it

However, the use of these drugs may vary from patient to patient, but generally there are side effects such as hair loss, suppression of bone marrow function, drug resistance, and organ damage (Rachel Airley (2009). Cancer chemotherapy. Wiley-Blackwell. ISBN 978-0-470-09254-5). Numerous efforts are being made all over the world to conquer cancer, and as one of these efforts, research to prevent the proliferation of cancer using natural products is active.

On the other hand, camellia is a medicinal plant belonging to the family Theaceae, which is native to China and Japan, including Korea, and about 200 species are known. Among them, about 70 species are related to the subgenus Camellia, that is, Camellia japonica and C. sasanqua . Camellia japonica L., native to Korea, is an arboreal tree in the family Theaceae and Camelliae. Camellia leaves are effective for psoriasis, sore throat, and burns, and branches and fruits are effective for hair dandruff, blood loss, epistaxis, eosinophilia, chondrosis, menstrual abnormalities, diuresis, sore throat, intestinal bleeding, and poisoning.毒), bruises, hematemesis and hemoptysis, haematuria, and burns are known to be effective. In addition, previous studies reported that camellia leaves and flowers contain triterpenes, flavonoids, tannins and fatty acids with antibacterial, antioxidant, and anti-inflammatory activities. These camellias have long been used in folk medicine to treat diseases such as bleeding and inflammation. In addition, in Korea, camellia was developed as a food material after 2004 and commercialized as a tea food called “camellia tea” in 2007.

However, research on camellia so far has been limited only to the leaves and stems of the camellia tree, or camellia oil, and not much research has been done on the fruit of the camellia.

Accordingly, the present inventors discovered that the raw peel extract of the immature fruit of the camellia tree had an effect of inducing the death of cancer cells while researching the fruit of the camellia tree, and completed the present invention.

Republic of Korea Patent Registration No. 10-1797072 (2017.11.14. Announcement) Republic of Korea Patent Registration No. 10-1723588 (2017.04.06. Announcement) Korean Patent Registration No. 10-0556524 (2006.03.06. Announcement) Korean Patent Publication No. 10-2016-0008774 (published on Jan. 1, 2016)

Therefore, the technical problem to be solved in the present invention is to provide a material having an effect of inducing apoptosis on cancer cells and having excellent human stability.

In order to solve the above technical problem, the present invention provides a pharmaceutical composition for anticancer comprising the raw fruit skin extract of the immature fruit of camellia as an active ingredient.

In addition, the present invention provides a food composition for an anticancer supplement comprising the raw fruit peel extract of the immature fruit of the camellia as an active ingredient.

The cancer may be oral cancer, colon cancer, breast cancer, kidney cancer, brain tumor, liver cancer, cervical cancer, lung cancer, and blood cancer, but is not limited thereto.

Camellia japonica L. of the present invention is a tree of the Camellia family (Theaceae), the genus Camellia (Camelliae).

The immature fruit of the present invention is a fruit before being matured and harvested. Usually, since the fruit is matured and harvested from September, it is the fruit before that, that is, the fruit before maturation from June to August.

The pericarp of the present invention refers to the portion remaining after removing the seeds in the fruit. In particular, the pericarp of an immature fruit refers to the portion remaining after removing the portion growing into the seeds (see FIG. 1 ).

The raw skin of the present invention refers to the peel of an immature fruit that has not been subjected to any processing such as heat treatment or drying.

The extract of the present invention refers to a preparation obtained by squeezing the raw pericarp of the immature fruit of the camellia tree with an appropriate leaching solution and evaporating the leachate to concentrate it, but is not limited thereto, but is not limited thereto. It may be a dried product obtained by drying a concentrate or an extract, a crude product or a purified product thereof.

In the present invention, the raw fruit skin extract of the immature fruit of camellia can be obtained using an extraction method and an extraction solvent known in the art, preferably water, anhydrous or hydrous lower alcohol having 1-4 carbon atoms (methanol , ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform or 1,3-butylene glycol, more preferably water, as the extraction solvent.

In addition, the raw fruit skin extract of the immature fruit of the camellia can be obtained through a conventional purification process in addition to the extraction method using the extraction solvent. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity, or affinity), etc. Through various purification methods additionally performed Through the obtained fraction, it is possible to obtain an extract of the raw fruit skin of the immature fruit of the camellia tree.

According to one specific embodiment of the present invention, after pulverizing the raw skin of the immature fruit of the camellia tree, 0.5 to 20 times, preferably 1 to 15 times the weight of the pulverized product of water is used in an amount of about 0.5 at room temperature. After extraction using an extraction method such as stirring extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction for 1 to 30 hours, the obtained extract is filtered, concentrated under reduced pressure, or dried to remove the camellia tree for 48 hours, preferably 1 to 30 hours. It is possible to obtain a raw fruit skin extract of an immature fruit.

In the present invention, the raw fruit peel extract of the immature fruit of Camellia includes all extracts, fractions and purified products obtained in each step of extraction, fractionation or purification, and dilutions, concentrates or dried products thereof.

According to one embodiment of the present invention, the raw peel extract of the immature fruit of camellia contains a specific component, unlike the dried fruit peel extract, and inhibits cell growth and induces death in cancer cells, thereby causing oral cancer, colon cancer, breast cancer, It has a preventive or therapeutic effect on cancers such as kidney cancer, brain tumor, liver cancer, cervical cancer, lung cancer, and blood cancer.

The composition of the present invention may contain 100 to 150 ug, preferably 110 to 140 ug, more preferably 120 to 130 ug, and most preferably 125 ug of the raw fruit peel extract of the immature fruit of camellia per 1 ml of the composition. . When the content is less than 100 ug/ml, the cancer cell growth inhibition and death-inducing effect is too weak, and when it exceeds 150 ug/ml, there is a problem of cytotoxicity.

According to one embodiment of the present invention, there is provided a pharmaceutical composition for anticancer comprising the raw fruit skin extract of the immature fruit of camellia as an active ingredient.

The pharmaceutical composition according to the present invention includes a pharmaceutically acceptable carrier in addition to the raw fruit skin extract of the immature fruit of Camellia. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes such as oral or parenteral, for example, oral, rectal or intravenous, muscle, subcutaneous, intrauterine dura mater or cerebrovascular It can be administered by intravenous injection.

The pharmaceutical composition is administered in a medically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level is determined by the type and severity of the disease, the activity of the drug, the patient age, weight, health and sex of the patient, the patient's sensitivity to the drug, the time of administration of the particular extract used, the route and rate of excretion, the duration of treatment, factors including drugs used in combination with or concurrently with the particular extract used, and other medicines It may be determined according to factors well known in the art. In general, a dose of 0.1 to 1,000 mg/kg, preferably 10 to 100 mg/kg, based on an adult, may be administered once to several times a day. In addition, in the case of an external preparation, it is recommended to apply it once to 5 times a day in an amount of 1.0 to 3.0 ml based on an adult and continue it for at least 1 month. However, the scope of the present invention is not limited by the above dosage.

The pharmaceutical composition may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It may be prepared by introduction into a dose container. In this case, the formulation may be formulated as a conventional formulation in the pharmaceutical field according to the purpose of treatment, for example, tablets, capsules, powders, granules, suspensions, emulsions, syrups, emulsions, warning agents, ointments, sprays, oils It can be formulated as a preparation, gel, alcohol, tincture, bath, liniment, lotion, patch, pad, cream, and the like. In addition, it can be preferably used as an external preparation for the skin for the purpose of topical administration applied directly to the affected area, in this case, the form of an ointment, lotion, spray, gel, etc. is preferable. These external preparations for skin may also be included in a support base or matrix that can be directly applied to the treatment site, and examples of the support base include gauze or a bandage.

According to one embodiment of the present invention, there is provided a food composition for an anticancer supplement comprising the raw fruit peel extract of the immature fruit of camellia as an active ingredient.

In the food composition according to the present invention, not only the raw fruit peel extract of the immature fruit of camellia as an active ingredient, but also ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents are added. can be included as

Examples of the carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides 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. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared as a drink, in addition to the raw fruit peel extract of the immature camellia fruit of the present invention, a pear concentrate, a plant mixed extract, for example, astragalus, cinnamon, Angelica, licorice, baekchul, dung beetle, reishi mushroom Extracts, polydextrose, vitamin C, nicotinic acid amide, trisodium citrate, citric anhydride, and the like may be further included.

The raw fruit skin extract of the immature fruit of the camellia tree can be usefully added to new functional medicines, beverages, health foods or health supplements.

On the other hand, the raw fruit peel extract of the immature fruit of the camellia tree of the present invention is a natural material, harmless to the human body, and has almost no toxicity and side effects, so it can be safely used even for long-term use. In particular, the pharmaceutical composition and food composition as described above can be safely applied to

As described above, the composition comprising the raw fruit skin extract of the immature camellia fruit according to the present invention exhibits an effect of inhibiting the growth of cancer cells and inducing apoptosis. In addition, the raw fruit skin extract of the immature camellia fruit of the present invention is a natural substance, has no cytotoxicity, and is harmless to the human body, so it can be safely used in pharmaceutical and food compositions.

1 is a diagram showing the pericarp of an immature fruit from which a part growing into a seed is removed from an immature fruit of a camellia tree according to an embodiment of the present invention.
2 is a diagram showing the cytotoxic effect on human normal cells (293) of the water extract, 80% alcohol extract, and 100% alcohol extract on the raw skin of the immature fruit of the camellia tree according to an embodiment of the present invention. .
3 is a diagram showing the death-inducing effect on cancer cells (FaDu) and human normal cells (293) of the raw fruit peel water extract and the dried fruit peel water extract of the immature fruit of the camellia tree according to an embodiment of the present invention.
4 is a diagram showing the results of HPLC analysis and comparison of water extracts from raw fruits and dried fruits of camellias according to an embodiment of the present invention.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Preparation Example 1: Preparation of raw fruit skin extract of immature camellia fruit

After harvesting the immature fruits of the camellia native to Wando-gun, Jeollanam-do in July, before the fruits of the camellia were mature, the part that became the seed at maturity was removed to obtain a rind of about 1-1.5 cm. After pulverizing the pericarp (fresh skin), 10 g was extracted in an accelerated solvent extraction device (Dionex™ ASE™ 350 Accelerated Solvent Extractor) using 200 ml each of water, 80% alcohol, and 100% alcohol as a solvent at 70° C. for 15 minutes. Thereafter, it was concentrated using a rotary evaporator (EYELA N-1000).

The concentrate was diluted by adding 10 ml of water, centrifuged at a speed of 1200 × g for 20 minutes, only the supernatant was taken, and then filtered through a 0.45 um syringe filter to be used as a sample for the following experiment.

Comparative Example 1: Preparation of dried fruit skin extract of immature camellia fruit

A sample was prepared in the same manner as in Preparation Example 1, except that the dried fruit skin (fresh skin) obtained in Preparation Example 1 was dried with hot air at 35° C. for 2 hours to prepare a dried fruit peel water extract of the immature camellia fruit. .

Example 1: Cell Culture

In order to test the efficacy of the raw or dried fruit peel extract of the immature camellia fruit according to the present invention, a human embryonic kidneys cell line 293 and a human hypopharyngeal carcinoma cell line FaDu were prepared. were cultured using DMEM (Dulbeccos modified Eagles medium) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 ug/ml streptomycin, respectively. The cells were cultured in an incubator of 37° C., 95% humidity, 5% CO ₂ conditions and subcultured with a new culture medium every 2-3 days.

Example 2: Cytotoxicity test

MTT analysis was performed to confirm the cytotoxicity of the raw or dried pericarp extract of the immature camellia fruit prepared in Preparation Example 1 and Comparative Example 1.

Specifically, human 293 cells were aliquoted to 2 × 10 ⁴ cells/well in a 96-well plate, and then cultured for 16 hours in a 37° C. cell incubator in the presence of _{5% CO 2 .} Then, the wells were treated with 62.5, 125, 250, 500, and 1000 ug/ml of each sample and reacted for 24 hours in a 37° C. cell incubator in the presence of _{5% CO 2 .} Then, add MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution to each well and incubate in a cell incubator at 37°C, then add 50% DMSO to lyse the cells. The absorbance was measured at 570 nm using a microplate reader. Then, the viability of 293 cells, which are normal cells, was expressed as a percentage using Equation 1 below. This cytotoxicity experiment was repeated three times, and the value was expressed as the mean ± standard deviation. The results for this are shown in FIG. 2 .

empirical formula 1

Cell viability (%) = (absorbance of sample treatment group / absorbance of control group) × 100

As can be seen in FIG. 2 , the water extract and 100% alcohol extract of the raw fruit of the immature camellia fruit did not show cytotoxicity at 125 ug/ml.

Example 3: Effect of inducing cancer cell death

In order to confirm the cancer cell death-inducing effect of the raw fruit skin extract of the immature camellia fruit without cytotoxicity according to Example 2 and the dried fruit skin water extract of the immature camellia fruit of Comparative Example 1, cultured in Example 1 One cancer cell (FaDu) and normal cells (293) were analyzed using the same MTT assay method as in Example 2. The results are shown in FIG. 3 .

As can be seen in FIG. 3 , it was confirmed that the raw fruit skin extract of the immature camellia fruit did not induce apoptosis of normal cells at a concentration of 125 ug/ml, but did induce apoptosis of cancer cells. On the other hand, the dried pericarp water extract of the immature camellia fruit did not induce apoptosis in both normal cells and cancer cells regardless of the concentration.

Example 4: HPLC analysis of extracts

HPLC analysis was performed on the extract to determine the cause of the effect of the raw fruit skin extract of Camellia immature fertilization inducing apoptosis of cancer cells. For specific HPLC analysis, agilent 1100 series instrument was used, the column was YMC-Pack ODS-AM (4.6x250mm, 5μm), the column oven temperature was 40 ℃, the flow rate was 0.8ml/min, and the UV detector was 254nm. Analyzed under the same concentration gradient conditions.

The results for this are shown in FIG. 4 . As can be seen in Figure 4, the raw pericarp extract of the immature fruit of camellia has two peaks significantly higher than that of the dried pericarp extract of the immature fruit of the camellia, It can be predicted that the apoptosis-inducing effect of cancer cells is generated by the components constituting these peaks.

Claims (8)

A pharmaceutical composition for anticancer comprising the raw fruit peel of the immature camellia fruit.
[Claim 2] The pharmaceutical composition for anticancer according to claim 1, wherein the immature fruit is a fruit before maturation, and the raw fruit skin is removed from the seed growing part.
The pharmaceutical composition for anticancer according to claim 1, wherein the extract is a water extract.
[Claim 2] The pharmaceutical composition for anticancer according to claim 1, wherein the raw fruit peel extract of the immature camellia fruit is contained in an amount of 100 to 150 ug/ml.
A food composition for anticancer supplementation comprising an extract of the raw fruit of the immature camellia fruit.
[Claim 2] The food composition for anticancer supplementation according to claim 1, wherein the immature fruit is a fruit before maturation, and the raw fruit skin has a seed-growing part removed.
According to claim 1, wherein the extract is an anticancer supplement food composition, characterized in that the water extract.
[Claim 2] The food composition for anticancer supplementation according to claim 1, wherein the raw fruit peel extract of the immature camellia fruit is contained in an amount of 100 to 150 ug/ml.

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