KR101446784B1 - Pharmaceutical composition for anticancer comprising extract of sea cucumber or its fraction as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for anticancer and food which contains a fraction of sea cucumber extract or sea cucumber extract as an active ingredient. It can be used not only as a pharmaceutical composition for prevention and treatment of cancer, but also as a functional food Can be usefully used.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for anticancer comprising an extract of sea cucumber or a fraction thereof as an active ingredient,

The present invention relates to a pharmaceutical composition and food for preventing or treating cancer, which comprises a fraction of sea cucumber extract or sea cucumber extract as an active ingredient.

Sea cucumbers are the 8 most important marine products of Korea that are set by the Ministry of Maritime Affairs and Fisheries. They are not only the best health and well-being food items called ginseng in the sea because they contain a large amount of active ingredients such as saponin.

Sea cucumbers are known to have been as much as the wild cucumbers in China. Sea cucumber is a medicinal herb that has been found in China and other Eastern countries for a long time. It has excellent effects on diabetes and asthma. It is a medicine that can regain energy when it is swollen due to temperature rise in the summer, It is known. The sea cucumbers have a remarkable resilience that the cutting site returns to the original state within 3 months when a part of the body is cut and a new intestine is formed within 1 month even if the intestines are removed. In the oriental medicine and folk remedies, It is known that it enhances immune function by enhancing phagocytic ability of phagocytes and is effective in the treatment of wounds.

According to modern science, sea cucumbers are abundant in protein and vitamin B, contain a large amount of iodine, and contain a large amount of chondroitin, which is used as a raw material for various foods and medicines. The major components of sea cucumber are glycoprotein and chondroitin sulfate, and glycoproteins are mainly composed of polysaccharides such as fucose, glucose, mannuronic acid and N-acetylglucosamine.

For the above reasons, sea cucumbers are used as food for children, foods for the elderly, food for recovery after the disease, foods for preventing anemia of pregnant women, foods for promoting bowel movement, etc. Also, since they have less fat and sugar, , And there is no research on the anticancer activity of sea cucumber extracts.

Korean Patent Registration No. 0979887 discloses a composition for the prevention and treatment of hyperlipidemia, which comprises dry powder or extract of sea cucumber as an active ingredient. However, the composition for preventing and treating hyperlipidemia of sea cucumber, It is different.

The present invention has been made in view of the above needs, and an object of the present invention is to reveal the various usefulness of the sea cucumber through the effect of the anticancer effect of the extract of methanol extract of sea cucumber and the fraction of extract of ethanol extract.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a fraction of sea cucumber extract or sea cucumber extract as an active ingredient.

The present invention also provides an anticancer food comprising a sea cucumber extract or a fraction of a sea cucumber extract as an active ingredient.

According to the present invention, an anticancer pharmaceutical composition comprising a sea cucumber extract and a fraction is useful as a pharmaceutical composition for prevention and treatment of cancer without causing side effects because it contains a substance obtained from a natural material. In addition, the sea cucumber extracts and fractions can be usefully used as functional foods in foods and tea, so that the added value of sea cucumbers can be expected to increase.

FIG. 1 is a graph comparing cell survival rates after treatment of 293T cells, which are normal cells, and AGS cells, which are gastric cancer cells, with different concentrations of sea cucumber extracts and fractions.
FIG. 2 shows the expression level of p53 protein after treating sea cucumber extract and fraction with gastric cancer cells.
FIG. 3 is a graph showing caspase activity after treatment of sea cucumber extract and fraction with gastric cancer cells.
FIG. 4 shows the expression level of LC3 gene after treatment with methanol extract of sea cucumber.
FIG. 5 shows GC analysis of components common to methanol extracts and hexane fractions of sea cucumber.
(A) fatty acid series, (B) organic acid series
FIG. 6 is a graph showing the GC analysis of the components existing only in the hexane fraction layer of the ethanol extract of sea cucumber.
(A) fatty acids, (B) per se

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a fraction of sea cucumber extract or sea cucumber extract as an active ingredient.

In the pharmaceutical composition of the present invention, the sea cucumber extract may be methanolic extract of sea cucumber. The methanolic extract of sea cucumber is prepared by adding 1000 to 1400 parts by weight of methanol to 4000 to 5000 parts by weight of lyophilized sea cucumber and heating at 55 to 65 ° C for 3 to 5 hours Preferably 1 to 3 times, and then concentrated under reduced pressure at 40 to 50 ° C. Preferably, 1,200 parts by weight of methanol is added to 4,500 parts by weight of lyophilized sea cucumber, extracted twice at 60 ° C for 4 hours, Followed by concentration under reduced pressure.

In the pharmaceutical composition of the present invention, the fraction of the sea cucumber extract may be a hexane fraction or a butanol fraction obtained by adding n-hexane or n-buthanol to an ethanol extract of sea cucumber. The hexane or butanol fraction may be, To 1200-1500 parts by weight of the lyophilized sea cucumber was added 3200-4000 parts by weight of ethanol and the mixture was extracted 1-3 times at 55-65 ° C for 1-3 hours and then concentrated under reduced pressure at 40-50 ° C to obtain a sea cucumber ethanol extract. n-hexane, or n-buthanol. Preferably, 3,600 parts by weight of ethanol is added to 1340 parts by weight of lyophilized sea cucumber, extracted twice at 60 ° C for 4 hours, concentrated under reduced pressure at 45 ° C And then adding hexane (n-hexane) or n-buthanol to the ethanol extract of sea cucumber.

Since the polymer material including polysaccharide (chondroitin sulphate) and glycoprotein is not extracted by the organic solvent extraction method, and the solvent extraction extracts only the low-molecular substance having molecular weight of 5000 or less, the fraction of the sea cucumber extract or sea cucumber extract of the present invention Does not include polysaccharides (chondroitin sulphate) and polymeric substances such as glycoproteins.

In the pharmaceutical composition of the present invention, the cancer may be gastric cancer, breast cancer, lymphoma, hepatoblastoma, cervical cancer, colorectal cancer, breast cancer or lung cancer, preferably gastric cancer.

The anticancer pharmaceutical composition of the present invention may contain 0.02 to 80% by weight, preferably 0.02 to 50% by weight of the extract or fraction, based on the total weight of the pharmaceutical composition.

The pharmaceutical compositions comprising the extracts or fractions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts or fractions 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 or the fraction according to the present invention can be administered orally or rectally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, Can be used. Examples of the carrier, excipient and diluent that can be contained in the pharmaceutical composition containing the extract or the fraction 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. have. 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, (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 suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dose of the extract or fraction of the present invention will vary depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract or fraction of the present invention is preferably administered at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg. 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 extracts or fractions of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides an anticancer food comprising a sea cucumber extract or a fraction of a sea cucumber extract as an active ingredient. The anti-cancer food may be preferably used for prevention or improvement of gastric cancer.

The food is not particularly limited as long as it can be ingested to increase anticancer activity.

When the extract or fraction of the present invention is used as a food additive, the extract or fraction may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the extract or fraction of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Examples of the food to which the extract or the fraction can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract or fraction of the present invention.

In addition to the above, the extract or the fractions of the present invention may be used as an extract or a fraction containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extract or fraction of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the extract or fraction of the present invention.

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

Extraction and Experimental Methods

(1) extraction of useful components of freeze-dried sea cucumber

1) Experimental method

Freeze - drying was performed to remove moisture, which is difficult to extract organic solvent, from sea cucumbers containing a large amount of water. To 4.5 kg of lyophilized sea cucumber, 12 L of methanol and 1.34 kg of freeze-dried sea cucumber were added 36 L of ethanol, and repeated extraction was performed twice at 60 ° C for 4 hours. The obtained extract was filtered to remove impurities. The filtered extract was concentrated under reduced pressure at 45 ° C to obtain methanol crude extract (128.23 g) and ethanol crude extract (379.71 g). Among them, the ethanolic crude extract was suspended in water, and the fraction was successively fractionated using n-hexane, chloroform (CHCl ₃ ), ethyl acetate (EtOAc) and butanol (n-BuOH).

(2) Cytotoxicity test of freeze-dried sea cucumber extract and fraction

1) Cell culture

The cancer cell lines used in this experiment were human gastric adenocarcinoma cells (AGS), which are human gastric cancer cells, and were distributed from the Korean Cell Line Bank (KCLB). The control kidney epithelium (293T) was cultured in DMEM medium, and the AGS cell line was cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 units / ml) And cultured in a monolayer in a 5% CO ₂ incubator.

2) Cytotoxicity experiment

2-yl-5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) thiophene was obtained from 293T (human kidney epithelial cell) 2H-tetrazolium (MTS, promega, USA) assay, which measures the viability of mitochondrial dehydrogenase in living cells, which converts MTS, a yellow substrate, to dark blue formazan. 293T (normal cell line) cells were seeded at 2 × 10 ⁴ cells / well in a 96-well plate at 37 ° C and 5% in an incubator, a CO ₂ condition were cultured for 24 hours. after the culture was added to MTS solution and the absorbance was measured at 490 nm using Microplate reader (microplate reader) and incubated for 4 hours. viability of the cells is the following formula Depending was calculated.

Cell survival rate (%) = (absorbance of sample-added group / absorbance of control group) x 100

 (3) Assessment of inhibition of cell growth on gastric cancer cells

(5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-methoxyphenyl) -sulfophenyl) -2H-tetrazolium (MTS, promega, USA) assay method. The AGS cells were plated at 1.5 × 10 ⁴ cells / well in a 96-well plate, The cells were cultured for 24 hours at 37 ° C and 5% CO _2. After incubation, the MTS solution was added, incubated for 4 hours, and the absorbance was measured at 490 nm using a microplate reader. .

Cell survival rate (%) = (absorbance of sample-added group / absorbance of control group) x 100

(4) Analysis of protein expression by Western blot analysis

Protein lysis buffer [10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM Na ₃ VO ₄ , 10 mM NaF, 10 mM sodium pyrophosphate, 0.4 mM EDTA, 0.5% Triton X- After incubation at 4 ° C for 2 hours, the cells were centrifuged at 12,000 rpm at 4 ° C for 20 minutes. The obtained protein was loaded on the SDS-PAGE gel by the same loading, separated, and transferred to a nitrocellulose membrane (Whatman). The membranes from which the proteins were transferred were blocked with TBST mixed with 5% skim milk, reacted with p53, β-actin primary antibody, washed with TBST, and incubated with AP-conjugated secondary The antibody is reacted at room temperature for 1 hour. After washing with TBST, BCIP / NBT solution was used to develop color in the dark room, and the activity of the protein was observed as a band on the membrane.

(5) Measurement of caspase activity

To investigate the apoptosis inducing effect of the extracts, Apo-ONE Homogeneous Caspase-3/7 assay kit (Promega, USA) was used. Cells were inoculated in 96-well plates at a concentration of 1 × 10 ⁴ cells / well, cultured in a thermostat incubator at 37 ° C and 5% CO ₂ , and then treated with appropriate concentrations for 1 to 3 hours . (Z-DEVD-R110) provided in a kit was added to the cells for 1 hour to measure the amount of caspase having increased activity in apoptotic cells, and the enzyme was reacted for 1 hour. Substrate The degree of protein degradation by caspase was measured using a microplate reader.

(6) RT-PCR and Real-time PCR

Total RNA was extracted from tumor cells treated with fractions using TRI reagent (MRC, Canada) according to the protocol of the kit. CDNA was obtained from each sample by reverse transcription of 1 μg of RNA using 1 unit of reverse transcriptase, 1 mM dNTP, and oligo (dT) and random primers. PCR was performed using a total volume of 50 μL containing 1 unit of Taq DNA polymerase, 0.2 mM dNTP, 10 × reaction buffer and 10 pmol of 5 'and 3' primers, and the PCR reaction was performed at 95 ° C. for 5 min denaturation). The cycle was repeated 30 times at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 seconds, and finally at 72 ° C for 7 minutes. The PCR product was confirmed by electrophoresis. Real-time PCR was used to confirm changes in gene expression. The cDNA used as a template was synthesized in the same manner as in the RT-PCR, and the synthesized cDNA was diluted with DNase-free water. The reaction mixture was incubated at 50 ° C for 2 min and 95 ° C for 10 min. The second step was performed at 95 ° C for 15 sec and at 54 ° C for 30 min. Sec, and 72 [deg.] C for 30 seconds was repeated 40 times. The results were analyzed and quantified using the Rotor-Gene 6000 series software program (Corbett), and the comparative Ct (threshold cycle) method was used for the analysis.

(7) Identification of components of sea cucumber by GC (Gas chromatography) analysis

The components of the peaks isolated by GC-Mass were analyzed by NIST chemical library (Wiley 275 L) and analyzed by GC / MS (Agilent 6890GC / 5973MS, Palo Alto, USA) Respectively. The organic solvent fraction extract of sea cucumber was prepared and the operating conditions of GC / MS for analysis of each component were as follows. GC was a Agilent 6890N (USA) equipped with a DB-5 (0.25 mm × 60 m, film thickness 0.25 μm) column and the injector temperature was 280 ° C. The temperature of the oven was maintained at 70 캜 for 3 minutes, then heated at a rate of 10 캜 / min and maintained at 300 캜 for 5 minutes. As the carrier gas, helium (He) was used at a flow rate of 1 mL / min, and 1 μL of the sample was injected and analyzed at a split ratio of 10: 1. GC / MS had an interface temperature of 250 ° C and an EI ionization voltage of 70eV. The analysis range of molecular weight was analyzed by SCAN mode at 50 ~ 550 m / z area, and all materials were purified through fragmentation of each material.

Example  1: sea cucumber extract and Fraction  Cytotoxic and anticancer activity

To remove the moisture in the sea cucumber, freeze - dried extracts were prepared using methanol and ethanol. Ethanol extracts were extracted with organic solvents. The fractions obtained from the sea cucumber ethanol extract were concentrated, resulting in a n-hexane fraction (205.63 g), CHCl ₃ The fractions (0.12 g), EtOAc fraction (0.24 g) and n-butanol fraction (6.73 g) were obtained (Table 1). The anticancer activity of gastric cancer was confirmed by using the n - hexane layer and the n - butanol layer, which had high yield among methanol extracts and ethanol fractions thus obtained.

Extraction yield of sea cucumber fraction Fraction n- Hexane CHCl ₃ EtOAc n- BuOH Weight (g) 205.628 0.1205 0.2434 6.73 yield(%) 15.345 0.008 0.018 0.502

In order to confirm the stability of the sample against cells before assaying the anticancer activity of the extracts and fractions of the freeze-dried sea cucumber, cytotoxicity tests were carried out on 293T cells as normal cells. Figure 1 shows cytotoxicity results when 293T cells were treated with various concentrations of extracts and fractions of freeze-dried sea cucumber up to 100 ug / ml. The freeze-dried sea cucumber extract, hexane fraction and butanol fraction were found to have no cytotoxic effect up to 100 ug / ml concentration in 293T cells.

The MTS assay was performed on AGS (gastric cancer cell line), a cancer cell, in order to confirm the anticancer effect of the three kinds of freeze-dried sea cucumber extracts and fractions which were confirmed to have cell stability in the normal cell line. Fig. 1 shows the anticancer efficacy results of three kinds of freeze-dried sea cucumber extracts against AGS (gastric cancer cell line). The ethanol extract of freeze-dried sea cucumber showed the highest anticancer ability with 60% or more cell growth inhibition ability at 60 ug / ml concentration. In the hexane layer and butanol layer of ethanol fraction, 57% And 38%, respectively, and showed anticancer efficacy.

Example  2: Western Blot ( Western blot ) Analysis of Protein Expression by Assay

The p53 gene, in the presence of damaged DNA in the G1 phase during cell differentiation, binds to the cell regulatory substances and inactivates these substances to inhibit entry into the S-phase from the G1 phase. In addition, To block the proliferation. In addition, it is known as a cancer suppressor gene that, when DNA damage is so severe that it can not be repaired, causes cell death (apoptosis) and removes cells that proliferate from damaged DNA in advance. Western blot analysis was performed to determine the expression of the p53 protein, which is a tumor suppressor gene, when three kinds of freeze-dried sea cucumber extracts and fractions were treated with gastric cancer cells. As a result, it was confirmed that the expression level of p53 protein was increased according to the treatment time of the methanol extract and the hexane layer and butanol layer as the ethanol fraction layer (FIG. 2).

Example  3: caspase ( caspase ) Active measurement

The effects of sea cucumber on the activities of caspase-3 and caspase-7, which are known to be important in the overall process of apoptosis induction, were investigated (FIG. 3). Three kinds of freeze-dried sea cucumber extracts and fractions were treated with 100 ug / ml of gastric cancer cells and the activity of kefaz-3 and 7 was measured for 1 to 3 hours. As a result, activity of caspase was induced at 1 hour in all samples , And the methanol extract showed a decrease in caspase activity at 3 hours.

Example  4: RT - PCR  And Real - time PCR Identification of gene expression through

In order to confirm the expression of LC3 (microtubule-associated protein light chain 3), the most important step during autophagy, one of the cell death processes, autophagosome formation, RT-RCR analysis Real-time PCR was performed using a real-time gene amplifier in order to know the change of gene expression level. As the child's action progresses, the expression level of the LC3 gene is increased. As a result of checking the methanol extract of the freeze-dried sea cucumber for 1 to 3 hours, the expression amount of LC3 is increased with the treatment time of the sample (Fig. 4). As a result of Examples 3 to 4, it was found that the sea cucumber extract and fractions exhibit excellent anticancer ability against gastric cancer cells by inducing apoptosis and autophagy through cell death.

Example  5: GC Mass  Analysis of components by analysis

The components of methanol extracts and hexane fraction layers, which showed high anticancer activity in stomach cancer cells, were analyzed. As a result of GC-Mass analysis, main components of the sea cucumber extract were mainly detected in fatty acid series and some sugar (FIG. 5). Unlike the methanol extract, oleic acid (unsaturated fatty acid) and butanedioic acid (organic acid) were further observed in the hexane fraction layer (FIG. 6). Oleic acid, commonly known as Omega-9, is a nutrient that helps lower cholesterol, improves the immune system, helps the fetus and infant to grow, inhibits gastric acid secretion, relieves constipation, prevents breast cancer, and activates nerve cells. Butanedioic acid is a so-called succinic acid which is used for energy metabolism and therefore used as a food additive or supplement. It is used as an excipient to control acidity in pharmaceuticals, rarely used for manufacturing effervescent tablets have.

Claims (7)

1000 to 1400 parts by weight of methanol are added to 4,000 to 5,000 parts by weight of lyophilized sea cucumber and extracted once or three times at 55 to 65 ° C for 3 to 5 hours and then concentrated at 40 to 50 ° C under reduced pressure to obtain 60 to 80 μg / Wherein the composition comprises methanol extract of sea cucumber as an active ingredient. delete delete delete delete delete 1000 to 1400 parts by weight of methanol are added to 4,000 to 5,000 parts by weight of lyophilized sea cucumber and extracted once or three times at 55 to 65 ° C for 3 to 5 hours and then concentrated at 40 to 50 ° C under reduced pressure to obtain 60 to 80 μg / A food for preventing or improving gastric cancer containing methanol extract of sea cucumber as an active ingredient.

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