KR101958370B1 - Composition for Preventing or Treating Cancer Containing Extract of chloromonas sp. - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer containing an extract of chloromonas species microalgae. More particularly, the present invention relates to the antioxidative effect of chloromonas extract and the anticancer effect through inhibition of cancer proliferation, invasion inhibition and cell death. The chloromonas species according to the present invention sp . ) The extract of microalgae is useful as an anticancer pharmaceutical composition, a food, an antioxidant food, a cosmetic product because it has excellent anticancer activity by inducing cancer cell proliferation, infiltration inhibition and cell death in addition to antioxidant activity.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating cancer containing an extract of microalgae of a chloromonas species.

The present invention relates to a composition for preventing or treating cancer containing an extract of chloromonas species microalgae. More particularly, the present invention relates to the antioxidative effect of chloromonas extract and the anticancer effect through inhibition of cancer proliferation, invasion inhibition and cell death.

Cancer Facts and Figures 2016 estimates that the incidence of childhood cancer accounts for a large proportion of all cancer cases. In 2016, 10,380 new cancers were found, of which 1,250 were estimated to have died from cancer. Many scientists are trying to develop new target therapies that selectively kill cancer cells and do not harm normal cells. Successful development of these therapies requires a cancer biologic mechanism that selectively targets molecular pathways critical for tumor survival, growth and metastasis (Mantovani et al., J Exp Med . 212: 435-445, 2015; Gore et al., Lancet Oncology 4: e70-78, 2013; Dossett et al., Expert Opin Drug Pure . 14: 559-570, 2015).

In recent decades, the incidence of cancer in humans has increased, leading to the development of a wide range of synthetic and natural water anticancer drugs (Kato et al., Oncoscience . 2: 576-80, 2015; Vazquez et al., BMC Syst Biol . 2013). More than 800 anticancer drugs have been shown to be effective in clinical trials, according to the Pharmaceutical Research and Manufacturers of America. Many of these drugs are derived from natural products such as plants and microorganisms and are effective against a wide variety of cancers such as prostate cancer, breast cancer, lung cancer and colon cancer. Further, as potential anticancer agents to improve cancer-specific targets and therapeutic efficacy study of a number of the current anti-cancer activity of the natural product (Basmadjian et al, Front Chem 2:20 , 2014;... Sawadogo et al, Molecules 20. : 7097-7142, 2015; Orang-Ojong et al., Mol Clin Oncol . 1: 610-620, 2013; Ko et al., Curr Med Chem. 21: 2346-2356, 2014; Giddings et al., J Ind Microbiol Biotechnol . 40: 1181-1210, 2013). In particular, organisms that are not characterized by extreme environments, such as the marine and Antarctic regions, develop self-defense mechanisms by releasing toxic metabolites. Such metabolites play an important role in anticancer drug research and therapeutic strategies (Younis et al., Environ Sci Pollut Res Int . 23: 4756-4767, 2016; Cragg et al., Chem Rev. 109: 3012-3043, 2009).

Microalgae are a diverse group of unicellular photosynthetic eukaryotes with at least 40,000 to 70,000 species belonging to various phyla such as Cyanophyta, Rhodophyta, Chlorophyta, Pyrrophyta, Cryptophyta, Haptophyta, Heterokontophyta and Streptophyta. (Gimpel et al., Font Microbiol. 6: 1376, 2015; Sharma et al., Environmental reviews. 19: 1-15, 2011). Microalgae adapt to various environments and are distributed around the world, so they are genetically and metabolically diverse, and as a result their secondary metabolites have various compatibility. Thus, the macroantigen of microalgae is useful in the treatment of human diseases (Shanab et al., Asian Pac J Trop Biomed . 2: 608-615, 2012; De Morais et al., Biomed Res Int . 2015: 835761, 2015 ). Recent studies have shown that the optimal development of algae-derived biomass is a major component of the pharmaceutical industry (Martins et al., Mar Drugs. 12: 1066-1101, 2014; Dias et al., Metabolites. 2: 303-336 , 2012; David et al., Phytother Res. 14: 299-315, 2015). In particular, some algae-derived compounds appear to have anticancer activity through modulation of multiple mechanisms such as cytotoxicity, inhibition of infiltration and promotion of apoptosis of cancer cells (Lee et al., Cancer Cell Int . 13:55, 2013 ; Farooqi et al., Cancer Cell Int . 12:50, 2012). These studies show that algae-derived compounds are potent natural anti-cancer agents that effectively prevent tumor formation (Talero et al., Mar Drugs . 13: 6152-6209, 2015; Kumar et al., Mar Drugs. 11: 5130-5147, 2013). For example, fucoxanthin is a carotenoid found in microalgae, diatoms, and brown algae. Although it does not induce apoptosis, it induces a cancer-suppressing gene and cell cycle arrest, thereby potentially inhibiting the growth of cancer cells in a concentration- (Takahashi et al., Oncol Lett . 10: 1463-1467,1205; Peng et al., Mar Drugs. 9: 1806-1828, 2011). Studies on algae-derived anticancer substances are helpful in developing effective targeted therapies for human cancer.

Accordingly, the present inventors have searched for a new microalgae-derived substance exhibiting anticancer activity and developed a potential anticancer drug using the microalgae. As a result, the microalgae Chloromonas sp . Was superior in antioxidative activity as well as anticancer activity through cancer cell proliferation, inhibition of invasion and induction of apoptosis, and the present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition and a food composition for preventing, ameliorating, and treating cancer containing an extract of microalgae as an active ingredient.

It is another object of the present invention to provide an antioxidant food composition and cosmetic composition containing an extract of microalgae as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an extract of chloromonas sp. Microalgae as an active ingredient.

The present invention also relates to the use of chloromonas species sp . ) A food composition for preventing or ameliorating cancer containing an extract of microalgae as an active ingredient.

The present invention also relates to the use of chloromonas species sp . ) An antioxidant food composition comprising an extract of microalgae as an active ingredient.

The present invention also relates to the use of chloromonas species sp . ) An antioxidative cosmetic composition comprising an extract of microalgae as an active ingredient.

The chloromonas species according to the present invention sp . ) The extract of microalgae is useful as an anticancer pharmaceutical composition, a food, an antioxidant food, a cosmetic product because it has excellent anticancer activity by inducing cancer cell proliferation, infiltration inhibition and cell death in addition to antioxidant activity.

Figure 1 is a graphical representation of the ETCH ( Chloromonas ethanol extracts of P. sp.).
Figure 2 shows the cytotoxicity and morphological changes of ETCH on human cancer cells.
FIG. 3 shows the effect of treatment of ETCH (0, 12.5 and 20 占 퐂 / ml) for 6 hours on the inhibition of colonization of cancer cells.
Figure 4 shows the effect of cancer cells treated with ETCH (0.16 and 3.2 占 퐂 / ml) for 24 hours on cell infiltration.
FIG. 5 is a graph comparing FACS analysis of the effect of ETCH on apoptosis in normal cells and cancer cells. (A) is a normal HaCaT cell, (B) is a HeLa cancer cell, and (C) is a graph.
Figure 6 shows Western blot analysis for apoptosis-related proteins caspase-3, Bcl-2, and p53.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Cancer occurs through a very complex process involving several mechanisms of tumorigenesis and a major cause of human mortality. Recent studies have shown that physiologically active compounds derived from natural sources can serve as potentially promising therapies in the treatment of cancer in humans (Lee et al., Cancer Cell Int . 13:55, 2013; Farooqi et al. Cancer Cell Int . 12: 50, 2012; Talero et al., Mar Drugs, 13: 6152-6209, 2015). In particular, the potential anticancer effects of microalgae-derived materials have received attention in relation to targeted therapy for human cancers, and studies on microalgae metabolites are useful in the treatment of human disease.

In the present invention, an Antarctic freshwater microalga, Chloromonas sp . (Ethanol extract of Chloromonas sp . ) From human cancer cells including breast, melanoma and cervical cancer cells. As a result, it was confirmed that ETCH has antioxidant ability, inhibits proliferation of cancer cells and induces apoptosis in a concentration dependent manner, but does not show cytotoxicity in normal cells. In addition, ETCH showed significant inhibitory effect on cell infiltration without cytotoxic effect. In other words, it has been demonstrated that ETCH induces anticancer activities such as antioxidation, antiproliferative, anti-infiltration and apoptosis in cancer cells through regulation of apoptosis-related genes such as caspase-3, Bcl-2 and p53. In the present invention, it was first reported that Antarctic microalgae has an anticancer activity in human cancer cells.

The ETCH of the present invention showed remarkable anti-proliferative effect in vitro (Figs. 2 and 3) and exhibited a broad anti-proliferative effect in various cancer cells. Although ETCH causes severe cytotoxicity to cancer cells, ETCH did not show cytotoxicity in normal cells. The microalgae extract, ETCH, showed less cytotoxicity in HaCaT than normal cells, HeLa, A375 and Hs578T, indicating that normal cells are more resistant to ETCH than cancer cells (Fig. 1). This indicates that ETCH can be a potentially promising candidate for target cancer treatment.

On the other hand, apoptosis, which is programmed apoptosis, is a biochemical process that occurs through a variety of distinct mechanisms involved in the activation of proteases such as caspases that degrade proteins (Aijl et al., Mutat Res. 728: 23-34, 2011 ). The caspase family, a cysteine protease, plays an important role as a functional group of apoptosis pathway. Among these, caspase-3 is considered to be the most important caspase and is activated by caspases such as caspase-8, -9 or -10 (Elmore et al., Toxicol Pathol . 35: 495-516, 2007). In addition, other important regulatory proteins involved in apoptosis are Bcl-2 and p53. Bcl-2 is considered to be a particularly important anticancer protein and is classified as an anticancer gene (Danial et al., Clin Cancer Res. 13: 7254-7263, 2007), while p53 acts as a tumor suppressor, (Amaral et al., Discov Med. 9: 145-52, 2010). To better understand the mechanism by which ETCH regulates apoptosis programmed cell death in cancer cells, caspase-3, Bcl-2 and p53 protein levels associated with the apoptosis signaling pathway were measured (Figure 6). As a result, it was confirmed that ETCH can induce early and late stage apoptosis of cancer cells through apoptosis pathway at least partially by caspase-3 activation.

In addition, during apoptosis, cells morphologically changed (Elmore et al., Toxicol Pathol. 35: 495-516, 2007; Saraste et al., Cardiovasc Res. 45: 528-537, 2000). For example, apoptotic cells are recognized by morphological changes such as contraction, deformation and condensation of chromatin in the nucleus. The cells exhibiting contractility are generally small in size, the cytoplasm is dense, and the cell organs are packed tightly. In the present invention, a characteristic change in morphology of cancer cells treated with ETCH was observed (FIG. 2). However, morphological changes did not occur in normal cells, and cell death was effectively inhibited.

In summary, the present invention showed that Antarctic microalgae chloromonas has antioxidant activity and induces apoptosis in cancer cells in vitro through a caspase-dependent pathway. Accordingly, the present invention provides evidence that the Antarctic microalgae chloromonas extract induces apoptosis and inhibits invasion, thereby acting as a novel anticancer agent for human cancer.

Therefore, the present invention relates to a method for producing chloromonas species sp . To a pharmaceutical composition for preventing or treating cancer containing an extract of microalgae as an active ingredient.

In another aspect, the present invention relates to a method for producing chloromonas species sp . To a pharmaceutical composition for inhibiting invasion or metastasis of cancer containing an extract of microalgae as an effective ingredient.

Cellular infiltration plays a crucial role in cancer metastasis, a major cause of cancer death, and has been shown to significantly reduce the patient's diagnosis and survival at infiltration (Re R et al., Free Radic Biol Med. 26: 1231-1237, 1999).

In the present invention, the chloromonas species sp . ) The microalgae can be characterized as KCTC 13280BP.

The chloromonas microalgae is preferably KCTC 13280BP derived from seawater in Sejong Antarctic, but is not limited thereto.

In the present invention, the extract is preferably extracted using ethanol, methanol or water as a solvent, but is not limited thereto.

In the present invention, the cancer is preferably selected from the group consisting of breast cancer, cervical cancer, malignant melanoma, colon cancer, liver cancer, ovarian cancer, brain tumor and lung cancer, but is not limited thereto.

The chloromonas microalgae extract of the present invention may be characterized in that it does not exhibit cytotoxicity to normal cells or human keratinocytes and induces cell proliferation inhibition, infiltration inhibition and apoptosis in cancer cells . The apoptosis may be characterized by a decrease in expression of procaspase-3 and Bcl-2 and an increase in caspase-3 and p53 cleavage.

The pharmaceutical composition of the present invention may further comprise, in addition to the above-described effective ingredient for administration, a pharmaceutically acceptable carrier, excipient or diluent. A " pharmaceutically acceptable carrier " is a substance that can be added to the active ingredient to help formulate or stabilize the formulation and does not cause significant toxic effects on the patient.

The carrier refers to a carrier or diluent that does not irritate the patient and does not interfere with the biological activity and properties of the chloromonas microalgae according to the present invention. Examples of the pharmaceutical carrier that is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Other carriers are described, for example, in Remington ' s Pharmaceutical Sciences (E. W. Martin).

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active materials is well known in the art. The composition is preferably formulated for parenteral use. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In some cases, the composition may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Sterile injectable solutions can be prepared by incorporating the required amount of chloromonas microalgae extract into a suitable solvent, optionally together with one or a combination of the ingredients described above followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, some preparative methods involve vacuum drying and freeze-drying (freeze-drying), which produces powders of the active ingredient and any additional desired ingredients from a pre-sterilized- )to be.

The term " administering " of the present invention means providing the desired composition of the invention to the patient in any suitable manner.

The term " patient " of the present invention means any animal such as a human, a monkey, a dog, a goat, a pig or a mouse having a disease whose symptoms can be alleviated by administration of the composition of the present invention.

The pharmaceutical composition of the present invention can be manufactured for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, and the like administration.

In another aspect, the present invention relates to a method for producing chloromonas sp . To a food composition for preventing or ameliorating cancer containing an extract of microalgae as an active ingredient.

In the present invention, the chloromonas sp. Microalgae may be KCTC 13280BP.

The chloromonas microalgae KCTC 13280BP is preferably derived from seawater in Sejong Antarctic, but is not limited thereto.

In the present invention, the extract is preferably extracted using ethanol, methanol or water as a solvent, but is not limited thereto.

In the present invention, the cancer is preferably selected from the group consisting of breast cancer, cervical cancer, malignant melanoma, colon cancer, liver cancer, ovarian cancer, brain tumor and lung cancer, but is not limited thereto.

In another aspect, the present invention relates to a method for producing chloromonas sp . ) An antioxidant food composition containing an extract of microalgae as an active ingredient.

Various spectrophotometric assays can be used to measure the antioxidative activity of the chloromonas extract of the present invention, the most common being 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). This method is based on electron transfer and involves the reduction of colored oxidizing agent, so that the reaction can be visually monitored, and the analysis is convenient.

In the present invention, the chloromonas species sp . ) The microalgae can be characterized as KCTC 13280BP.

The chloromonas microalgae KCTC 13280BP is preferably derived from seawater in Sejong Antarctic, but is not limited thereto.

In the present invention, the extract is preferably extracted using ethanol, methanol or water as a solvent, but is not limited thereto.

The food composition may preferably be a health functional food, and may be a food additive. The health functional food or food additive is preferably powder, granule, tablet, capsule or beverage, but is not limited thereto.

The food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of the food. Examples of the foods to which the chloromonas extract according to the present invention can be added include meat products, sausages, breads, chocolate, candies, snacks, confectionery, pizza, ramen noodles, other noodles, dairy products including ice- , Tea, a drink, an alcoholic beverage, and a vitamin complex, and includes foods in a conventional sense.

The food composition of the present invention may preferably comprise a beverage composition. The beverage composition may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and 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 composition of the present invention.

The chloromonas extract according to the present invention as an essential ingredient that can be contained in the food composition of the present invention may contain various herbal medicine extracts, food-aid additives, natural carbohydrates and the like as additional components such as food on the chloromonas extract according to the present invention can do. In addition, the food-aid additive may further include food-aid additives. Examples of the food-aid additive include food-aid additives customary in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers and the like. Examples of such 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 (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . In addition to the above, the food composition of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. Besides natural fruit juices and fruit juice drinks and vegetable drinks These additives may be used independently or in combination. Although the ratio of such additives is not critical, 100 parts by weight of the composition of the present invention It is generally selected from the range of 0.01 to 0.1 wt.

In another aspect, the present invention relates to a method for producing chloromonas sp . ) An antioxidative cosmetic composition containing an extract of microalgae as an active ingredient.

In the present invention, the chloromonas species sp . ) The microalgae can be characterized as KCTC 13280BP.

The chloromonas microalgae KCTC 13280BP is preferably derived from seawater in Sejong Antarctic, but is not limited thereto.

In the present invention, the extract is preferably extracted using ethanol, methanol or water as a solvent, but is not limited thereto.

When the composition of the present invention is prepared with a cosmetic composition, the composition of the present invention may contain not only the above-mentioned chloromonas microalgae but also components commonly used in cosmetic compositions, for example, antioxidants, stabilizers, Vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing agent, or a surfactant-free cleansing agent, it may be applied to the skin and then wiped off or removed or washed with water. The surfactant-containing cleansing formulation is a cleansing foam, a cleansing water, a cleansing towel, and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Sample collection and preparation

The Antarctic freshwater microalgae Chloromonas sp . collected in fresh water near Sejong Antarctic area (62 ° 13 'S, 58 ° 47'W) in 2014 and deposited the strain at the Korea Biological Resources Center on June 1, 2017 (Accession number: KCTC 13280BP).

Microalga Chloromonas sp . was dried and 10 g of the dried material was stored in a 50 mL tube and 20 mL of a solvent such as ethanol was added. The mixture was continuously stirred in a reciprocating shaker at a speed of 150 rpm for 24 hours to dissolve completely in the solvent. The tube was centrifuged at 4 째 C for 15 minutes at 4000 rpm to obtain a supernatant, which was then filtered with Wattman filter paper No. 1. The residual extract (ETCH) obtained by evaporating the solvent from the extract using a rotary vacuum evaporator with a bath temperature of 50 ° C was used in the examples.

Example  1: antioxidant activity of ETCH

In the present embodiment, ethanol extract of Chloromonas sp .; To determine the free radical scavenging properties of the ethanol extract of chloromonas, two analytical methods were used: DPPH radical and ABTS radical cation analysis.

1-1: DPPH  Radical elimination analysis

DPPH analysis was performed using the previous method (Brand-Williams et al., Food Sci Technol . 28: 25-30, 1995). 1.2 mg of DPPH free radical was dissolved in 50 mL of methanol to prepare a stock solution. The thus-obtained DPPH stock solution was diluted with methanol until absorbance became 0.650 ± 0.020 at 517 nm, and the prepared DPPH solution was mixed with a sample Respectively. The mixture was incubated in the dark room for 30 minutes and then the absorbance was measured at 517 nm. Ascorbic acid was used as a control.

The IC50 value, which is the concentration of the sample required to inhibit 50% of the radicals, was calculated by plotting the percentage of scavenging activity for different concentrations (0.2-1.0 [mu] g / mL).

DPPH elimination effect (%) = 100 x (A _Control - A _Sample ) / A _Control

As a result, the DPPH radical scavenging activity of ETCH increased in a concentration-dependent manner, showing an EC50 value of 0.97 mg mL, ranging from 5.97 to 54.35% (FIG. 1). As a control, ascorbic acid showed an EC50 value of 0.12 mg / mL and an elimination activity of 96.8% at 0.6 mg / mL.

1-2: ABTS  Radical elimination analysis

The ABTS analysis is based on the previous method (Re R et al., Free Radic Biol Med . 26: 1231-1237, 1999). ABTS (Sigma) was dissolved in ethanol to a final concentration of 7 mM and then mixed with potassium sulfate solution to a final concentration of 2.45 mM. The mixture was left at room temperature in the dark for 12-16 hours to produce the radical. ABTS · ^{+ The} stock solution was diluted with 95% ethanol to maintain the absorbance at 0.70 ± 0.02 at 734 nm as measured by a spectrophotometer. To measure the scavenging activity, 10 μL of each sample was mixed with 990 μL of ABTS · ⁺ solution, reacted at 25 ° C. for 6 minutes, and absorbance was measured at 734 nm. Ascorbic acid was used as a control.

ABTS cancellation effect (%) = 100 x (A _Control - A _Sample ) / A _Control

As a result, ETCH was able to abolish ABTS ⁺ radicals with a concentration-dependent activity of about 56.4% at 1 mg / mL (FIG. 1). The EC50 values of the ascorbic acid and the extract of the positive control group were 0.95 and 0.09 mg / mL, respectively. Antioxidant activity by ABTS method was slightly higher than antioxidant activity by DPPH assay.

Example  2: Anti-proliferative effect of ETCH on human cancer and normal keratinocyte

Microalga Chloromonas sp. To investigate the possibility of using the extract as a source of anticancer drugs, three cancer cell lines (A375, HeLa and Hs578T) were cultured for 48 hours with various concentrations of ETCH. Human keratinocyte line (HaCaT) and cancer cell lines (HeLa, A375 and Hs578) were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Dulbecco's modified Eagle's) medium (Gibco / Invitrogen) containing 10% FBS in a 5% CO ₂ incubator at 37 ° C. Smear the cells with 0.5 × 10 ⁶ cell density / well in 6-well plates and incubated with the culture and replacing the culture medium every 2-3 days over night. The cells were inoculated at a density of 14 × 10 ¹³ / cm ² .

2-1: Cytotoxicity analysis and morphological change

Cytotoxic effects were assessed using MTT assay.

Cells were seeded in 96-well plates at a density of 1 x 10 ³ cells / well and cultured at 37 ° C in 5% CO ² . Cell viability was measured by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyetetrazolium bromide (MTT) -cell titer 96AQueous One Solution cell proliferation assay (Promega) and analyzed for 3 days. 20 μl of MTT was added every 24 hours to detect metabolic activated cells. Plates were incubated for 1 hour and analyzed on a Multilabel Counter (Bio-Rad Laboratories).

As a result, melanoma A375 cells and cervical cancer HeLa cells showed cytotoxicity in a dose dependent manner when the concentration was above 6.25 ㎍ / ㎖, and Hs578T, which is a breast cancer cell, showed anti-proliferative effect at a high concentration (12.5 μg / mL) (Fig. 2).

There was no significant difference in viability between normal cells and HaCaT when compared with the control group. This suggests that ETCH extract is selectively toxic to human cancer cells.

Although ETCH has different degrees of cytotoxicity for different types of cancer cells, it exhibits extensive inhibition of cancer cell growth. This was confirmed by examining the morphology of the cells with a microscope. Cell morphological changes were recorded directly using a phase-contrast microscope equipped with a digital camera (Nikon), after which the plates were stained with 0.5% crystal violet and the recordings were scanned.

As a result, changes in cell morphology such as contraction and deformation were found in cancer cells, not normal cells, which was consistent with cytotoxicity analysis.

2-2: Colony  Formation analysis

Colony formation analysis revealed that microalgae Chloromonas sp. The long-term effect of the extract on cell proliferation was evaluated.

HaCaT and cancer cells were seeded into 6-well plates at a density of 2000 cells / well. After 24 h adherent cultures, cells were treated with 12.5 and 25 ug / mL ETCH for 6 h. Then the cells were replaced with fresh medium and replaced every 3 days until a regularly changing colony appeared. Colonies were stained with 0.4% crystal violet (Sigma) in 50% methanol and visible colonies were counted.

As a result, three cancer cell lines (A375, HeLa and Hs578T) treated with ETCH for 6 hours for 14 days of culture with regular replacement of the normal medium every 3 days inhibited colony formation. The number of colonies was significantly reduced in cancer cells treated with ETCH (12.5 and 25 / / mL), while the number of colonies was not significantly changed in normal cells (Fig. 3).

Example  3: inhibition of cell infiltration by ETCH

To investigate the effect of ETCH on cancer cell infiltration capacity, Boyden chamber analysis was performed at a concentration of 1.6-3.2 μg / mL, which does not affect the cell proliferation of cancer cells HeLa and A375.

A Transwell insert chamber with 8 micrometer porous membranes (Cell Biolabs) was used for the invasion assay. 1.6 x 10 ⁶ cells / mL cells were washed three times with PBS and placed in an upper chamber with serum-free medium containing ETCH at a concentration of 1.6 or 3.2 μg / mL. The lower chamber was filled with medium containing 10% FBS. ETCH-treated cells were cultured in a 5% CO ² incubator at 37 ° C for 48 hours. To quantify infiltrating cells, cells in the upper chamber were removed using a cotton swab, infiltrated cells were stained with Cell Stain Solution, visualized with a phase contrast microscope, and pictures were taken. The stained cells were solubilized in the extraction solution and OD 560 nm was measured on a plate reader.

As a result, ETCH showed a significant inhibitory effect on cell infiltration depending on the concentration between 1.6-3.2 μg / mL. The inhibitory effect of ETCH on cancer cell infiltration was not due to the cytotoxic effect of ETCH, and the infiltration analysis results showed that the infiltration activity through the basement membrane was significantly inhibited in the ETCH treated cells as compared with the control cells. That is, this result suggests that ETCH is involved in the inhibition of HeLa and A375 cell infiltration.

Example  4: Cell death by ETCH apoptosis ) Related gene regulation

We investigated whether the cytotoxic effect of ETCH on cancer cells could be induced by apoptosis.

4-1: Flow cytometry

For apoptosis analysis, the apoptosis effect of ETCH was evaluated using the Annexin V-FITC apoptosis detection kit (BD Biosciences). For Annexin V staining, HaCaT and HeLa cells (control group, ETCH treated group) were harvested by trypsinization, washed twice with cold PBS, and then plated in 100 μl 1x binding buffer containing 5 μl annexin V-FITC and 2 μl PI 1 x 10 < ⁵ > cells / mL). After incubating the cells in the dark room for 15 minutes at room temperature, 400 의 of 1X binding buffer was added to each tube and the cells were detected using a flow cytometer within 1 hour.

Evaluation of healthy (AnnexinV- / FITC-), early cell death (Annexin + / FITC), late cell death (AnnexinV + / FITC +) and remnant (AnnexinV- / FITC +) cell populations in normal and cancer cells Respectively. Annexin V positive (early and late cell death) cells were considered apoptosis populations.

As a result, the apoptosis population of HeLa cells was significantly increased as the dose of ETCH was increased; In the ETCH-treated group, 6.57% (12.5 μg / mL) and 16.67% (25 μg / mL), respectively, compared with the control group (3.72%). Apoptosis did not occur in normal cells (FIG. These results suggest that the extract induces apoptosis selectively in cancer cells rather than normal cells. Because of the similar range of absorbance between PI and green ETCH, a healthy population of cells treated with ETCH can be transferred entirely to the PI staining region.

4-2: Western Blot

Next, the assay was expanded to examine the effect of ETCH on the molecular markers of apoptosis. To better understand the mechanism of ETCH-induced apoptosis, we examined the major regulators of apoptosis signaling.

The sample was dissolved in RIPA buffer (50 mM Tris Cl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate). Total protein (50 μg) of each sample was separated on a 4-20% Criterion ^™ Tris.HCl precast gel (BioRad) and transferred to a nitrocellulose (NC) membrane. Membranes were incubated with primary antibodies (anti-caspase-3, anti-Bcl-2 and anti-p53; After HRP (Santa Cruz Biotechnology) probed with conjugated secondary antibody IgG, the membrane was developed with enhanced chemiluminescence (Amersham Pharmacia).

As a result, the expressions of apoptosis-related proteins procaspase-3 and Bcl-2 were remarkably decreased, and the cleaved caspase-3 and p53 increased in a concentration-dependent manner (FIG. 6), consistent with flow cytometry analysis. Overall, these results suggest that ETCH activates apoptosis-related signaling pathways leading to apoptosis in cancer cells.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC13280BP 20170601

Claims (18)

Chloromonas sp. KCTC 13280BP A pharmaceutical composition for preventing or treating cancer containing an extract of microalgae as an active ingredient.
delete The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the extract is extracted using ethanol, methanol or water as a solvent.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, malignant melanoma, colon cancer, liver cancer, ovarian cancer, brain tumor and lung cancer.
Chloro Pseudomonas species (chloromonas sp.) KCTC 13280BP microalgae extract the active ingredient, pharmaceutical compositions for the inhibition of metastasis or invasion of cancer containing as the.
delete [Claim 6] The pharmaceutical composition according to claim 5, wherein the extract is extracted using ethanol, methanol or water as a solvent.
The pharmaceutical composition according to claim 5, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, malignant melanoma, colon cancer, liver cancer, ovarian cancer, brain tumor and lung cancer.
Chloromonas sp. KCTC 13280BP Food composition for prevention or improvement of cancer containing extract of microalgae as an active ingredient.
delete [Claim 11] The composition for preventing or improving cancer according to claim 9, wherein the extract is extracted using ethanol, methanol or water as a solvent.
10. The composition for preventing or improving cancer according to claim 9, wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, malignant melanoma, colon cancer, liver cancer, ovarian cancer, brain tumor and lung cancer.
Chloromonas sp. KCTC 13280BP An antioxidant food composition containing an extract of microalgae as an active ingredient.
delete The antioxidative food composition according to claim 13, wherein the extract is extracted using ethanol, methanol or water as a solvent.
Chloromonas sp. KCTC 13280BP An antioxidant cosmetic composition containing an extract of microalgae as an active ingredient.
delete The antioxidative cosmetic composition according to claim 16, wherein the extract is extracted using ethanol, methanol or water as a solvent.

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