KR102054391B1 - Composition for Preventing or Treating Inflammation or Cancer Containing Extract of Micractinium sp. - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases or cancer containing an extract of Micractinium sp. microalgae. More specifically, the present invention relates to an anti-inflammatory effect and an anticancer effect through inhibition of cell proliferation of cancer cells and induction of cell cycle arrest of the extract of Micractinium. The extract of Micractinium sp. microalgae according to the present invention is excellent in anti-inflammatory activity, as well as anticancer activity by inhibiting cell proliferation and inducing cell cycle arrest, thereby being useful as a pharmaceutical composition for anti-inflammation, and a pharmaceutical composition and a food composition for anticancer.

Description

Composition for Preventing or Treating Inflammation or Cancer Containing Extract of Micractinium sp.

The present invention is a microlactinium species ( Micractinium) sp . The present invention relates to a composition for the prevention or treatment of inflammatory diseases or cancer containing the extract of microalgae, and more particularly, to the anti-inflammatory effect of microlactinium extract and the anti-cancer effect by inhibiting cell proliferation and inducing cell cycle arrest of cancer cells. It is about.

Inflammation is a protective response mediated by the innate immune system to restore cell homeostasis against foreign pathogenic agents that interfere with the integrity of cell homeostasis (Garrett WS, Gordon JI, Glimcher LH.Homeostasis and inflammation in the Cell 2010 intestine; 140:. 859-870 )... The fundamental biological mechanism of the inflammatory process consists of three stages: initiation, regulation, and resolution, and is tightly regulated to maintain physiological and cellular homeostasis in the organism (Cekici A, Kantarci A, Hasturk). H, Van Dyke TE.Inflammatory and immune pathways in the pathogenesis of periodontal disease.Periotontol . 2000; 64: 57-80.). When tissues are damaged by pathogenic infections, cells in our bodies activate immune pathways by activating signaling pathways, including cytokines and chemokines. For example, macrophages located primarily in infected areas recognize the infection and induce inflammation by secreting proinflammatory cytokines that attract other immune cells, such as white blood cells and lymphocytes, to the site of infection. The manned immune cells contributes to the strengthening of the immune response to infections, destruction by infection homeostasis status is quickly restored to a normal state (G Dugue Arango, A. Descoteaux Macrophage cytokines:. Involvement in immunity and infectious diseases Front Immunol . 2014; 5: 491.).

If acute inflammation does not resolve for any reason and progresses to chronic inflammation, the cause of many diseases including cancer, inflammatory cells are destroyed and tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin- 6 (IL-6) it is secreted is to cause mutations to form the DNA damage, or tumor (Godkin a, Smith KA Chronic infections with viruses or parasites: breaking bad to make good Immunology 2017; 150:... 389-396.) . Inflammatory cells also contain inflammatory cytokines or enzymes such as epidermal growth factor (EGF), matrix metalloproteinases (MMPs), and monocyte chemo-attractant proteins (MCPs). Overexpression (Mostofa AG, Punganuru SR, Madala HR, Al-Obaide M, Srivenugopal KS.The process and regulatory components of inflammation in brain oncogenesis. Biomolecules . 2017; 7: E34.). The substance changes the inflammatory microenvironment around the tumor to help tumors multiply and progress rapidly, thereby facilitating metastasis by destroying nearby tissue.

To better understand the mechanisms of inflammation progression and cancer development associated with inflammation, the process involves cytokines, nuclear factor (NF) -κB, cyclooxygenase (COX) -2 and inducible nitric oxide synthase (iNOS). It is necessary to determine the role of such key regulatory molecules (Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, et. al . Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol pharm Bull . 2007; 30: 2345-2351.). Cytokines produced by immune / inflammatory cells, including IL and TNF-α, are regulated through the regulation of transcription factors such as NF-κB, signal transducer and activator of transcription 3 (STAT3), and activator protein 1 (AP-1) (cells By inducing regulatory genes that promote proliferation and survival) plays an important role in tumorigenesis. NF-κB, another major regulatory molecule in inflammation-mediated tumor development, serves as a transcription factor that regulates the expression of regulatory molecules in response to inflammation such as IL-6, TNF-α, iNOS, COX-2, and chemokines (Lawrence T. The nuclear factor NF-κB pathway in inflammation. Cold Spring Harb Perspect Biol . 2009; 1: a001651.). Indeed, NF-κB and its downstream target genes, iNOS and COX-2, are constantly active in a variety of cancer types, by regulating cell proliferation, antiapoptotic activity and angiogenesis and increasing metastasis. (Rinkenbaugh AL, Baldwin AS. The NF-κB pathway and cancer stem cells. Cells . 2016; 5: E16.).

Recently microalgae have attracted attention as a source of bioactive molecules with pharmaceutical and therapeutic potential for inflammation and cancer. Many studies have shown the antioxidant, anti-inflammatory and anticancer potential of microalgae species and their secondary metabolites, including carotenoids, fatty acids, glycolipids and polysaccharides (Talero E, Garcia-Maurino S, Avila-Roman J, Rodriguez-Luna A). , Alcaide A, Motilva V. Bioactive compounds isolated from microalgae in chronic inflammation and cancer. Mar Drugs . 2015; 13: 6152-6209.). However, the physiological and molecular mechanisms that support the biological activity of microalgae-derived metabolites are not clear.

Therefore, the present inventors have searched for a new microalgae-derived material exhibiting anti-inflammatory and anti-cancer activity, and as a result of diligent efforts to develop a potential anti-cancer agent using the same, Micractinium , antarctic freshwater microalgae sp . Ethanol extract of the anti-inflammatory and anti-proliferative and cancer cells to confirm the effect, and completed the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

An object of the present invention to provide a pharmaceutical composition and food composition for the prevention, improvement, treatment of inflammatory diseases containing the extract of microalgae as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition and food composition for the prevention, improvement, treatment of cancer containing the extract of microalgae as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing an extract of Micractinium sp .

The invention also relates to microlactinium species ( Micractinium) sp . ) Provides a food composition for the prevention or improvement of inflammatory diseases containing the extract of microalgae as an active ingredient.

The invention also relates to microlactinium species ( Micractinium) sp . ) Provides a pharmaceutical composition for the prevention or treatment of cancer containing the extract of microalgae as an active ingredient.

The invention also relates to microlactinium species ( Micractinium) sp . ) Provides a food composition for preventing or improving cancer containing the extract of microalgae as an active ingredient.

Micractinium species according to the invention sp . ) Since the extract of microalgae has excellent anti-cancer activity through inhibition of cell proliferation and cell cycle arrest of cancer cells in addition to anti-inflammatory activity, it is useful as an anti-inflammatory pharmaceutical composition, an anti-cancer pharmaceutical composition and food.

1 is Micractinium sp . As a result of morphological observation of (KCTC 13536BP), (A) is an optical microscope and (B) is a scanning electron microscope.
2 is ETMI ( Micractinium) for cell viability and NO production in lipopolysaccharide (LPS) -induced RAW 264.7 macrophages. sp . Ethanol extract) is shown. (A) evaluated cell viability using MTT assay 24 hours after treatment, and (B) measured NO production by Griess reaction. Three experimental mean ± SD values are indicated. * p <0.05, ** p <0.01, #p <0.001.
Figure 3 shows the effect of ETMI on the expression level of proinflammatory mediators, where (A) is transcriptional level and (B) is translational level of iNOS and COX-2 genes for ETMI treatment. will be. Three experimental mean ± SD values are indicated. * p <0.05, ** p <0.01, #p <0.001.
Figure 4 shows the effect of ETMI on the expression and production of proinflammatory cytokines, (A) of the cytokines TNF-α and IL-6, the level of transcription, (B) shows the production. Three experimental mean ± SD values are indicated. * p <0.05, ** p <0.01, #p <0.001.
Figure 5 shows the effect of ETMI on the growth of human colorectal cancer cells HCT116, (A) inhibits cell proliferation, (B) shows colony formation on ETMI treatment.
FIG. 6 shows (A) cell cycle arrest induction and (B) expression of cell cycle-regulated genes for ETMI treatment. Three experimental mean ± SD values are indicated. * p <0.05, ** p <0.01.

Unless defined otherwise, 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.

Chronic inflammation is known to cause various diseases including cancer (Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell . 2010; 140: 883-899.). Thus, if the inflammatory response is not controlled early, the body may develop into chronic inflammation, autoimmune disease or metabolic disease due to loss of function and disruption of homeostasis. Recent studies investigating whether chronic inflammation increases the incidence of cancer have shown that inflammation and cancer have a common signaling pathway involving vicious ring linkages at the molecular level (Zhang Q, Zhu B, Yongsheng L. Resolution of .. cancer-promoting inflammation: a new approach for anticancer therapy Front Immunol 2017; 8: 71.). Inflammatory substances secreted from inflammatory cells are closely related to the proliferation, survival and metastasis of cancer cells. In the inflammatory tumor microenvironment, inflammation not only promotes the survival and proliferation of cancer cells, but also induces angiogenesis and metastasis by destroying adaptive immune responses and altering the response to hormones and drugs. For example, if acute inflammation does not resolve and progresses to chronic inflammation, inflammatory cells are destroyed and TNF-α, IL-1 and IL-6 cause DNA damage or mutation. Inflammatory cells overexpress inflammatory cytokines or enzymes such as EGF, MMP and MCP (Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, et al . Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol Pharm Bull . 2007; 30: 2345-2351). Further, in the inflammatory tumor microenvironment hypoxia-induced factor produces a vascular endothelial growth factor (VEGF) (Finger EC, Giaccia AJ. Hypoxia, inflammation, and the tumor microenvironment in metastatic disease. Cancer Metastasis Rev. 2010; 29: 285-293.). The substance changes the inflammatory microenvironment around the tumor and makes it easier for tumor cells to destroy nearby tissues and spread to other organs, helping tumors to proliferate and induce metastasis.

Inflammation mediated by the innate immune system is an organism's defense mechanism against infectious environmental risks. It also causes the etiology of various human diseases, including the onset and progression of cancer. Microalgae is attracting attention as a source of bioactive molecules with therapeutic potential for a wide variety of diseases. In addition, the antioxidant, anti-inflammatory and anticancer potential of microalgae and secondary metabolites has been widely reported. But the underlying mechanism is still unknown. The main object of the present invention is antarctic freshwater microalgae Micractinium sp . Ethanol extract (et hanol extract of Mi cractinium sp . To evaluate the anti-inflammatory and cytotoxic effects of ETMI). Many using RAW 264.7 macrophages and human colon cancer cells in HCT16 By in vitro analysis, the molecular mechanisms underlying the anti-inflammatory and anticancer activity of ETMI were studied.

In the present invention, ETMI is concentration dependent, and major inflammatory markers such as cyclooxygenase (COX) -2, interleukin (IL) -6, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF) -α and nitric oxide (NO). By regulating, it was confirmed that the anti-inflammatory activity was exhibited. In particular, anti-inflammatory activity was observed at relatively low concentrations of ETMI (5 μg / mL), indicating a decrease in inflammatory mediator expression levels compared to previously reported (Samarakoon KW, Ko JY, Shah MR, Lee JH, Kang MC). ...., Nam ON et al In vitro studies of anti-inflammatory and anticancer activities of organic solvent extracts from cultured marine microalgae Algae 2013; 28: 111-119, Sanjeewa KKA, Fernando IPS, Samarakoon KW, Lakmal HHCL, Kim EA , Kwon ON et al . Anti-inflammatory and anti-cancer activities of sterol rich fraction of cultured marine microalga Nannochloropsis oculata . Algae . 2016; 31: 277-287.). ETMI may include bioactive molecules with potent anti-inflammatory activity required for adaptation in harsh environments such as Antarctica. In general, to adapt to extreme conditions such as the Antarctic, microalgae and other organisms have developed specific self-defense mechanisms by synthesizing toxic secondary metabolites that have been found to be pharmaceutically useful. In this respect, bioactive compounds of Antarctic organisms can be promising drug targets for drug development. According to data according to an embodiment of the present invention for the inflammatory molecular mechanism, ETMI effectively regulated the transcription level of proinflammatory mediators. However, ETMI treatment significantly inhibited the transcription level and production of proinflammatory cytokine IL-6, whereas TNF-α production decreased slightly in a dose dependent manner compared to a significant decrease in mRNA levels. This difference between TNF-α mRNA and production can occur because the rapid conversion of TNF-α mRNA does not significantly affect TNF-α biosynthesis of macrophages. Indeed, TNF-α mRNA is present in the short term even under conditions of maximum TNF-α production during tumor necrosis, suggesting that rapid conversion of TNF-α mRNA is not associated with TNF-α biosynthesis regulatory mechanisms (Mijatovic T, Houzet L). ., Defrance P, Droogmans L, Huez G, Kruys V. Tumor necrosis factor-alpha mRNA remains unstable and hypoadenylated upon stimulation of macrophages by lipopolysaccharides Eur J Biochem 2000; 267:. 6004-6012)..

Accordingly, the present invention provides in one aspect a microlactinium species ( Micractinium) sp . The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases, containing the extract of microalgae as an active ingredient.

In the present invention, the microlactinium species ( Micractinium sp . ) Microalgae may be characterized as KCTC 13536BP.

The microlactin microalgae KCTC 13536BP is preferably derived from Sejong Antarctic freshwater, 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 inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative It is preferably selected from the group consisting of neuro-inflammatory, but is not limited thereto.

The term "inflammation" of the present invention is a defense mechanism of biological tissues against a certain stimulus, and is a biological defense mechanism that is intended to remove the injury caused by various harmful stimuli and restore the original state. There are two types of irritation: inflammation or chemical and physical stimulation, and the process of inflammation can be divided into two types: acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are associated with the removal of foreign bodies by posting a microcirculatory system. Chronic inflammation has a long duration, and tissue growth is seen.

In another aspect, the present invention, Micractinium species ( Micractinium) sp . The present invention relates to a food composition for preventing or improving an inflammatory disease containing an extract of microalgae as an active ingredient.

In the present invention, the microlactinium species ( Micractinium sp . ) Microalgae may be characterized as KCTC 13536BP.

The microlactin microalgae KCTC 13536BP is preferably derived from Sejong Antarctic freshwater, 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 inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative It is preferably selected from the group consisting of neuro-inflammatory, but is not limited thereto.

In the present invention, it was also confirmed that ETMI exhibits a concentration-dependent cytotoxic effect on HCT116 cells, thereby significantly reducing cancer cell proliferation. ETMI induced G1 cell cycle arrest through the regulation of G1 / S phase-change arousal genes, including CDKN1A (p21), cyclin-dependent kinase 4 (CDK4), and 6 (CDK6). The expression of CDKN1A at the level of transcription gradually increased with ETMI treatment, while the expression of CDK4 and CDK6 decreased. This is the Antarctic freshwater microalgae Micractinium sp . And the anti-inflammatory and anticancer activity of ETMI, providing new clues to understanding the molecular link between inflammation and cancer, indicating that ETMI may be a potentially promising candidate for targeted cancer treatment.

In summary, in the present invention, Antarctic freshwater microalgae Micractinium sp . Ethanol extract has been shown to have anti-inflammatory and anti-cancer effects on macrophages and colon cancer cells. The identification of bioactive compounds that mediate common signaling pathways between inflammation and cancer and the development of drugs that interfere with the function of these proteins can be an effective approach to cancer treatment by eliminating the link between inflammation and cancer. The present invention thus indicates that ETMI may be a promising candidate for the development of pharmacologically effective drugs for the treatment of cancer.

Accordingly, the present invention provides in another aspect a Micractinium species sp . The present invention relates to a pharmaceutical composition for preventing or treating cancer, which contains an extract of microalgae as an active ingredient.

In the present invention, the microlactinium species ( Micractinium sp . ) Microalgae may be characterized as KCTC 13536BP.

The microlactin microalgae KCTC 13536BP is preferably derived from Sejong Antarctic freshwater, 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, colorectal cancer, liver cancer, ovarian cancer, brain tumor and lung cancer, but is not limited thereto.

The extract of microlactin microalgae of the present invention may be characterized in that it does not exhibit cytotoxicity to normal cells, and cancer cells may be characterized by inducing cell proliferation inhibition and cell cycle arrest.

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

The carrier refers to a carrier or diluent that does not irritate the patient and does not inhibit the biological activity and properties of the microlactinium microalgae according to the present invention. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, 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 preparation of sterile injectable solutions or dispersions for immediate administration. The use of such media and agents for pharmaceutically active substances is known in the art. The composition is preferably formulated for parenteral injection. 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, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. In some cases, it is possible to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of microlactinium microalgae extract in an appropriate solvent with one or a combination of ingredients described above as needed followed by sterile microfiltration. . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation include vacuum drying and freeze-drying (freeze-drying), which produce a powder of the active ingredient and any further desired ingredients from its presterilized-filtered solution. )to be.

As used herein, the term "administration" means providing a patient with a composition of the present invention in any suitable manner.

As used herein, the term "patient" means any animal, such as a human, monkey, dog, goat, pig, or rat, having a disease in which symptoms may be improved by administering the composition of the present invention.

The pharmaceutical composition of the present invention may be prepared for the purpose of oral, topical, parenteral, nasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like.

In another aspect, the present invention, Micractinium species sp . ) Relates to a food composition for preventing or improving cancer containing the extract of microalgae as an active ingredient.

In the present invention, the microlactinium species ( Micractinium sp . ) Microalgae may be characterized as KCTC 13536BP.

The microlactin microalgae KCTC 13536BP is preferably derived from Sejong Antarctic freshwater, 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, colorectal cancer, liver cancer, ovarian cancer, brain tumor and lung cancer, but is not limited thereto.

In the present invention, the food composition may preferably be a health functional food, it may be a food additive. The health functional food or food additive is preferably a powder, granules, tablets, capsules or beverages, but is not limited thereto.

The food of the present invention may be used as it is, or may be used together with other food or food ingredients, or the microlactinium microalgae extract according to the present invention may be suitably used according to a conventional method.

There is no particular limitation on the kind of food. Examples of the food to which the microlactinium extract according to the present invention may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups. , Beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., and includes all foods in the usual sense.

The food composition of the present invention may preferably comprise a beverage composition. The beverage composition may contain various flavors, natural carbohydrates, and the like as additional ingredients, as in general beverages. Natural carbohydrates described above are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion 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.

As an essential ingredient that may be included in the food composition of the present invention, the microlactinium extract according to the present invention may contain various herbal extracts, food supplement additives or natural carbohydrates, such as the food on the microlactin extract according to the present invention. It may contain as an additional component. In addition, the food supplement may also be added to the food supplement additives include conventional food supplements, such as flavors, flavors, colorants, 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 conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example

Sample Collection and Preparation

Antarctic freshwater microalgae Micractinium used in the present invention sp . Was obtained near Sejong Antarctic (62˚13'S, 58˚47'W). Microalgae samples were deposited (# KSF105) at the Korea Polar Research Institute (Republic of Korea) (Fig. 1), and strains were deposited at the Korea Institute of Bioscience and Biotechnology Resource Center on May 29, 2018. : KCTC 13536BP). To identify microalgae, 18S rDNA sequences were analyzed with the Basic Local Alignment Search Tool (BLAST), which analyzed sequence similarity using the NCBI GenBank database.

Microalgae Micractinium sp . 10 g of the dried product was stored in a 50 mL tube, and 20 mL of a solvent such as ethanol was added thereto. Stirring was continued for 24 hours at a rate of 150 revolutions / minute on a reciprocating shaker for mixing. The tube was centrifuged at 4 ° C., 4000 rpm for 15 minutes to give a supernatant, then filtered with Whatman No. 1 filter paper. The residual extract (the et hanol extract from the polar microalga Mi cractinium sp., ETMI) obtained by evaporating the solvent from the extract using a rotary vacuum evaporator having a water bath temperature of 50 ° C. was used in the examples. The preparation of ETMI can be referred to published papers (Suh SS, Yang EJ, Lee SG, Youn UJ, Han SJ, Kim IC, et. al . Bioactivities of ethanol extract from the Antarctic freshwater microalga, Chloromonas sp. Int J Med Sci . 2017; 14: 560-569.).

Cell culture

RAW 264.7 macrophages and HCT116 cells were cultured in Dulbecco's modified Eagle's (DMEM) medium containing 10% fetal bovine serum and 1% penicillin in a humidified CO ₂ incubator at 37 ° C. RAW 264.7 macrophages were incubated with or without varying concentrations of ETMI for 1 hour prior to LPS (0.5 μg / mL) stimulation. HCT116 cells were seeded in 96-well plates at a density of 5 Х 10 ³ cells / well and incubated at 37 ° C. in 5% CO ₂ .

RNA  Extraction and qRT - PCR

To measure mRNA expression of COX-2, IL-6, iNOS and TNF-α, total RNA was isolated from ETMI-treated cells according to the manufacturer's instructions for TRIzol Reagent (Invitrogen). First strand copy DNA (cDNA) was synthesized using the Super Script first-strand cDNA synthesis kit (Invitrogen). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR green real-time PCR master mixes (ThermoFisher Inc., USA) with gene expression primers. Relative mRNA levels were normalized to the levels of β-actin, the housekeeping gene.

The primers used are as follows:

iNOS: (F) 5′-GGAGCCTTTAGACCTCAACAGA-3 ′ (SEQ ID NO: 1)

      (R) 5′-TGAACGAGGAGGGTGGTG-3 (SEQ ID NO: 2)

COX-2: (F) 5′-GAAGTCTTTGGTCTGGTGCGTG-3 ′ (SEQ ID NO: 3)

       (R) 5′-GTCTGCTGGTTTGGAATAGTTGC-3 ′ (SEQ ID NO: 4)

IL-6: (F) 5′-GAGGATACCACTCCCAACAGACC-3 ′ (SEQ ID NO: 5)

      (R) 5′-AAGTGCATCGTTGTTCATACA-3 ′ (SEQ ID NO: 6)

TNF-α: (F) 5′-CATCTTCTCAAAATTCGAGTGACAA-3 ′ (SEQ ID NO: 7)

        (R) 5′-TGGGAGTAGACAAGGTACAACCC-3 ′ (SEQ ID NO: 8)

β-actin: (F) 5′-TGTTTGAGACCTTCAACACC-3 ′ (SEQ ID NO: 9)

          (R) 5′-CGCTCATTGCCGATAGTGAT-3 ′ (SEQ ID NO: 10)

Weston Blot ( Western blotting )

RAW 264.7 cells were seeded in 6-well plates at a density of 5 Х 10 ⁵ cells / mL. After incubation overnight, ETMI was pretreated at different concentrations for 1 hour before adding LPS (0.5 μg / mL). After incubation for 24 hours, cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer. Western blots may be carried out with reference to the preceding paper (Talero E, Garcia-Maurino S , Avila-Roman J, Rodriguez-Luna A, Alcaide A, Motilva V. Bioactive compounds isolated from microalgae in chronic inflammation and cancer. Mar Drugs . 2015; 13: 6152-6209.). Anti-iNOS antibodies were purchased from Sigma-Aldrich (Cat # N7782) and anti-COX-2 and anti-β-actin antibodies were obtained from Cell Signaling (Cat # 4842 and Cat # 3700, respectively). Immune blot signal was compared to β-actin signal and relative protein expression was measured.

Statistical analysis

Results are expressed as mean ± SEM of 3 independent biological experiments. Statistically significant differences between each treatment and control group were determined using a one-way analysis of variance followed by Student's t- test. p values <0.05 were considered to represent statistically significant differences.

Example  One: ETM RAW  264.7 cell viability ( viability ) Impact

To measure the cytotoxicity of ETMI in RAW 264.7 cells, cells were exposed to various ETMI concentrations from 5 μg / mL to 320 μg / mL and then treated with 1 μg / mL LPS for 24 hours. Cell growth rates were then measured using MTT (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) assay.

As shown in FIG. 2 (A), the proliferation of RAW 264.7 cells was not significantly affected by the treatment of ETMI below 40 μg / mL concentration, as compared with cells without LPS or ETMI treatment.

Example  2: ETMI LPS -Judo NO  Generated and iNOS Of COX -2 effect on expression

Example  2-1: iNOS  And COX Expression measurement of -2

To confirm the inhibitory effect of ETMI on LPS-induced proinflammatory responses, the expression levels of iNOS and COX-2 genes, the major proinflammatory mediators, were measured in ETMI-treated RAW 264.7 cells. Cells exposed to ETMI at 5, 10, 30 and 40 μg / mL for 1 hour showed no cytotoxic effect. Cells were then treated with 1 μg / mL LPS for 24 hours.

As shown in FIG. 2 (B) and FIG. 3, LPS-stimulated expression of iNOS and COX-2 showed transcriptional levels (FIG. 3 (A)) as compared to control cells with only LPS stimulation and no ETMI pretreatment. There was a significant decrease in concentration dependence at both translation levels (FIG. 3 (B)).

Example  2-2: NO  Produce measure

Levels of NO, one of the important mediators of inflammation, in ETMI-treated RAW 264.7 cells were measured. Cells exposed to ETMI at a concentration of 5, 10, 30, 40 μg / mL for 1 hour did not show a cytotoxic effect, and the cells were then treated with 1 μg / mL LPS for 24 hours.

The NO concentration of the RAW 264.7 cell culture supernatant was measured using Griess reagent. 100 μL of the supernatant was mixed with the same amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N- (1-naphthyl) ethylenediamine). After incubating the mixture for 10 minutes at room temperature, the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. Nitrite concentrations were determined using sodium nitrite calibration curve (0-100 μM).

As a result, it was confirmed that ETMI significantly reduced LPS-stimulated NO production in a concentration dependent manner (FIG. 2 (B)).

The results of Examples 2-1 and 2-2 suggest that ETMI has high anti-inflammatory activity even at low concentrations through the regulation of key inflammatory regulators at both transcription and translation levels.

Example  3: ETMI LPS Stimulated Proinflammatory  Impact on cytokine production

Many pro-inflammatory cytokines, including TNF-α and IL-6 may be a key regulator of inflammatory processes and diseases (Rinkenbaugh AL, Baldwin AS The NF -κB pathway and cancer stem cells Cells 2016; 5...: E16.). Thus, the effect of ETMI on LPS-stimulated proinflammatory cytokine production was confirmed. RAW 264.7 macrophages were seeded in 24-well plates at a density of 5 x 10 ⁵ cells / well and treated with varying concentrations of ETMI (0, 5, 10, 20 and 40 μg / mL) for 1 hour, followed by 0.5 μg / Stimulated with mL LPS for 24 hours.

Compared with ETMI untreated cells, with or without LPS stimulation, it was confirmed that the expression of LPS-induced TNF-α and IL-6 significantly decreased in ETMI-treated cells (FIG. 4 (A)). Cell culture medium was then collected and the levels of TNF-α and IL-6 were analyzed using the enzyme linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The 40 μg / mL concentration of ETMI in RAW 264.7 cells significantly reduced LPS-mediated increased IL-6 levels by about 5 times compared to control. In contrast, LPS-stimulated TNF- [alpha] production was slightly reduced in a concentration dependent manner despite a significant decrease in mRNA levels (Figure 4 (B)).

These results suggest that ETMI can modulate the inflammatory process through the regulation of proinflammatory cytokine production.

Example  4: HCT116  For cells Of ETMI  Cytotoxic effect

Chronic inflammation has been reported to cause cancer through modulation of tumor formation pathway (Multhoff G, Molls M, Radons J. Chronic inflammation in cancer development. Front Immunol . 2011; 2: 98.). Therefore, we tried to determine whether ETMI can exert anti-inflammatory action and inhibit tumor formation.

For cytotoxicity analysis, RAW 264.7 macrophages and HCT116 cells were seeded in 96-well plates at a density of 1 × 10 ⁵ cells / well. Macrophages were incubated with various concentrations of ETMI (12.5, 25, 50 μg / mL) for 1 hour and then stimulated with LPS (1 μg / mL) for 24 hours. After that CellTiter 96AQ _ucous Cell proliferation was analyzed by One Solution Cell Proliferation Assay (Promega).

HCT116 cells were seeded in 6-well culture plates at densities of 1 × 10 ³ or less for colony formation assays. Cells were incubated for 24 hours at 37 ° C. for cell attachment and then treated with various concentrations of ETMI (12.5, 25, 50 and 100 μg / mL) for 12 hours. The ETMI containing the culture medium was then removed. That is, cells were washed with PBS and cultured in normal medium until viable colonies were observed. Cells were fixed with methanol-acetic acid (3: 1), stained with staining solution, and measured (Suh SS, Yang EJ, Lee SG, Youn UJ, Han SJ, Kim IC, et al . Bioactivities of ethanol extract from the Antarctic freshwater microalga, Chloromonas sp. Int J Med Sci . 2017; 14: 560-569.).

As a result, it was confirmed that at concentrations of 25 μg / mL or more, ETMI inhibited proliferation of HCT116 cells in a concentration-dependent manner (FIG. 5 (A)), indicating that ETMI exhibited concentration-dependent inhibitory activity against HCT116 cells. In addition, the number of ETMI-treated cancer cell colonies was reduced compared to control cells (FIG. 5 (B)). Thus, ETMI-treated cancer cell colony formation supports this conclusion.

Example  5: ETMI -process HCT116  Induction of cell cycle arrest in cells

To better understand the potential mechanism of the inhibitory activity of ETMI, the effect of extracts on cell cycle distribution in HCT116 cells was investigated using flow cytometry.

HCT116 cells were treated with ETMI at various concentrations (0, 25, 50 and 100 μg / mL) and then incubated for 24 hours. Cells were then harvested, washed with PBS and fixed in 70% ice-cold ethanol for at least 4 hours. Cells were then stained with PBS solution containing 50 g / mL propidium iodide and 50 g / mL RNase A. Cell cycle distribution was measured by flow cytometry (Becton Dickinson).

As a result, as shown in Figure 6, the cell population of the G0 / G1 phase significantly increased in a concentration-dependent, while the S phase decreased. 38.61% of control cells were in G0 / G1 phase, 36.93% in S phase and 24.45% in G2 / M phase. As the concentration of extract increased to 100 μg / mL, the proportion of extract-treated cells (38.61%) gradually increased (38.61% at 0 μg / mL, 43.83% at 25 μg / mL, 46.77% at 50 μg / mL). , 53.83% at 100 μg / mL). S cell population decreased at the same concentration (36.93% at 0 μg / mL, 35.78% at 25 μg / mL, 31.46% at 50 μg / mL, 27.16% at 100 μg / mL) (FIG. 6 (A)). These results provide strong evidence of cell cycle arrest at stage G1 and, eventually, evidence of inhibition of HCT116 cell division by ETMI.

To further study the effect on cell cycle distribution, the transcription levels of marker proteins of G1 phase change were analyzed by qRT-PCR. As a result, it was shown that ETMI treatment significantly increased the level of p21 mRNA while decreasing the expression of CDK4 and CDK6 mRNA (FIG. 6 (B)). These results suggest that ETMI at least partially inhibits proliferation by modulating the expression level of regulatory genes at the G1 / S change stage.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13536BP 20180529

<110> Korea Institute of Ocean Science and Technology <120> Composition for Preventing or Treating Inflammation or Cancer          Containing Extract of Micractinium sp. <130> P18-B089 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 ggagccttta gacctcaaca ga 22 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 tgaacgagga gggtggtg 18 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gaagtctttg gtctggtgcg tg 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gtctgctggt ttggaatagt tgc 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gaggatacca ctcccaacag acc 23 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aagtgcatcg ttgttcatac a 21 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 catcttctca aaattcgagt gacaa 25 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 tgggagtaga caaggtacaa ccc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 tgtttgagac cttcaacacc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cgctcattgc cgatagtgat 20

Claims (17)

Micractinium Species sp . ) A pharmaceutical composition for the prevention or treatment of inflammatory diseases, containing the extract of microalgae as an active ingredient.
The method of claim 1, wherein the microlactinium species ( Micractinium) sp . ) Microalgae is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that KCTC 13536BP.
According to claim 1, wherein the extract is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that extracted using ethanol, methanol or water as a solvent.
The method of claim 1, wherein the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis Pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that selected from the group consisting of degenerative neuro-inflammatory.
Micractinium Species sp . ) Food composition for the prevention or improvement of inflammatory diseases containing the extract of microalgae as an active ingredient.
The method of claim 5, wherein the microlactinium species ( Micractinium) sp . ) Microalgae is a food composition for the prevention or improvement of inflammatory diseases, characterized in that KCTC 13536BP.
The method of claim 5, wherein the extract is a food composition for the prevention or improvement of inflammatory diseases, characterized in that extracted using ethanol, methanol or water as a solvent.
The method of claim 5, wherein the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis Food composition for the prevention or improvement of inflammatory diseases, characterized in that selected from the group consisting of degenerative neuro-inflammatory.
Micractinium Species sp . ) A pharmaceutical composition for preventing or treating cancer containing the extract of microalgae as an active ingredient.
10. The method of claim 9, wherein the Micractinium species ( Micractinium) sp . ) Microalgae is a pharmaceutical composition for the prevention or treatment of cancer, characterized in that KCTC 13536BP.
The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the extract is extracted using ethanol, methanol or water as a solvent.
The pharmaceutical composition for preventing or treating cancer of claim 9, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, malignant melanoma, colorectal cancer, liver cancer, ovarian cancer, brain tumor and lung cancer.
The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the cancer cell inhibits cell proliferation and induces cell cycle arrest.
Micractinium Species sp . ) Food composition for the prevention or improvement of cancer containing the extract of microalgae as an active ingredient.
15. The method of claim 14, wherein the microlactinium species ( Micractinium) sp . ) Microalgae is a food composition for the prevention or improvement of cancer, characterized in that KCTC 13536BP.
15. The method of claim 14, wherein the extract is a food composition for preventing or improving cancer, characterized in that extracted using ethanol, methanol or water as a solvent.
The food composition of claim 14, 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.

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