KR102207449B1 - Composition for growth inhibition of colorectal cancer stem cells comprising extracts of novel endolichenic fungi derived from Cetraria laevigata lichen - Google Patents

Abstract

The present invention relates to a composition for inhibiting the growth of colon cancer stem cells comprising an extract of a novel lichen endogenous fungus as an active ingredient, and a novel lichen endogenous fungus isolated from cetraria laevigata lichen EL001668 (KACC 83020BP ) The extract inhibits the expression of colorectal cancer stem cell markers ALDH-1, CD133, CD44, Lgr-5 and Msi-1, induces the death of colorectal cancer stem cells, and self-renewal of colorectal cancer stem cells and into cancer cells. Since it exhibits an effect of inhibiting the differentiation of, it can be usefully used as an active ingredient of a composition for inhibiting tumor stem cell growth.

Description

Composition for growth inhibition of colorectal cancer stem cells comprising extracts of novel endolichenic fungi derived from Cetraria laevigata lichen}, comprising extracts of new lichen endogenous fungi isolated from Cetraria laevigata lichen}

The present invention relates to a composition for inhibiting the growth of colorectal cancer stem cells comprising an extract of a novel lichen endogenous fungus as an active ingredient, and more specifically, a novel lichen endogenous fungus isolated from cetraria laevigata lichens. It relates to a composition for inhibiting the growth of colon cancer stem cells comprising EL001668 and an extract thereof as an active ingredient.

Cancer (malignant tumor) is caused by continuous division and proliferation of normal cells genetically or acquired through one or more mutations, and in this process, tumors are characterized by a variety of biological characteristics due to the population of cells formed by secondary mutations. It has been known to show.

Recently, the hypothesis that cancer stem cells are the cause of cancer has attracted attention thanks to the results of several experiments (G Dontu et al. , cell prolif., 2003, 36, 59; M Al-Hajj et al. . , Proc. Natl. Acad. Sci. USA, 2003, 100, 3983; D Bonnet and JE Dick, Nat. Med, 1997, 3, 730; W Matsui et al. , Blood, 2004, 103, 1668; T Reya et al. , Nature, 2001, 414, 1051; SK Singh et al. , Cancer Res., 2003, 63, 5821; AT Collins et al. , Cancer Res., 2003, 63, 5821). Specifically, in tumor cells, there are tumor stem cells that have the ability to generate cancer cells of various traits through differentiation as well as self-renew, and these cells proliferate to influence the growth and biological characteristics of malignant tumors. It is reported to be. However, the tumor stem cells differentiate into cells having various biological characteristics while proliferating infinitely, facilitating invasion and metastasis of tumor cells, and are higher for chemotherapy (anticancer agents and radiation) than differentiated tumor cells. It has been reported that it has endogenous life and can adversely affect the prognosis of patients such as tumor recurrence (Yu Z, et al. , The inter. Jour&biochem. and cell biology, 2012, 44(12):2144-2151; Wu C. and Alman BA, Cancer letters, 2008, 268(1): 1-9; Sampieri K and Fodde R, Seminars in cancer biology, 2012, 22(3):187-193; Peitzsch C, et al. , Radiotherapy and oncology , 2013, 108(3):378-387). The existence of tumor stem cells was first demonstrated in acute myelogenous leukemia, and recently, it was confirmed that stem cells exist in solid tumors by proving the existence of tumor stem cells in general solid tumors including breast cancer. Therefore, the tumor stem cell model is expected to be of great help in the identification of molecular biological characteristics of tumor occurrence, development of new diagnostic techniques, and improvement of treatment methods.

Research on stem cells is receiving a lot of attention while suggesting the potential for treatment of intractable diseases, and it is contributing not only to the field of regenerative medicine, but also to various fields from understanding the pathophysiology of cancer diseases to treatment.

The most important condition in stem cell research is a specific stem cell marker. Recently, several major discoveries have been made using lineage-tracing mouse experiments on intestinal stem cell markers present at the base of the intestinal tract. Leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) is a target molecule for WNT signaling that marks crypt base columnar cells (CBC cells) located between the intestinal gland and the basal Paneth cells, and Blymphoma Mo-MLV insertion region 1 (BMI1) and telomerase reverse transcriptase (TERT) are stem cell markers expressed in +4 LRC cells of the Jangsundae. Since the intestinal stem cells represented by these intestinal stem cell markers have different locations and characteristics, the intestinal stem cells cannot be described with any one marker, and stem cells of different characteristics are mixed. Due to various characteristics, +4 LRC cells are considered to be inactive stem cells, and CBC cells are considered to be active stem cells, but even in the same +4 LRC cells, BMI1-expressing cells and TERT-expressing cells show different characteristics, and the up-down relationship with LGR5 cells. As they appear differently, there is still a lot of controversy about the interaction between these different characteristics of intestinal stem cells, the vertical relationship, and the mechanism by which the monoclonal enterophytes are formed (Lopez-Garcia C, et al., Science 2010;330:822-825). ; Snippert HJ, et al., Cell 2010;143:134-144).

In addition, musashi 1 (Msi-1), OLFM4, and ASCL2 have long been known as other markers of long stem cells. Recently, CD133 (Prominin-1), CD166, CD44, CD24, aldehyde dehydrogenase-1 (ALDH- 1), doublecortin and CAM kinase-like 1 (DCAMKL-1), Ephrin B2 (EphB2), leucine-rich repeats and immunoglobulin-like domain1 (Lrig1), and LGR5 have also been reported as major long-stem cell markers (Zhu L, et al. al., Nature 2009;457:603-607; Huang EH, et al., Cancer Res 2009;69:3382-3389; Vaiopoulos AG, et al., Stem Cells 2012; 30:363-371; Powell AE, et al., Cell 2012;149:146-158).

Treatment that targets cancer stem cells can be one of the ways that can ultimately overcome cancer. A method for inhibiting the self-renewal proliferation of cancer stem cells is proposed through understanding the various factors and signaling systems related to the proliferation and differentiation of normal stem cells within the intestinal tract, and signals similar to those in the maintenance, proliferation and differentiation of normal stem cells. Since the delivery system also acts on cancer stem cells, studies are being conducted to inhibit the proliferation of cancer stem cells by controlling signaling substances such as WNT, Notch, and bone morphogenetic protein (BMP), and by controlling the influence of the microenvironment (Vermeulen L, De Sousa E Melo F, van der Heijden M, et al. Nat Cell Biol 2010;12:468-476).

The maintenance, proliferation, and differentiation of stem cells requires a very complex signal transduction system, and it is known that not only the regulation by the stem cells themselves, but also signal transduction from the surrounding environment such as niche plays a very important role. Since many factors and signal transduction systems involved in the regulation of normal intestinal stem cells are involved as major factors in the regulation of cancer stem cells, recent research developments on normal intestinal stem cell regulation factors have contributed a lot to studies on the regulation of cancer stem cells. WNT, Hedgehog (Hh), Notch, BMP, etc. are known as signaling systems related to intestinal stem cells and intestinal gland and formation.

WNT signaling has been studied a lot in the gastrointestinal tract because mutations in the APC gene, one of the WNT signaling systems, are importantly involved in the development of colorectal cancer, and are involved in embryonic development, maintenance of cell homeostasis, and the occurrence of various diseases. In particular, it plays an important role in stem cell maintenance, proliferation and differentiation in the intestine (Brittan M, et al., Gut 2004;53:899-910).

The process from stem cells to differentiation is determined by the stem cell's differentiation program and various signals from the surrounding environment. Activation of the Notch signal increases the expression of the hairy/enhancer of split (Hes) gene, and the activation of Hes-1 expression is related to secretory cell differentiation, such as Math-1 and Neu\-rogenin-3, which are basic helix-loop-helix ( bHLH) Inhibits the activity of transcription factors. Inhibition of γ-secretase, an intermediate mediator of Notch signaling, induces hyperproliferation of secretory cells in the intestinal mucosa by Hes-1 inhibition, and mice lacking Hes-1 in developing embryos show early development of endocrine cells in the stomach and small intestine. The number of goblet cells and enterocytes in the intestine is increased (Milano J, et al., Toxicol Sci 2004;82:341-358; Jensen J, et al., Nat Genet 2000;24:36-44).

Notch molecules are activated by binding with ligands known as delta and jagged, and activated cells inhibit Notch activation of surrounding cells by inhibiting their Notch ligand, and this mechanism contributes to the determination of differentiation of surrounding cells. In the intestinal tract, Notch activation determines the differentiation between absorbent and secretory cells. Inhibition of Notch signaling causes proliferation of goblet cells, and Notch activation causes goblet cell defect. In addition, when acting together with WNT signal, it promotes cell proliferation, and when acting alone without WNT signal, it induces differentiation into absorbent intestinal cells, which is involved in maintenance of intestinal gland and homeostasis. Since WNT signal is increased in most colorectal cancer tumors, it is reported that cancer stem cell proliferation by Notch signal is much related, and the differentiation of stem cells by Notch signal regulation is well known, so it is a major target for cancer stem cell inhibition and differentiation treatment. (Crosnier C, et al., Nat Rev Genet 2006;7:349-359; Fre S, et al., Nature 2005;435:964-968; van Es JH, van Gijn ME, Riccio O, et al. Nature 2005;435:959-963; van Es JH, de Geest N, van de Born M, Clevers H, Hassan BA. Nat Commun 2010; 1:18).

Shh plays a very important role in cell proliferation and morphogenesis in the development of various body tissues such as the skeletal system, central nervous system, and face, and is a factor that plays an important role in the proliferation and differentiation of neural stem cells (Chiang C, et al., Nature. 1996; 383, 407-13). The transcriptional regulatory gene Gli, which plays an important role in the development of cancer and embryos, plays a crucial role in transducing and substantiating Shh signals in the Shh signaling pathway.In fact, activating the Gli1 gene in normal cells increases cell growth. The apoptosis mechanism is inhibited, resulting in cancer (Kawai S, et al., Bone. 2001; 29, 54-61).

On the other hand, lichens are symbiotic organisms in which fungi (mycobionts) and algae (algae, photobionts) live in a complex, and are very diverse in shape, size and color, and mainly live in rocks, bark and soil. Within lichens, there are various fungi that exist together without causing visible damage to lichens, which are called endolichenic fungi (ELF) and are recently attracting attention as a new biological resource. It is known that secondary metabolites produced by these lichen endogenous fungi have various physiological activities such as antioxidant, antibacterial, anti-inflammatory and anti-cancer (Kramer et al ., 1995). Therefore, since there is a very high possibility that substances with various physiological activities and high novelty and rareness can be found in endogenous molds of lichens, molecular biology studies using endogenous molds of lichens are actively progressing (Hwang HG et al. , The Korean Journal of microbiology, 2011, 47(1), 97-101).

Accordingly, the present inventors have tried to develop a natural product-derived colorectal cancer therapeutic agent or a composition for inhibiting the growth of colorectal cancer without toxicity and side effects, and as a result, a novel endogenous mold of lichens was isolated from Cetraria laevigata lichens, The extract thereof exhibits cytotoxicity to cancer cells, induces the death of colorectal cancer stem cells, and in particular, found that it inhibits the regeneration of colorectal cancer stem cells and differentiation into tumor cells, and completed the present invention.

The technical problem to be solved in the present invention is to provide a novel lichen endogenous mold EL001668.

Another technical problem to be solved in the present invention is to provide a composition for inhibiting the growth of colon cancer stem cells comprising the extract of the novel lichen endogenous fungus EL001668 as an active ingredient.

Another technical problem to be solved in the present invention is to provide an anti-colorectal cancer adjuvant containing the extract of the novel lichen endogenous fungus EL001668 as an active ingredient.

Another technical problem to be solved in the present invention is to provide a pharmaceutical or food composition comprising the extract of the novel lichen endogenous fungus EL001668.

Another technical problem to be solved in the present invention is to provide a method for inhibiting the growth of colorectal cancer stem cells present in an organ in the individual by administering the composition for inhibiting colorectal cancer stem cells to an individual.

Another technical problem to be solved in the present invention is to provide a method for treating colon cancer as well as inhibiting recurrence of colon cancer by using the anti-colorectal cancer adjuvant in combination with other conventional chemotherapy.

In order to solve the above technical problem, the present invention relates to a novel lichen endogenous mold EL001668.

In the present invention, "extract" refers to a metabolite of lichen endogenous fungi extracted from or isolated from novel lichen endogenous fungus EL001668 by a conventional method. In addition, in the present invention, "extract" is used as a meaning including not only the extract, but also dry powder or all forms formulated using the extract.

In the present invention, the novel lichen endogenous fungus EL001668 is a lichen endogenous fungus isolated from Cetraria laevigata lichens, characterized in that it contains ITS consisting of SEQ ID NO: 3. Here, the lichen endogenous fungus is a fungus belonging to the lichenized fungi. It is not a fungus that forms lichens. It is a fungus that lives in the lichen without causing any special damage, and is a fungus isolated from the lichens of lichens. Say.

Specifically, after complete surface sterilization of the lichen tissues of Cetraria laevigata lichens, they are plated and cultured on potato dextrose agar (PDA) to isolate lichen endogenous fungi. As a result of sequencing the ITS site, a new strain was discovered, and the new strain is the lichen endogenous fungus EL001668 containing ITS consisting of a 584 bp nucleotide sequence, which was reported to the Korean Agriculural Culture Collection (KACC). It was deposited as KACC 83020BP on September 20, 2018.

In order to solve the above-described other technical problem, the present invention relates to a composition for inhibiting growth of colon cancer stem cells comprising an extract of the novel lichen endogenous fungus EL001668 as an active ingredient.

In the present invention, the extract of the lichen endogenous fungus EL001668 refers to a substance exhibiting the efficacy of inhibiting the growth of colon cancer stem cells obtained by extracting the lichen endogenous fungus EL001668 with an organic solvent.

In the present invention, the organic solvent is not necessarily limited thereto, but methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof, preferably methanol, ethanol, butanol, hexane, dichloromethane, ethyl acetate, or a mixed solvent thereof. , The active ingredient of the extract can be extracted at room temperature or heated under conditions that are not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the extract may be different, so select and use an appropriate organic solvent. The extraction method is not particularly limited, and includes, for example, cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like.

In addition, the extract of the lichen endogenous fungus EL001668 can be obtained through a conventional purification process in addition to extraction with an organic solvent. For example, separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity, or affinity), etc., obtained through various additional purification methods. It can also be obtained through fractionation.

In the present invention, the colorectal cancer stem cell growth inhibition is apoptosis of tumor stem cells, self-renewal of tumor stem cells, inhibition of differentiation into cancer cells, and tumor stem cell markers ALDH-1, CD133, CD44, Lgr-5 and Msi It means including inhibition of expression of -1. Here, the tumor stem cells are undifferentiated cells having both self-renewal and differentiation into cancer cells.

On the other hand, tumor stem cell markers are proteins involved in metastasis of tumor stem cells, growth and proliferation into cancer cells, etc., and are frequently expressed in patients with recurrence of cancer. Accordingly, suppression of the expression of the tumor stem cell marker can inhibit the metastasis of tumor stem cells, growth and proliferation into cancer cells, and regeneration of cancer cells, thereby treating cancer.

As a specific example, ALDH-1 (aldehyde dehydrogenase-1) is an intracellular enzyme that catalyzes the oxidation of aldehydes in a physiological or pathological environment, and is a marker of tumor stem cells such as head and neck cancer, colon cancer, breast cancer, acute leukemia, and lung cancer. CD133 (prominin-1; AC133) is a representative cell adhesion molecule that plays an important role in the adhesion and migration of cancer cells. Brain tumors, colon cancer, liver cancer, lung cancer, thyroid cancer, ovarian cancer, laryngeal cancer, endometrial or uterine cancer, It is used as a marker for tumor stem cells such as head and neck cancer, and CD44 (hyaluronate receptor P-glycoprotein 1) is a protein that plays a similar role to CD133. Pancreatic cancer, liver cancer, prostate cancer, ovarian cancer, colon cancer, biliary tract cancer, cervical cancer, It is used as a marker for tumor stem cells in head and neck cancer, breast cancer, flat cell cancer, and stomach cancer. Msi-1 (RNA-binding protein; musahi-1) is a protein that helps the growth of tumor stem cells, and is used as a marker for tumor stem cells and cancer cells such as breast cancer, stomach cancer, brain cancer, colon cancer, and skin cancer, and Lgr-5 (Wnt The targeting gene (APR49) is a target molecule for Wnt signaling and is used as a marker for tumor stem cells such as rectal cancer and colon cancer.

In the present invention, the lichen endogenous fungus EL001668 extract inhibits the expression of tumor stem cell markers ALDH-1, CD133, CD44, Lgr-5, and Msi-1, induces the death of tumor stem cells, and induces tumor stem cells. Because it inhibits the self-renewal and differentiation of cancer cells, it enables chemotherapy that selectively targets tumor stem cells, and can be used as a composition that can prevent recurrence of cancer.

In one specific implementation, extracts of various lichen endogenous fungi sold at the Korean Lichen Center at Sunchon National University showed cytotoxicity against human colorectal cancer stem cells, and among them, the extract of the lichen endogenous fungus EL001668 showed the best cytotoxicity. In addition, when the extract of the lichen endogenous fungus EL001668 was treated with human colorectal cancer stem cells (CSC221), the number of killed colorectal cancer stem cells increased, and ALDH-1, known as a marker promoting the differentiation or self-renewal of colorectal cancer stem cells The expression of CD133, CD44, Lgr-5 and BMsi-1 was decreased.

As another aspect, the present invention relates to a composition for inhibiting the growth of tumor stem cells comprising the extract of endogenous lichen EL001668 as an active ingredient.

Lichen endogenous fungus EL001668 extract contained in the composition for inhibiting growth of colorectal cancer stem cells of the present invention may be included in an amount of 0.001 to 10% by weight, preferably 0.001 to 5% by weight, based on the total weight of the composition. When the content is less than 0.001% by weight, the effect of inhibiting the growth of colorectal cancer stem cells is too weak, and when the content exceeds 10% by weight, the increase in the effect due to the increase in the content is insufficient, which is uneconomical.

The lichen endogenous fungus EL001668 extract according to the present invention exhibits toxicity to colon cancer cells and decreases the growth rate and size of colon cancer tissues, and thus can be used as a composition capable of preventing or treating colon cancer.

In one specific implementation, when the lichen endogenous fungus EL001668 is isolated from Cetraria laevigata lichen, and the extract extracted therefrom is treated with colon cancer cells (DLD1 and HT29), the lichen endogenous fungus EL001668 extract is It was toxic to cancer cells and cancer stem cells.

The composition for inhibiting colorectal cancer stem cells comprising the extract of the lichen endogenous fungus EL001668 of the present invention can be used in combination with other conventional chemotherapy.

Specifically, the anticancer effect can be maximized by using it in combination with conventional radiation therapy or anticancer treatment. In particular, by administering together with a chemical anticancer agent, the conventional anticancer agent and radiation resistance are reduced through the selective targeting effect of tumor stem cells, thereby inhibiting recurrence of cancer, as well as treating cancer at the source.

The anticancer agent can be used as long as it is any drug that has been previously used. For example, mustard gas derivatives (mechloretamine, cyclophosphamide, chlorambucil, melphalan, and ifosfamide, etc.), ethyleneimines (thiotepa and hexamethylmelamine), and acylalkylsulfonates (Busulfan), hydrazines and triazines (altretamine, procarbazine, dacarbazine and temozolomide), nitrosureas (carmustine, lomustine, streptozosine, etc.), metal salts (carboplatin) , Cisplatin and oxaliplatin), vinca alkaloids (vincristine, vinblastine and vinorelbine), taxanes (paclitaxel and docetaxel), grapephyllotoxins (etoposide and tenisopid), camptothecan analogues (irinotecan And topotecan), anthracyclines (doxorubicin, daunorubicin, epirubicin, mitoxantrone and idarubicin), chromomycins (dactinomycin and plicamycin), anti-tumor antibiotics (mitomycin And bleomycin), folic acid antagonists (methotrexate), pyrimidine antagonists (5-fluorouracil, poxuridine, cytarabine, capecitabine and gemcitabine), purine antagonists (6-mercaptopurine and 6 -Thioguanine), adenosine deaminase inhibitors (cladribine, fludarabine, nelarabine and pentostatin), topoisomerase I inhibitors (ironotecan and topotecan), topoisomerase II inhibitors (Amsaclean, etoposide, etoposide phosphate and teniposide), ribonucleotide reductase inhibitors (hydroxyurea), adrenocortic steroid inhibitors (mitotan), enzymes (asparaginase and fecal acid) Gaspargase), antimicrobial agents (estramustine), and retinoids (bexarotene, isotretinoin, and tretinoin (ATRA)), one or more selected from the group consisting of, but are not limited thereto.

In yet another aspect, the present invention relates to an anti-colorectal cancer adjuvant comprising an extract of endogenous mold EL001668 of lichens as an active ingredient.

In the present invention, the anti-colorectal cancer adjuvant can be used in combination with a conventional chemical anti-cancer agent and the like, thereby enhancing the effect of the anti-cancer agent for killing colon cancer cells.

Specifically, by administering the anti-colorectal cancer adjuvant of the present invention together with a conventional chemical anti-cancer agent, the conventional anti-cancer agent and radiation resistance are reduced through the selective targeting effect of tumor stem cells, thereby inhibiting the recurrence of cancer as well as preventing cancer. Make it curable.

The anticancer agent can be used as long as it is any drug that has been previously used. For example, mustard gas derivatives (mechloretamine, cyclophosphamide, chlorambucil, melphalan, and ifosfamide, etc.), ethyleneimines (thiotepa and hexamethylmelamine), and acylalkylsulfonates (Busulfan), hydrazines and triazines (altretamine, procarbazine, dacarbazine and temozolomide), nitrosureas (carmustine, lomustine, streptozosine, etc.), metal salts (carboplatin) , Cisplatin and oxaliplatin), vinca alkaloids (vincristine, vinblastine and vinorelbine), taxanes (paclitaxel and docetaxel), grapephyllotoxins (etoposide and tenisopid), camptothecan analogues (irinotecan And topotecan), anthracyclines (doxorubicin, daunorubicin, epirubicin, mitoxantrone and idarubicin), chromomycins (dactinomycin and plicamycin), anti-tumor antibiotics (mitomycin And bleomycin), folic acid antagonists (methotrexate), pyrimidine antagonists (5-fluorouracil, poxuridine, cytarabine, capecitabine and gemcitabine), purine antagonists (6-mercaptopurine and 6 -Thioguanine), adenosine deaminase inhibitors (cladribine, fludarabine, nelarabine and pentostatin), topoisomerase I inhibitors (ironotecan and topotecan), topoisomerase II inhibitors (Amsaclean, etoposide, etoposide phosphate and teniposide), ribonucleotide reductase inhibitors (hydroxyurea), adrenocortic steroid inhibitors (mitotan), enzymes (asparaginase and fecal acid) Gaspargase), antimicrobial agents (estramustine), and retinoids (bexarotene, isotretinoin, and tretinoin (ATRA)), one or more selected from the group consisting of, but are not limited thereto.

On the other hand, the extract of the lichen endogenous fungus EL001668 of the present invention is derived from a natural lichen and has very little side effects on the human body, induces apoptosis of tumor stem cells, inhibits the self-renewal of tumor stem cells and differentiation into cancer cells. When used in combination with chemotherapy (anticancer drugs and radiation therapy), it induces the death of tumor stem cells along with the death of cancer cells, thereby reducing the existing anticancer drugs and radiation resistance, thereby enhancing the anticancer treatment effect. It can be usefully used as a food composition.

In one specific embodiment, when the extract of the lichen endogenous fungus EL001668 is used as a pharmaceutical composition, components commonly used in the pharmaceutical composition may be additionally included in addition to the extract of the lichen endogenous fungus EL001668. For example, lubricants, wetting agents, flavoring agents, emulsifying agents, suspending agents, preservatives and carriers.

Carriers suitable for the pharmaceutical composition are those commonly used in formulation, and carbohydrate compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.), gum acacia, calcium phosphate, Alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton seed oil, soy milk, olive oil, coconut oil ), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. Pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The lichen endogenous fungus EL001668 extract contained in the pharmaceutical composition of the present invention may be included in an amount of 0.001 to 10% by weight, preferably 0.001 to 5% by weight, based on the total weight of the composition. When the content is less than 0.001% by weight, the effect of inhibiting the growth of colorectal cancer stem cells is too weak, and when the content exceeds 10% by weight, the increase in the effect due to the increase in the content is insufficient, which is uneconomical.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, etc. by various routes such as oral or parenteral, and all modes of administration can be expected, for example, oral, rectal or intravenous It can be administered by infusion, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration.

A suitable dosage of the pharmaceutical composition may be variously prescribed depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, administration time, route of administration, excretion rate and response sensitivity of the patient. More specifically, it is preferable to administer an amount effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of the disease. For example, the dosage of the composition administered to the patient may be about 0.5-1,000 mg/kg of the patient's body weight. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can be manufactured by enclosing it in a dose container. In this case, the formulation may be in the form of a suspension, granules, tablets, powders, emulsions or capsules, and may additionally include a dispersing agent or a stabilizer.

As another specific embodiment, when the extract of the lichen endogenous fungus EL001668 of the present invention is used as a food composition, it may include a component commonly added during food production in addition to the lichen endogenous fungus EL001668 extract. Includes, for example, proteins, carbohydrates, fats, nutrients, flavoring and flavoring agents. The above-described carbohydrates are monosaccharides of glucose and fructose sugars, disaccharides such as maltose, sucrose and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

There is no particular limitation on the type of food. Examples of foods to which the lichen endogenous fungus EL001668 extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, There are teas, drinks, alcoholic beverages and vitamin complexes, and all health foods in the usual sense are included.

When the food of the present invention is a beverage, it may contain various flavoring agents or natural carbohydrates as an additional component like a normal beverage. As the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. .

In addition to the above-described ingredients, the food includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, Carbonating agents used in carbonated beverages may be contained. In addition, the health food of the present invention may contain pulp for the manufacture of natural fruit juices, beverages and vegetable beverages. These components may be used independently or in combination.

As described above, the composition for inhibiting the growth of colorectal cancer stem cells or the anticancer adjuvant of the present invention is used in combination with conventional chemotherapy (anticancer agent and radiation treatment), thereby inducing the death of tumor stem cells together with the death of cancer cells. It can enhance the anticancer treatment effect by reducing the anticancer drug and radiation resistance. In addition, the active ingredient contained in the composition of the present invention is derived from a natural product, lichens, and has very few side effects to the human body, so it can be used very safely in pharmaceutical and food compositions.

In yet another aspect, the present invention relates to a method for inhibiting the growth of colon cancer stem cells present in the organs of the individual by administering a composition for inhibiting the growth of colon cancer stem cells comprising an extract of the lichen endogenous fungus EL001668 to an individual. .

In one specific embodiment, the composition for inhibiting the growth of colon cancer stem cells containing the extract of the lichen endogenous fungus EL001668 of the present invention is administered to an individual to induce the death of colon cancer stem cells present in the organs of the individual, and the colon cancer stem cells self It provides a method of inhibiting regeneration, differentiation into colon cancer cells, and inhibiting growth and proliferation.

In yet another aspect, the present invention is a method of inhibiting recurrence of colon cancer as well as treating colon cancer by using an anticancer adjuvant comprising the extract of the lichen endogenous fungus EL001668 in combination with other conventional chemotherapy (anticancer agent and radiation treatment) It is about.

In one specific embodiment, the anti-colorectal cancer adjuvant comprising the extract of the lichen endogenous fungus EL001668 of the present invention is administered to an individual with a conventional anti-cancer agent or used in combination with radiation therapy to reduce cancer recurrence by reducing the conventional anti-cancer agent and radiation resistance In addition, it provides a method for fundamentally treating cancer through selective targeting of colorectal cancer stem cells present in an individual's organ.

In the present invention, the term "individual" refers to monkeys, cattle, horses, pigs, sheep, dogs, including humans with diseases that can improve symptoms by administering the composition for inhibiting tumor stem cells or anticancer adjuvants of the present invention. It refers to mammals such as cats, rats, mice, and chimpanzees.

In the present invention, the term "administration" means introducing a predetermined substance into an individual by any suitable method, and the administration route of the composition for inhibiting tumor stem cells or the anticancer adjuvant of the present invention is as long as it can reach the target tissue. It can be administered orally or parenterally through any common route.

In the present invention, the term "inhibition" or "treatment" means (a) inhibition of the development of a disease or disease, (b) alleviation of a disease or disease, and (c) elimination of a disease or disease in an individual.

The above-described disease or disease of the individual by the method of the present invention can be achieved by the administration of the composition for inhibiting tumor stem cell growth or an anticancer adjuvant of the present invention into the individual to achieve the purpose of inhibition or treatment. It goes without saying that it will be understood by those of ordinary skill in the art through the examples described below.

As used herein, the term "prevention" refers to inhibiting the occurrence of a disease or disease in an individual who has not been diagnosed with a disease or disease, but is prone to such a disease or disease. As used herein, the term "treatment" means (a) inhibition of the development of a disease or disease (b) alleviation of the disease or disease and (c) elimination of the disease or disease in an individual. In the present specification, the term "individual" refers to monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, etc., including humans with diseases that can improve symptoms by administering the composition of the present invention. It means mammal.

The above-described disease or disease of the individual by the method of the present invention can achieve the purpose of inhibition or treatment by the intra-individual administration of the composition for inhibiting the growth of colorectal cancer stem cells or an anticancer adjuvant of the present invention. And it will be understood by those of ordinary skill in the art through the examples described below, and the like.

The novel lichen endogenous fungus EL001668 extract isolated from Cetraria laevigata lichens of the present invention inhibits the expression of colon cancer stem cell markers ALDH-1, CD133, CD44, Lgr-5 and Msi-1. , Induces the death of colorectal cancer stem cells, and exhibits the effect of inhibiting the self-renewal and differentiation of colorectal cancer stem cells into cancer cells, so it can be usefully used as an active ingredient of a composition for inhibiting tumor stem cell growth. In addition, the composition for inhibiting the growth of colorectal cancer stem cells is an anticancer adjuvant that is administered together with an existing chemical anticancer agent, or when used in combination with radiation therapy, through the selective targeting effect of the colorectal cancer stem cells, the anticancer agent and radiation resistance of the tumor stem cells By reducing it, it not only suppresses recurrence of cancer, but also makes it possible to treat cancer at the source. In addition, the active ingredients included in the composition of the present invention are derived from natural lichens, and have very few side effects to the human body, so they can be used very safely in pharmaceutical and food compositions.

1 is a graph showing the inhibition of Gli promoter activity of an ELF library (55 species) extract.
Figure 2 is a graph showing the Wnt / β-catenin signaling pathway inhibitory activity of ELF extract.
Figure 3 is a graph showing the inhibitory activity of the Notch signaling pathway of ELF extract.
4 is a graph showing the cytotoxicity of the ELF extract to CSC221 cells.
5 is a graph showing the mRNA expression reduction effect of the tumor stem cell markers ALDH-1, CD133, CD44, Lgr5, and Msi-1 of the ELF extract.
6 is a result of analysis of spheroid-forming ability of ELF extract using the colon cancer cell line DLD1.
7 is a result of analysis of spheroid-forming ability of ELF extract using the colon cancer cell line HT29.
8 is a result of analyzing the ITS sequence of a novel lichen endogenous fungus EL001668 isolated from Cetraria laevigata lichens.

Hereinafter, the present invention will be described in more detail through manufacturing examples and examples. These preparation examples and examples are only for describing the present invention in more detail, and it is common in the technical field to which the present invention pertains that the scope of the present invention is not limited by these preparation examples and examples according to the gist of the present invention. It will be self-evident to those who have the knowledge of.

Example 1: Collection of endogenous molds of lichens and preparation of extracts thereof

A library of Endolichenic fungi (ELF) was obtained from Sunchon National University's Korean Lichen Research Institute (KOLRI). The hyphae of these pre-sold lichen endogenous fungi were inoculated into potato dextrose agar medium (PDA, 24g/L of potato extract, 10g/L of Agar) and then cultured for 1 month and recovered. Then, after lyophilizing the cultured lichen endogenous fungi, acetone was added and extracted with a Soxhlet extractor. Then these extracts were filtered and vacuum dried at 45°C.

The prepared lichen endogenous fungus extract was dissolved in DMSO (dimethylsulfoxide) and used in all subsequent experiments.

Example 2: Screening ELF extracts having inhibitory activity of colon cancer stemness

2-1. Measurement of inhibition of Gli promoter activity

In this experiment, an extract that lowered the Gli promoter activity was screened. DMSO was used as a control.

It is known that Shh, Wnt, and Notch signaling pathways play an important role in the acquisition and maintenance of cancer cell tumor stem cell ability. Of these, when the Shh signaling pathway activates Gli-promoter by Gli, the transcription and translation of proteins required to express tumor stem cell function are increased.

The extract that inhibits the activity of the Gli promoter was screened using the ELF extract library of Example 1. At this time, DMSO was used as a control group, and Sticta weigelii extract (the second sample in FIG. 1) was used as a positive control group.

Specifically, transformed murine fibroblasts (NIH3T3) cells made permanently with a reporter vector having a Gli promoter and a pRL-TK vector were used. At this time, the pRL-TK vector was a vector expressing renila luciferase, and was used as an internal control indicating cell viability in each well.

First, the transformed murine fibroblasts (NIH3T3) cells were aliquoted into a 24-well plate at 500 µl/well, respectively, and 10% FBS and 1% penicillin/streptomycin were included in an incubator at 37° C. and 5% CO _2. Incubated for 12 hours in DMEM medium. Thereafter, 54 kinds of endogenous fungal extracts of lichens prepared in Example 1 were added, respectively, and then cultured for 48 hours. Cells after culture were lysed and luciferase activity was measured using a luciferase reporter assay system (Promega, USA). The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, significance was verified by one-way analysis of variance (One-way ANOVA, Tukey's HSD) at p<0.05 significance level, and SPSS 17.0 (Chicago, USA) statistical program was used.

The results are shown in FIG. 1. Here, it was confirmed that the lichen endogenous fungus extract of the bar graph indicated in red was an extract showing significant inhibition of Gli promoter activity (less than 75%).

2-2. Measurement of Wnt and Notch activity

Inhibitory activity was measured using TOPFLASH and HES-Luc, which measure Wnt and Notch activities, respectively, with respect to the extracts showing the inhibitory activity of the Gli promoter.

Specifically, human embryonic kidney (HEK293T) cells were transformed with the TOPFLASH reporter vector or the HES-1 reporter vector. At this time, the pRL-TK vector was a vector expressing renila luciferase, and was used as an internal control indicating transformation efficiency in each of the transformed wells.

First, cultured human embryonic kidney (HEK293T) cells were dispensed into a 24-well plate at 500 µl/well, respectively, and then 10% FBS and 1% penicillin/streptomycin in an incubator with 5% CO ₂ at 37°C. Incubated for 12 hours in DMEM medium containing this. After incubation for 12 hours, a mixture of a transfection reagent and a vector of each group was added to each well, followed by incubation in a 37°C, 5% CO ₂ incubator for 24 hours. Thereafter, the medium was carefully removed and replaced with a DMEM medium containing 10% FBS and 1% penicillin/streptomycin, while 13 kinds of endogenous fungal extracts of lichens showing inhibition of Gli promoter activity in Example 2-1 were respectively added. Incubated for the next 48 hours. Cells after culture were lysed and luciferase activity was measured using a luciferase reporter assay system (Promega, USA). The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, significance was verified by one-way analysis of variance (One-way ANOVA, Tukey's HSD) at p<0.05 significance level, and SPSS 17.0 (Chicago, USA) statistical program was used.

The results are shown in FIGS. 2 and 3. 2 is a graph showing the Wnt inhibitory activity of the ELF extract. 3 is a graph showing the Notch inhibitory activity of the ELF extract.

As a result, it was confirmed that the ELF extract, which showed inhibitory activity on the Gli promoter, had various degrees of inhibitory activity on the Wnt/β-catenin signaling pathway and the Notch signaling pathway.

Example 3: Spheroid Analysis of Lichen Endogenous Fungus Extracts on Human Colon Cancer Stem Cells

A spheroid analysis for CSC221 (cancer stem cell enriched human colorectal adenocarcinoma cell line) cells (colorectal cancer stem cells) of the ELF extract having the activity of inhibiting tumor stemness found in Example 2 (Int. J. Mol. Sci. 2017, 18, 1888).

Specifically, colon cancer stem cells (CSC221) were suspended as single cells using trypsin-EDTA. Thereafter, the colon cancer stem cells were cultured with nitrogen-added DMEM/F12 containing human transformed epithelial cell growth factor (hrEGF; 20 ng/mL; Biovision) and human-derived fibroblast growth factor (hbEGF; 10 ng/mL; Invitrogen). The medium (Invitrogen, Carlsbad, CA) was inoculated. Then, after dispensing the colorectal cancer stem cells to 1×10 ⁴ cells/well in a 24-well plate, 5 μg/mL concentration of lichen endogenous fungus extract was added and cultured for 10 to 14 days to determine the degree of colonization by IMT iSolution. It was observed using software (IMT i-Solution Inc., Northampton, NJ, USA). The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, to verify the significance of the experiment, one-way variance analysis (One-way ANOVA, Tukey's HSD) was used to analyze at a significance level of p<0.05, and SPSS 17.0 (Chicago, USA) statistical program was used. As a positive control, a cell line treated with 0.01% DMSO was used. The results are shown in Table 1 below.

Based on the results of Table 1 above, a total of five lichen endogenous fungal extracts of EL001541, EL001558, EL001609, EL001668 and EL001672 that have inhibitory activity (1.0 or less) on all of Gli, HES1, and TOPFLASH and exhibit significant inhibitory activity against CSC221 cells. I chose

Example 4: Measurement of cytotoxicity of endogenous fungal extracts of lichens against human colon cancer stem cells

CSC221 cells were cultured using DMEM (Dulbecco's modified eagle medium, Gen Depot, USA) containing 10% FBS (fetal bovine serum, Gen Depot, USA), 1% penicillin and streptomycin. Incubation was carried out in an incubator with a temperature of 37°C and 5% CO ₂ .

Then, colon cancer stem cells (CSC221) were dispensed into a 96-well plate to be 1×10 ⁴ cells/well and cultured for 16 hours in a 37°C cell incubator in the presence of 5% CO ₂ . Thereafter, each well was treated with 100 µg/mL of 5 kinds of EL001541, EL001558, EL001609, EL001668 and EL001672 extracts showing significant inhibitory activity against CSC221 cells in Example 3, and then 37°C cells in the presence of 5% CO ₂ It was reacted for 60 hours in the incubator. Then, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well and cultured in a cell incubator at 37°C, and 50% DMSO and 20% SDS were included. Each cell was lysed by adding a cell lysis solution, and absorbance was measured at 570 nm using a microplate reader (VERSAmax, molecular devices, minnesota, USA). Then, the sample concentration when the degree of inhibition of cell growth was 50% was expressed as an IC ₅₀ value. The toxicity test for the cells was repeated three times, and the values were expressed as mean±standard deviation. In addition, to verify the significance of cytotoxicity, one-way variance analysis (One-way ANOVA, Tukey's HSD) was used to analyze at a significance level of p<0.05, and SPSS 17.0 (Chicago, USA) statistical program was used. The results are shown in FIG. 4. As shown here, the extracts other than the EL001558 extract did not show cytotoxicity to CSC221 cells.

Accordingly, the ELF extract having effective activity is as shown in Table 2 below.

Example 5: Measurement of changes in the amount of mRNA of tumor stem cell markers by extracts of endogenous fungi from lichens

In Example 4, it was confirmed whether the amount of mRNA of the tumor stem cell markers Aldh1, CD133, CD44, Lgr5 and Msi-1 was reduced with respect to the ELF extract without cytotoxicity to human colorectal cancer stem cells.

Colon cancer stem cells were dispensed into a 6-well plate and cultured for 16 hours in a 37°C cell incubator in the presence of 5% CO ₂ . Then, each well was treated with an ELF extract at a concentration of 5 μg/mL, and then reacted in a cell incubator at 37° C. in the presence of 5% CO ₂ for 48 hours. Then, after removing the medium, RNA was extracted using trizol, and cDNA was synthesized using MMLV revese transcriptase kit (invitrigen, Carlsbad, CA, USA) and SYBR green (Enzynomics, Seoul, Korea). . Then, the resulting cDNA and PCR reaction buffer and primers of ALDH-1, CD133, CD44, Lgr5, Msi-1, and GAPDH were added to amplify. The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, to verify the significance of the experiment, one-way variance analysis (One-way ANOVA, Tukey's HSD) was used to analyze at a significance level of p<0.05, and SPSS 17.0 (Chicago, USA) statistical program was used. As a control, a colon cancer stem cell line treated with DMSO was used. The results are shown in FIG. 5. As shown here, the EL001668 and EL001672 extracts significantly reduced the mRNA levels of the tumor stem cell markers Aldh1, CD133, CD44, Lgr5 and Msi-1.

Example 6: Spheroid analysis of lichen endogenous fungus extract using colon cancer cell lines DLD1 and HT29

In Example 5, the ELF extract having reduced the amount of mRNA of the tumor stem cell marker was subjected to spheroid analysis using the colon cancer cell lines DLD1 and HT29.

Colorectal cancer cell lines DLD-1 and HT29 were suspended as single cells using trypsin-EDTA. Then, the colon cancer stem cells were transformed into human transformed epithelial cell growth factor (hrEGF; 20 ng/mL; Biovision) and human-derived fibroblast growth factor (hbEGF; 10 ng/mL; Invitrogen) containing nitrogen-added DMEM/F12 It was inoculated in culture medium (Invitrogen, Carlsbad, CA). Then, after dispensing the colon cancer cell line to be 1 X 10 ⁴ cells/well in a 24-well plate, EL001668 and EL001672 extracts at concentrations of 2.5, 5, and 10 μg/mL were added and cultured for 10 to 14 days to form colonies. Was observed using IMT iSolution software (IMT i-Solution Inc., Northampton, NJ, USA). The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, to verify the significance of the experiment, one-way variance analysis (One-way ANOVA, Tukey's HSD) was used to analyze at a significance level of p<0.05, and SPSS 17.0 (Chicago, USA) statistical program was used. As a positive control, a cell line treated with 0.01% DMSO was used. The results are shown in FIGS. 6 and 7.

6 and 7 are spheroid analysis results using the colon cancer cell lines DLD1 and HT29 of EL001668 and EL001672 extracts. As a result, EL001668 and EL001672 extracts inhibited the spheroid-forming ability of DLD1 and HT29 in a concentration-dependent manner.

The experimental results are summarized in Table 3 below, and from these results, the lichen endogenous fungi EL001668 and EL001672 have excellent effective activity to inhibit the ability of colon cancer tumor stem cells, so that the composition for inhibiting the growth of colon cancer stem cells and the prevention, treatment and It would be useful as a composition for improvement.

Example 7: Identification of endogenous mold in lichens

The lichen endogenous mold EL001668 whose significance was confirmed by the above Examples 1 to 6 was identified.

Specifically, the hyphae of the lichen endogenous fungus EL001668 was inoculated into potato dextrose agar medium (PDA, BD Difco, Sparks, MD, USA) and cultured at 25°C. The total DNA of the cultured lichen endogenous fungus EL001668 was extracted using the DNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS) site of the rNDA gene is universal primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 1) (Gardes and Bruns 1993) and LR5 (5'-ATCCTGAGGGAAACTTC-3', SEQ ID NO: 2) (Vilgalys and Hester 1990) was used to amplify.

The PCR reaction cycle conditions were 30 seconds after denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds as one cycle. It was carried out twice and finally a stretching step was performed at 72° C. for 10 minutes. Subsequently, the obtained PCR product was concentrated and purified using a PCR quick-spin PCR product purification kit (INTRON biotechnology, Seongnam, Korea).

As a result of the experiment, it was confirmed that the ITS sequence of the lichen endogenous fungus EL001668 isolated from Cetraria laevigata lichen was 584 bp (SEQ ID NO: 3), which shows a large difference from the ITS sequence of the conventional lichen endogenous fungus (Fig. 8). Reference), classified as a novel lichen endogenous fungus.

Accordingly, the present inventor deposited the lichen endogenous fungus EL001668 as KACC 83020BP to the National Institute of Agricultural Sciences Microbial Bank (KACC) on September 20, 2018.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION OF SUNCHON NATIONAL UNIVERSITY <120> Composition for growth inhibition of colorectal cancer stem cells comprising extracts of novel endolichenic fungi derived from Cetraria laevigata lichen <130> PA-18-0211 <150> KR 10-2018-0097708 <151> 2018-08-21 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> universal primers ITS1F <400> 1 cttggtcatt tagaggaagt aa 22 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> universal primers LR5 <400> 2 atcctgaggg aaacttc 17 <210> 3 <211> 584 <212> DNA <213> Unknown <220> <223> ITS sequence of endolichenic fungi EL001668 derived from Cetraria laevigata lichen <400> 3 tatgcttaag ttcagcgggt attcctacct gatccgaggt caacctagaa gatttggggt 60 gttttacggc aggggaccgg gtccaagcta caggcgatat gtaaaagcta ctacgtctgg 120 agtgcgaacc ggctccgcca ctgactttgg agagctacag gagagttcca gtaggctccc 180 aacgctaagc aacaggggct taagggttga aatgacgctc gaacaggcat gcccaccaga 240 atactagtgg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc tgcaattcac 300 attacttatc gcatttcgct gcgttcttca tcgatgccag aaccaagaga tccgttgttg 360 aaagttttaa cttatttagt tatcttttca gaagtccaat gctataaaaa cagagtttcg 420 ggggccgtcg gcagggtcgc ctaccgggta ggtcctacag ggtaggtctc acggggtagg 480 acgccacctg ccgaggcaac gcgaggtatg ttcacatggg tttgggagtt ttggtaactc 540 tgtaatgatc cctccgctgg ttcaccaacg gagaccttgt tacg 584

Claims (4)

A novel lichen endogenous fungus EL001668 (KACC 83020BP) isolated from Cetraria laevigata lichens.
A composition for inhibiting the growth of colorectal cancer stem cells comprising an acetone extract extracted from the lichen endogenous fungus EL001668 (KACC 83020BP) as an active ingredient.
A pharmaceutical composition for the prevention or treatment of colon cancer comprising an acetone extract extracted from the lichen endogenous fungus EL001668 (KACC 83020BP) as an active ingredient.
A food composition for preventing or improving colorectal cancer comprising an acetone extract extracted from lichen endogenous fungus EL001668 (KACC 83020BP) as an active ingredient.

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