KR102283102B1 - Composition comprising frugoside as an effective ingredient for preventing or treating of lung cancer - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or improving lung cancer comprising frugoside or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

A composition for preventing or treating lung cancer comprising frugoside as an active ingredient {COMPOSITION COMPRISING FRUGOSIDE AS AN EFFECTIVE INGREDIENT FOR PREVENTING OR TREATING OF LUNG CANCER}

The present invention relates to a composition for preventing or treating lung cancer in patients with lung cancer, in particular, NRF2 overexpressing, comprising frugoside as an active ingredient.

According to data from the Korea Central Cancer Registry, the most common cancers in Koreans are stomach cancer, thyroid cancer, colorectal cancer, lung cancer, liver cancer, and breast cancer in that order, and the mortality rate is confirmed to be pancreatic cancer, lung cancer, and liver cancer in that order. Due to rapid industrial development, westernized lifestyle, entry into an aging society, and changes in disease structure, cancer-related cancer-related deaths in Korea are continuously increasing.

In particular, the overall 5-year survival rate of lung cancer is about 14%, and the 5-year survival rate after surgery is less than about 50% from stage 2B (about 39%), which corresponds to a cancer with a very low survival rate. About 85% of lung cancers are non-small cell lung cancer (such as squamous cell carcinoma, adenocarcinoma, and large cell cancer), and about 15% are small cell lung cancer (small cell cancer, etc.). These lung cancers have a very poor prognosis and are excellent targeted anticancer drugs It is a situation that is urgently required, and it is known to have a characteristic in which NRF2 is overexpressed. Moreover, in a recent study, it was reported that mutations in the kelch-like ECH-associated protein 1 (KEAP1) gene and mutations in the liver kinase B1 (LKB1) frequently occur together in non-small cell lung cancer among lung cancers. KEAP1 mutation induces activation of NRF2 (Nuclear factor (erythroid-derived 2)-like 2), and it has been reported that cancer cell growth is effectively inhibited when NRF2 activation is inhibited in KEAP1 mutant lung cancer. is a big situation.

Japanese Patent Publication No. 4944380 (2012.03.09)

An object of the present invention is to provide a pharmaceutical composition for preventing or treating lung cancer comprising frugoside or a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating lung cancer comprising frugoside represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The lung cancer may be lung cancer of a patient in which NRF2 is overexpressed.

The lung cancer may be non-small cell lung cancer selected from the group consisting of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.

The lung cancer may be an anticancer drug-resistant lung cancer.

The frugoside or a pharmaceutically acceptable salt thereof may increase active oxygen in lung cancer cells.

The pharmaceutical composition may be treated in combination with cisplatin.

The concentration ratio of the frugoside or a pharmaceutically acceptable salt thereof and the cisplatin may be 1:5 to 1:100.

In one embodiment of the present invention, there is provided a health functional food for preventing or improving lung cancer comprising frugoside represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for preventing or treating lung cancer in patients with lung cancer, in particular, NRF2 overexpressing, comprising frugoside or a pharmaceutically acceptable salt thereof as an active ingredient, the frugoside (Frugoside) By reducing the expression of antioxidant protein through inhibition of NRF2 activation, increasing the free radicals in lung cancer cells, it shows a lung cancer apoptosis effect.

In addition, the frugoside (Frugoside) has the advantage of overcoming the resistance caused by the anticancer drug (in particular, cisplatin) as it exhibits an apoptosis effect on lung cancer with resistance by the anticancer agent (cisplatin). In addition, when combined with cisplatin, it shows a synergistic effect on lung cancer cell death.

Figure 1 (A) shows the results of confirming the activity of the NRF2 target gene when treated with frugoside (Frugoside; FS-1), and Figure 1 (B) is frugoside (Frugoside; FS-1) when treated Shows the results of measuring active oxygen, and FIG. 1 (C) shows the results of confirming the expression of NRF2 according to concentration and time during frugoside (FS-1) treatment, and FIG. 1 (D) is Shows the results of confirming the expression of the NRF2 target gene during treatment with frugoside.
2(A) and (B) show that NRF2 degradation is promoted during treatment with frugoside (FS-1) by treatment with a protein synthetase inhibitor (cycloheximide) and a proteasome 26S proteasome inhibitor (MG132). Whether or not NRF2 is degraded during treatment with frugoside (FS-1) is verified through overexpressed NRF2 in FIG. 2(C).
Figures 3 (A) and (B), using the NRF2 cleavage model, frugoside (Frugoside; FS-1), it was confirmed whether or not the stability of NRF2 is reduced.
Figure 4 (A) shows the expression of NRF2 in lung cancer cell lines (A549, H460) and anticancer drug-resistant lung cancer cell lines (A549-CR, H460-CR), Figure 4 (B) is frugoside (Frugoside; FS-1) ) It is a comparison of lung cancer cell death in lung cancer cell line (A549) and anticancer drug-resistant lung cancer cell line (A549-CR) when treated alone or in combination with frugoside (FS-1) and cisplatin. , FIG. 4(C) shows whether colon cancer cells are killed in colorectal cancer cell line (HT29) when frugoside (FS-1) is treated.

The present inventors confirmed that frugoside exhibits apoptotic effect on both lung cancer and anticancer drug-resistant lung cancer while screening for a substance having an excellent therapeutic effect on intractable cancer (particularly, NRF2 overexpressing lung cancer). The invention was completed.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating lung cancer

The present invention provides a pharmaceutical composition for preventing or treating lung cancer comprising frugoside represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The pharmaceutical composition for preventing or treating lung cancer according to the present invention is characterized in that it contains frugoside or a pharmaceutically acceptable salt thereof as an active ingredient.

IUPAC name of the program Lugo side (Frugoside) (Cannogenol-3- O-beta-D-allomethyloside) is 3 - [(3 S, 5 R, 8 R, 9 S, 10 R, 13 R, 14 S, 17 R) -14-hydroxy-10- ( hydroxymethyl) -13-methyl-3 - [(3 R, 4 R, 5 S, 6 R) -3,4,5-trihydroxy-6-methyloxan-2-yl] oxy-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta [a] phenanthren-17-yl] and -2 H -furan-5-one , the molecular formula is C ₂₉ H ₄₄ O ₉ , and the CAS number is 546-02-1 (ChemlDplus) or 56324-39-1 (EPA DSSTox).

The side profile Lugo (Frugoside) is a second balloon (Cardiospermum halicacadum ) may be isolated from the extract. Specifically, the frugoside (Frugoside) may be one separated from the fraction of the balloon vinegar extract, first, the balloon vinegar extract is the balloon seconds dried products extracted with water, C1 to C4 lower alcohol or a mixture thereof. and extraction with methanol is preferable, but is not limited thereto. In addition, the frugoside (Frugoside) may be prepared by chemical synthesis.

Meanwhile, the "pharmaceutically acceptable salt" in the present specification may be an acid addition salt formed by a free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol late, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the invention is prepared by a conventional method, for example, by dissolving the compound in an aqueous solution of an excess of acid, and precipitating the salt using a water-miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. It can be manufactured by It can also be prepared by heating equal amounts of the above compound and an acid or alcohol in water, followed by evaporating the mixture to dryness or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. Also, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

In addition, the compound of the present invention includes all salts, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically acceptable salts.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and adding an excess of an organic acid or an inorganic acid It can be prepared by precipitation or crystallization after adding an aqueous solution. Subsequently, after evaporating the solvent or excess acid from this mixture, it can be dried to obtain an addition salt, or it can be prepared by suction filtration of the precipitated salt.

The frugoside or a pharmaceutically acceptable salt thereof may inhibit NRF2 activation or degrade NRF2. Accordingly, the frugoside (Frugoside) or a pharmaceutically acceptable salt thereof can increase the active oxygen in lung cancer cells. Specifically, the degradation of NRF2 may be mainly performed in the Neh domain corresponding to the β-TRCP binding domain of NRF2.

In other words, the frugoside decreases the expression of antioxidant protein through inhibition of NRF2 activation (NRF2 degradation), thereby increasing free radicals in lung cancer cells, thereby exhibiting a lung cancer apoptosis effect.

Meanwhile, the concentration of frugoside is preferably 0.001 μg/ml to 10 mg/ml, more preferably 0.1 μg/ml to 5 mg/ml, but is not limited thereto. Although the frugoside is included in a small concentration in the composition, it has the advantage of being able to show a sufficient effect for preventing or treating lung cancer. At this time, when the concentration of frugoside is less than the above range, there is a problem in that it is difficult to properly exert an apoptosis effect on lung cancer, and when the concentration of frugoside exceeds the above range, cytotoxicity is reduced There may be toxicity concerns, including:

The lung cancer includes non-small cell lung cancer and small cell lung cancer, and specifically, the lung cancer may be selected from the group consisting of squamous cell carcinoma, adenocarcinoma, large cell cancer, and small cell cancer.

Furthermore, the lung cancer may be the lung cancer of a patient in which NRF2 is overexpressed, and the frugoside decreases the expression of antioxidant protein through inhibition of NRF2 activation, thereby increasing free radicals in lung cancer cells, lung cancer cell death seems to work However, the apoptosis effect is only insignificant for cancers in which NRF2 is not overexpressed, such as colorectal cancer.

As used herein, the term "patient" broadly refers to an individual in need of prevention, improvement or treatment, and more specifically, a human or non-human primate, mouse, rat, dog, means mammals such as cats, horses, and cattle.

In particular, among the lung cancers, it is preferable that a mutation in the kelch-like ECH-associated protein 1 (KEAP1) gene, which induces NRF2 activation, occurs, and squamous cell carcinoma, adenocarcinoma and large intestine mutation in the KEAP1 gene are common. It is more preferably non-small cell lung cancer selected from the group consisting of cell cancer, but is not limited thereto.

In addition, the lung cancer may include a state in which anticancer drug resistance has not occurred, as well as a state in which anticancer drug resistance has occurred. In this case, the anticancer drug resistance may be cisplatin resistance.

Specifically, the cisplatin (cisplatin) as an alkylating agent used in lung cancer, it is involved in the process of DNA replication, transcription, binding and blocking this process, thereby exhibiting an anticancer effect. More specifically, the cisplatin (cisplatin) is a compound in which two chlorines and ammonia are coordinated to a platinum atom, and it is known that platinum complex salt inhibits the division of E. coli. The mechanism of action is to form a bridge between or within DNA chains in response to DNA, thereby inhibiting DNA synthesis and causing cellular disorders. Although it is an excellent anticancer drug that is effective against many cancers, it also has side effects such as toxicity to the kidneys.

The pharmaceutical composition may be treated in combination with cisplatin, and in this case, a synergistic effect in preventing, treating or improving lung cancer can be expected.

In the case of the combined treatment, the concentration ratio of the frugoside or a pharmaceutically acceptable salt thereof and the cisplatin may be 1:5 to 1:100, preferably 1:30 to 1:100 However, the present invention is not limited thereto.

The pharmaceutical composition for preventing or treating lung cancer according to the present invention is in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. It may be formulated and used, and may include suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions for formulation.

The carrier or excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, unknown. various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, weight agents, binders, wetting agents, disintegrants, and surfactants.

A solid formulation for oral administration may be prepared by mixing Frugoside with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, lyophilized preparations, and suppositories. As a non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for preventing or treating lung cancer according to the present invention varies depending on the patient's condition, weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for preventing or treating lung cancer according to the present invention may be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Health functional food for preventing or improving lung cancer

In addition, the present invention provides a health functional food for preventing or improving lung cancer containing frugoside represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The health functional food for preventing or improving lung cancer according to the present invention contains frugoside or a pharmaceutically acceptable salt as an active ingredient, and the frugoside and the lung cancer are as described above. .

In addition, the health functional food may also be treated in combination with cisplatin, and in this case, a synergistic effect in preventing, treating or improving lung cancer can be expected.

In the case of the combined treatment, the concentration ratio of the frugoside or a pharmaceutically acceptable salt thereof and the cisplatin may be 1:5 to 1:100, preferably 1:30 to 1:100 However, the present invention is not limited thereto.

In the health functional food for preventing or improving lung cancer according to the present invention, when the frugoside is used as an additive in a health functional food, it can be added as it is or used with other foods or food ingredients, and a conventional method can be used appropriately. The mixing amount of the active ingredient may be appropriately determined according to each purpose of use, such as prevention, health or treatment.

The formulation of health functional food may be in the form of powder, granules, pills, tablets, and capsules, as well as in the form of general food or beverages.

There is no particular limitation on the type of food, and examples of food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, and the like, and may include all foods in a conventional sense.

In general, when preparing food or beverage, the frugoside may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material. However, in the case of long-term ingestion for health and hygiene or health control, the amount may be less than the above range, and in the present invention, there is no problem in terms of safety in terms of using a natural product. can also be used.

Beverages among health functional foods according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food for preventing or improving lung cancer according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stability It may contain fire retardants, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. In addition, the composition for preventing or improving lung cancer of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the health functional food of the present invention.

Accordingly, the present invention relates to a composition for preventing or treating lung cancer, in particular, NRF2 overexpressing lung cancer comprising frugoside or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the frugoside (Frugoside) By reducing the expression of antioxidant protein through inhibition of NRF2 activation, increasing the free radicals in lung cancer cells, it shows a lung cancer apoptosis effect.

In addition, the frugoside (Frugoside) has the advantage of overcoming the resistance caused by the anticancer drug (in particular, cisplatin) as it exhibits an apoptosis effect on lung cancer with resistance by the anticancer agent (cisplatin). In addition, when combined with cisplatin, it shows a synergistic effect on lung cancer cell death.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

frugoside detach

Balloon candle ( Cardiospermum) halicacadum ) was purchased at the Gyeongdong Traditional Oriental Medicine Market. Dried whole plants of C. halicacadum (5 kg) were extracted with 80% MeOH (25 L) at room temperature for 3 hours. The extract was filtered and concentrated using a rotary vacuum dryer to give the MeOH extract. The MeOH extract (1.0 kg _{) was suspended in 2,500 ml of H 2} O and an equal volume of ethyl acetate (EA). The EA soluble fraction (110 g) was separated into 21 fractions (CHM1-21) by silica gel column chromatography eluting with a gradient of _{CHCl 3 and MeOH (10:1 to 2:1).} CHM6 (1.2 g) was separated by an octadecyl silica column (MeOH:H ₂ O = 10:1) to obtain 9 fractions (CHM 6-1 to CHM 6-9). A subfraction of CHM 6-3 _{was purified by silica gel column eluted with isocratic conditions of CHCl 3} and MeOH (5:1) to give Frugoside (87 mg). The structure of frugoside was determined by comparison of spectroscopic data with data reported in [Elgamal et al., 1999].

Example One: frugoside by processing NRF2 Confirmation of protein expression reduction and activity thereby amount of oxygen increase confirmation

(1) ARE- luciferase ( luciferase ) reporter analysis

To confirm the effect of reducing NRF2 by frugoside using a luciferase reporter containing an ARE (antioxidant responsive elemnent) moiety that binds to the NRF2 transcription factor, pGL4.14-ARE-luc in lung cancer (A549) cell line After introducing the plasmid, 150 ug/ml of hygromycin was added. Through this, an A549-ARE-Luc cell line in which ARE-luciferase was inserted into the chromosome was established. After the A549-ARE-Luc cell line was treated with frugoside for 15 hours at each concentration as shown in FIG. 1(A), a luciferase assay was performed using Promega's luciferase assay system.

(2) active amount of oxygen for measurement FACS analysis

In order to confirm the NRF2 inhibitory effect of frugoside treatment in lung cancer (A549) cell line, the amount of active oxygen was measured, and the reduction of frugoside effect by treatment with the antioxidant compound N-acetyl cysteine (Nac) was measured. The amount of active oxygen was treated for 6 hours by concentration as shown in FIG. 1(B), and then cells were treated with CM-DCF-DA (10 μM) reagent reacting with hydrogen peroxide for 30 minutes, and then measured using FACS analysis. Nac (30 mM) was pretreated 1 hour before frugoside treatment.

(3) Western blot analysis

In order to confirm the decrease in NRF2 protein by frugoside treatment, Western blot was performed. Collect the sample, and lysis buffer A [HEPES(pH7.5)(20mM), NaCl(150mM), EDTA(1mM), EGTA(2mM), 1% Triton X-100, 10% Glycerol (glycerol), protease cocktail (protease cocktail) I, II (Sigma, USA)] was added and then left at 4 ℃ for 20 minutes. After the cell lysate was transferred to a new tube, the protein was obtained by centrifugation at 4°C and 15000 rpm for 10 minutes. The cell lysate was heated at 95° C. for 10 minutes by adding 5X sample buffer to make a gel loading sample and electrophoresed on an SDS PAGE gel. Protein bands separated according to molecular weight by electrophoresis were transferred to a nitrocellulose membrane using a transfer kit (biorad, USA), and then transferred with 5% skim milk for 1 hour. blocked (blocking). Then, each protein was detected by treatment with the antibody. Frugoside was treated with 0.4 μM for 24 hours and hourly for each concentration as shown in FIG. 1(C).

(4) mRNA Expression analysis

In order to confirm the change in the level of NRF2 mRNA by frugoside treatment in lung cancer (A549) cell line, real-time RT-PCR analysis was performed. As shown in FIG. 1(D), frugoside was treated at 0.4 uM for each hour, RNA was extracted from the cells, and cDNA was synthesized and analyzed by real-time reverse transcription-polymerization chain reaction.

As a result, FIG. 1(A) shows the result of confirming the activity of the NRF2 target gene during frugoside (FS-1) treatment, and from the result, the expression of antioxidant proteins (Srx, Prxs, etc.) The cause is confirmed to be NRF2 degradation.

1(B) shows the results of measuring active oxygen during frugoside (FS-1) treatment, and it was confirmed from the results that the expression of antioxidant proteins (Srx, Prxs, etc.) was reduced.

Figure 1 (C) shows the results of checking the expression of NRF2 according to the concentration and time during frugoside (FS-1) treatment, and it can be confirmed from the result that the decrease in NRF2 expression is concentration and time dependent there was.

FIG. 1(D) shows the results of confirming the expression of the NRF2 target gene during frugoside treatment. From the results, it was confirmed that the decrease in NRF2 expression was made at the protein level, not the mRNA level.

In other words, according to FIGS. 1(A) to (D), frugoside (FS-1) reduces the expression of antioxidant proteins (Srx, Prxs, etc.) through the degradation of NRF2, thereby reducing active oxygen in lung cancer cells. By increasing it, it may be effective in killing lung cancer cells.

Example 2: frugoside Antioxidant protein by treatment modulator NRF2 amount of protein Identification of the mechanism of reduction

(One) western blot using analysis frugoside function check

It was confirmed that frugoside promotes the degradation of NRF2 protein through pretreatment with a protein synthesis inhibitor (cycloheximide: CHX) and a proteasome inhibitor (MG-132). CHX was pretreated with 10uM for 1 hour, MG-132 was also pretreated with 10uM for 1 hour, and then frugoside was treated as shown in FIGS. 2(A) and (B). Western blot analysis was performed in the same manner as in Example 1.

(2) NRF2 Overexpression through introduction of an overexpression vector

In order to prove that the decrease in the amount of NRF2 protein by frugoside treatment is a decrease due to promotion of degradation of NRF2 protein, not an effect due to transcriptional regulation, an overexpression vector was prepared and introduced into cells to confirm the frugoside effect. The NRF2 overexpression vector was prepared by cutting the XbaI and KpnI restriction enzyme sites into the pCGN-HA plasmid and inserting the amplified NRF2 gene using cDNA as a template to construct a pCGN-HA-NRF2 vector. In addition, the prepared plasmid was used with Theromo's Lipofectamine 3000 reagent, and was overexpressed by transfection into cells. After incubation for 48 hours, the amount of endogenous and exogenous expressed NRF2 protein was analyzed by western blot analysis after treatment with frugoside at 0.4uM hourly as shown in FIG. 2(C). was performed in the same manner as in Example 1.

(3) frugoside to identify the mechanism of action NRF2 Construction of protein fragment expression vector

The NRF2 expression vector was amplified by PCR as a template, cut into a glutathione-s-transferase (GST) fusion vector (pEBG-GST) with restriction enzymes BamHI and NotI, and then inserted into pEBG-GST-NRF2 as shown in FIG. 3(A). -FL (full length), D1, D2, D3, and D4 were manufactured. In addition, after each overexpression, frugoside (0.4uM) was treated for each time as shown in FIG. 3(B), and then the expression level was analyzed through Western blot analysis, and the analysis was performed in the same manner as in Example 1. .

As a result, Figures 2 (A) and (B) by the treatment of protein synthesis inhibitor (cycloheximide) and protease 26S proteasome inhibitor (MG132), frugoside (Frugoside; FS-1) treatment, It was verified that NRF2 degradation is promoted, and FIG. 2(C) is through overexpressed NRF2, which verified the degradation of NRF2 when treated with frugoside (FS-1). According to Figure 2 (A) to (C), frugoside (Frugoside; FS-1) by reducing the expression of antioxidant proteins (Srx, Prxs, etc.) through the degradation of NRF2, to increase the active oxygen in lung cancer cells Therefore, it may be effective in apoptosis of lung cancer cells.

In addition, Figures 3 (A) and (B) using the NRF2 cleavage model, frugoside (Frugoside; FS-1), it was confirmed whether NRF2 stability is reduced during treatment. As shown in Figure 3 (A) and (B), frugoside (Frugoside; FS-1), NRF2, which is mainly degraded by treatment, is confirmed to be the Neh domain corresponding to the β-TRCP binding domain bar, The specific mechanism involved in the inhibition of NRF2 activation by Frugoside was investigated.

Example 3: Antioxidant protein in anticancer drug-resistant lung cancer modulator NRF2 amount of protein Confirmation and induction of apoptosis of anticancer drug-resistant lung cancer by treatment with frugoside

(One) western blot analysis

As shown in Fig. 4(A), Western blot analysis of NRF2 expression in cisplatin-sensitive lung cancer (A549 and H460) cell lines and cisplatin-resistant lung cancer (A549-CR and H460-CR) cell lines derived from these cell lines, respectively. The expression level was analyzed through, and the analysis was performed in the same manner as in Example 1.

(2) Annexin V- FITC /PI after dyeing for FACS analysis apoptosis assay by

As shown in FIG. 4(B), frugoside and cisplatin were co-treated with cisplatin-sensitive and resistant lung cancer (A549 and H460) cell lines to promote frugoside apoptosis in lung cancer and NRF2-overexpressing resistant cancer The effect was verified. Each sample was analyzed using FACScanto after staining cells induced by necrotic cell death and apoptotic cell death using Annexin V-FITC and propidium iodide (PI). In addition, as shown in FIG. 4(C), the apoptosis-inducing effect of frugoside was confirmed in NRF2 expression-independent cancer (colon cancer; HT-29) cell lines. After staining with AnnexinV-FITC/PI as above, apoptosis was measured using FACScanto equipment.

As a result, FIG. 4(A) shows whether NRF2 is expressed in lung cancer (A549, H460) cell lines and anticancer drug-resistant lung cancer (A549-CR, H460-CR) cell lines, and NRF2 is overexpressed in both lung cancer cell lines and anticancer drug-resistant lung cancer cell lines. However, it is confirmed that the degree of overexpression of NRF2 is large in antibiotic-resistant lung cancer cell lines. That is, antibiotic resistance is induced according to NRF2 overexpression, and thus frugoside (FS-1) can exhibit an effective apoptosis effect even for anticancer drug-resistant lung cancer with a large degree of NRF2 overexpression.

Figure 4 (B) shows the lung cancer (A549) cell line and anticancer drug-resistant lung cancer (A549-CR) when treated with frugoside alone or in combination with frugoside (FS-1) and cisplatin. ) to compare whether lung cancer apoptosis or not in cell lines, frugoside (FS-1) shows a synergistic effect on lung cancer cell death when combined with cisplatin, so it can serve as an adjuvant for anticancer drugs .

Figure 4 (C) shows whether or not frugoside (Frugoside; FS-1) treatment, colorectal cancer (HT29) cell line, colorectal cancer cell death, frugoside (Frugoside; FS-1) is NRF2 overexpressed It is confirmed that the apoptosis effect is insignificant for colorectal cancer with a small degree.

Hereinafter, formulation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to limit the present invention, but to describe it in detail.

Formulation example One: powdery manufacturing

Frugoside 20 mg

Lactose hydrate 100 mg

talc 10 mg

The above ingredients were mixed and filled in an airtight bag to prepare a powder.

Formulation example 2: Preparation of tablets

Frugoside 10 mg

100 mg cornstarch

Lactose hydrate 100mg

2mg magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional method for manufacturing tablets.

Formulation example 3: Preparation of capsules

Frugoside 10 mg

Microcrystalline Cellulose 3 mg

Lactose hydrate 14.8 mg

0.2 mg magnesium stearate

After mixing the above ingredients, the capsules were prepared by filling in gelatin capsules according to a conventional manufacturing method of capsules.

Formulation example 4: Preparation of injections

Frugoside 10 mg

mannitol 180mg

2974 mg of sterile distilled water for injection

Sodium monohydrogen phosphate 26 mg

After mixing the above ingredients, the content of the above components per 1 ampoule (2 mL) was prepared according to a conventional method for preparing injections.

Formulation example 5: liquid manufacturing

Frugoside 10 mg

10 g isomerized sugar

5 g of mannitol

Purified water appropriate amount

Lemon flavored amount

The above components are dissolved by adding each component to purified water according to a conventional manufacturing method. After adding an appropriate amount of lemon flavor, purified water is added to adjust the total volume to 100 mL, sterilized, and filled in a brown bottle to prepare a solution.

Formulation example 6: Manufacture of health functional food

Frugoside 10 mg

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health functional food, but the composition is mixed in a preferred embodiment, but the mixing ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Then, the granules can be prepared and used in the manufacture of health functional foods according to a conventional method.

Formulation example 7: Manufacture of health drinks

Frugoside 10 mg

15 g vitamin C

100 g vitamin E (powder)

19.75 g of iron lactate

3.5 g zinc oxide

3.5 g of nicotinic acid amide

0.2 g vitamin A

₁ 0.25 g of vitamin B

Vitamin B ₂ 0.3g

Purified water quantitative

After mixing the above ingredients according to the usual health drink manufacturing method, after stirring and heating at 85 ° C for about 1 hour, the resulting solution is filtered and acquired in a sterilized 2 liter container, sealed and sterilized, then refrigerated. It is used to prepare the health drink composition of the invention.

Although the composition ratio is prepared by mixing ingredients suitable for comparatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as the demanding class, the demanding country, and the use.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (8)

A pharmaceutical composition for preventing or treating cisplatin-resistant lung cancer in a patient overexpressing NRF2 comprising frugoside represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
delete According to claim 1,
The lung cancer is a non-small cell lung cancer selected from the group consisting of squamous cell carcinoma, adenocarcinoma and large cell carcinoma, a pharmaceutical composition for preventing or treating cisplatin-resistant lung cancer in a patient overexpressing NRF2.
delete According to claim 1,
The frugoside (Frugoside) or a pharmaceutically acceptable salt thereof is a pharmaceutical composition for preventing or treating cisplatin-resistant lung cancer in a patient in which NRF2 is overexpressed, which increases active oxygen in lung cancer cells.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating cisplatin-resistant lung cancer in a patient in which NRF2 is overexpressed, in which the pharmaceutical composition is treated in combination with cisplatin.
7. The method of claim 6,
The concentration ratio of the frugoside or a pharmaceutically acceptable salt thereof and the cisplatin is 1:5 to 1:100, in a mass ratio of 1:5 to 1:100, cisplatin-resistant lung cancer prevention or treatment of a patient overexpressing NRF2 pharmaceutical composition for use.
A health functional food for preventing or improving cisplatin-resistant lung cancer in a patient overexpressing NRF2 comprising frugoside represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.

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