KR102187951B1 - Pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, comprising an extract of Ganoderma lucidum and quercetin as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of Epstein-Barr virus (EBV)-positive gastric cancer, and the like, comprising an Epstein-Barr virus (EBV)-positive gastric cancer comprising an extract of antler ganoderma lucidum mushroom extract and quercetine as an active ingredient, wherein quercetin is a cell of EBV-positive gastric cancer cells. It has an effect of promoting apoptosis, and in the case of treatment by mixing with a low concentration of Nogak Reishi mushroom extract, there is an advantage that the apoptosis effect of EBV-positive gastric cancer tumors is more excellent than that of each single treatment. In addition, the toxicity of quercetin can be remarkably reduced by mixing quercetin and P. ganoderma lucidum extract, and it is expected to be used as a specific preventive or therapeutic agent for EBV infection diseases of the mixed composition of Quercetin extract and quercetin according to the present invention. . In addition, the composition of the present invention is expected to be used as a health functional food for the prevention or improvement of EBV-infected diseases, particularly EBV-positive gastric cancer.

Description

Pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, comprising an extract of Ganoderma lucidum and quercetin as an active ingredient ingredient}

The present invention relates to a pharmaceutical composition for the treatment of Epstein-Barr virus (EBV)-positive gastric cancer, and the like, comprising an extract of antler reishi mushroom and quercetine as an active ingredient.

Epstein-Barr virus (EBV) is a human gamma-1 herpesvirus, a life-long infectious opportunistic virus that affects more than 90% of the world's population. During the incubation period, most viruses are present in conjunction with chromatin, and are multicopy episomes in the nuclei of various tumor cells such as B cells, T cells, natural killer cells (NK cells) and epithelial cells (epithelial cells). ) Is reported to exist. During the incubation period, it can cause a variety of malignancies, such as Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disease, and malignant lymphoma (NK/T). -cell lymphoma), nasopharyngeal carcinoma, and gastric carcinoma have been reported.

As a reported cause of gastric cancer, about 10% of gastric cancer patients are diagnosed as being associated with EBV. In cases of EBV-positive gastric cancer, almost all gastric cancer cells are infected with EBV, and more than 50,000 cases of EBV-associated gastric cancer worldwide are reported annually. Therefore, gastric cancer associated with EBV is considered to be the most common case among tumors caused by EBV.

Recently, researchers working on gastric cancer classified gastric cancer into four subtypes in a paper published by the Cancer Genome Atlas Research Network (Nature 513, 202-209): EBV-tumor positive for Epstein. -Barr virus), microsatellite unstable tumors, genomically stable tumors and tumors with chromosomal instability. Among the four subtypes, EBV-positive gastric cancer has characteristics such as repeated mutation of PIK3CA, remarkable DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2), so other types of gastric cancer It has been reported to have distinctive features from the family. Therefore, since EBV-positive gastric cancer shows a remarkable DNA hypermethylation pattern in the genome, developing a drug or composition that induces DNA demethylation is an important factor in discovering a new treatment and diagnosis method for EBV-positive gastric cancer. It is expected to be available. Quercetin is a compound well known as a flavonoid component contained in licorice extract, and its IUPAC name is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromene. 4-one (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one). In fact, quercetin belongs as a kind of flavonol, but it is a kind of flavonoid that has a 3-hydroxyflavone backbone (3-hydroxy-2-phenylchromen-4-one) and contains licorice. It is known to be included as an active ingredient in various kinds of medicinal plants (Committee of Pharmacognosy Publication. (2013). Pharmacognosy. Pharmacognosy. (Seoul Korea: Dong Myoung), pp. 102-105).

Various studies have reported that quercetin has various bioactive effects such as antiviral activity, anti-asthma activity, anticancer activity, anti-inflammatory activity, and monoamine-oxidase inhibitor. Quercetin can impair oncoviral replication, and specifically, it is reported that it plays a role in inhibiting the secretion of HBsAg and HBeAg in cells infected with Hepatitis Virus B (HBV). In addition, quercetin has an effective inhibitory ability against HCV replication among most flavonoids, so it can be used together with interferon a as an anti-HCV therapeutic agent. In addition, quercetin and its analogs exhibit a strong inhibitory ability against HIV-1 reverse transcriptase, and studies to use it as an HIV treatment are also being conducted.

In addition, there are reports that quercetin can be used as an anticancer treatment. This is reported to be due to the antioxidant or anti-inflammatory activity of quercetin, and also has an activity capable of inhibiting the growth of cancer cells and inducing apoptosis (Dajas F. Life or death: neuroprotective and anticancer effects). of quercetin.Journal of ethnopharmacology. 2012; 143:383-396). However, specific evidence that quercetin has a protective effect in human cancer has not been studied, and in particular, whether quercetin exhibits anticancer activity for gastric cancer induced by EBV has not been studied.

In addition, nokgak ganoderma lucidum is a type of ganoderma lucidum karsten, which is known to have the effect of replenishing energy and blood when the body is weak, stabilizing the mind and body, and improving the digestive system. Through these effects, it is used for exhaustion (虛勞), heart system (心悸), symptoms of poor sleep, dizziness, mental fatigue, and old cough. As pharmacological action, central nervous system suppression, immunity enhancement, sleep time extension, blood pressure lowering, antitussive expectorant action, addictive hepatitis reduction effect, intestinal excitation, anti-tumor action (animal experiment), etc. have been reported, but for EBV infection diseases It has not been studied.

Accordingly, the present inventors have tried to develop an effective therapeutic agent for gastric cancer caused by infection with Epstein-Barr virus (EBV). As a result, quercetin induces soluble proliferation of EBV to kill infected cells and In addition to effectively inhibiting the horizontal infection of EBV progeny virus dissolved with normal cells, it effectively inhibits the growth of gastric carcinoma caused by EBV infection in vivo, and its anticancer activity is further enhanced by mixing with Nogak Reishi mushroom extract. It was improved and confirmed that the expression of EBV-related genes was suppressed, and it was confirmed that the mixed composition of Nogak Reishi mushroom extract and quercetin can be usefully used as an active ingredient of a pharmaceutical composition for treating gastric cancer among EBV infection diseases, and the present invention Completed.

Korean Registered Patent Publication 10-1432974

The present invention is to provide a composition for preventing, treating, or improving Epstein bar virus-positive gastric cancer, comprising a mixed composition of an extract of nogak ganoderma lucidum and quercetin 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 that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of Nogak Reishi mushroom as an active ingredient.

In addition, the present invention provides a health functional food composition for improving Epstein-Barr virus-positive gastric cancer, containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum as an active ingredient.

According to an embodiment of the present invention, the ankalicornia mushroom extract is water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and these It may be extracted with one or more solvents selected from the group consisting of a mixed solvent of.

According to another embodiment of the present invention, the Nogak Reishi mushroom extract may be contained in a concentration of 0.01 mg/ml to 0.10 mg/ml.

According to another embodiment of the present invention, the quercetin or a pharmaceutically acceptable salt thereof may be characterized in that it is contained in a concentration of 0.1 μM to 100 μM.

According to another embodiment of the present invention, the composition may induce apoptosis of Epstein-Barr virus-positive gastric cancer cells.

According to another embodiment of the present invention, the composition may induce lytic reactivation of Epstein-Barr virus.

According to another embodiment of the present invention, the extract of green gakura reishi mushroom may include Ganoderic Acid A (Ganoderic Acid A).

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of Epstein-Barr virus infection disease, containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving Epstein-Barr virus infectious diseases, containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum as an active ingredient.

In addition, the present invention provides a method for treating an Epstein-Barr virus infectious disease comprising administering to an individual a composition containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum as an active ingredient.

In addition, the present invention provides a use of a composition containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of Pseudomonas reticulum as an active ingredient for the treatment of Epstein-Barr virus infection.

According to an embodiment of the present invention, the Epstein-Barr virus infection disease may be EBV-positive gastric cancer.

The composition according to the present invention is a mixed composition in which quercetin is mixed with an extract of antler Ganoderma lucidum, and quercetin can be specifically used for diagnosis and treatment of EBV-positive gastric cancer, and a combination treatment of a low concentration of Ganoderma lucidum extract and quercetin In each case, the apoptosis effect of EBV-positive gastric cancer tumors is better than that of each treatment alone. In addition, the toxicity of quercetin can be remarkably reduced by mixing quercetin and P. ganoderma lucidum extract, and it is expected to be used as a specific preventive or therapeutic agent for EBV infection diseases of the mixed composition of Quercetin extract and quercetin according to the present invention. .

1A shows a schematic diagram of an anti-tumor assay performed using a xenograft mouse model and a high concentration (30 mg/kg) drug.
1B is a high concentration (30 mg/kg) of antler reishi mushroom extract (Ganoderma lucidum extracts, hereinafter GLE), a high concentration (30 mg/kg) of quercetin (QCT) in nude mice with tumors derived from MKN1-EBV, and It shows the appearance of a mouse when each of isoliquiritigenin (hereinafter referred to as ISL) was treated, a combination of GLE and QCT, and a combination of GLE and ISL were treated.
Figure 1c is a result of comparing the anti-tumor effect when a high concentration of GLE and QCT, respectively, and when a mixture of GLE and QCT is administered in an MKN1-EBV-derived tumor.
Figure 1d is a result of comparing the anti-tumor effect when a high concentration of each of GLE and ISL is administered and when a mixture of GLE and ISL is administered in MKN1-EBV-derived tumors.
Figure 2a shows a schematic diagram of an anti-tumor assay performed using a xenograft mouse model and low concentration (10 mg/kg) drug.
Figure 2b is a case of treatment with low concentration (10 mg/kg) of GLE, low concentration (10 mg/kg) of each of QCT and ISL in MKN1-EBV-derived tumors, a combination of GLE and QCT, and a combination of GLE and ISL. In case, this is the result of comparing the reduction in tumor volume.
2C is a result of comparing the anti-tumor effect of the administration of low concentrations of GLE and QCT, respectively, and administration of a mixture of GLE and QCT in MKN1-EBV-derived tumors.
Figure 2d is a result of comparing the anti-tumor effect when a low concentration of each of GLE and ISL was administered and a mixture of GLE and ISL was administered in an MKN1-EBV-derived tumor.
Figure 3a is a result of measuring the CD ₅₀ of GLE in SNU719 cells.
Figure 3b is a result of measuring the cytotoxic effect of the simultaneous treatment of 0.013 mg / mL GLE and various concentrations of QCT in SNU719 cells.
Figure 3c is a result of measuring the cytotoxic effect of simultaneous treatment with 0.96 μM QCT and various concentrations of GLE in SNU719 cells.
4A is a result of analysis of proapoptotic and antiapoptotic proteins through Western blot in SNU719 cells.
4B is a result of analysis of proapoptotic and antiapoptotic proteins in cytoplasmic and nuclear proteins derived from SNU719 cells through Western blot.
5A is a result of evaluating the stimulating effect on reactivation of EBV lysis by treating SNU719-BHLF1 luciferase cells with low concentration GLE and various concentrations of QCT.
5B is a result of evaluating the stimulating effect on reactivation of EBV lysis by treating SNU719-BHLF1 luciferase cells with low concentration QCT and various concentrations of GLE.
5C is a result of evaluating the stimulating effect on reactivation of EBV lysis by treating SNU719-BHLF1 luciferase cells with QCT or GLE of CD ₅₀ .
6A is a result of measuring EBV gene expression when simultaneously treated with 0.013 mg/mL GLE and various concentrations of QCT in SNU719 cells.
6B is a result of measuring EBV gene expression when simultaneously treated with 0.96 μM QCT and various concentrations of GLE in SNU719 cells.
7A is a result of measuring CD ₅₀ of Ganoderic Acid A (GAA) in SNU719 cells.
Figure 7b is a result of measuring the cytotoxic effect of simultaneous treatment with various concentrations of 0.49 mM GAA and QCT in SNU719 cells.
Figure 7c is a result of measuring the cytotoxic effect of simultaneous treatment with various concentrations of 0.96 μM QCT and GAA in SNU719 cells.
Figures 8a and 8b show the effect of the simultaneous treatment of GAA and QCT on the dissolution reactivation of EBV.
9A is a result of evaluating the effect of various concentrations of QCT and 0.49 mM GAA on EBV gene expression in SNU719 cells.
9B is a result of evaluating the effect of various concentrations of GAA and 0.96 μM QCT on EBV gene expression in SNU719 cells.

As described above, EBV infection has been reported as a carcinogenic cause of gastric cancer, but no research has been reported on a therapeutic agent for gastric cancer caused by EBV infection. Accordingly, the present inventors conducted a thorough study to find a therapeutic agent for gastric cancer caused by EBV infection. As a result, the cells of the SNU719 cells were mixed with quercetin's apoptosis-promoting effect in the EBV-positive gastric cancer cell line, SNU719 cell line. The present invention confirmed that the effect of quercetin to promote apoptosis is remarkably increased, and quercetin alone treatment in SNU719 cells increases the expression of EBV gene, whereas when mixed with P. ganoderma lucidum extract, the expression of EBV gene is suppressed. Was completed.

In one embodiment of the present invention, as a result of evaluating the effect of inhibiting tumor growth using high concentrations of GLE and QCT, it was confirmed that there was no significant difference in tumor size between single treatment and simultaneous treatment (see Example 2).

In another embodiment of the present invention, as a result of evaluating the effect of tumor growth inhibition using low concentrations of GLE and QCT, it was confirmed that the simultaneous treatment of GLE and QCT inhibited tumor development most effectively (see Example 3).

In another embodiment of the present invention, as a result of performing a cell viability test using GLE and QCT, it was confirmed that QCT exhibits a higher cytotoxic effect than GLE (see Example 4).

In another embodiment of the present invention, as a result of evaluating the apoptosis induction effect by GLE and QCT treatment, the addition of GLE to QCT significantly increased cleavage of PARP1 protein compared to that induced by QCT alone. Was confirmed (see Example 5).

In another embodiment of the present invention, it was confirmed that EBV lytic reactivation was induced by the simultaneous treatment of GLE and QCT (see Example 6).

In another example of the present invention, it was confirmed that EBV gene expression was upregulated by simultaneous GLE and QCT treatment (see Example 7).

In another embodiment of the present invention, it was confirmed that simultaneous treatment of GAA with low concentration QCT showed a decrease in cell viability and a strong cytotoxic effect (see Example 8).

In another embodiment of the present invention, the effect of dissolution and reactivation of EBV by simultaneous treatment of GAA and QCT was confirmed (see Example 9).

In another example of the present invention, it was confirmed that EBV lysis gene expression was upregulated by simultaneous GAA and QCT treatment (see Example 10).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the treatment of an Epstein-Barr virus (EBV) infectious disease containing quercetin or a pharmaceutically acceptable salt thereof, and an Epstein-Barr virus (EBV) infection as an active ingredient, and the EBV The infectious disease may be EBV positive gastric cancer.

In the present invention, "Ganoderma lucidum" is a type of Ganoderma lucidum, and its appearance resembles antlers of deer, and its appearance resembles a deer antler, and its appearance resembles a deer antler. It is also called nokgak youngji mushroom. It is known that the beta-glucan content (40%), which is known to have excellent anticancer effect, is more than twice that of the general reishi mushroom (19%), but the therapeutic effect of EBV infection is unknown.

The composition for the treatment or improvement of Epstein-Barr virus-positive gastric cancer of the present invention includes an extract of Nogak Reishi mushroom, and the extract of Noggak Reishi mushroom is 0.001 mg/ml to 1.00 mg/ml, preferably 0.01 mg/ml to 0.10 mg/ml. Ml, more preferably 0.01 mg/ml to 0.0532 mg/ml concentration, but is not limited thereto.

The nogak ganoderma lucidum extract of the present invention can be extracted using a conventional solvent under conditions of a normal temperature and pressure according to a conventional method known in the art for extracting an extract from a natural product. For example, in the present invention, the Ganoderma lucidum extract is a group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof It may be extracted using one or more solvents selected from, preferably, it may be extracted using ethanol, and more preferably, it may be extracted using 99% ethanol. In addition, a method of extracting the extract from Nogak Reishi mushroom may be extracted through various methods such as hot water extraction, cold needle extraction, reflux extraction, ultrasonic extraction, and stationary extraction, but is not limited thereto.

The prepared extract may be filtered, concentrated, or dried to remove the solvent, and filtration, concentration, and drying may be performed. For example, a filter paper or a reduced pressure filter may be used for filtration, a vacuum concentrator may be used for concentration, and a spray drying method, a freeze drying method, and the like may be performed for concentration, but are not limited thereto.

In addition, the extract extracted with the solvent may be further fractionated with a solvent selected from the group consisting of butanol, n-hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. .

In the present invention, "Quercetin" is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (2-(3,4-dihydroxyphenyl)-3 ,5,7-trihydroxy-4H-chromen-4-one; Quercetin; Quercetin; QCT) has the structure of the following [Formula 1];

[Formula 1]

In the present invention, "Quercetin" is preferably isolated from licorice (Glycyrrhiza uralensis) extract or a fraction thereof, but is not limited thereto.

The composition for the treatment or improvement of Epstein-Barr virus-positive gastric cancer of the present invention comprises quercetin, and the quercetin is contained in a concentration of 0.01 μM to 1000 μM, preferably 0.1 μM to 100 μM, more preferably 0.1 μM to 62 μM. However, it is not limited thereto.

In the present invention, "Epstein-Barr virus (EBV)" is also known as human herpesvirus 4 (HHV-4) and is a dsDNA virus. Diseases caused by or caused by EBV infection include infectious mononucleosis, Burkitt's lymphoma, Ho Chicken disease, and gastric cancer, and the present inventors described the present invention in EBV-positive cell line SNU719 cell line. According to the treatment of the mixed composition of SNU719 cells, overexpression of factors related to apoptosis and inhibition of expression of genes related to EBV proliferation were confirmed.

In the present invention, the "EBV infection disease" is a disease caused by EBV infection, for example, infectious mononucleosis, Burkitt's lymphoma, Ho Chicken's disease, and gastric cancer, and preferably gastric cancer.

In the present invention, "gastric cancer" or "EBV-positive gastric cancer" is preferably a gastric cancer caused by an Epstein-Barr virus (EBV) infection.

In the present invention, "prevention" refers to any action that inhibits or delays the occurrence, spread, or recurrence of EBV infection and EBV infection by administration of the composition of the present invention, and the EBV infection may be gastric cancer, but is limited thereto. It is not. In addition, in the present invention, "treatment" refers to any action in which the symptoms of the disease are improved or advantageously changed by administration of the composition of the present invention.

Accordingly, the present invention can provide a method for treating EBV infection and diseases caused by EBV infection, comprising administering to an individual a composition comprising an extract of P. ganoderma lucidum and quercetin or a pharmaceutically acceptable salt thereof, The disease caused by the EBV infection may be gastric cancer, but is not limited thereto.

In the present invention, the "individual" may be a mammal, such as a mouse, livestock, mouse, or human, and specifically, may be a companion dog, racehorse, human, etc., in need of treatment for EBV infection, and preferably human.

The quercetin can be used in the form of a pharmaceutically acceptable salt in the mixed composition of the extract and quercetin of the present invention, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Do. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous 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, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, quercetin is dissolved in an excess acid aqueous solution, and the salt is precipitated using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Can be manufactured. It can also be prepared by heating the same amount of quercetin and an acid or alcohol in water, and then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

The acid addition salt according to the present invention is prepared by a conventional method, for example, quercetin is dissolved in an excess acid aqueous solution, and the salt is precipitated using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Can be manufactured. It can also be prepared by heating the same amount of quercetin and an acid or alcohol in water, and then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be made 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. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

In addition, quercetin of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

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

When the composition of the present invention is used as a pharmaceutical, a pharmaceutical composition containing the Pseudomonas reticulum composition and quercetin or a pharmaceutically acceptable salt thereof as an active ingredient is formulated in various oral or parenteral dosage forms at the time of clinical administration. It may be administered, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like.These formulations include diluents (e.g., lactose, dexte) in addition to the active ingredients. Rose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or sodium salts thereof. Disintegrants or boiling mixtures and/or absorbents, colorants, flavors, and sweeteners.

The pharmaceutical composition containing quercetin of the present invention or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum as an active ingredient can be administered parenterally, and parenteral administration is subcutaneous injection, intravenous injection, intramuscular injection, or chest It depends on how I inject my injections. At this time, in order to formulate a formulation for parenteral administration, a pharmaceutical composition containing quercetin or a pharmaceutically acceptable salt thereof as an active ingredient is mixed in water with a stabilizer or buffer to prepare a solution or suspension, and this is an ampoule or vial It can be prepared in unit dosage form. The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances, which are conventional methods of mixing, granulation. It can be formulated according to the method of painting or coating.

In addition, the dosage of the composition of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and when based on an adult patient weighing 60 kg, generally It is 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, and may be dividedly administered once a day or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

In addition, the present invention provides a health functional food for improving gastric cancer, containing quercetin or a pharmaceutically acceptable salt thereof, and an extract of nogak ganoderma lucidum as an active ingredient.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, There are beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and all health functional foods in the usual sense are included.

Quercetin of the present invention or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum can be added to food as it is or can be used with other foods or food ingredients, and can be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined depending on the purpose of use (for improvement). In general, the amount of the compound in the health food may be added in 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional beverage composition of the present invention is not particularly limited to other ingredients other than those containing the compound of the present invention as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates, etc. I can. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, quercetin or a pharmaceutically acceptable salt thereof, and an extract of Nogak Reishi mushroom of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents, and thickening agents (cheese , Chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, the quercetin of the present invention or a pharmaceutically acceptable salt thereof, and an extract of P. ganoderma lucidum may contain flesh for the manufacture of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected from the range of 0.1 to about 20 parts by weight per 100 parts by weight of quercetin or a pharmaceutically acceptable salt thereof of the present invention, and P. ganoderma lucidum extract.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental method

Example 1.1. GLE Ready

GLE was obtained from the Korea Institute of Horticultural Science, Korea Mushroom Cultivation (KMCC). Fresh fruiting bodies of G. lucidum were harvested from G. lucidum KMCC 02955 strain. Molecules were extracted from the fruiting bodies at 25° C. for 3 days using a mixture of 30% ethanol and 70% sterile distilled water, and the extract was filtered, concentrated, Sterilized and dried.

GLE was dissolved in sterile distilled water to prepare a GLE stock solution (8 mg/mL) and stored at -20°C until use. GAA is Sigma-Aldrich Co. (St. Louis, Mo, USA) was purchased and dissolved in dimethyl sulfoxide (DMSO) to prepare a GAA stock solution (100 mM) and stored at -20°C until use.

Example 1.2. Cell lines and reagents

SNU719 (EBVaGC) and MKN1-EBV (EBVaGC), gastric cancer cell lines containing the EBV genome as an episome, are 10% fetal calf serum (Hyclone, MA, USA), antibiotics / antibacterial agents (Gibco, MD, USA) , GlutaMAX® (Gibco, MD, USA) and 25 mmol/mL HEPES (Sigma-Aldrich, MO, USA) added Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, MA, USA) and 5% CO ₂ , It was cultured in an incubator under conditions of 95% humidity and 37°C.

Example 1.3. Xenograft ( xenograft ) Chemotherapy in mouse model

A nude mouse (female, 5 weeks old, Raonbio Co., Ltd., Seoul, Korea) was used as a xenograft animal model for evaluation of antitumor effects. Mice were individually housed in a controlled environment free of pathogens (23-27°C under 12 hours light / 12 hours dark cycle) and were provided with food and water freely.

Mice were first divided into two groups (n = 30), and 5 x 10 ⁶ MKN1-EBV cells were subcutaneously implanted on the back side of the right hind limb of the two groups of mice. After 14 days, one group was used to test the anticancer effect upon simultaneous treatment with relatively high concentrations of GLE and QCT or ISL, and the other group was used to test the synergistic effect upon simultaneous treatment with relatively low concentrations of GLE and QCT or ISL. Became.

Relative low and high concentrations were defined based on previous studies (Cordyceps militaris extract 100 mg/kg, quercetin and isoliquiritigenin. 30 mg/kg). Mice in the high concentration group administered with water orally with water were GLE (30 mg/kg), QCT (30 mg/kg), ISL (30 mg/kg), GLE + QCT (30 mg/kg + 30 mg/kg). ) Or GLE + ISL (30 mg/kg + 30 mg/kg), orally, GLE (10 mg/kg), QCT (10 mg/kg), ISL (10 mg/kg) orally administered at a low concentration. ), GLE + QCT (10 mg/kg + 10 mg/kg), or GLE + ISL (10 mg/kg + 10 mg/kg) was repeatedly administered 10 times every 2 days. Tumor size, weight of mice, and survival rate of mice were measured every 2 or 3 days from 23 to 30 days after drug administration.

After identifying the tumor, the size of the tumor was measured using a standard caliper, and it was calculated using the tumor size = [tumor length (mm) × tumor width (mm) ² ]/2. After the tumor size reached 2000 mm ³ , Mice were euthanized and tumors were harvested.

Example 1.4. Cytotoxicity assay

The cytotoxic effects of GLE and GAA in SNU719 cells were evaluated by performing a cytotoxicity assay according to the manufacturer's protocol using cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan).

To summarize the protocol, 100 μl of cell suspension (1 × 10 ⁴ cells/well) was seeded in 96-well plates and the next day GLE or GAA was added to various concentrations (0-2000 μg/mL for GLE, 0- for GAA). 5000 ug/mL). For 48 hours after treatment, 10 µl of CCK-8 solution was added to each sample, and the samples were incubated again for 3 hours, and then at 450 nm using an enzyme-linked immunosorbent assay reader. The absorbance of the cell suspension was measured.

Example 1.5. Cell viability analysis

The effects of GLE, QCT and GAA on SNU719 cell viability were tested according to the manufacturer's protocol using the MUSE count & viability kit (Merck Millipore, Darmstadt, Germany).

To summarize the protocol, cells (2 × 10 ⁶ cells/well) were seeded in 6-well plates and GLE and QCT or a mixture of GLE and GAA were mixed at various concentrations (0-0.053 mg/mL for GLE, 0 for QCT). -62 μM and 0-0.49 μM for GAA) were treated for 48 hours, and the negative control group was treated with the same volume of DMSO for 48 hours. Treated cells were treated with trypsin and harvested and resuspended with 10 ⁶ -10 ⁷ cells/mL of fresh serum. The cell suspension (20 μL) was mixed with 380 μL of MUSE count & viability reagent, and then incubated at 25° C. for 5 minutes, and then cell viability was measured using a MUSE cell analyzer (Millipore, Burlington, MA, USA).

Example 1.6. Luciferase ( Luciferase ) analysis

SNU719 cells were transfected using a neon system (Invitrogen, Carlsbad, CA, USA) having a luciferase reporter configuration, containing the Renilla luciferase gene transcribed by the promoter of the EBV soluble gene, BHLF1. Subsequently, the transfected SNU719 cells were selected with blasticidin (Invitrogen) for about 30 days, and SNU719-BHLF1 luciferase cells were established.

SNU719-BHLF1 luciferase cells at 37°C for 48 hours at various concentrations (0-0.053 mg/mL for GLE, 0-62 μM for QCT, and 0-0.49 μM for GAA) of GLE and QCT, or GLE and GAA. Treated with the mixture. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's protocol.

Example 1.7. Western Blot analysis

To evaluate the proapoptotic effect of co-treatment with GLE and QCT or ISL, Western blot was performed at 37°C for 48 hours at various concentrations of GLE (0-0.053 mg/mL) and QCT (0-62 μM). This was done using SNU719 cells treated with a mixture of. Total protein was harvested using trypsin, and cells (10 ⁷ ) using 100 μL of reporter lysis buffer (Promega, Madison, WI) added with a protease inhibitor and phenylmethylsulfonyl fluoride. Was dissolved and further crushed through sonication (3min, 30s on/off pulses). In addition, the cytoplasm and nuclear proteins of the fractionated SNU719 cells were additionally recovered from the cell lysate using a NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific, Waltham, MA, USA). The obtained total protein, cytoplasm, and nuclear protein contents were measured using Bradford analysis, and the same amount of protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moved to the membrane. The membrane was probed with antibodies against the protein of interest. Anti-poly [ADP-ribose] polymerase 1 (PARP1), anti-Bcl-2, anti-Bax, anti-caspase3, anti-cytochrome Anti-cytochrome C (C), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and anti-proliferative nuclear antigen (PCNA) antibodies were tested in Cell Signaling Technology (Danvers, MA, USA). It was purchased and used to detect the protein. The expression patterns of GAPDH and PCNA were used as internal controls, and the antibody-binding protein was visualized by an enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Chicago, IL, USA), and the membrane was stripped, followed by other antibodies. It was re-robe (reprobe) again and the used antibody is shown in Table 1 below.

Manufacture Catalog Number 1st Antibody Anti-PARP1 Cell Signaling Technology 9542S Anti-Bcl2 Cell Signaling Technology 2870S Anti-Bax Cell Signaling Technology 5023S Anti-cleaved caspase3 Cell Signaling Technology 9664S Anti-cytochrome C Cell Signaling Technology 11940S Anti-GAPDH Cell Signaling Technology 2118S 2nd Antibody goat anti-rabbit IgG-HRP Santa Cruz Biotechnology

Example 1.8. qRT - PCR (Quantitative reverse transcription- PCR )analysis

To evaluate the effect of co-treatment of QCT and GLE or GAA on gene expression, qRT-PCR analysis was performed using SNU719 cells treated with QCT and GLE or QCT and GAA. RNA was isolated from cells using an RNeasy mini kit (Qiagen, Germantown, MD, USA), and purified RNA was converted to cDNA using superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and is specific for EBV genes. Typical primers were used, and information on the used primers is shown in Table 2 below.

Gene Primer Sequence (5'→ 3') Sequence number EBNA1 Forward TAC AGG ACC TGG AAA TGG CC One Reverse TCT TCT TTG AGG TCC ACT G 2 LMP1 Forward TCT GCC CTC GTT GGA GTT AGA 3 Reverse CCC TCA ACA AGC TAC CGA TGA T 4 EBER Forward CCT ACG CTG CCC TAG AGG TTT 5 Reverse ACC GAA GAC GGC AGA AAG C 6 LMP2 Forward AGC TGT AAC TGT GGT TTC CAT GAC 7 Reverse GCC CCC TGG CGA AGA G 8 BNRF1 Forward GCG GCC TTC ACG AAT GC 9 Reverse GGA ACG TGT TGT CCC TAA CCT C 10 BRLF1 Forward GTG TCA CTG TTG CCC GAG TC 11 Reverse GAA GCC CGG TGC CCA AAG 12 BLLF1 Forward AGA ATC TGG GCT GGG ACG TT 13 Reverse ACA TGG AGC CCG GAC AAG T 14 BZLF1 Forward CTT GGC CCG GCA TTT TCT 15 Reverse ACG ACG CAC ACG GAA ACC 16

Example 2. High concentration GLE And QCT Tumor growth inhibition

Triterpenoids isolated from GLE can inhibit telomerase, and QCT can induce strong EBV lysis and reactivation. Therefore, it was investigated whether the simultaneous treatment of GLE and QCT could exert a synergistic effect on the inhibition of EBV-positive tumor development. To test this hypothesis, a xenograft model using human EBVaGC MKN1-EBV cells was used (see Fig. 1A), MKN1-EBV cells were injected into nude mice, GLE (30 mg/kg), QCT and ISL. It was administered orally as single treatment or simultaneous treatment.

As a result, tumor development was observed on the 23rd day after oral administration as shown in FIG. 1B, and tumor size of the thigh of the xenograft mouse was measured as shown in FIGS. 1C and 1D. Tumor development was significantly suppressed by GLE, QCT and ISL alone treatment, and tumors of mice treated with a single drug showed mutations up to 300 mm ³ , but tumors of the control group developed up to 700 mm ³ . In addition, simultaneous treatment of GLE and QCT or ISL showed a significant delay in tumor development, and tumors of mice receiving concurrent treatment did not develop well up to 250 mm ³ . However, no significant difference was observed in the tumor size of MKN1-EBV between tumors of mice treated with single treatment (30 mg / kg) and concurrent treatment (30 mg / kg each). These results indicate that simultaneous treatment of GLE and QCT or ISL at high concentrations did not synergistically inhibit EBVaGC development.

Example 3. Low concentration GLE And QCT Tumor growth inhibition

Co-treatment of 30 mg/kg of GLE and QCT in constant Example 2 resulted in a non-significant reduction in tumor size compared to the case of single GLE treatment at 30 mg/kg. Therefore, since the possibility of cytotoxicity cannot be ruled out for the simultaneous treatment of 30 mg/kg to show a significant synergistic effect, a lower concentration of the compound was used. That is, GLE, QCT, and ISL (10 mg / kg) were orally administered to nude mice by single treatment or simultaneous treatment (see Fig. 2A).

As a result, tumor development was observed 30 days after tumor injection as shown in FIG. 2B, and tumor size of the thigh of the xenograft mouse was measured as shown in FIGS. 2C and 2D. QCT and ISL alone treatment at 10 mg/kg significantly delayed tumor incidence, and tumors of mice treated with QCT or ISL alone grew up to 700 mm ³ and 600 mm ³ , respectively, but GLE alone and control mice. Tumors grew up to 1200 mm ³ . Interestingly, co-treatment of 10 mg/kg of GLE and QCT showed significant inhibition of tumor development compared to either GLE or QCT single treatment. On the other hand, the simultaneous treatment of GLE and QCT suppressed tumor development by 400 mm ³ , whereas the simultaneous treatment of 10 mg/kg of GLE and ISL showed a significant difference in suppressing tumor development compared to the case of GLE or ISL single treatment. Did not show. These results imply that the simultaneous treatment of GLE and QCT at low concentrations can exhibit a synergistic antitumor effect on EBVaGC development.

Example 4. GLE And QCT Reduced cell viability due to simultaneous treatment

As observed in Example 3 above, the simultaneous treatment of GLE and QCT at low concentrations exhibited a synergistic anti-tumor effect, so the potential molecular mechanisms were further studied. First, the 50% cytotoxic dose (CD50) of GLE in SNU719 cells was determined to be 0.0532 mg/mL (see FIG. 3A). Subsequently, a cell viability test was performed according to the method of Example 1.5 to evaluate whether simultaneous treatment of GLE and QCT can exhibit a synergistic cytotoxic effect in SNU719 cells.

As a result, as shown in Fig. 3b, the addition of GLE (0.0133 mg/mL) to QCT at a series of increasing concentrations did not significantly increase cell death compared to QCT alone. As a result of simultaneous treatment with 0.96 μM QCT and 0.013 mg/mL GLE, cell death was reduced by 0.59 times compared to the cell death by 0.96 μM QCT single treatment. In contrast, as shown in Figure 3c, the addition of QCT (0.96 μM) to GLE at a series of increasing concentrations slightly increased apoptosis compared to GLE alone, and 0.013 mg/mL GLE and 0.96 μM QCT simultaneous treatment was 0.013 It induced a 1.33 fold increase in apoptosis compared to that induced by mg/mL GLE single treatment. Taken together, the above results mean that QCT can exhibit a higher cytotoxic effect than GLE.

Example 5. With GLE QCT Cell death by simultaneous treatment ( apoptosis ) Judo

Since the addition of QCT improved the cytotoxicity of GLE in SNU719 cells, it was investigated whether cytotoxicity was related to apoptosis in SNU719 cells, and specifically, Western blot was performed according to the method of Example 1.7.

As a result, it was confirmed that the apoptosis of SNU719 cells significantly increased by the addition of GLE, as shown in FIG. 4A. The addition of GLE (0.0133 mg/mL) to QCT at a series of increasing concentrations promoted PARP1 cleavage, inhibited Bcl-2 expression, and increased caspase 3 and cytochrome C expression.

In addition, in order to confirm the effect of simultaneous treatment of GLE and QCT on PARP1 cleavage, nuclear proteins were isolated from cytoplasmic proteins, and Western blot was performed according to the method of Example 1.7.

As a result, as shown in Fig. 4b, the addition of GLE increased QCT-induced PARP1 cleavage in a concentration-dependent manner. The addition of GLE (0.0133 mg/mL) to QCT at a series of increasing concentrations significantly increased cleavage of the PARP1 protein compared to that induced by QCT alone. Results inconsistent with the cell viability analysis results (see FIG. 3) mean that QCT may exhibit a more potent cell killing effect than GLE.

Example 6. GLE And QCT Due to simultaneous processing EBV Dissolution reactivation ( lytic reactivation) induction

Apoptosis can be activated through EBV lysis reactivation, and BZLF1, a representative EBV soluble protein, induces apoptosis through indirect inhibition of nuclear factor κ-B (NF-κB) family protein, down-regulation of p65 and CD74. . Therefore, it was investigated whether the increase in QCT-mediated apoptosis by GLE is related to the reactivation of EBV lysis.

First, luciferase analysis was performed according to the method described in Example 1.6. The structure of the luciferase reporter contains a Renilla luciferase gene transcribed under the promoter of BHLF1, an EBV soluble gene, and SNU719-BHLF1 luciferase cells were treated with different concentrations of QCT and 0.0133 mg/mL GLE. As a result, as shown in FIG. 5A, the addition of GLE to a series of increasing concentrations of QCT significantly increased luciferase activity in low concentrations of QCT. Simultaneous treatment of 0.96 μM QCT and 0.0133 mg/mL GLE improved luciferase activity by 1.9 times compared to 0.96 μM QCT single treatment.

In addition, SNU719-BHLF1 luciferase cells were treated with different concentrations of GLE and 0.96 μM QCT. As a result, as shown in FIG. 5B, the addition of QCT to a series of increasing concentrations of GLE did not improve GLE-mediated EBV lysis reactivation at low GLE concentrations.

In addition, luciferase activity was compared in SNU719 cells treated with QCT or GLE of CD ₅₀ . As a result, as shown in Figure 5c, compared to the control group, QCT (62 μM) single treatment increased luciferase activity by 22.92 times, and when simultaneously treated with 62 μM QCT and 0.0133 mg/mL GLE, 62 μM QCT single treatment The luciferase activity increased 1.99 times more than that. However, 0.053 mg/mL GLE alone increased luciferase activity by 2.33 times compared to the control, and as a result of simultaneous treatment with 0.053 mg/mL GLE and 0.96 μM QCT, the luciferase activity increased 2.59 times more than QCT alone. did. Taken together, the above results mean that the addition of GLE during QCT treatment may be more effective in inducing dissolution reactivation of EBV.

Example 7. GLE And QCT Up-regulated by simultaneous processing EBV Gene expression

Since simultaneous treatment of GLE and QCT increases the activity of the EBV lytic gene promoter, we investigated whether it is possible to increase EBV lytic gene expression to induce EBV lytic gene reactivation.

First, after simultaneous treatment with GLE and QCT, qRT-PCR analysis was performed according to the method described in Example 1.8 to determine EBV gene expression, and SNU719 cells were treated with different concentrations of QCT and 0.0133 mg/mL GLE. As a result, as shown in FIG. 6A, the overall expression levels of EBV potential and soluble genes were significantly increased after simultaneous treatment with low concentrations of GLE and QCT. Interestingly, gene expression of EBNA1, LMP1, EBBER and BRLF1 reached a maximum after adding 0.0133 mg/mL of GLE, and decreased with increasing QCT concentration.

In addition, SNU719 cells were treated with different concentrations of GLE and 0.96 μM QCT. As a result, as shown in FIG. 6B, the potential of EBV and the overall expression level of the lytic gene significantly increased after treatment with low concentrations of QCT and GLE. Gene expressions of EBNA1, LMP1, EBBER, BRLF1, BNRF1, and BLLF1 reached a maximum at 0.96 μM after QCT addition and decreased with increasing GLE concentration. Interestingly, co-treatment of QCT and GLE increased BZLF1 expression until a GLE concentration of 0.05 mg/ml was reached. Taken together, these results indicate that QCT and GLE are almost equally effective in upregulating EBV lytic gene expression.

Example 8. GAA And QCT Reduced cell viability due to simultaneous treatment

GLE contains GAA and GAF as major bioactive compounds. According to prior studies, GLE contains 0.43 mg of GAA per 1 mg of fruiting body of G. lucidum strain KMCC 02955, and GAA can be regarded as an index for selecting a high-quality fruiting body for cancer treatment. In addition to its anti-tumor effect, GAA has a strong antiviral effect against EBV and HIV. Therefore, it was expected that the simultaneous treatment of GAA and QCT would result in enhanced antiviral and antitumor effects. To test this hypothesis, first, the CD ₅₀ value of GAA in SNU719 cells was determined to be 1.96 mM (see Fig. 7A). Subsequently, a cell viability test was performed according to the method described in Example 1.5 to investigate whether the simultaneous treatment of GAA and QCT could exert a synergistic cytotoxic effect in SNU719 cells.

As a result, as shown in Fig. 7b, the addition of 0.49 mM GAA in QCT at a low concentration significantly increased cell death by 2.73 times compared to that induced by QCT alone. In contrast, as shown in Fig. 7c, the addition of 0.96 μM QCT at the low concentration of GAA did not induce a significant increase in cell death compared to that induced by GAA alone (1.18 times). Taken together, the above results mean that a stronger cytotoxic effect can be exhibited when GAA co-treatment with low concentration QCT.

Example 9. GAA And QCT By simultaneous processing EBV Dissolution reactivation

Since the addition of GLE enhanced the dissolution reactivation of QCT-mediated EBV, a luciferase assay was performed according to the method described in Example 1.6 to investigate whether the addition of GAA could also enhance dissolution reactivation of EBV. . First, SNU719-BHLF1 luciferase cells were treated with different concentrations of QCT and GAA, and luciferase assay was performed 48 hours after treatment.

As a result, as shown in FIG. 8A, the addition of 0.49 mM GAA to a series of increasing concentrations of QCT increased luciferase activity by 1.83 times compared to that induced by QCT alone. In contrast, addition of 0.96 μM QCT to a series of increasing concentrations of GAA, as shown in FIG. 8B, did not cause a significant increase in luciferase activity compared to that induced by GAA alone (1.07 fold).

Taken together, the above results mean that the addition of GAA during QCT treatment may be more effective in inducing dissolution reactivation of EBV.

Example 10. GAA And QCT By simultaneous processing EBV Upregulation of gene expression

After the addition of GAA increased the activity of the EBV soluble gene promoter, it was further tested whether the addition of GAA could increase and regulate EBV soluble gene expression to induce EBV lysis reactivation.

qRT-PCR analysis was performed with the method described in Example 1.8 to determine the expression of EBV gene in SNU719 cells treated with different concentrations of QCT and GAA.

As a result, as shown in FIG. 9A, the addition of 0.49 mM GAA to a series of increasing concentrations of QCT induced marked upregulation of EBNA1 and BZLF1 gene expression depending on the concentration of QCT. In particular, the addition of GAA increased BZLF1 expression by 1201.0 times compared to 0.96 μM QCT alone.

Similarly, qRT-PCR analysis was performed to measure the expression of the EBV lytic gene in SNU719 cells treated with various concentrations of GAA and 0.96 μM QCT.

As a result, as shown in FIG. 9B, when 0.96 μM QCT was added to a series of increasing concentrations of GAA, BZLF1 (20.4 times) was expressed in a GAA concentration-dependent manner, but EBNA1 did not. Taken together, the results indicate that the addition of GAA during QCT treatment may be more effective in upregulating BZLF1 expression.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to 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 limiting.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, comprising an extract of Ganoderma lucidum and quercetin as an active ingredient <130> MP18-241 <150> KR 10-2017-0156679 <151> 2017-11-22 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EBNA1 Forward Primer <400> 1 tacaggacct ggaaatggcc 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EBNA1 Reverse Primer <400> 2 tcttctttga ggtccactg 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LMP1 Forward Primer <400> 3 tctgccctcg ttggagttag a 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LMP1 Reverse Primer <400> 4 ccctcaacaa gctaccgatg at 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EBER Forward Primer <400> 5 cctacgctgc cctagaggtt t 21 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> EBER Reverse Primer <400> 6 accgaagacg gcagaaagc 19 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LMP2 Forward Primer <400> 7 agctgtaact gtggtttcca tgac 24 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> LMP2 Reverse Primer <400> 8 gccccctggc gaagag 16 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> BNRF1 Forward Primer <400> 9 gcggccttca cgaatgc 17 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BNRF1 Reverse Primer <400> 10 ggaacgtgtt gtccctaacc tc 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BRLF1 Forward Primer <400> 11 gtgtcactgt tgcccgagtc 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BRLF1 Reverse Primer <400> 12 gaagcccggt gcccaaag 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BLLF1 Forward Primer <400> 13 agaatctggg ctgggacgtt 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BLLF1 Reverse Primer <400> 14 acatggagcc cggacaagt 19 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BZLF1 Forward Primer <400> 15 cttggcccgg cattttct 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> BZLF1 Reverse Primer <400> 16 acgacgcaca cggaaacc 18

Claims (14)

Quercetin or a pharmaceutically acceptable salt thereof at a concentration of 0.1 μM to 62 μM, and an Epstein-Barr virus (Epstein-) containing as an active ingredient an extract of P. ganoderma lucidum at a concentration of 0.01 mg/ml to 0.0532 mg/ml Barr virus; EBV) A pharmaceutical composition for the treatment of benign gastric cancer.
According to claim 1
The Pseudomonas Ganoderma lucidum extract is at least one selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof A pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, characterized in that extracted with a solvent.
delete delete The method of claim 1,
The composition is a pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, characterized in that to induce apoptosis (apoptosis) of Epstein-Barr virus-positive gastric cancer cells.
The method of claim 1,
The composition is a pharmaceutical composition for the treatment of Epstein-Barr virus-positive gastric cancer, characterized in that to induce lytic reactivation of Epstein-Barr virus.
The method of claim 1,
Wherein the nokgak ganoderma lucidum extract is characterized in that it contains Ganoderic Acid A (Ganoderic Acid A), Epstein-Barr virus positive gastric cancer treatment pharmaceutical composition.
For improvement of Epstein-Barr virus (EBV) positive gastric cancer, containing as an active ingredient Quercetin at a concentration of 0.01 mg/ml to 0.0532 mg/ml and Quercetin at a concentration of 0.1 μM to 62 μM Health functional food composition.
According to claim 8
The Pseudomonas Ganoderma lucidum extract is at least one selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof Epstein-Barr virus positive gastric cancer improvement health functional food composition, characterized in that extracted with a solvent.
delete delete The method of claim 8,
The composition is characterized in that inducing apoptosis (apoptosis) of Epstein-Barr virus-positive gastric cancer cells, Epstein-Barr virus-positive health functional food composition for improving gastric cancer.
The method of claim 8,
The composition is a health functional food composition for improving Epstein-Barr virus-positive stomach cancer, characterized in that to induce lytic reactivation of Epstein-Barr virus.
The health functional food composition according to claim 8, wherein the extract of Nogak Reishi mushroom comprises Ganoderic Acid A (Ganoderic Acid A), Epstein-Barr virus-positive gastric cancer improvement.

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