KR101236233B1 - Pharmaceutical compositions and health functional foods compositions for the improvement of liver functions containing the extract of Youngia denticulata, fraction of thereof or compound isolated therefrom as an active ingredient - Google Patents

Abstract

The present invention is a young indel ( Youngia denticulata ) extract, fractions thereof or compounds isolated therefrom, chlorogenic acid, 3,5-di-o-caffeoylquinic acid (3,5-Di-O-caffeoylquinic acid), ganodermaid A Pharmaceutical composition for preventing cancer or improving liver function, comprising as an active ingredient at least one compound selected from the group consisting of (Youngiaside A), Youngiaside B, and Youngiaside C The present invention relates to an anti-oxidant response according to the present invention, wherein the extract of perilla extract, a fraction thereof or a compound separated therefrom are quinone reductase (QR), which is an anti-cancer indicator, and an antioxidant reaction factor (Antioxidant response). It enhances the activity of liver detoxifying enzymes including element, ARE), is not cytotoxic, and protects the liver from liver damage induced by ethanol and acetaminophen. The composition according to the invention may be useful in medicine, health supplements for cancer prevention or improvement of liver function.

Description

Pharmaceutical compositions and health functional foods compositions for the improvement of liver functions containing liver extract extract, fractions thereof or compounds separated therefrom as active ingredients the extract of Youngia denticulata, fraction of lilies or compound isolated there from as an active ingredient}

The present invention is a young indel ( Youngia denticulata ) extract, a fraction thereof or a compound isolated therefrom relates to a composition for preventing cancer or improving liver function, comprising, in more detail, an excellent detoxifying enzyme activity and no cytotoxicity. At least one compound selected from the group consisting of extracts, fractions or chlorogenic extracts, 3,5-di-o-cafeoylquinic acid, ganodermaid A, ganodermaid B and ganodesiac C as an active ingredient It relates to a pharmaceutical composition for preventing cancer or improving liver function, and a health functional food composition.

Even after more than 30 years of cancer research, the incidence of cancer continues to increase due to environmental pollution and poor dietary habits, causing about 10 million people worldwide each year. The World Health Organization (WHO) is one of the leading causes of death. Cancer is counted as one. Cancer is a middle-aged disease, and it is inevitable that more than 70% of new clinical cases will be diagnosed at age 60 or older and the incidence of cancer increases as the world population ages. According to the Ministry of Health and Welfare, about 100,000 cancer patients occur every year.

Common treatments for cancer include surgery, radiation, and chemotherapy. In order to reliably treat cancer, it is necessary to diagnose and operate cancer early, and to combine radiation or chemotherapy. However, when the diagnosis of cancer is delayed or the cancer has spread, the treatment methods are complicated and the mental and economic burden of the treatment is much greater. Therefore, chemical cancer prevention (cancer chemoprevention) that prevents normal cells from being converted into cancer cells using safe chemicals and development of diagnostic methods to detect cancer early are important areas of recent cancer research.

The field of anti-cancer is the field of treating cancer by killing previously generated cancer cells and the field of preventing cancer by inhibiting the occurrence of carcinogens by reacting with intracellular targets or reaching targets or by inhibiting the occurrence of cancer by releasing carcinogens in vitro. Can be divided into: In the case of a substance known to be used for cancer treatment, there may be a case in which it cannot be used for cancer prevention. On the contrary, there is a substance that can be used for cancer prevention but cannot be used for cancer treatment. The trend is approaching.

Conventional anticancer drugs have been used for the treatment of cancer, which has the disadvantage of showing serious toxicity and side effects by damaging not only cancer cells but also normal cells.

In contrast, the composition for cancer prevention is a new strategy of cancer research to prevent the conversion of normal cells into cancer cells by artificially inhibiting, delaying or reversing the carcinogenesis process using safe chemicals that are relatively non-toxic. In order to be a candidate for a chemical cancer prevention substance, the compound should be non-toxic or weak and vegetable compounds are mainly used except for some vitamin metabolites because chemicals that can be easily separated or synthesized from natural products are used for widespread use. Sur YJ, Nature Rev Cancer , 3, 768-780, 2003).

Cancer has multiple stages, such as initiation, promotion, progression and metastasis. Chemical cancer prevention aims primarily at suppressing cancer initiation and promotion. It is divided into two types according to the mechanism. That is, blocking agents and carcinogens that block the carcinogens from reaching the target cells or attack DNA of the target cells through metabolic activation to induce structural damage and mutation. It is a suppressing agent that inhibits the process of exposing and initiating previously developed cells into benign and malignant cancers through promotion and progression (Surh YJ, Nature). Rev Cancer , 3, 768-780, 2003).

Blocking agents include diallyl sulfide, isothiocyanate, and ellagic acid, inhibitors of cytochrome P-450 enzymes. Phenylethyl isothiocyanate, sulforaphane, oltipraz, and the like, which are inducers of a phase detoxification enzyme, are known.

Suppressing agents include DFMO as a polyamine metabolism inhibitor, retinoid family as a cell differentiation promoter, genistein as a protein kinase C inhibitor, and EGCG as an oxidative DNA damage inhibitor. Known.

Phase II detoxification enzymes have been shown to exhibit specific cytotoxicity against various cancer cells and have protection against DNA damage caused by carcinogens, mutagenic substances, and reactive oxygen species such as those having anticancer function. Of the two-phase detoxifying enzymes, quinone reductase (QR) is a type of second-phase enzyme mainly produced in hepatocytes. Prevents tumoration and makes carcinogens poisonous. More specifically, quinone reductase is an enzyme that blocks interaction with DNA by mutations or carcinogens, and is an enzyme that catalyzes the reduction of quinones using NADP (H). A homodimer having a molecular weight of 54,000 Da and two identical structures, each of which consists of 273 amino acids, and the active site contains flavin adenine dinucleotide (FAD), It is called menadione reductase, vitamin K reductase, DT-diaphorase, and the like. Therefore, quinone reductase improves liver function by enhancing liver detoxification function, and can prevent cancer by blocking the action of carcinogens that can cause cancer in advance.

Gene regulation of the detoxification enzyme system by chemical cancer prevention has been reported to involve two regulators, Antioxidant response element (ARE) and Xenobiotics-responsive element (XRE) (Okey AB et. al . , Toxicol Lett , 70, 1-22). Among them, the antioxidant reaction factor (ARE) regulates the induction of the biphasic detoxifying enzyme system, so it can be used to test the effect of a single function inducer.

Through the mechanism of enhancing the detoxification function of the liver through the enhancement of the activity of the two-phase detoxification enzymes it can protect the liver by preventing liver damage due to ethanol intake. On the other hand, acetaminophen, a drug that is metabolized into quinone compounds in vivo, is a representative antipyretic analgesic drug frequently used in place of aspirin. However, long-term administration of acetaminophen results in severe hepatotoxicity (AJ Ware et al., Int. Med., 88: 267 (1978); and HL Bonkowsky et al., Lancet, 2: 1016 ( 1978). Enhancement of liver detoxification enzymes, including quinone reductase, can protect the liver from hepatotoxicity caused by quinone-based drugs such as acetaminophen.

At present, attempts are being made to find a composition for preventing cancer or improving liver function that induces the activity of the detoxifying enzyme system worldwide, and a study for separating a natural composition from plants is underway. However, in Northeast Asia, including Korea, research is mainly limited to medicinal plants, and research on wild vegetables, vegetables, and fruits native to each country has not been reported until now. The present inventors also endeavored to develop a substance having a purpose for preventing cancer or improving liver function from plants that have not been known at all, and among them, it has become an interest in wild ginseng native to Korea.

Youngia denticulata is a perennial or biennial plant of the dicotyledonous prunus asteraceae. It grows in the dry place of the mountains and fields. The height is 30 ~ 70 ㎝ and the stem is thin and purple. Branches spread and squeeze out juice. The leaves on the roots are spatula-shaped, fall off when the flowers bloom, and the leaves on the stems are shifted, without petioles. The leaf length is 6-11 cm, the butterfly is 3-7 cm and the tip is dull. The lower part is like an ear, half the stem is wrapped around, and the sawtooth is sparse at the edge. Flowers bloom in yellow in August-September and doehwa are about 15 ㎜ in diameter and run in the order of inflorescence. When flowers bloom, they stand upright, hang down, and sag below. The guns are shaped like narrow barrels, and the pieces of the guns are long oval-shaped bassos arranged in two rows. Fruits are achene, brown or black, with 12 ridges. The tube is white, about 3.5 mm long, eats young shoots as herbs, and is distributed in Korea, Japan, China, and India.

However, there is no known cancer preventing use or liver protection effect of the extracts of the perilla extract, fractions thereof or compounds isolated therefrom, and no specific pathway is disclosed in detail. Accordingly, the present inventors are searching for detoxifying enzyme activity in edible natural products to develop a chemical cancer preventive agent or liver function improving agent that is non-toxic or weak in humans, and the extract of hepatic perilla extract, fractions thereof or a compound isolated therefrom is detoxifying enzyme of liver Induces the activity of quinone reductase, a cancer prevention indicator enzyme, has no cytotoxicity, protects liver function by inducing the activity of detoxification enzyme system, and also shows chemical cancer prevention effect and prevents cancer containing composition containing it as an active ingredient Or completed the present invention by revealing that it can be used as a medicine and health functional food for improving liver function.

It is an object of the present invention to provide a pharmaceutical composition for preventing cancer, comprising the extract of perilla extract, fractions thereof, or a compound of Formula 1 to 5 as an active ingredient.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

It is another object of the present invention to provide a nutraceutical extract, a fraction thereof or a health functional food composition for preventing or improving cancer containing the compound of Formulas 1 to 5 as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for improving liver function, which comprises an extract of cypressus, a fraction thereof, or the compound of Formulas 1 to 5 as an active ingredient.

Further, another object of the present invention to provide a health functional food composition for improving liver function containing the extract of perilla extract, fractions thereof or the compound of Formula 1 to 5 as an active ingredient.

In order to achieve the above object, the present invention is an extract of the extract of Indica which induces the activity of quinone reductase, increases the activity of the antioxidant reaction factor (ARE) and protects the liver from liver damage induced by ethanol and acetaminophen , A fraction thereof or the chlorogenic acid of Chemical Formula 1, the 3,5-di-o-cafeoylquinic acid of Chemical Formula 2, the Youngiaside A of Chemical Formula 3, the Youngiacide B of Chemical Formula 4, and the Chemical Formula 5 Provided is a pharmaceutical composition for preventing cancer, comprising as an active ingredient at least one compound selected from the group consisting of Ganoderma c.

In addition, the present invention is an extract, fractions thereof, or the like of the inflorescence which induces the activity of quinone reductase, increases the activity of the antioxidant reaction factor (ARE), and protects the liver from liver damage induced by ethanol and acetaminophen. Chlorogenic acid of Chemical Formula 1, 3,5-di-o-cafeoylquinic acid of Chemical Formula 2, Young Giaside A of Chemical Formula 3, Young Giaside B of Chemical Formula 4 and Young Giaside C of Chemical Formula 5 It provides a dietary supplement for cancer prevention or improvement containing one or more compounds selected from the group consisting of as an active ingredient.

The present invention is an extract of the extract, the fraction thereof or the formula of the quinone reductase activity to increase the activity of the antioxidant reaction factor (ARE) and protect the liver from liver damage induced by ethanol and acetaminophen Chlorogenic acid of, the 3,5-di-o-caoylquinic acid of the formula (2), the ungiaside A of the formula (3), the ungiaside B of the formula (4) and the ungiaside C of the formula (5) It provides a pharmaceutical composition for improving liver function containing at least one compound selected from the group as an active ingredient.

In addition, the present invention is an extract, fractions thereof, or the like of the inflorescence which induces the activity of quinone reductase, increases the activity of the antioxidant reaction factor (ARE), and protects the liver from liver damage induced by ethanol and acetaminophen. Chlorogenic acid of Chemical Formula 1, 3,5-di-o-cafeoylquinic acid of Chemical Formula 2, Young Giaside A of Chemical Formula 3, Young Giaside B of Chemical Formula 4 and Young Giaside C of Chemical Formula 5 It provides a health functional food composition for improving liver function containing at least one compound selected from the group consisting of as an active ingredient.

Cypressus extract, fractions thereof, or chlorogenic extract of Chemical Formula 1, 3,5-di-o-cafeoylquinic acid extract of Chemical Formula 2, Ganodermaid A of Chemical Formula 3, and Composition containing an organoside B and at least one compound selected from the group consisting of the organoside C of Formula 5 as an active ingredient induces the activity of quinone reductase, a cancer prevention indicator enzyme, It activates the antioxidant response factor (ARE) that induces activity, not only does not have cytotoxicity, but also protects the liver from liver damage induced by ethanol and acetaminophen. The compound of 5 may be usefully used for preventing cancer or improving liver function.

1 is a graph showing the relative quinone reductase relative inactivation and cell viability of the extract of perilla extract according to the present invention;
Figure 2 is a graph showing the relative quinone reductase relative inactivation of four of the perilla fraction fraction according to the present invention;
Figure 3 is a graph showing the cell survival rate of four kinds of perilla fractions according to the present invention;
4 is a graph showing the relative quinone reductase relative inactivity of the compounds of Formulas 1 to 5 according to the present invention;
5 is a graph showing the relative quinone reductase relative inactivation and cell viability of the chlorogenic acid of Formula 1 according to the present invention;
6 is a graph showing the relative quinone reductase relative inactivation and cell viability of 3,5-di-o-cafeoylquinic acid of Formula 2 according to the present invention;
7 is a graph showing the relative quinone reductase relative inactivation and cell viability of Ganodermaid A of Formula 3 according to the present invention;
8 is a graph showing quinone reductase relative inactivation and cell viability of Ganodermaid B of formula 4 according to the present invention;
9 is a graph showing the relative quinone reductase relative inactivation and cell viability of Ganodermaid C of formula 5 according to the present invention;
10 is a graph showing the effect on the antioxidant response element (ARE) of the ethyl acetate fraction of the perilla extract according to the present invention;
11 is a chlorogenic acid of Chemical Formula 1, 3,5-di-o-cafeoylquinic acid of Chemical Formula 2, Ganodermaid A of Chemical Formula 3, Ganodermaid B of Chemical Formula 4, and Ganoderma lucidum according to the present invention. A graph showing the effect of Aside C on the Antioxidant Response Element (ARE);
12 is a graph showing the effect of reducing the toxicity induced by ethanol in CYP2E1 overexpressing cells of the extract of perilla extract according to the present invention;
Figure 13 is a graph showing the effect of reducing the toxicity induced by acetaminophen of the extract of perilla extract according to the present invention.

The present invention provides a pharmaceutical composition for preventing cancer, comprising the extract of perilla as an active ingredient.

Hereinafter, the present invention will be described in detail.

The perilla extract according to the present invention can be obtained by extracting the perilla extract or dried products thereof, the perilla extract can be used without limitation, such as harvested, cultured or commercially available.

The extract of perilla according to the present invention is conventional in the art, such as a method using an extraction device such as supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction or ultrasonic extraction, or a method using an adsorption resin including XAD and HP-20. Phosphorus extraction method may be used, and preferably, it may be prepared by heating at reflux extraction or extraction at room temperature, but is not limited thereto. The number of extraction is preferably 1 to 5 times, and more preferably 3 times of extraction, but is not limited thereto.

The extract may be extracted using water, an organic solvent or a mixed solvent thereof. The organic solvent is preferably any one or a mixed solvent selected from the group consisting of C ₁ to C ₄ alcohols, ethyl acetate, methylene chloride and chloroform, more preferably C ₁ to C ₄ alcohols, Most preferred is extraction with methanol or ethanol.

For example, the extract of perilla extract according to the present invention is pulverized dried perilla extract to an appropriate size, put into an extraction container, put the extraction solvent, boil the solution and extracted under reflux, and then allowed to stand for a certain time and then filtered with a filter paper and alcohol extract. You can get it. The extraction time is preferably 2 to 12 hours, more preferably 3 to 5 hours. Thereafter, a method such as concentration or lyophilization may be additionally performed.

In addition, the present invention provides a pharmaceutical composition for preventing cancer, which contains the perilla fraction as an active ingredient.

The perilla extract according to the present invention can be obtained by sequentially performing the phylogenetic fraction using the extract of the perilla extract using n-hexane, ethyl acetate and n-butanol.

Furthermore, the present invention is a chlorogenic acid represented by the following formula (1), 3,5-di-o-caoylquinic acid represented by the formula (2), ganodermaid A represented by the formula (3), ganoderma represented by the formula (4) Provided is a pharmaceutical composition for preventing cancer, comprising at least one compound selected from the group consisting of side B and ganodermaid C represented by Formula 5 as an active ingredient.

The compound of Formula 1 to Formula 5

Adding perilla extract to water, an organic solvent or a mixture thereof to obtain a perilla extract (step 1);

Sequentially fractionating the extract obtained in step 1 with n-hexane, ethyl acetate and n-butanol to obtain a fraction (step 2); And

Silica gel chromatography may be performed on the fraction obtained in step 2 to separate and purify the compound of Formula 1 to Formula 5 (Step 3).

Hereinafter, the manufacturing method according to the present invention will be described in more detail step by step.

First, step 1 according to the present invention is a step of obtaining a perilla extract with an extraction solvent. The extract may be used without limitation, such as harvested, cultured or commercially available. Dried dried perilla is crushed to a suitable size and placed in an extraction container. As the extraction solvent, C ₁ to C ₄ alcohols, ethyl acetate, methylene chloride, chloroform or a mixed solvent thereof can be used, and among them, methanol or ethanol is preferable. After extraction for 4 hours by ultrasonic at room temperature, and filtered with a filter paper can be obtained extract according to the present invention.

Next, step 2 is a step of obtaining fractions by sequentially fractionating the perilla extract obtained in step 1 with a solvent having a different polarity. N-hexane, ethyl acetate, and n-butanol may be sequentially used as the solvent.

Next, step 3 is a step of separating and purifying the compound of Formula 1 to Formula 5 by performing silica gel chromatography on the fraction obtained in Step 2.

The silica gel chromatography may be performed using a column for bulk exclusion chromatography, and preferably, a column packed with Sephadex LH-20 may be used. The fraction obtained in step 2 is subjected to silica gel chromatography using a Sephadex LH-20 column with 100% methanol as the mobile phase. At this time, the obtained fractions may be subjected to high performance liquid chromatography to separate the compounds of Formulas 1 to 5.

The high performance liquid chromatography may be performed using a mixed solvent of water and acetonitrile gradient gradient of 20% by volume to 30% by volume, 25% by volume to 40% by volume, and 25% by volume of acetonitrile as a developing solvent. At this time, the flow rate of the mobile phase is preferably 2-15 ml / min, the execution time is preferably 0.5 to 1 hour.

The perilla extract, fractions or compounds of Formulas 1 to 5 according to the present invention can be used for preventing cancer or improving liver function by detoxifying carcinogens by increasing the enzymatic activity of quinone reductase. The use of these cancers to prevent or improve liver function is explained based on specific experimental results.

Experiment for measuring the effect of inhibiting quinone reductase activity, a cancer prevention indicator enzyme, in hepatic cancer cell line (Hepa1clc7) of rats against the extract of perilla extract, fractions thereof or the compounds of Formulas 1 to 5 according to the present invention (Example 1 , See Example 2 and Example 3), the experiment to derive a cancer prevention table based on measuring cytotoxicity (see Examples 4, 5 and 6), human colon cancer cell line (HCT116) To examine the effects of ethanol and acetaminophen-induced toxicity on human liver cancer cell lines (HepG2) and experiments measuring ARE activity inducing biphasic detoxification enzyme activity in humans. A summary of the results of the experiments (see Example 9 and Example 10) for measurement are as follows.

First, the perilla extract according to the present invention increased the quinone reductase activity by 2.3 times, 2.7 times and 2.4 times, respectively, compared to the untreated control at concentrations of 12.5, 25 and 50 μg / ml (see FIG. 1). It can be seen that the cytotoxicity was not induced at a concentration of 12.5 μg / ml, which induces an increase of quinone reductase activity more than two times (see FIG. 1).

In addition, the ethyl acetate fraction of perilla extract according to the present invention increased quinone reductase activity by 2.1-fold, 2.7-fold, 3.2-fold, respectively, compared to the untreated control at concentrations of 12.5, 25, 50 μg / ml (Fig. 2), do not induce cytotoxicity at a concentration of 12.5 μg / ml, which induces an increase in quinone reductase activity more than twofold (see FIG. 3), and biphasic detoxification at concentrations of 10, 50, 100 μg / ml It can be seen that the concentration-dependent effect on the ARE activity inducing enzyme-based activity (see Figure 10).

In addition, the chlorogenic acid of Formula 1 increased the quinone reductase activity by 1.54 times compared to the untreated control at a concentration of 250 μM (see FIG. 5), and induces an increase in quinone reductase activity by 1.5 times or more. It can be seen that it does not induce cytotoxicity at the concentration of μM (see FIG. 5), and increases the ARE activity that induces two-phase detoxifying enzyme activity at a concentration of 50 μM (see FIG. 11) (see FIG. 11).

In addition, the 3,5-di-o-cafeoylquinic acid of Formula 2 increased the quinone reductase activity by 1.39 times compared to the untreated control at a concentration of 250 μM (see FIG. 6), and quinone reductase activity. It can be seen that it does not induce cytotoxicity at a concentration of 250 μM that induces an increase of 1.3 times or more (see FIG. 6), and increases the ARE activity that induces biphasic detoxifying enzyme activity at a concentration of 50 μM by 1.22 times ( See FIG. 11).

In addition, Ganodermaid A of Formula 3 increased quinone reductase activity by 2.96 and 3.91 times, respectively, compared to the untreated control at concentrations of 125 μM and 250 μM (see FIG. 7), and increased quinone reductase activity. Does not induce cytotoxicity at gastric concentrations that induce more than twofold (see FIG. 7), and it can be seen that the ARE activity that induces two-phase detoxifying enzyme activity at a concentration of 50 μM increases by 1.65 times (see FIG. 11). ).

In addition, ungiaside B of Formula 4 increased the quinone reductase activity by 2.71 and 3.61 times, respectively, compared to the untreated control at concentrations of 125 μM and 250 μM (see FIG. 8), and increased quinone reductase activity. Does not induce cytotoxicity at the above concentrations that induce more than two times (see FIG. 8), and it can be seen that the ARE activity that induces two-phase detoxifying enzyme activity at a concentration of 50 μM increases by 1.22 times (see FIG. 11). ).

In addition, ungiaside C of Formula 5 increased quinone reductase activity by 2.26, 3.19, 3.88-fold, respectively, compared to the untreated control at concentrations of 62.5 μM, 125 μM, and 250 μM (see FIG. 9). It can be seen that it does not induce cytotoxicity at the gastric concentration that induces an increase in ductase activity more than two times (see FIG. 9), and increases the ARE activity that induces the biphasic detoxifying enzyme activity at a concentration of 50 μM by 1.67 fold. (See FIG. 11).

In addition, the cell viability was reduced by about 32% when treated with 250 mM ethanol, and the cell survival was about 100% when treated with concentrations of 3.125, 6.25, 12.5 and 25 μg / ml By increasing the above it can be seen that the perilla extract according to the present invention effectively inhibits the death of hepatocytes by ethanol (see Fig. 12).

In addition, the treatment of acetaminophen 40 mM cell survival rate is reduced by about 60%, the treatment of the perilla extract according to the present invention increases the cell survival rate to about 120% depending on the concentration, according to the present invention It can be seen that the perilla extract ethyl acetate fraction exhibits a higher cell viability than the butanol fraction, thereby effectively inhibiting the killing of hepatocytes by acetaminophen (see FIG. 13).

Therefore, the extract of the wild horse meat extract, fractions thereof or the compounds of formulas 1 to 5 according to the present invention enhance the activity of liver detoxifying enzymes, including quinone reductase, which is a cancer prevention indicator enzyme, and have no cytotoxicity, Since the liver is protected from hepatic damage induced by acetaminophen, pharmaceutical compositions containing these as active ingredients can be useful for preventing cancer or improving liver function by showing a chemical cancer prevention effect.

Preferably, the pharmaceutical composition according to the present invention may be usefully used for preventing liver cancer or colon cancer, but is not limited thereto.

In another aspect, the present invention provides a health functional food composition for preventing or improving cancer containing the above-mentioned extract and extracts, fractions or compounds of Formula 1 to 5 as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition for improving liver function, which comprises the above-mentioned extract of perilla extract, fractions thereof, or a compound of Formula 1 to 5 as an active ingredient.

In another aspect, the present invention provides a health functional food composition for improving liver function, containing the above-mentioned extract of wild horse extract, fractions thereof or the compound of Formula 1 to 5 as an active ingredient.

Cancer preventive extract, fractions thereof, or a pharmaceutical composition for preventing cancer, comprising a compound of Formula 1 to 5 as an active ingredient, liver composition for improving the liver function in the following various oral or parenteral dosage forms It may be formulated, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules. Liquid. Suspensions, emulsifiers, syrups. Granules, elixirs, and the like, these formulations may be used one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. that are commonly used in addition to the active ingredient. As a disintegrant, agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydride, etc. may be used, and as lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As a binder, magnesium aluminum silicate. Starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like can be used. In addition to lactose, dextrose, sucrose, mannitol, sorbitol, cellulose. Glycine or the like may be used as a diluent, and in some cases, generally known boiling mixtures, absorbents, colorants, flavors, sweeteners, and the like may be used together.

In addition, the extract of cynthia, fractions thereof, or a pharmaceutical composition for preventing cancer containing the compound of Formulas 1 to 5 as an active ingredient, a pharmaceutical composition for improving liver function may be administered parenterally, and parenteral administration is a subcutaneous injection. , Intravenous injection, intramuscular injection or intrathoracic injection. At this time, in order to formulate into a formulation for parenteral administration, the extract of perilla extract, fractions thereof or the compound of formula 1 to 5 are mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is administered in unit doses of ampoules or vials. It can be manufactured in a mold.

The composition may be sterile or contain preservatives, stabilizers, hydrating or emulsifiers, salts for controlling osmotic pressure, buffers and other auxiliaries and other therapeutically useful materials, which are conventional methods of mixing, granulating or coating methods. It can be formulated accordingly.

In the case of formulating a pharmaceutical composition for preventing cancer, the pharmaceutical composition for improving liver function, which comprises an extract of the extract of the present invention, fractions thereof, or a compound of Formulas 1 to 5 as an active ingredient, in an effective dosage form, As an extract, the extract of perilla, a fraction thereof, or the compound of Formulas 1 to 5 are preferably contained in a unit dose of about 0.1-1,500 mg. The dosage depends on the physician's prescription depending on factors such as the patient's weight, age and the particular nature and severity of the disease. However, the dosage required for adult treatment usually ranges from about 1-500 mg per day, depending on the frequency and intensity of administration. For intramuscular or intravenous administration to adults, a single dose separate from the usual dosage of about 5-300 mg per day will suffice, but for some patients a higher daily dose may be desirable.

The extract of the perilla extract, fractions thereof, or the compound of Formulas 1 to 5 according to the present invention may be added to health supplements or functional foods such as foods and beverages for the purpose of preventing or improving cancer and improving liver function. In this case, the extract of perilla extract, fractions thereof or the compound of formulas 1 to 5 may be added in an amount of 0.01-20% by weight, preferably 0.1-5% by weight based on the raw materials when used as a food additive. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene purposes or health control purposes, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range. The extracts, fractions or compounds of formulas (1) to (5) can be used together with other foods or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of food. Examples of foods to which the extract, fractions separated therefrom, or the compound of Formulas 1 to 5 may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. Dairy products, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and the like, including all of the health food in the usual sense.

The food supplement additive of the present invention may use various flavors or natural carbohydrates. The natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol, and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01-0.04 parts by weight, preferably 0.02-0.03 parts by weight per 100 parts by weight of the composition according to the invention.

In addition to the above, the composition according to the present invention contains various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition according to the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination, and the proportion of additives is not critical, but is usually selected in the range of 0.01-0.1 parts by weight per 100 parts by weight of the composition according to the invention.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

< Manufacturing example  1> Indelion  Preparation of extract

The dried perilla was collected from Pyeongchang Sanchae Test Center in Gangwon-do, dried in the shade, and cut into small pieces. The extract was ultrasonically added to the extracting container with 1 kg of dried perilla and 5% of 94% ethanol. The extract was filtered through a filter paper. . The extraction process was repeated three times, and then the solvent was concentrated under reduced pressure and dried to obtain 70 g of ethanol extract.

< Manufacturing example  2> Indelion Fraction  Produce

The ethanol extract was subjected to phylogenetic fractionation and sequentially partitioned into n-hexane, ethyl acetate and n-butanol, respectively, 18.7 g in n-hexane, 2.3 g in ethyl acetate, 5.8 g in n-butanol and 34.4 g in water. Fractions were obtained.

< Manufacturing example  3> Indelion From fractions  Preparation of compounds

<3-1> Of Chlorogenic Acid  Produce

The ethyl acetate fraction obtained in Preparation Example 2 was subjected to chromatography using a Sephadex LH-20 column using 100% methanol as a mobile phase to obtain fractions 1 to 9. Fraction 4 (324 mg) in the fraction was dissolved in methanol, and then subjected to high performance liquid chromatography to obtain chlorogenic acid. At this time, a mixed solvent of water and acetonitrile was used as a mobile phase, and the concentration was gradually gradientd from 20% by volume to 30% by volume acetonitrile for 40 minutes, and the flow rate was 10 ml / min, YMC Hydrosphere ODS (φ20 × 250 mm, 5 μm) column was used.

¹ H-NMR (DMSO-d ₆ ) δ 2.02-1.74 (4H, m, H-2,6), 3.54 (1H, m, H-3), 3.92 (1H, m, H-4), 5.07 ( 1H, m, H-5), 6.98 (1H, dd, J = 8.2, 2.0 Hz, H-2 '), 6.77 (1H, d, J = 8.2 Hz, H-3'), 7.03 (1H, d , J = 2 Hz, H-6 '), 7.43 (1H, d, J = 15.9 Hz, H-7'), 6.16 (1H, d, J = 15.9 Hz, H-8 ')

¹³ C-NMR (DMSO-d ₆ ) δ 73.96 (C-1), 36.76 (C-2), 70.74 (C-3), 68.49 (C-4), 71.35 (C-5), 37.64 (C- 6), 126.04 (C-1 '), 121.79 (C-2'), 116.17 (C-3 '), 148.75 (C-4'), 145.98 (C-5 '), 115.21 (C-6') , 145.37 (C-7 '), 114.75 (C-8'), 166.20 (C-9 '), 175.42 (COOH)

<3-2> 3,5- di -O - caffeoylquinic acid (3,5- Di- O- caffeoylquinic acid )

Dissolve fraction 5 (584 g) obtained in Preparation Example 3-1 in methanol, perform chromatography using a Sephadex LH-20 column using 100% methanol as a mobile phase, and then perform high performance liquid chromatography. , 5-di-o-cafeoylquinic acid was obtained. At this time, a mixed solvent of water and acetonitrile was used as the mobile phase, and the concentration was gradientd to 25% by volume acetonitrile for 40 minutes, and the flow rate was 2 ml / min, using a YMC Hydrosphere ODS (φ10 × 250 mm, 5 μm) column. It was.

¹ H-NMR (DMSO-d ₆ ) δ 2.23-2.04 (4H, m, H-2,6), 5.27 (1H, m, H-3), 3.90 (1H, m, H-4), 5.20 ( 1H, m, H-5), 7.08 (2H, dd, J = 2.3, 8.3 Hz, H-2 ', 2 "), 6.86 (2H, d, J = 8 Hz, H-3', 3") , 7.13 (2H, d, J = 2 Hz, H-6 ′, 6 ″), 7.57 (1H, d, J = 16.2 Hz, H-7 ′), 6.33 (1H, d, J = 16.0 Hz, H -8 '), 7.53 (1H, d, J = 16.3 Hz, H-7 "), 6.25 (1H, d, J = 16.0 Hz, H-8")

¹³ C-NMR (DMSO-d ₆ ) δ 73.04 (C-1), 36.44 (C-2), 71.47 (C-3), 68.10 (C-4), 71.48 (C-5), 35.29 (C- 6), 126.12 (C-1 '), 121.72 (C-2'), 116.38 (C-3 '), 148.96 (C-4'), 146.14 (C-5 ', 5 "), 115.31 (C- 6 ', 8'), 145.32 (C-7 '), 166.67 (C-9'), 126.20 (C-1 "), 121.96 (C-2"), 116.37 (C-3 "), 148.83 (C -4 "), 115.40 (C-6"), 145.69 (C-7 "), 114.69 (C-8"), 166.14 (C-9 "), 175.94 (COOH)

<3-3> Young Gia Said  A ( Youngiaside  A) manufacture

Fraction 3 (621 mg) obtained in Preparation Example 3-1 was dissolved in methanol, and then subjected to high performance liquid chromatography to obtain gamma-side A. At this time, a mixed solvent of water and acetonitrile was used as the mobile phase, and concentration gradient was given sequentially from 40% by volume to 40% by volume of acetonitrile for 40 minutes, and the flow rate was 10 ml / min, YMC Hydrosphere ODS (φ20 × 250 mm, 5 μm) column was used.

¹ H-NMR (DMSO-d ₆ ) δ 2.12 (1H, dd, J = 2.4, 13.9 Hz, H-2) 2.45 (1H, d, J = 13.9 Hz, H-2), 5.96 (1H, m, H-3), 3.67-3.59 (4H, m, H-5,6,9, Glc-6), 2.93 (1H, m, H-7), 3.06 (2H, m, H-8), 6.11 ( 1H, dd, J = 1.7, 3.0 Hz, H-13), 5.97 (1H, m, H-13), 1.69 (3H, s, H-14), 4.35 (2H, quar, J = 1.6, 14.3 Hz , H-15), 4.13 (1H, d, J = 7.8 Hz, Glc-1), 3.00 (1H, m, Glc-2), 3.12 (1H, m, Glc-3), 3.06 (2H, m, Glc-4,5), 3.45 (1H, m, Glc-6)

¹³ C-NMR (DMSO-d ₆ ) δ 136.51 (C-1), 45.81 (C-2), 128.49 (C-3), 141.26 (C-4), 51.88 (C-5), 82.24 (C- 6), 57.70 (C-7), 37.28 (C-8), 68.06 (C-9), 126.56 (C-10), 139.23 (C-11), 169.73 (C-12), 121.35 (C-13 ), 23.42 (C-14), 67.52 (C-15), 102.22 (Glc-1), 73.95 (Glc-2), 77.09 (Glc-3), 70.46 (Glc-4), 77.23 (Glc-5) , 61.43 (Glc-6)

<3-4> Young Gia Said  B ( Youngiaside  B) manufacture

Fraction 3 (621 mg) obtained in Preparation Example 3-1 was dissolved in methanol, and then subjected to high performance liquid chromatography to obtain gamma-side B. At this time, a mixed solvent of water and acetonitrile was used as the mobile phase, and concentration gradient was given sequentially from 40% by volume to 40% by volume of acetonitrile for 40 minutes, and the flow rate was 10 ml / min, YMC Hydrosphere ODS (φ20 × 250 mm, 5 μm) column was used.

¹ H-NMR (DMSO-d ₆ ) δ 2.14 (1H, dd, J = 2.4, 13.9 Hz, H-2) 2.45 (1H, d, J = 13.9 Hz, H-2), 5.96 (1H, m, H-3), 3.67-3.63 (4H, m, H-5,6,9, Glc-6), 2.93 (1H, m, H-7), 3.00 (2H, m, H-8), 6.12 ( 1H, dd, J = 1.7, 3.0 Hz, H-13), 5.98 (1H, m, H-13), 1.72 (3H, s, H-14), 4.37 (2H, quar, J = 1.6, 14.3 Hz , H-15), 4.25 (1H, d, J = 7.8 Hz, Glc-1), 3.21-3.17 (1H, m, Glc-2,5), 4.79 (1H, t, J = 9.4 Hz, Glc- 3), 3.30 (1H, m, Glc-4), 3.49 (1H, m, Glc-6), 3.35 (2H, s, H-β), 7.06 (2H, d, J = 8.6 Hz, H-2 ', 6'), 6.68 (2H, d, J = 8.6 Hz, H-3 ', 5')

¹³ C-NMR (DMSO-d ₆ ) δ 136.49 (C-1), 45.90 (C-2), 129.06 (C-3), 139.23 (C-4), 51.73 (C-5), 82.24 (C- 6), 57.70 (C-7), 37.31 (C-8), 68.06 (C-9), 126.58 (C-10), 136.49 (C-11), 167.74 (C-12), 121.36 (C-13 ), 23.42 (C-14), 67.53 (C-15), 101.64 (Glc-1), 71.85 (Glc-2), 78.46 (Glc-3), 68.22 (Glc-4), 76.90 (Glc-5) , 61.09 (Glc-6), 171.47 (C-α), 40.14 (C-β), 125.18 (C-1 '), 130.80 (C-2', 6 '), 115.43 (C-3', 5 ' ), 156.48 (C-4 ')

<3-5> Young Gia Said  C ( Youngiaside  C) manufacture

Fraction 3 (621 mg) obtained in Preparation Example 3-1 was dissolved in methanol, and then subjected to high performance liquid chromatography to obtain a Youngiacide C. At this time, a mixed solvent of water and acetonitrile was used as the mobile phase, and concentration gradient was given sequentially from 40% by volume to 40% by volume of acetonitrile for 40 minutes, and the flow rate was 10 ml / min, YMC Hydrosphere ODS (φ20 × 250 mm, 5 μm) column was used.

¹ H-NMR δ 2.15 (1H, dd, J = 2.4, 13.9 Hz, H-2) 2.46 (1H, d, J = 13.9 Hz, H-2), 5.97 (1H, m, H-3), 3.67 -3.61 (3H, m, H-5, 6, 9), 2.93 (1H, m, H-7), 3.00 (2H, m, H-8), 6.12 (1H, dd, J = 1.3, 3.0 Hz , H-13), 5.98 (1H, m, H-13), 1.70 (3H, s, H-14), 4.37 (2H, quar, J = 1.6, 14.3 Hz, H-15), 4.24 (1H, d, J = 7.8 Hz, Glc-1), 3.11-3.07 (1H, m, Glc-2), 3.40-3.31 (3H, m, Glc-3,5,6), 4.58 (1H, t, J = 9.6 Hz, Glc-4), 3.30 (1H, m, Glc-4), 3.24 (1H, m, Glc-6), 3.53 (2H, d, J = 2.6 Hz, H-β), 7.05 (2H, d, J = 8.6 Hz, H-2 ', 6'), 6.69 (2H, d, J = 8.6 Hz, H-3 ', 5')

¹³ C-NMR (DMSO-d ₆ ) δ 136.46 (C-1), 45.81 (C-2), 128.57 (C-3), 141.06 (C-4), 51.90 (C-5), 82.23 (C- 6), 57.69 (C-7), 37.31 (C-8), 68.05 (C-9), 126.58 (C-10), 139.24 (C-11), 169.72 (C-12), 121.35 (C-13 ), 23.43 (C-14), 67.73 (C-15), 102.13 (Glc-1), 74.04 (Glc-2), 74.27 (Glc-3), 72.05 (Glc-4), 74.53 (Glc-5) , 61.03 (Glc-6), 171.26 (C-α), 40.01 (C-β), 124.87 (C-1 '), 130.74 (C-2', 6 '), 115.51 (C-3', 5 ' ), 156.58 (C-4 ')

< Example  1> Indelion  Quinones in Extract Reductase  Activity Induction Effect Measurement

In order to measure the effect of quinone reductase activity, a cancer preventive index enzyme, of the extract of perilla extract according to the present invention, the following experiment was performed on a hepatic cancer cell line (Hepa1clc7) of rats. First, a-MEM (minimum essential medium) solution in which 10% fetal bovine serum (FBS) was added was mixed with a liver cancer cell culture medium to prepare a cell number of 1 × 10 ⁵ cells / ml. Thereafter, 100 μl of the 96-well plate was treated and incubated at 5% carbon dioxide and 37 ° C. for 24 hours. After the cells were stabilized, the extract obtained in Preparation Example 1 was treated with 7 concentrations between 3.125 and 200.000 µg / ml with a 2-fold gradient, and then cultured at 5% carbon dioxide and 37 ° C. for 24 hours. . After the incubation, the cells were washed with phosphate buffered saline (PBS) solution, and then the cell membrane was lysed with 80 µl of a solution containing 0.08% digitonin and 2 mM EDTA to obtain a protein solution. 50 μl of the protein solution and reaction solution A (49 mL 25 mM Tris buffer, 34 mg BSA, 0.34 mL 1.5% Tween-20 solution, 0.34 mL coenzyme solution, 100 units of glucose-6-phosphate) 200 μl of dehydrogenase (glucose-6-phosphate dehydrogenase), 15 mg MTT, 50 μl of 50 mM menadione was measured and the absorbance increase was measured at 610 nm with a microplate reader. The amount of protein was Bradford (Bradford). It was measured and measured at 595 nm using a solution. The crude enzyme solution in the reaction solution A was composed of 150 mM glucose-6-phosphate, 4.5 mM NADP, 0.75 mM FAD, and the MTT was 3- (4,5-dimethylthiazo-2- yl) -2,5-diphenyltetrazolium bromide. The quinone reductase activity was calculated by the following equation.

[Equation 1]

Quinone reductase activity = A / B

A: nmol MTT / min (increase in absorbance at 610 nm of the intracellular quinone reductase treated with the test sample)

B: mg protein (amount of protein in the whole cell treated with the test sample)

The results are shown in Fig.

As can be seen in Figure 1, the extract of the present invention, the extract of quinone reductase activity in the concentration of 6.25, 12.5, 25 ㎍ / ㎖, 2.2 times, 2.3 times, 2.6 times increase compared to the untreated control, respectively I was.

Meanwhile, the cancer prevention index (Chemoprevention Index, CI) of the extract was calculated by the following Equation 2. The calculation results are shown in Table 1 below.

&Quot; (2) &quot;

Cancer Prevention Indicators (CI) = A / B

A: IC ₅₀ (concentration of the sample at which the survival rate of the cells treated with the test sample becomes 50%)

B: CD (the concentration of the sample doubling the quinone reductase activity of the cells treated with the test sample)

Wild vegetables
Hepa1clc7 CD (μg / ml) IC ₅₀ (μg / ml) CI Indelion 9.43 〉 200.00 21.20 Sulforaphane 1.17 14.96 12.83

As shown in Table 1, the cancer prevention table (CI) for the extract of perilla extract according to the present invention is 21.20, which is very good when compared to a sulforaphane (Sulforaphane, CI = 12.83) known as a conventional cancer prevention functional food material. It can be seen that the effect, and as shown in Figure 1, it was confirmed that there is little toxicity at the concentration to increase the quinone reductase activity more than two times.

< Example  2> Indelion Fraction  Quinone Reductase  Activity Induction Effect Measurement

In order to determine the effect of quinone reductase activity, a cancer preventive index enzyme of the perilla extract according to the present invention, the rat liver cancer cell line (Hepa1clc7) was treated in the same manner as in Example 1, and then obtained in Preparation Example 2. One fractions were treated at 7 concentrations between 3.125 and 200.000 μg / ml with a 2-fold gradient, followed by incubation at 5% carbon dioxide and 37 ° C. for 24 hours. After the incubation, the cells were washed with phosphate buffered saline (PBS) solution, and the cell membrane was lysed with 80 µl of a solution containing 0.08% digitonin and 2 mM EDTA to obtain a protein solution. The protein solution was measured for quinone reductase protein activity in the same manner as in Example 1. The measurement results are shown in FIG. 2.

As can be seen in Figure 2, the extract of ethyl acetate fraction of the present invention was 2.1 times, 2.7 times, 3.2 times the quinone reductase activity, respectively, compared to the untreated control at concentrations of 12.5, 25, 50 ㎍ / ㎖ Fold increased.

Meanwhile, the cancer prevention index (Chemoprevention Index, CI) of the fraction was calculated using Equation 2. The calculation results are shown in Table 2 below.

Wild vegetables
Fraction
Hepa1clc7 CD (μg / ml) IC ₅₀ (μg / ml) CI
And subtract

Hexane 26.26 89.12 3.39 Ethyl acetate (EA) 9.90 151.10 15.27 Butanol (BuOH) 16.81 〉 200.00 11.90 Water (H ₂ O) 123.82 〉 200.00 1.62

As shown in Table 2, the cancer prevention table (CI) for the ethyl acetate fraction of the perilla fractions according to the present invention was 15.27 was confirmed to have better efficacy than the fractions of other solvents.

<Example 3> induced quinone reductase dehydratase activity of the compounds of formula 1 to 5 effect the measurement

In order to measure the quinone reductase activity inducing effect of the compounds of Formulas 1 to 5 according to the present invention, the treatment was performed in the same manner as in Example 1 to the liver cancer cell line (Hepa1clc7) of rats, and then Preparation Example 3 Compounds obtained at were treated at 7 concentrations between 7.81 and 250.00 μg / ml with a 2-fold gradient, followed by incubation at 5% carbon dioxide and 37 ° C. for 24 hours. After the incubation, the cells were washed with phosphate buffered saline (PBS) solution, and the cell membrane was lysed with 80 µl of a solution containing 0.08% digitonin and 2 mM EDTA to obtain a protein solution. The protein solution was measured for quinone reductase protein activity in the same manner as in Example 1. The measurement results are shown in Fig.

As can be seen in Figure 4, in the compounds of the formula 1 to 5 according to the present invention, the gyanoside A is 2.96 times, 3.92 times quinone reductase activity, respectively, compared to the untreated control at the concentration of 125.00, 250.00 μM Increased (see FIG. 7). Ganodermaid B also increased quinone reductase activity by 2.71 and 3.61 fold, respectively, compared to the untreated control at concentrations of 125.00 and 250.00 μM (see FIG. 8). Ganoderma C also increased quinone reductase activity by 2.26 fold, 3.18 fold and 3.88 fold, respectively, at concentrations of 62.50, 125.00 and 250.00 μM compared to untreated controls (see FIG. 9).

Meanwhile, the cancer prevention index (Cemoprevention Index, CI) of the compound was calculated using Equation 2. The calculation results are shown in Table 3 below.

compound CD (μg / ml) IC ₅₀ (μg / ml) CI Chlorogenic EX 〉 500.00 〉 500.00 0.27 3,5-di-o-cafeoylquinic acid 〉 500.00 〉 500.00 0.64 Infant Side A 54.30 〉 500.00 9.21 Young Gaia Side B 61.51 〉 500.00 8.13 Infant Side C 38.26 〉 500.00 13.07

As shown in Table 3, among the compounds of Formulas 1 to 5, the cancer prevention table (CI) of Youngiaside A, Youngiaside B, and Youngiaside C was 9.21, 8.13, and 13.07, respectively, compared to the other two compounds. It was found that the effect of increasing quinone reductase activity was very excellent, and among them, Ganodermaid C had the best efficacy. In addition, as shown in Figures 7, 8 and 9 it was confirmed that there is little toxicity at a concentration that increases the quinone reductase activity more than two times.

< Example  4> Indelion  Cytotoxicity of Extracts

In order to measure the cytotoxicity of the extract of perilla extract prepared in Preparation Example 1, the following experiment was performed on a rat liver cancer cell line (Hepa1c1c7). First, a-MEM (minimum essential medium) solution in which 10% fetal bovine serum (FBS) was added was mixed with a liver cancer cell culture medium to prepare a cell number of 1 × 10 ⁵ cells / ml. Thereafter, 100 μl of the 96-well plate was treated and incubated at 5% carbon dioxide and 37 ° C. for 24 hours. After the cells were stabilized, the extract obtained in Preparation Example 1 was treated with 7 concentrations of 3.125 to 200 µg / ml at a 2-fold gradient and incubated at 5% carbon dioxide and 37 ° C for 24 hours. After the incubation was completed, the CCK-8 (Dojindo laboratory, Japan) reagent was added and incubated at 5% carbon dioxide and 37 ° C. for 1 hour, and then absorbance was measured at 450 nm.

The measurement results for the extract of wild grapes are shown in FIG. 1.

As can be seen in Figure 1, the perilla extract was found to be cytotoxic at a concentration of 12.5-50 ㎍ / ㎖ inducing quinone reductase activation effect.

< Example  5> Indelion Fraction  Cytotoxicity Measurement

After treatment in the same manner as in Example 4 to the hepatic cancer cell line (Hepa1c1c7) of the rat in order to measure the cytotoxicity of the fractions prepared in Preparation Example 2, the fraction obtained in Preparation Example 2 2 times gradient Were treated at 7 concentrations of 3.125 to 200 μg / ml and incubated at 5 ° C. and 37 ° C. for 24 hours. After the incubation was completed, the CCK-8 (Dojindo laboratory, Japan) reagent was added and incubated at 5% carbon dioxide and 37 ° C. for 1 hour, and then absorbance was measured at 450 nm.

The measurement results for the perilla fractions are shown in FIG. 3.

As can be seen in Figure 3, the ethyl acetate fraction having the most quinone reductase activation effect among the perilla fractions is not cytotoxic at a concentration of 12.5 to 50 ㎍ / ㎖ inducing quinone reductase activation effect Confirmed.

< Example  6> Cytotoxicity Measurement of Compounds of Formulas 1-5

In order to measure the cytotoxicity of the compounds of Formulas 1 to 5 according to the present invention after treatment in the same manner as in Example 4 to the hepatic cancer cell line (Hepa1c1c7) of the rat, the compounds isolated in Preparation Example 3 at a 2-fold gradient Treated at 7 concentrations between 7.81 and 250.00 μg / ml and incubated at 5% carbon dioxide and 37 ° C. for 24 hours. After the incubation was completed, the CCK-8 (Dojindo laboratory, Japan) reagent was added and incubated at 5% carbon dioxide and 37 ° C. for 1 hour, and then absorbance was measured at 450 nm.

The measurement results are shown in FIGS. 5, 6, 7, 8 and 9.

Young Giaside C, which has the best quinone reductase activation effect among the compounds of Formulas 1 to 5, was found to be cytotoxic at concentrations up to 250.00 μM inducing quinone reductase activation effects (see FIG. 9).

< Example  7> Indelion Fraction Antioxidant response factor (ARE)  Measure effectiveness

In order to measure the effect on the antioxidant response element (ARE) that induces the activity of the two-phase detoxification enzyme system of the perilla extract fraction according to the present invention, the antioxidant response factor of human colon cancer cell line (HCT116) Reporter gene assays were performed. First, a-MEM (minimum essential medium) solution in which 10% fetal bovine serum (FBS) was added was mixed with the colorectal cancer cell culture medium, and the number of cells was prepared at 1 × 10 ⁵ cells / ml. Thereafter, 100 ml of each 96-well plate was incubated for 24 hours at 5% carbon dioxide and 37 ℃. After the cells were stabilized, a vector phARE-luc in which a promoter including an antioxidant reaction factor and a luciferase protein expression gene was inserted was transformed using Fugene (Roche Biochemicals). The vector was prepared by inserting a promoter region including the antioxidant reaction factor of the human quinone reductase gene into pGL3-Basic vector (Promega) into which luciferase protein expression gene was inserted. After the cells were stabilized, the ethyl acetate fractions obtained in Preparation Example 1 were treated at concentrations of 10, 50, and 100 μM, incubated at 5% carbon dioxide and 37 ° C for 24 hours, and then luciferase assay kit (Luciferase assay). Cell lysate was analyzed using Kit (Promega). The results are shown in FIG.

As shown in FIG. 10, it was confirmed that the activity of the antioxidant reaction factor (ARE) increased in a concentration-dependent manner as the treatment concentration of the fraction increased. Thus, it can be seen that the ethyl acetate fraction of the present invention has an effect of improving liver function and preventing chemical carcinogenesis by increasing the activity of the detoxifying enzyme system.

<Example 8> measuring effect on antioxidant response factor (ARE) of the compound of formula 1-5

In order to measure the effect on the antioxidant response element (ARE) that induces the activity of the two-phase detoxifying enzyme system of the compounds of Formulas 1 to 5 according to the present invention carried out on human colon cancer cell line (HCT116) After the same treatment as in Example 7, the compounds isolated in Preparation Example 3 were treated at a concentration of 50 μM and incubated at 5% carbon dioxide and 37 ° C. for 24 hours. After the incubation, the cell lysate was analyzed using a luciferase assay kit (Promega). The results are shown in FIG.

As shown in FIG. 11, the compounds of Formulas 1 to 5 showed an increase in the activity of antioxidant response factor (ARE) at a concentration of 50 μM compared to the untreated control. Therefore, it can be seen that the compounds of Formulas 1 to 5 according to the present invention have an effect of improving liver function and preventing chemical carcinogenesis by increasing the activity of the detoxifying enzyme system.

< Example  9> Indelion  Extract CYP2E1 In overexpressing cells  Determination of the effect on ethanol-induced toxicity

Of perilla extract according to the present invention The effect on ethanol-induced toxicity was measured in human liver cancer cell line (HepG2). A cell line overexpressing the CYP2E1 gene in human liver cancer cell line (HepG2) was made and treated with 250 mM ethanol to induce liver damage, while simultaneously extracting the extract prepared in Preparation Example 1 between 3.125 and 50.000 µg / ml. 4 concentrations of and were incubated for 48 hours at 5% carbon dioxide and 37 ℃. After the incubation, the MTT reagent was added to incubate at 5% carbon dioxide and 37 ° C. for 1 hour, and then absorbance was measured at 450 nm.

The results are shown in FIG.

As can be seen in Figure 12, when treated with ethanol 250 mM cell viability is reduced by about 32%, and when the extract of the present invention in the concentration of 3.125, 6.25, 12.5 and 25 ㎍ / ㎖ It was confirmed that the cell survival rate increased to about 100% or more. In addition, it was confirmed that the perilla extract according to the present invention effectively inhibits the death of hepatocytes by ethanol.

< Example  10> Indelion  Extract and Fraction To acetaminophen  Determination of effects on toxicity induced by

Extract of Perilla Extract and Fractions According to the Present Invention The effect on the toxicity induced by acetaminophen was measured in human liver cancer cell line (HepG2). HepG2 cell lines were treated with 5 to 40 μg / ml for 24 hours with a 2-fold gradient in FBS-free medium. At this time, sulforaphane (5 μM) and glutathione (1 mM) were also treated with the positive control group to compare the activity. After removing the medium, washed twice with PBS, and then cultured for an additional 24 hours by adding 40 mM acetaminophen to the medium without FBS. After the incubation, the MTT reagent was added to incubate at 5% carbon dioxide and 37 ° C. for 1 hour, and then absorbance was measured at 450 nm.

The results are shown in FIG.

As can be seen in Figure 13, when treated with acetaminophen 40 mM cell viability is reduced by about 60%, and when treated with red pepper extract according to the present invention the cell viability up to about 120% concentration-dependently It was confirmed that the increase, and the perilla ethyl acetate fraction according to the present invention showed a higher cell viability than the butanol fraction. In addition, it was confirmed that the extract and fractions according to the invention effectively inhibits the death of hepatocytes by acetaminophen.

From this, the extract of perilla extract, fractions thereof, or the compound of formulas 1 to 5 show an effect of increasing the activity of quinone reductase, a representative second phase detoxifying enzyme and a cancer prevention marker, and an antioxidant response factor ( ARE) is not only cytotoxic but also protects the liver from liver damage induced by ethanol and acetaminophen, making it a safe substance for cancer prevention drugs, liver function drugs, and health foods. Can be used.

Examples of preparations for the prophylactic or cancer function improving agent of cancer of the present invention are illustrated below.

Formulation Example 1 Preparation of Pharmaceutical Formulation

1-1. Manufacture of powder

And subtract 2 g of extract, fractions thereof, or compound of formulas 1-5

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

And subtract 100 mg of extract, fractions thereof or compounds of formulas 1-5

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

1-3. Preparation of capsules

100 mg of perilla extract, fractions thereof or compounds of formulas 1-5

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

1-4. Injection preparation

100 mg of perilla extract, fractions thereof or compounds of formulas 1-5

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

Formulation Example 2: Preparation of Health Functional Food

1-1. Manufacturing of beverages

522 mg of honey

Chioctosanami 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Perilla extract, fractions thereof or compounds of Formulas 1-5

                                               0.48-1.28 mg

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

1-2. Preparation of Chewing Gum

Gum Base 20%

Sugar 76.36-76.76%

Perilla extract, fractions thereof or compounds of Formulas 1-5

               0.24-0.64%

1% of fruit flavor

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

1-3. Manufacture of candy

50-60% sugar

Starch syrup 39.26-49.66%

Perilla extract, fractions thereof or compounds of Formulas 1-5

            0.24-0.64%

Orange flavor 0.1%

The composition and the content of the candy were prepared using a conventional method.

1-4. Manufacture of flour food products

Add 0.5 to 5 parts by weight of the perilla extract, fractions thereof, or the compound of Formulas 1 to 5 to 100 parts by weight of flour, and prepare bread, cake, cookies, crackers and noodles using this mixture to promote health Dragon food was prepared.

1-5. Manufacture of dairy products

5 to 10 parts by weight of perilla extract, fractions thereof, or the compound of Formulas 1 to 5 were added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

1-6. Manufacture of wires

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted.

Black soybeans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.

The grains and seeds prepared above and the grains and seeds according to the invention Extracts, fractions, or compounds of Formulas 1 to 5 were prepared by combining the following proportions.

30% brown rice

15% rate

Barley 20%

Perilla 7%

Black Bean 7%

Black Sesame 7%

Perilla perilla extract, fractions thereof, or compound 3-1

Ganoderma 0.5%

Foxglove 0.5%

Claims (30)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete A pharmaceutical composition for improving liver function, which comprises an extract of perilla extract extracted using water, an organic solvent or a mixed solvent thereof as an active ingredient.
Hepatic function containing the extract of the fruit extract obtained by sequentially fractionating the extract of the fruit extract of claim 22 with n-hexane, ethyl acetate and n-butanol as an active ingredient Improvement pharmaceutical composition.
A health functional food composition for improving liver function, which contains a perilla extract, which is extracted using water, an organic solvent or a mixed solvent thereof as an active ingredient.
Hepatic function comprising the extract of the extract of the 24, which is obtained by sequentially fractionating the extract of the perilla extract of n-hexane (n-hexane), ethyl acetate (ethyl acetate) and n-butanol (n-butanol) as an active ingredient Functional food composition for improvement.
The method of claim 22 or 24,
The organic solvent is any one selected from the group consisting of C ₁ to C ₄ alcohol, ethyl acetate, methylene chloride and chloroform or a mixed solvent thereof.
The method of claim 26,
The organic solvent is a composition, characterized in that the C ₁ to C ₄ alcohol.
28. The method of claim 27,
The C ₁ to C ₄ alcohol is a composition, characterized in that methanol or ethanol.
The method according to any one of claims 22 to 25,
The perilla extract or perilla fraction is characterized in that the detoxification of carcinogens by increasing the activity of quinone reductase.
The method according to any one of claims 22 to 25,
The perilla extract or perilla fraction is characterized in that the detoxification of carcinogens by increasing the activity of the antioxidant reaction factor (ARE) that induces two-phase detoxification enzyme-based activity.

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