KR20200124086A - Composition for preventing or treating renal disease comprising Zizyphus jujuba MILL extract - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating kidney diseases, a food composition for preventing or alleviating kidney diseases, and quasi-drug composition for preventing or alleviating kidney diseases, all of which contain a compound isolated from a Zizyphus jujuba root extract as an active component; a method for manufacturing the Zizyphus jujuba root extract or a fraction thereof; and a method for preventing or treating kidney diseases of an individual. A composition containing the Zizyphus jujuba root extract according to an aspect can inhibit kidney cell death from kidney cell damaging substances, so that the extract can be effectively used for preventing, alleviating, or treating kidney diseases, thereby being able to be used as the pharmaceutical composition or the food composition when containing the extract as the active component. In addition, a 3-dihydroxyceanothetric acid 2-methyl ester compound contained in the fraction of the extract as the active component also exhibits kidney cell protection activity, so that when the compound is contained, it is possible to be effectively used as a pharmaceutical material or a health functional food material.

Description

Composition for preventing or treating kidney disease comprising jujube tree root extract {Composition for preventing or treating renal disease comprising Zizyphus jujuba MILL extract}

A pharmaceutical composition for preventing or treating kidney disease, comprising a compound isolated from the extract of jujube tree root as an active ingredient, a food composition for preventing or improving kidney disease, a quasi-drug composition, a method for preparing a jujube tree root extract or a fraction thereof And a method of preventing or treating kidney disease in an individual.

In addition to the main function of filtering out metabolites and waste products in the body, the kidneys keep the body's water content, electrolytes, and acidity constant within a narrow range, and various important functions for maintaining blood pressure, correcting anemia, and metabolizing calcium and phosphorus. It is an organ that has endocrine functions that produce and activate eggplant hormones. Renal diseases that cause the weakening of the kidney function include glomerulonephritis, chronic kidney failure, acute kidney failure, nephrotic syndrome, pyelonephritis, kidney stones, and kidney cancer. Depending on the rate at which kidney function deterioration progresses, it can be divided into acute kidney injury (AKI) and chronic kidney disease (CKD).

Despite the advancement of modern medicine, many patients admitted to the hospital suffer a lot from the decline in renal function, and in particular, patients with high severity of the disease often require renal replacement therapy due to the decline in renal function. The prevalence of acute renal injury has been reported from about 5% of hospitalized patients to about 30-50% of patients admitted to the intensive care unit, and this prevalence is steadily increasing despite the development of new treatments.

Although studies on such various kidney diseases and their mechanisms have been actively conducted, problems such as gastrointestinal damage caused by drugs have been pointed out in the case of conventional drug treatment. Therefore, in order to treat kidney disease, which is rapidly increasing, many efforts have been made to develop substances from plants that can effectively protect kidney cells while inhibiting the occurrence of gastrointestinal damage.

One aspect relates to a pharmaceutical composition for preventing or treating kidney disease comprising a root extract of Jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

One aspect relates to a method for preparing a jujube tree root extract or a fraction thereof, comprising the step of extracting the root of the jujube tree (Zizyphus jujuba MILL.) with water, a lower alcohol of C1 to C4, or a mixed solvent thereof to obtain an extract. .

One aspect relates to a method for preventing or treating kidney disease in an individual comprising administering a root extract or a fraction thereof to an individual other than a human being, a jujube tree (Zizyphus jujuba MILL.).

Another aspect relates to a food composition for preventing or improving kidney disease, including the root extract of the jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

Another aspect relates to a quasi-drug composition for preventing or improving kidney disease, comprising a root extract of jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

One aspect provides a pharmaceutical composition for preventing or treating kidney disease, comprising a root extract of jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

One aspect relates to a method for preparing a jujube tree root extract or a fraction thereof, comprising the step of extracting the root of the jujube tree (Zizyphus jujuba MILL.) with water, a lower alcohol of C1 to C4, or a mixed solvent thereof to obtain an extract. .

One aspect relates to a method for preventing or treating kidney disease in an individual comprising administering a root extract or a fraction thereof to an individual other than a human being, a jujube tree (Zizyphus jujuba MILL.).

Another aspect provides a food composition for preventing or improving kidney disease, including the root extract of the jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

Another aspect provides a quasi-drug composition for preventing or improving kidney disease, comprising a root extract of jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

Since the composition of the root extract of Zizyphus jujuba MILL. according to one aspect can inhibit renal cell death from renal cell damage substances, the use of the extract can effectively be used for preventing, improving or treating kidney disease, When the extract is included as an active substance in the composition, it can be used as a pharmaceutical composition or a food composition. In addition, the 3-dihydroxyceanothetric acid (dehydroxyceanothetric acid) 2-methyl ester compound contained in the extract fraction also exhibits renal cell protective activity as an active ingredient. It can be effectively used as a food material.

1 is a result of confirming the appearance and apoptosis of LLC-PK1 cells treated with 3-dihydroxyceanothetric acid 2-methyl ester compound and cisplatin (FIG. 1A), which was quantified and confirmed as a graph. It is shown in the results (Fig. 1B).
Figure 2 shows the expression of P-P38, P38, P-JNK, JNK, P-ERK, ERK in LLC-PK1 cells treated with 3-dihydroxyceanothetric acid 2-methyl ester compound and cisplatin It is a diagram showing the results of confirming (Fig. 2A) and the results of confirming the expression levels of CC-3, -8, -9, Bax, Bcl-2 and GAPDH (Fig. 2B).

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. The contents of all publications referred to herein by reference are incorporated herein by reference in their entirety.

One aspect provides a pharmaceutical composition for preventing or treating kidney disease, comprising a root extract of jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

The jujube tree root extract may be extracted using a hydrophilic solvent. The hydrophilic solvent may be water, a C ₁ to C ₁₀ alcohol, or a mixture thereof. The alcohol may be a compound having at least one -OH group of C ₁ to C ₁₀ , C ₁ to C ₆ or C ₁ to C ₄ . The alcohol may be methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, or a mixture thereof. In one embodiment, the extraction solvent of the jujube tree root extract may be water, a C ₁ to C ₄ lower alcohol, or a mixture thereof. In one embodiment, the extraction solvent of the jujube tree root extract may be methanol or hot water.

In one aspect, the composition may include a fraction prepared from a date palm root extract using at least one solvent selected from the group consisting of chloroform, ethyl acetate, butanol, and water. In a specific example, a compound having the following chemical formulas 1 to 8 derived from a fraction obtained using a chloroform fraction and an ethyl acetate solvent was isolated.

[Formula 1]

The compound represented by Formula 1 is 2α-methylcarboxy-A(1)-norlup-20(29)-en-27,28-dioic acid or 3-dihydroxyceanothetric acid 2-methyl ester It may be referred to as and the CAS number corresponds to 2002461-52-9.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

The jujube tree root extract may be a crude extract, a fraction, or a combination thereof. The crude extract is obtained by contacting the roots of the jujube tree with an extraction solvent, and refers to a substance containing a specific component and not separated. The fraction refers to the separation of a substance containing a specific component from the crude extract. The separation may be separation using an organic solvent, chromatography or filtration. The chromatography may be, for example, silica gel column chromatography, Sephadex LH-20 chromatography, ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, or a combination thereof.

The extraction may be by adding and incubating the jujube tree root in the extraction solvent. The jujube tree root may be dried or washed. The incubation may be performed without stirring or stirring at room temperature to reflux temperature. The incubation temperature may be appropriately selected depending on the selected solvent. For example, it may be room temperature to reflux temperature, 30°C to reflux temperature, 40°C to reflux temperature, 50°C to reflux temperature, 60°C to reflux temperature, or 65°C to reflux temperature. The extraction time can be appropriately selected according to extraction conditions. The extraction time may be 2 to 4 hours, for example 2.5 to 3.5 hours. The extraction solvent may be 5 to 15 times, for example, 5 to 13 times, 5 to 11 times, 8 to 15 times, 8 to 13 times, 8 to 11 times, or 9 to 11 times of the jujube tree root. The extraction may be extracted once or more, for example, 1 to 5 times, 1 to 4 times, 1 to 3 times, 2 to 5 times, or 2 to 4 times. The obtained crude extract can be filtered, concentrated and dried. The drying may be dried under reduced pressure.

The extract may be extracted under ultrasonic irradiation. The ultrasonic irradiation is at room temperature, 10 to 25 ℃, 10 to 30 ℃, 10 to 40 ℃, 10 to 45 ℃, 10 to 50 ℃, 10 to 55 ℃, 10 to 60 ℃, 10 to 65 ℃, or 10 to 70 It may be performed at ℃. Irradiation time is 10 to 30 minutes. 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 3 hours, 10 minutes to 4 hours, 10 minutes to 5 hours, 10 minutes to 6 hours, 10 minutes to 9 hours, 10 minutes to 12 hours, or 10 It may be performed for minutes to 24 hours. The ultrasonic irradiation may be performed in a sealed container.

In one embodiment, the extract may be extracted using an organic solvent. The organic solvent may be ethyl acetate, chloroform, methanol, hexane, dichloromethane, butanol, acetone, acetonitrile, or a combination thereof. In one embodiment, the compounds represented by Formulas 1 to 8 may be isolated from the ethyl acetate or chloroform fraction of the jujube tree root extract. In one embodiment, the compound represented by Formula 1 may be obtained from a chloroform fractionated layer obtained by extracting a date palm root with methanol and then fractionating with chloroform.

The extraction may further include performing silica gel column chromatography or Sephadex LH-20 chromatography on the crude extract.

The jujube tree root extract, fraction, or a composition containing the compound represented by Formulas 1 to 8 isolated from the fraction prevents kidney disease or secondary diseases caused therefrom by protecting kidney cells by inhibiting apoptosis. Or it may be to cure. In one embodiment, the composition may prevent or treat kidney disease by inhibiting kidney damage caused by cisplatin.

The pharmaceutical composition may contain the jujube tree root extract, fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction in an amount effective for the purpose of treating, preventing or improving kidney disease. The effective amount can be appropriately selected by a person skilled in the art depending on the cells or individuals selected. The effective amount is, for example, 0.1 μg/mL to 1,000 μg/mL, for example, 0.1 μg/mL to 500 μg/mL, 0.1 μg/mL to 100 μg/mL, 0.1 μg/mL to 50 μg per composition. /mL, 0.1 μg/mL to 25 μg/mL, 1 μg/mL to 1,000 μg/mL, 1 μg/mL to 500 μg/mL, 1 μg/mL to 100 μg/mL, 1 μg/mL to 50 μg /mL, 1 μg/mL to 25 μg/mL, 5 μg/mL to 1,000 μg/mL, 5 μg/mL to 500 μg/mL, 5 μg/mL to 100 μg/mL, 5 μg/mL to 50 μg/ mL, 5 μg/mL to 25 μg/mL, 10 μg/mL to 1,000 μg/mL, 10 μg/mL to 500 μg/mL, 10 μg/mL to 100 μg/mL, 10 μg/mL to 50 μg/mL , Or 10 μg/mL to 25 μg/mL, 10 μg/mL to 350 μg/mL, 12.5 μg/mL to 300 μg/mL, 25 μg/mL to 270 μg/mL, 50 μg/mL to 150 μg/mL, or 100 It may be included in μg/mL to 200 μg/mL.

In the present specification, the term kidney disease may be a disease due to kidney damage, chronic kidney disease or acute kidney disease, for example, the chronic kidney disease or acute kidney disease may be selected from the group consisting of renal failure and nephritis, It is not limited thereto.

The composition according to an aspect may include any one or more of the compounds represented by Formulas 1, 2, 3, 4, 5, 6, 7, and 8 or a pharmaceutically acceptable salt thereof, and specifically It may contain two or more active ingredients, and may also include a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the compound represented by Chemical Formulas 1, 2, 3, 4, 5, 6, 7, and 8 or a pharmaceutically acceptable salt thereof may be extracted and isolated from the roots of jujube trees, and may also be artificially synthesized. I can.

The composition according to one aspect is the composition of the compounds represented by Formulas 1, 2, 3, 4, 5, 6, 7, and 8 or a pharmaceutically acceptable salt thereof, single, 2, 3, It may be composed of 4, 5, 6, 7 or 8 active ingredients.

In the composition, the jujube tree root extract, fraction, or a composition comprising a compound represented by Formulas 1 to 8 isolated from the fraction prevents or treats kidney disease, or is selected from the group consisting of kidney damage, renal failure, and nephritis. It may be included in an amount effective to prevent or treat kidney disease, or to prevent or treat secondary diseases resulting therefrom. The composition may be used for prevention, treatment, or improvement of kidney disease related to kidney damage, or kidney disease caused by direct or indirect cause of kidney disease.

One aspect relates to a method for preparing a jujube tree root extract or a fraction thereof, comprising the step of extracting the root of the jujube tree (Zizyphus jujuba MILL.) with water, a lower alcohol of C1 to C4, or a mixed solvent thereof to obtain an extract. .

The manufacturing method according to an aspect may further include obtaining a fraction from the obtained extract using at least one solvent selected from the group consisting of chloroform, ethyl acetate, butanol, and water.

The extraction may be by adding and incubating the jujube tree root in the extraction solvent. The jujube tree root may be dried or washed. The incubation may be performed without stirring or stirring at room temperature to reflux temperature. The incubation temperature may be appropriately selected depending on the selected solvent.

The fraction refers to the separation of a substance containing a specific component from the crude extract. The separation may be separation using an organic solvent, chromatography or filtration. The chromatography may be, for example, silica gel column chromatography, Sephadex LH-20 chromatography, ion exchange chromatography, affinity chromatography, size exclusion chromatography, HPLC, or a combination thereof.

Another aspect provides a food composition for preventing or improving kidney disease, including the root extract of the jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

Another aspect provides a quasi-drug composition for preventing or improving kidney disease, comprising a root extract of jujube tree ( Zizyphus jujuba MILL.) as an active ingredient.

The composition may be a pharmaceutical composition, a food composition, or a quasi-drug composition. The composition may further include an excipient or carrier acceptable pharmaceutically, food, or quasi-drug. The composition may be a pharmaceutical, food or quasi-drug composition, or may be a composition added as an active ingredient of a pharmaceutical, food or cosmetic. In one embodiment, the composition is any one compound selected from the group consisting of the flower jujube tree root extract, a fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction, or a mixture thereof, or a pharmaceutically acceptable It may be a pharmaceutical composition or a food composition for preventing or treating kidney disease comprising a salt as an active ingredient.

The food composition may be used alone or in combination with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present invention is based on the total weight of 15 parts by weight or less, 10 parts by weight or less, 1 to 15 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, Alternatively, it may be added in an amount of 0.1 to 15 parts by 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.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, Alcoholic beverages and vitamin complexes, and all foods in the usual sense are included. The food composition may be used, for example, as a food material such as milk and yoplait, food and beverage, and functional food. The food composition may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

The food composition is also used in nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages Carbonation agent, or a combination thereof. The food composition may also contain natural fruit juice, fruit juice beverage, pulp for the production of vegetable beverages, or a combination thereof. These additives may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition.

The composition may be formulated as an oral or parenteral dosage form. The oral dosage form may be a granule, powder, liquid, tablet, capsule, dry syrup, or a combination thereof. The parenteral dosage form may be an injection or an external preparation for skin. The external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof.

The composition may contain a pharmaceutically acceptable diluent or carrier. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, for example by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. I can. The administration can be administered systemically or locally. The administration may be topically administered to a wound site.

The composition may be administered in combination with drugs such as steroids, anti-inflammatory agents, and antibiotics in order to effectively prevent or treat kidney disease in an individual or secondary disease resulting therefrom. The combined administration may be sequential, simultaneous, or individually administered to the individual.

The administration may be performed by using a jujube tree root extract, a fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction per individual per day from 0.001 μg/mL to 1,000 μg/mL, for example, 0.01 μg/mL to 500 μg/mL , 0.1 μg/mL to 100 μg/mL, 0.1 μg/mL to 50 μg/mL, μ0.1 μg/mL to 25 μg/mL, 1 μg/mL to 1,000 μg/mL, 1 μg/mL to 500 μg /mL, 1 μg/mL to 100 μg/mL, 1 μg/mL to 50 μg/mL, 1 μg/mL to 25 μg/mL, 5 μg/mL to 1,000 μg/mL, 5 μg/mL to 500 μg/ mL, 5 μg/mL to 100 μg/mL, 5 μg/mL to 50 μg/mL, 5 μg/mL to 25 μg/mL, 10 μg/mL to 1,000 μg/mL, 10 μg/mL to 500 μg/mL , 10 μg/mL to 100 μg/mL, 10 μg/mL to 50 μg/mL, or 10 μg/mL to 25 μg/mL may be administered.

The term "comprised as an active ingredient" is meant to include an amount sufficient to achieve the efficacy or activity of a jujube root extract, a fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction. The present invention is a composition containing an extract extracted from the root of a natural plant material or a compound isolated therefrom, and there is no side effect to the human body even if an excessive amount is administered. The quantitative upper or lower limit can be selected and implemented within an appropriate range.

The jujube tree root extract of the present invention, the fraction or the compound represented by Formulas 1 to 8 isolated from the fraction may be added to the quasi-drug composition for the purpose of preventing or improving kidney disease. When using the jujube tree root extract of the present invention, a fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction as a quasi-drug additive, the extract may be added as it is or used with other quasi-drug components, according to a conventional method. Can be used appropriately. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

One aspect relates to a method for preventing or treating kidney disease in an individual comprising administering a root extract or a fraction thereof to an individual other than a human being, a jujube tree (Zizyphus jujuba MILL.).

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, for example by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. I can. The administration can be administered systemically or locally. The administration may be topically administered to a wound site.

The composition may be administered in combination with drugs such as steroids, anti-inflammatory agents, and antibiotics in order to effectively prevent or treat kidney disease in an individual or secondary disease resulting therefrom. The combined administration may be sequential, simultaneous, or individually administered to the individual.

The administration may be performed by using a jujube tree root extract, a fraction, or a compound represented by Formulas 1 to 8 isolated from the fraction per individual per day from 0.001 μg/mL to 1,000 μg/mL, for example, 0.01 μg/mL to 500 μg/mL , 0.1 μg/mL to 100 μg/mL, 0.1 μg/mL to 50 μg/mL, μ0.1 μg/mL to 25 μg/mL, 1 μg/mL to 1,000 μg/mL, 1 μg/mL to 500 μg /mL, 1 μg/mL to 100 μg/mL, 1 μg/mL to 50 μg/mL, 1 μg/mL to 25 μg/mL, 5 μg/mL to 1,000 μg/mL, 5 μg/mL to 500 μg/ mL, 5 μg/mL to 100 μg/mL, 5 μg/mL to 50 μg/mL, 5 μg/mL to 25 μg/mL, 10 μg/mL to 1,000 μg/mL, 10 μg/mL to 500 μg/mL , 10 μg/mL to 100 μg/mL, 10 μg/mL to 50 μg/mL, or 10 μg/mL to 25 μg/mL may be administered.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Jujube tree ( Zizyphus jujuba MILL.) Obtaining Triterpenoids Compounds Derived from Roots

1.1 Jujube tree ( Zizyphus jujuba MILL.) Obtaining extracts and fractions from roots

The roots of the dried jujube tree ( Zizyphus jujuba MILL . ) obtained from the pearl material were provided from the Herbal Medicine Institute of the College of Pharmacy at Seoul National University, and 7.5 kg of the dried jujube tree roots were used twice for 3 hours each with 30 L of methanol at room temperature. Through ultrasonic extraction, a methanol extract was prepared. The methanol extract was concentrated under vacuum to prepare 630.4 g of a crude extract. The crude extract was suspended in water (H ₂ O) and then sequentially extracted with chloroform and ethyl acetate to obtain 103.5 g of chloroform fraction and 75.0 g of ethyl acetate fraction, respectively.

1.2 Obtaining the compound from the chloroform fraction

Using Kiesgel 60 silica gel (40-60㎛, 230-400 mesh, Merck, Whitehouse, NJ, USA) as the chloroform fraction, chloroform and methanol were gradually increased in a volume ratio of 100:1 to 3:1, and sequentially Silica gel column chromatography was performed to obtain 10 fractions (C1 to C10).

Among them, silica gel column chromatography was performed on the C3 fraction. Specifically, Kiesgel 60 silica gel (40-60㎛, 230-400 mesh, Merck, Whitehouse, NJ, USA), chloroform and methanol were gradually increased in a volume ratio of 200:1 to 20:1 and sequentially carried out, 6 fractions (c3a ~ c3f) was obtained. Among them, the c3e and c3f fractions were subjected to recrystallization using methanol to obtain 124.6 mg of the compound of Formula 7 and 26.4 mg of the compound of Formula 8, respectively.

Next, from the C5 fraction, a white solid substance insoluble in methanol was obtained, and then recrystallization was performed using methanol to obtain 12.5 g of the compound of Formula 6.

Using Kiesgel 60 silica gel (40-60㎛, 230-400 mesh, Merck, Whitehouse, NJ, USA) for the rest of the C5 fraction, chloroform and methanol were gradually increased from 100:1 to 10:1 by volume. By sequentially performing silica gel column chromatography, 10 fractions (c5a to c5j) were obtained. Among them, the c5e fraction was subjected to recrystallization using methanol to obtain 45.8 mg of the compound of Formula 1.

Thereafter, Sephadex LH-20 chromatography (Sephadex LH-20 (25-100 μm, Pharmacia, Piscataway, NJ, USA) was performed on the c8 fraction in a 1:1 volume ratio of chloroform and methanol, and 5.8 mg of the compound of Formula 5 Was obtained.

1.3 Obtaining the compound from the chloroform fraction

The ethyl acetate fraction was gradually increased in a volume ratio of chloroform and methanol from 50:1 to 1:1 with Kiesgel 60 silica gel (40-60㎛, 230-400 mesh, Merck, Whitehouse, NJ, USA) to sequentially silica gel column Chromatography was performed to obtain 14 fractions (e1 to e14).

Among them, the e7 fraction was subjected to recrystallization using methanol to obtain 14.5 g of the compound of Formula 2.

Thereafter, the e8 fraction was subjected to recrystallization using methanol to obtain 276.4 mg of the compound of Formula 3.

Next, Sephadex LH-20 chromatography (Sephadex LH-20 (25-100㎛, Pharmacia, Piscataway, NJ, USA) in a 1:1 volume ratio of dichloromethane (CH ₂ Cl ₂ ) and methanol for the e9 fraction ) To obtain 39.6 mg of the compound of Formula 4.

Example 2. Jujube tree ( Zizyphus jujuba MILL.) Structure analysis of root-derived compounds

2.1 Analysis of the structure of the compound of Formula 1

The molecular structure of the compound of Formula 1 isolated in Example 1 was determined by obtaining ¹ H NMR and ¹³ C NMR spectra through nuclear magnetic resonance (NMR) analyzers (Bruker AMX 600 spectrometers), and the structure of the compound identified in the following c5e fraction Was determined by the following Chemical Formula 1, and the results of the structure analysis are summarized in Table 1, and it was confirmed that the compound of Chemical Formula 1 was 3-dehydroxyceanothetric acid 2-methyl ester.

Compound of Formula 1:

No. δ _H
(600MHz,
pyridine- d ₅ ) δ _c
(150MHz,
pyridine- d ₅ ) No. δ _H
(600MHz,
pyridine- d ₅ ) δ _c
(150MHz,
pyridine- d ₅ ) One 2.69, d (7.6) 55.7 17 57.0 2 176.8 18 2.28, m 52.7 3 1.85, m
1.78, m 42.6 19 3.71, m 48.3 4 38.6 20 151.6 5 1.80, m 56.5 21 2.18, m
1.53, m 31.6 6 1.31, m 19.1 22 2.29, m
1.53, m 38.1 7 1.99, m
1.81, m 38.1 23 1.17, s 31.9 8 41.7 24 0.89, s 27.0 9 2.17, m 46.5 25 0.89, s 19.8 10 51.6 26 1.16, s 18.5 11 1.89, m
1.55, m 24.4 27 178.9 12 2.62, m
2.05, m 27.2 28 179.8 13 2.98, dt
(4.8, 12.9) 40.7 29 5.08, s
4.82, s 110.7 14 60.8 30 1.92, s 19.8 15 2.50, m
1.91, m 29.3 -OMe 3.78, s 51.3 16 2.91, m
1.95, m 35.8

2.2 Structural analysis of compounds of formulas 2, 3, 4 and 5

The compounds of Formulas 2, 3, 4 and 5 isolated in Example 1 were obtained through a nuclear magnetic resonance (NMR) analyzer (Bruker AMX 500 and 600 spectrometers) to obtain ¹ H NMR and ¹³ C NMR spectra, which were obtained from literature values and By comparison, the molecular structure was determined as follows.

Compounds of Formulas 2, 3, 4 and 5 were identified as ceanothic acid (Chemical Formula 2), epiceanothic acid (Chemical Formula 3), 24-hydroxy ceanothic acid (Chemical Formula 4), and 3- O- protocatechuoylceanothic acid (Chemical Formula 5), respectively.

Compound of Formula 2:

Compound of Formula 3:

Compound of Formula 4:

Compound of Formula 5:

2.3 Structural analysis of compounds of formulas 6, 7 and 8

¹ H NMR and ¹³ C NMR spectra were obtained from the compounds of Formulas 6, 7, and 8 isolated in Example 1 through a nuclear magnetic resonance (NMR) analyzer (Bruker AMX 500 and 600 spectrometers), and compared with the literature values. The molecular structure was determined as follows.

Compounds of Formulas 6, 7 and 8 were confirmed to be betulinic acid (Chemical Formula 6), 2- O - trans - p- coumaroylalphitolic acid (Chemical Formula 7), and 2- O - cis - p- coumaroylalphitolic acid (Chemical Formula 8), respectively. .

Compound of Formula 6:

Compound of Formula 7:

Compound of Formula 8:

Example 3. Jujube tree ( Zizyphus jujuba MILL.) Confirmation of the protective effect of extracts, fractions and compounds derived from roots against kidney toxicity

An experiment was conducted to confirm whether the compound isolated from the extract and fraction of the jujube tree root can exhibit a protective effect against cisplatin-induced renal tubular cell damage. Renal tubular epithelial cells LLC-PK1 (ATCC CL-101 ^TM ) were used to evaluate the protective effect on renal toxicity as follows. LLC-PK1 cells were added with 10% fetal bovine serum (Gibco BRL Life Technologies), 100 units/ml of penicillin G (Gibco BRL Life Technologies) and 100 ug/ml of streptomycin (Gibco BRL Life Technologies) It was cultured in a 95% air/5% carbon dioxide incubator maintained at 37°C using a DMEM (Cellgro, Manassas, VA, USA) medium. After introducing 10 ⁴ cells of LLC-PK1 cultured into each well of a 96-well culture plate each including 100 μl of the same medium, and allowing the cells to stabilize for 24 hours, the sample was added to 90 μl of the same medium at the specified concentration and added for 2 hours. During incubation. Subsequently, 10 µM cisplatin dissolved in the same medium was added each and cultured for 24 hours, and then 10 µl of CCK-8 (CCK-8, Dojindo Laboratories, Japan) reagent was added to each well. After incubation at 37° C. for 30 minutes, renal tubular epithelial cells treated with 25 uM cisplatin by measuring absorbance using a 450 nm detection wavelength in a BIO-TEK (Winooski, VT, USA) microplate reader Table shows the survival rate of renal tubular epithelial cells that appear when the ethanol and hot water extracts of jujube tree roots, chloroform, ethyl acetate, butanol, and water fractions and the compounds of formulas 1 to 8 isolated from the fractions are contacted with LLC-PK1 at various concentration It is shown in 2.

division Concentration (μg/mL) Cell viability (%) Ethanol extract 0 57.2±4.1 12.5 58.3±3.4 25 59.5±2.9 50 66.3±1.4 100 83.1±0.3 200 88.8±3.5 Hot water extract 0 57.3±1.7 12.5 61.4±3.4 25 63.6±4.1 50 68.1±3.7 100 86.4±2.1 200 88.2±3.5 Chloroform fraction 0 58.7±4.2 12.5 60.7±2.6 25 70.1±2.7 50 78.8±0.9 100 81.1±1.4 200 93.0±1.7 Ethyl acetate fraction 0 58.7±4.2 12.5 61.9±4.5 25 77.6±2.0 50 78.8±0.7 100 80.7±1.6 200 91.2±2.3 Butanol fraction 0 58.7±4.2 12.5 66.5±2.4 25 66.8±1.5 50 73.0±3.4 100 72.7±3.1 200 74.0±3.1 Water fraction 0 58.7±4.2 12.5 62.4±3.3 25 65.0±4.5 50 69.8±2.5 100 67.5±4.8 200 72.5±2.7 division Concentration (μM) Cell viability (%) Compound of formula 1 0 47.9±2.4 10 49.1±4.0 25 58.6±3.4 50 63.9±4.5 100 73.8±2.8 200 81.8±4.6 Compound of formula 2 0 53.8±1.4 10 41.9±4.6 25 34.7±4.9 50 31.0±2.7 100 30.2±2.3 200 26.0±4.0 Compound of formula 3 0 55.5±2.1 10 61.5±1.5 25 55.3±4.0 50 40.2±3.9 100 34.5±3.1 200 33.6±2.9 Compound of formula 4 0 53.8±3.9 10 53.5±2.0 25 62.5±3.0 50 62.5±1.5 100 67.7±1.5 200 76.5±4.3 Compound of formula 5 0 47.9±2.4 10 48.4±4.7 25 69.3±0.9 50 71.8±4.8 100 71.4±2.9 200 70.9±3.5 Compound of formula 6 0 52.9±2.0 10 58.6±0.8 25 59.6±1.0 50 59.2±3.0 100 58.4±3.1 200 61.0±2.6 Compound of formula 7 0 52.9±2.0 10 59.1±2.5 25 60.6±2.0 50 64.5±1.6 100 71.1±2.5 200 72.8±4.3 Compound of formula 8 0 52.9±2.0 10 58.8±2.0 25 57.5±2.4 50 61.7±1.8 100 63.4±2.0 200 67.3±1.4

When the LLC-PK1 was treated with cisplatin, the number of viable cells was reduced to 50% or less than that of the non-treated group without treatment, so that damage to renal tubular cells due to cisplatin could be confirmed. However, as shown in Table 1 above, the cell viability reduced by cisplatin treatment increased significantly depending on the treated concentration when treated with ethanol and hot water extracts of jujube tree roots, chloroform, ethyl acetate, butanol, and water fractions, respectively. I could confirm. In particular, the ethanol and hot water extracts of jujube tree roots showed that the ethanol, hot water extract, chloroform, and ethyl acetate fractions showed a survival rate of 80% or more at a concentration of 100 μg/mL or more, showing excellent renal toxicity protection.

As for the compound, it was confirmed that both the compounds isolated from the chloroform fraction or the ethyl acetate fraction increased the survival rate of renal tubular cells. In particular, it was confirmed that the compounds of Formulas 1, 4, 5, 7 and 8 obtained from the chloroform fraction significantly increased the cell viability. In particular, it was confirmed that the compound of Formula 1 increased the viability of cells by 80% or more at a concentration of 200 μM, thereby exhibiting excellent protective effects against renal toxicity.

Example 4. Confirmation of the protective effect on kidney toxicity of the compound of formula 1 derived from the root extract of jujube tree

An experiment was carried out to confirm whether compound 1 of the compounds derived from the extract of jujube tree roots exhibits a protective effect against renal toxicity by cisplatin. The LLC-PK1 cells cultured in each well of a 6-well culture plate were introduced into a medium containing 3 ml of the medium composition of Example 3 each 4x10 ⁵ and cultured for 24 hours to stabilize the cells, and then the compound of Formula 1 was added to 100 , 200 μM was added to 2.7 ml of the same medium composition and incubated for 2 hours. Subsequently, the dissolved 25 μM cisplatin was added to the same medium by 0.3 ml each and cultured for 24 hours, and then the cells were cultured so that the number of cells was ⁵ ×10 ⁵ to ⁵ ×10 ⁶ cells/mL using trypsin. Discard the supernatant by centrifugation, and then resuspend the cultured cells in 100 μL of annexin binding buffer containing 100 mM HEPES, 140 mM NaCl, and 25 mM CaCl ₂ at pH 7.4, and then resuspend the cultured cells in 5 μL Nexin V Alexa Fluor 488 was added to fix and stain in a dark room at room temperature for 30 minutes. Thereafter, the cell suspension was centrifuged to discard the supernatant, and the precipitated cell pellet was resuspended in 100 μL of an annexin binding buffer, and 1 μL of propidium was added to fix and stain the cells at room temperature and dark room for 1 minute. Analysis of the stained cells was performed using a Tali Image-based cytometer and TaliPCApp (version 1.0) to confirm the appearance and apoptosis of LLC-PK1 cells treated with the compound of Formula 1 and cisplatin. The results confirmed by the graph are shown in Fig. 1B and Table 3.

division Concentration (μM) Apoptosis (%) Compound of formula 1 0 31.3±0.5 100 12.0±1.7 200 6.0±0.0

As shown in Fig. 1A, when most of the cells were treated with 25 μM cisplatin compared to the control group, which is the non-cisplatin group, which has a normal round nucleus and an intact plasma membrane, the plasma membrane is damaged with concentration of chromatin and condensation of the nucleus. Cells that were stained with annexin V and exhibited green fluorescence, that is, cells that died out were identified. In the group treated with the compound of Formula 1 at 100 and 200 μM, it was confirmed that cells exhibiting green fluorescence were reduced. By quantifying the cell death rate, it was confirmed that the compound of Formula 1 significantly reduced apoptosis by cisplatin in LLC-PK1 cells, as confirmed in FIGS. 1B and 2. Therefore, it was confirmed that the compound of Formula 1 markedly reduces the apoptosis rate of the kidney cells, thus effectively exhibiting a kidney protective effect.

Example 5. Confirmation of the inhibitory effect on kidney cell death of the compound of formula 1 derived from a jujube tree root extract

An experiment was conducted to confirm that the apoptosis inhibitory effect of Compound 1 among the compounds derived from the extract of jujube tree root was due to changes in the expression of signaling substances involved in cell death. The LLC-PK1 cells cultured in each well were introduced into a medium containing 3 ml of the medium composition of Example 3 each ⁴ ×10 ⁵ and cultured for 24 hours to stabilize the cells, and then the compound of Formula 1 was added to a concentration of 100, 200 μM. It was added to 2.7 ml of the same medium composition and incubated for 2 hours. Subsequently, the lysed 25 μM cisplatin was added to the same medium in 0.3 ml each and cultured for 24 hours, and then the cells were collected using a cell scraper. After washing the collected cells once with phosphate-buffered saline (PBS), the cell suspension was centrifuged to discard the supernatant, and the precipitated cell pellet was repa containing 1 mM phenylmethylsulfonyl fluoride. A buffer (RIPA buffer, Cell Signaling, MA, USA) was added and left at 4° C. for 20 minutes. Protein was quantified using a protein analysis kit (BCA protein detection kit, Thermo Scientific, Rockford, USA) on the supernatant obtained by centrifuging the reaction product at 12,000 rpm for 20 minutes to remove cell residues from the separated cell lysate. Was carried out. Cell lysates of 20 μg per well were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to develop the denatured protein. The developed protein was transferred to a polyvinylidene difluoride membrane (PVDF membrane) (Merck Millipore, Germany). To prevent non-specific binding of the antibody to the polyvinylidene fluoride membrane, a TBST buffer containing 5% skim milk (20 nM tris-HCl, 150 mM NaCl, and 0.05% Tween-20, pH 7.5) ) Was added and allowed to stand at room temperature for 1 hour to block non-specific binding sites. After that, the membrane was washed 3 times for 10 minutes each with TBST buffer. Next, the expression levels of P-P38, P38, P-JNK, JNK, P-ERK, ERK, cleaved caspase-3, -8, -9, Bax, Bcl-2 and GAPDH To measure, the rabbit primary antibody (Cell Signaling, Danvers, USA) that binds to each protein was diluted 1:1,000, reacted at room temperature for 1 hour, and washed 3 times for 10 minutes with TBST buffer. Afterwards, a goat anti-rabbit IgG secondary antibody (Calbiochem, La Jolla, CA, USA) was diluted 1:2,000 and reacted with the membrane for 2 hours at room temperature, and the protein was confirmed with an ECL detection kit (GE healthcare). . The results of confirming the expression of P-P38, P38, P-JNK, JNK, P-ERK, ERK are shown in Figs. 2A and 3, -8, -9, Bax, Bcl-2, and GAPDH expression levels are shown in Fig. 2B. Shown in.

As shown in FIGS. 2A and B, when cisplatin was treated in LLC-PK1 cells, it was confirmed that a signal transducer protein inducing cell death was expressed compared to the non-cisplatin-treated group, but when the compound of Formula 1 was treated It was confirmed that protein expression was similar to that of the cisplatin-treated control group, and in particular, when compound 1 was treated at a concentration of 200 μM, it was confirmed that protein expression was almost similar to that of the control group not treated with cisplatin. Therefore, it was confirmed that the compound of Formula 1 contained in the extract extracted from the roots of jujube trees effectively inhibits signaling substances that induce apoptosis by renal cell-damaging substances, thereby protecting renal cells.

Claims (20)

Jujube tree ( Zizyphus jujuba MILL.) A pharmaceutical composition for preventing or treating kidney disease comprising a root extract as an active ingredient. The pharmaceutical composition for preventing or treating kidney disease according to claim 1, wherein the extraction solvent of the jujube tree root extract is extracted with water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof. The pharmaceutical composition for preventing or treating kidney disease according to claim 2, wherein the lower alcohol is methanol. The pharmaceutical composition for preventing or treating kidney disease according to claim 1, wherein the composition comprises one or more solvent fractions selected from the group consisting of chloroform, ethyl acetate, butanol, and water of a jujube tree root extract. The pharmaceutical composition for preventing or treating kidney disease according to claim 4, wherein the chloroform fraction comprises a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

The pharmaceutical composition for preventing or treating kidney disease according to claim 1, which comprises 50 μg/mL to 200 μg/mL of the jujube tree root extract. The pharmaceutical composition for preventing or treating kidney disease according to claim 1, wherein the jujube tree root extract inhibits apoptosis. The pharmaceutical composition for preventing or treating kidney disease according to claim 1, wherein the jujube tree root extract inhibits cell damage caused by cisplatin. The pharmaceutical composition for preventing or treating kidney disease according to claim 1, wherein the kidney disease is selected from the group consisting of kidney damage, kidney failure, and nephritis. A composition for preventing or treating kidney disease comprising any one or more active ingredients of a compound represented by the following Formulas 1, 2, 3, 4, 5, 6, 7, and 8 or a pharmaceutically acceptable salt thereof.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

The method according to claim 10, comprising two or more active ingredients, a pharmaceutical composition for preventing or treating kidney disease. The pharmaceutical composition for preventing or treating kidney disease according to claim 10, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition for preventing or treating kidney disease according to claim 10, wherein the compound or a pharmaceutically acceptable salt thereof is extracted and isolated from the roots of jujube trees. The pharmaceutical composition for preventing or treating kidney disease according to claim 10, wherein the compound or a pharmaceutically acceptable salt thereof is artificially synthesized. The pharmaceutical composition for preventing or treating kidney disease according to claim 10, wherein the active ingredient is composed of single, 2, 3, 4, 5, 6, 7 or 8 active ingredients. Jujube tree ( Zizyphus jujuba MILL.) A food composition for preventing or improving kidney disease, comprising a root extract as an active ingredient. Jujube tree ( Zizyphus jujuba MILL.) A quasi-drug composition for preventing or improving kidney disease comprising a root extract as an active ingredient. Jujube tree ( Zizyphus jujuba MILL.) Method for producing a jujube tree root extract or a fraction thereof, comprising the step of extracting the roots with water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof to obtain an extract. The method of claim 19, further comprising obtaining a fraction from the obtained extract using at least one solvent selected from the group consisting of chloroform, ethyl acetate, butanol, and water. Jujube tree ( Zizyphus jujuba MILL.) A method of preventing or treating kidney disease in an individual comprising administering a root extract or a fraction thereof to an individual other than humans.

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