KR101833332B1 - n-Hexane fractions of Morning glory seeds for nephroprotection and treating acute renal failure - Google Patents

Abstract

The present invention relates to a normal hexane fraction of pharbitis seeds which is effective for renal protection and acute renal failure therapy. The normal hexane fraction of the pharbitis seeds has an effect of recovering renal tissues which are damaged by administering an anticancer drug such as cisplatin, and is effective in inhibiting production of NO in renal cells and inhibiting apoptosis to inhibit an increase of blood urea nitrogen (BUN), serum creatine, and uric acid, thereby treating, preventing, and alleviating acute renal failure by pharmaceutical mechanism which hinders deformation of glomeruli and renal tubule. Therefore, the normal hexane fraction of pharbitis seeds can be applied as an active ingredient for the production of renal-protective health food and medicine for preventing and treating the acute renal failure.

Description

{N-Hexane fractions of Morning glory seeds for nephroprotection and treating acute renal failure effective in renal protection and acute renal failure therapy}

The present invention relates to a normal hexane fraction of Schizophrenia effective for renal protection and acute renal failure therapy.

The main function of the kidneys is to filter out urine and adjust the balance of body water, electrolytes, acids and bases to maintain the body's fluid condition at all times. This kidney function is not normally performed is called kidney failure. Renal failure can be classified as acute or chronic depending on the time of onset.

Acute renal failure is a clinical syndrome that causes a rapid decline in renal function caused by various causes such as decreased renal blood flow, glomerulonephritis, nephrotoxic antibiotics, or the use of anticancer drugs. Such acute renal failure is accompanied by a decrease in glomerular filtration rate (GFR), a decrease in urine volume, a hyperglycemia due to accumulation of nitrogen waste, or an imbalance of body fluids and electrolytes. Renal insufficiency in acute renal failure is a high-risk disease with a mortality rate of 50%, which is difficult to recover if failure of initial cause removal fails. In particular, hyperlipidemia, also known as uremic manifestation in acute renal failure, is caused by a sudden loss of renal function as a result of damage to the tubules or a decrease in glomerular filtration rate (GFR).

Causes of acute renal failure can be divided into three major categories, renal (renal) and renal (postrenal) based on the kidney. Renal failure is a renal failure caused by obstruction of blood flow to the kidney. Renal failure is kidney failure due to direct damage of the kidney itself due to disease or neoplastic cytotoxicity. Examples of the diseases that are associated with renal failure include glomerulonephritis, renal microvascular disease, prolonged systemic renal failure, or acute renal tubular necrosis caused by drugs, thrombosis, trauma, atherosclerosis, and nephropathic renal failure, Is a renal failure caused by obstruction of any part of the urinary tract.

As described above, acute renal failure can be caused by various causes. It has been reported that acute renal failure caused by exogenous cisplatin, which accounts for most of acute renal failure, occurs when renal tubular cells absorb cisplatin. Cisplatin (cisplatin, cis-diaminedichloroplatinum II), which induces acute renal failure, is one of the commonly used anticancer drugs and causes acute renal failure due to structural abnormality of the tubule. Tubular damage caused by cisplatin is known to be mainly caused by free radicals. In addition, the increase of lipid peroxidation in the kidney is also involved in the renal toxicity by cisplatin, and it is known that cisplatin itself reduces the intrinsic content of GSH (glutathione), which suppresses the antioxidant effect in the kidney and shows antioxidant effect.

On the other hand, Pharbitidis Semen refers to seeds of Pharbitis nil Choisy. The black seed of morning glory is called black 牛, and the white seed is called white 牛. It is used when the body is swollen and has a strong diuretic effect and a diuretic effect and the hypochlorous action and chronic pyelonephritis and cirrhosis when the body is poured. It is known that it contains Pharbitin, Nilic acid and Gallic acid. Among them, parvitin has an anti-inflammatory effect as an important ingredient, and has been reported to be effective also in alleviation of edema and anti-obesity.

In addition, many patent applications for medicines using herbal medicines have been filed. Specifically, Korean Patent Publication No. 10-0991857 (Patent Document 1) discloses a pharmaceutical composition using a Zygote extract as a therapeutic agent for periodontal disease, and Korean Patent Laid-Open Publication No. 10-2013-0023172 (Patent Document 2) (Patent Document 3) discloses a pharmaceutical composition using a black shark extract as a therapeutic agent for cardiovascular diseases.

However, to date no reports have been reported on the use of the fraction of the respondent for the purpose of treatment, prevention or amelioration of acute renal failure.

Korean Patent Publication No. 10-0991857 Korean Patent Publication No. 10-2013-0023172 Korean Patent Publication No. 10-2013-0023172

The present inventors have continuously studied to increase the usability of the testosterone. As a result, it has been shown that the normal hexane fraction of the kidney protects the renal tissue from damage caused by cisplatin in the kidney protective mechanism, The present invention has been accomplished by confirming that it has an effect of inhibiting apoptosis and inhibiting renal glomeruli and tubular tubular degeneration.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating and preventing acute renal failure including a normal hexane fraction of Morning glory seed or Pharbitidis Semen .

Another object of the present invention is to provide a health food composition for the protection of kidneys comprising a normal hexane fraction of Morning glory seed or Pharbitidis Semen .

In order to solve the above-mentioned problems, the present invention provides a preventive and therapeutic agent for acute renal failure, which comprises an effective component of the fragrant northern hexane fraction obtained by fractionating C _1-4 alcohol extract of Morning glory seed or Pharbitidis Semen with normal hexane There is provided a pharmaceutical composition for oral administration.

In addition, the present invention provides a health food composition for the protection of kidneys, wherein the fraction of normal nile hexane obtained by fractionating C _1-4 alcohol extract of Morning glory seed or Pharbitidis Semen with normal hexane is contained as an active ingredient.

In the composition of the present invention, the normal nematic fraction of Schizophyllum, which is contained as an active ingredient, inhibits nitric oxide increased by cisplatin, inhibits intracellular inflammation, inhibits kidney cell death, It is also effective in the treatment or prevention of acute renal failure due to side effects of cisplatin.

Thus, the normal fraction of normal hexane can be used as an active ingredient in medicines or health foods for the protection of the kidney or for the treatment, prevention or amelioration of acute renal failure.

FIG. 1 is a graph showing the recovery rate of kidney cells obtained by treating the normal hexane fraction of pufferfish in kidney cells induced by cisplatin. FIG.
FIG. 2 is a graph showing the recovery rate of kidney cells obtained by treating the ethylacetate fractions of the responder in kidney cells induced by cisplatin. FIG.
FIG. 3 is a graph comparing the recovery rates of kidney cells according to treatment concentrations of the normal hexane fraction of pseudomonas.
FIG. 4 is a graph comparing the inhibitory effect of nitric oxide against the dose of the normal hexane fraction in the kidney cells induced by nitric oxide production by cisplatin.
FIG. 5 is a graph comparing the inhibitory effect of apoptosis-related factors on the dose of normal hexane fractions obtained from papillomavirus.
FIG. 6 is a graph showing changes in blood urea nitrogen (BUN), serum creatine, and uric acid according to the dose of the normal hexane fraction.
FIG. 7 is a photograph comparing histopathologic changes according to the doses of the normal hexane fractions of the insect.

Hereinafter, the present invention will be described in detail so that those skilled in the art can easily carry out the present invention.

The present invention relates to a pharmaceutical composition or a health food composition for treating, preventing or ameliorating kidney-protective function or acute renal failure by containing a normal fraction of nasal hexane as an active ingredient.

The crude normal hexane fraction according to the present invention is a natural product obtained from a natural raw material. In addition, the normal hexane fraction has the effect of restoring the renal tissue damaged by the anticancer medication such as cisplatin, and inhibiting the generation of nitric oxide (NO) in the kidney cells and inhibiting apoptosis. In addition, the normal hexane fraction inhibits renal glomeruli and tubular degeneration by effectively inhibiting the increase of blood urea nitrogen (BUN), serum creatine and uric acid in the blood, It has the treatment, prevention and improvement efficacy of kidney failure. Particularly normal normal hexane fractions are effective for the treatment, prevention and improvement of renal renal failure induced by cisplatin.

The method for obtaining the normal hexane fraction from Morning glory seed or Pharbitidis Semen is as follows:

I) extracting the insecticide with at least one extraction solvent selected from the group consisting of C _1-4 alcohol and C _1-4 alcohol aqueous solution to obtain an alcoholic extract;

Ii) suspending the alcohol extract in water and then fractionating it with n-hexane to obtain a fraction.

The method for obtaining the fragrant fraction is described in more detail as follows.

Step i) is a step of obtaining a protracted extract.

In the present invention, Morning glory seed or Pharbitidis Semen is used as a natural raw material. The rats were cleaned with water, dried and shredded. And extracted by immersing in at least one extraction solvent selected from the group consisting of C _1-4 alcohol and C _1-4 alcohol aqueous solution. The extraction solvent may be selected from methanol, ethanol, isopropanol, propanol, butanol, and an aqueous solution thereof. Preferably, ethanol, ethanol aqueous solution or alcohol is used, more preferably an ethanol aqueous solution of 50 to 90% , And most preferably an ethanol aqueous solution having a concentration of 70% by weight is used. The amount of the extraction solvent is preferably 1: 1 to 30 times by weight, more preferably 1:10 to 20 times by weight, based on the dry weight of the sweet potato, and the amount of the extraction solvent is not particularly limited I do not. The extraction temperature may be in the range of from room temperature to the reflux temperature of the extraction solvent, and specifically, it may be extracted at a temperature of 20 to 90 ° C. The extraction time is preferably 1 to 10 days, but is not limited thereto. The number of times of extraction may be one or more times, but since the yield of the active ingredient is significantly reduced as the extraction continues, it may not be economical to repeat the extraction more than five times. Therefore, the extraction is preferably repeated two to five times Do.

The extraction method is a method commonly known in the art, for example, a method using an extraction device such as a supercritical extraction, a subcritical extraction, a high-temperature extraction, a high-pressure extraction or an ultrasonic extraction, or a method using an adsorption resin containing XAD and HP- And the like can be used.

The extract is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. The resulting extract may also be subjected to vacuum drying, vacuum drying, boiling drying, spray drying, room temperature drying, freeze drying, and the like, if necessary. In particular, when the freeze-drying method is applied, there is an advantage that loss of volatile organic substances in the extract can be reduced.

Step ii) is a step of extracting the crude extract with normal hexane to obtain fractions.

Specifically, the slough extract obtained in the step (i) is suspended in water and then fractionated with n-hexane to obtain fractions. The above fraction can be obtained by repeating from 1 time to 5 times, preferably 3 times. The fraction is concentrated under reduced pressure using a vacuum rotary evaporator or the like to obtain an extract. The resulting extract may also be subjected to vacuum drying, vacuum drying, boiling drying, spray drying, room temperature drying, freeze drying, and the like, if necessary. In particular, when the freeze-drying method is applied, there is an advantage that the loss of volatile organic substances in the fraction can be reduced.

The fragile n-hexane fraction obtained through the above method can be used as an effective ingredient in a health food for the purpose of kidney protection and a drug for the treatment and prevention of acute renal failure.

The pharmaceutical composition according to the present invention contains the normal hexane fraction as an active ingredient and may be contained in a liquid or lyophilized powder form. The above-described normal hexane fraction may be contained in an amount of 0.1 to 50% by weight based on the total weight of the pharmaceutical composition, but the present invention is not limited thereto.

When the pharmaceutical composition is used clinically, it may be formulated together with carriers customary in the pharmaceutical field to prepare pharmaceutical preparations customary in the pharmaceutical field, such as tablets, capsules, powders, granules, pills, liquids and suspensions Preparations for oral administration; Injectable preparations in the form of injectable solutions or suspensions, or ready-to-use injectable dry powders which can be used as distilled water for injection for injection; Or ointments, and the like. The pharmaceutical preparations comminuted with conventional carriers may be administered orally or parenterally, for example intravenously, subcutaneously, intraperitoneally or topically. Accordingly, the pharmaceutical composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, hydroxymethylcellulose, microcrystalline cellulose, silicified microcrystalline cellulose, povidone, crospovidone, croscarmellose sodium, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, Colloidal silicon dioxide, lactose, talc, magnesium stearate, colloidal stearyl magnesium, mineral oil, and the like.

 In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, troches, rosin, capsules and the like, which may contain at least one excipient such as lactose, saccharose, sorbitol , Mannitol, starch, amylopectin, cellulose, calcium carbonate, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, elixirs, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are used in addition to water and liquid paraffin, May be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used. Parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intra-thoracic injection.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the age, body weight, degree of disease, drug form, route of administration, and duration of the patient, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention may be administered at a dose of 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration can be administered several times a day, preferably once or six times, at a predetermined time interval according to the judgment of a doctor or a pharmacist.

In addition, the health food composition according to the present invention contains a normal hexane fraction as an active ingredient for the purpose of preventing or improving renal protection or acute renal failure, and may be contained in a liquid or lyophilized powder form.

The active ingredient may be ingested as food prepared from tablets, capsules, powders, granules, pills, liquid preparations, suspensions, etc., or may be added to general foods. Unlike conventional medicines, the health food uses food as a raw material, so there is no side effect that may occur when a medicine is taken for a long time. The content of the active ingredient can be suitably determined according to its use purpose (for prevention or improvement). Generally, the active ingredient in the health food composition may be contained in an amount of 0.1 to 90% by weight. However, in the case of long-term ingestion intended for health and hygiene purposes or for health control purposes, 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 exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the active ingredient can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolates, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, , Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense. The shape of the food is not particularly limited, and it may be a solid, semi-solid, gel, liquid, or powder.

The beverage can be prepared using the health food composition of the present invention. There are no particular restrictions on the selection of other ingredients other than the active ingredient as a component to be contained in the beverage, and it may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As other flavoring agents, natural flavoring agents (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the health food composition of the present invention may contain various additives such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.) Salts, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. It can also contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to 20% by weight in the health food composition of the present invention.

The present invention as described above is explained in more detail based on the following examples, but the present invention is not limited thereto.

[Example]

Production example. Preparation of extracts and solvent fractions

Pharbitidis Semen was washed and dried and then shredded. 1 kg of crushed slough was immersed in 15 kg of 70% ethanol and extracted at 80 to 90 ° C for 5 to 6 hours. The extract was filtered and concentrated under reduced pressure to obtain 110 g (yield: 11%) of the ethanol extract of the crude oil.

100 g of the above extract obtained above was suspended in 2 L of distilled water, followed by fractionation in turn using 20 L of n-hexane, dichloromethane, ethyl acetate and n-butanol and distillation under reduced pressure to obtain respective solvent fractions. The yield of each solvent fraction is as follows: 5.2 g of the n-hexane fraction, 37 g of the dichloromethane fraction, 29.5 g of the ethyl acetate fraction, 16.3 g of the n-butanol fraction and 6.3 g of the remaining water fraction.

Example 1: Cytotoxicity measurement for extracts and solvent fractions

Cytotoxicity was measured on the slug extract and each solvent fraction obtained in the above Preparation Example.

The degree of cell survival or proliferation was measured by MTT method. Human embryonic kidney cells (HEK-293) were cultured in a DMEM medium containing 10% serum and antibiotics and in an incubator maintained at 37 ° C with 5 vol.% Carbon dioxide feed. Cells were seeded in 96-well plates at a concentration of 1 × 10 ⁴ cells / well and preincubated for 24 hours. The cells were then seeded at a given concentration (1, 5, 25, 125, 200, 300, / mL) for 24 hours. 50 μL of a 0.5 mg / mL MTT solution was added to each well and incubated at 37 ° C. for 4 hours. The supernatant was removed and the non-aqueous MTT-formazan crystals were dissolved in dimethylsulfoxide (DMSO ) Was added and dissolved. The amount of formazan was measured at 570 nm using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, Calif.).

Table 1 below summarizes the concentration (IC ₅₀ ) that inhibits 50% kidney cytotoxicity for the insecticidal extract and each solvent fraction.

division IC ₅₀ ([mu] g / mL) Ethanol Extract 8.0 ± 0.32

Fraction N-hexane fraction 152.0 + - 7.20 Dichloromethane fraction 27.9 ± 1.88 Ethyl acetate fraction 139.3 ± 5.00 Normal butanol fraction 7.9 ± 2.75 Water fraction 3.7 ± 1.69

According to the above Table 1, it can be seen that the ethanol extract of Pseudomonas aeruginosa is highly toxic and difficult to use as a therapeutic agent for renal failure. Among the five fractions, the fraction of normal hexane and the fraction of ethyl acetate has low cytotoxicity in renal cells, and thus it can be confirmed that it can be used as a therapeutic agent for renal cell protection and renal disease.

Example 2. Recovery efficacy of toxic-induced kidney cells by cisplatin

As a result of the experiment of Example 1, renal cell protection efficacy was measured by the following method for the normal hexane fraction or ethyl acetate fraction of Rhizoma, which was confirmed to have low cytotoxicity in kidney cells.

The degree of cell survival or proliferation was measured by MTT method. Human embryonic kidney cells (HEK-293) were cultured in a DMEM medium containing 10% serum and antibiotics and in an incubator maintained at 37 ° C with 5 vol.% Carbon dioxide feed. Cells were plated in 96-well plates at a concentration of 1 x 10 ⁴ cells / well and pre-cultured for 24 hours. Then, cisplatin (5 μM) was pretreated for 2 hours at a concentration of 25, 50, 100, 200 μg / mL in the normal hexane fraction and the ethyl acetate fraction, respectively, for 24 hours. 50 μL of MTT solution at a concentration of 0.5 mg / mL was added to each well and incubated at 37 ° C. for 4 hours. After that, the supernatant was removed, and the MTT-formazan crystals of water-insoluble were dissolved in dimethylsulfoxide DMSO) was added and dissolved. The amount of formazan was measured at 570 nm using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, Calif.). The cell survival rate when the normal hexane fraction or the ethyl acetate fraction was treated was calculated by the following equation (1), and the results are shown in FIG. 1 and FIG.

[Equation 1]

FIGS. 1 and 2 are graphs showing the recovery rates of kidney cells obtained by treating the fraction of normal hexane or the fraction of ethyl acetate, respectively, in response to toxic-induced kidney cells induced by cisplatin. 2, it can be confirmed that the ethyl acetate fraction of Pseudomonas aeruginosa has almost no effect of inhibiting the renal cytotoxicity induced by cisplatin. On the other hand, FIG. 1 shows that the normal hexane fraction inhibiting the renal cytotoxicity induced by cisplatin was effective. In particular, when treated with 100 μg / mL of normal hexane fraction, approximately 43% .

Example 3. Effective concentration of the normal hexane fraction for the protection of kidney cells

The following experiments were carried out in order to ascertain the titratable concentration of the normal hexane fraction of the deficient kidney cells recovering toxic-induced kidney cells by cisplatin.

The degree of cell survival or proliferation was measured by MTT method. Human embryonic kidney cells (HEK-293) were cultured in a DMEM medium containing 10% serum and antibiotics and in an incubator maintained at 37 ° C with 5 vol.% Carbon dioxide feed. Cells were plated in 96-well plates at a concentration of 1 x 10 ⁴ cells / well and pre-cultured for 24 hours. The normal hexane fraction was pretreated at 50, 60, 70, 80, 90, and 100 ㎍ / mL for 2 hours and cisplatin (5 μM) was treated for 24 hours.

50 μL of MTT solution at a concentration of 0.5 mg / mL was added to each well and incubated at 37 ° C. for 4 hours. After that, the supernatant was removed, and the MTT-formazan crystals of water-insoluble were dissolved in dimethylsulfoxide DMSO) was added and dissolved. The amount of formazan was measured at 570 nm using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, Calif.). In addition, the cell viability at the treatment concentration of normal hexane fraction was calculated by the above Equation 1, and the result is shown in FIG.

As shown in FIG. 3, the kidney cells induced by cisplatin showed excellent recovery effects of about 48.51, 62.76 and 60.05% when treated with the normal hexane fraction at 80, 90, 100 ㎍ / mL concentration .

Example 4 Inhibitory Effect of Nitric Oxide (NO) on Normal Hexane Fractions

In order to confirm the inhibitory effect of the production of nitric oxide (NO) obtained by treating the normal hexane fraction in response to the kidney cells induced by the production of nitric oxide (NO) by cisplatin, the following experiment was conducted.

The degree of inhibition of nitric oxide produced by cisplatin was measured using the Griess test method. Human embryonic kidney cells (HEK-293) were cultured in a DMEM medium containing 10% serum and antibiotics and in an incubator maintained at 37 ° C with 5 vol.% Carbon dioxide feed. Cells were plated in 96-well plates at a concentration of 1 x 10 ⁴ cells / well and pre-cultured for 24 hours. The normal hexane fraction was treated with cisplatin (5 μM) for 24 hours at a concentration of 80, 90 and 100 μg / mL for 2 hours. After 24 hours, the medium was mixed with the same amount of a Griess reagent (Sigma, St Louis, MO, USA), transferred to a 96-well plate and analyzed using a SpectraMAX 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, Calif.) At 540 nm Respectively. At this time, sodium nitrite (Sigma, St Louis, MO, USA) was diluted stepwise to obtain standard values. The measurement results are shown in the graph of Fig.

As shown in FIG. 4, the amount of nitric oxide (NO) induced by cisplatin in kidney cells was significantly reduced by treating the normal hexane fraction. Specifically, treatment with normal hexane fractions at 80, 90, and 100 ㎍ / mL showed about 50.82, 66.09, and 66.47% inhibition of nitric oxide compared with cisplatin alone.

Example 5. Measurement of cell death-suppressing effect of normal hexane fraction

In order to confirm the cell death-suppressing effect obtained by treating the normal hexane fraction in response to the kidney cells induced by apoptosis by cisplatin, the following experiment was conducted.

Human embryonic kidney cells (HEK-293) were cultured on a 60-well plate at a concentration of 5 × 10 ⁵ cells / well for 24 hours, and the normal hexane fraction was pretreated for 2 hours at 80, 90 and 100 μg / Cisplatin (5 [mu] M) was treated for 24 hours. After 24 hours, the cells were washed with ice-cold phosphate buffered saline (PBS, pH 7.4), suspended in RIPA buffer containing 1% proteinase inhibitor mixture, and dissolved on ice for 30 minutes. Cell lysates were centrifuged at 14,000 rpm for 15 minutes at 4 ° C and protein concentration was measured using BCA (Bicinchoninic acid) assay. A total of 40 μg of the protein sample was separated by SDS-PAGE gel, and the protein was transferred onto the nitrocellulose membrane at 290 mA for 2 hours. Protein-transferred membranes were incubated with Trisbuffered saline + Tween-20 (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20) containing 5% After blocking, anti-cleaved caspase-3, 8, and 9 antibodies (1: 1000) were ligated. Immunoreactivity was detected by SuperSignal West Pico Chemiluminescent (Pierce, Rockford, IL, USA) using a peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1: 2000). FluorChem E System (ProteinSimple, San Jose, CA, USA).

FIG. 5 is a graph showing the inhibitory effect on the apoptosis-related factors according to the throughput of the normal hexane fraction. FIG. 5 shows that cleaved caspases 3, 8, and 9 were important factors for inducing apoptosis. The kidney cells were killed by cleaved caspases 3, 8, and 9, which were increased by cisplatin, The concentration of cleaved caspases 3, 8 and 9 decreased in a concentration-dependent manner when the hexane fraction was treated.

Example 6. Assessment of kidney protection of human normal hexane fractions in an animal model of renal toxicity

Five-week-old ICR mice (males) were obtained from Raon Bio and purified for one week with free, regular solid feed and water. The environmental conditions of the animal room were set at a temperature of 23 ° C and 3 ° C, a relative humidity of 50 10%, an illumination time of 12 hours (8:00 am to 8:00 pm), a ventilation frequency of 10 to 20 times / . During the experiment, the temperature and humidity of the animal room were automatically controlled by constant temperature humidification.

The experimental animals were divided into two groups: 1) control group, 2) cisplatin administration group, 3) cisplatin administration group, 50 mg / kg administration group of normal hexane fraction, 4) cisplatin administration group and 100 mg / kg of normal hexane fraction, And fractionated 200 mg / kg.

The hexane fraction was suspended in corn oil (50, 100, 200 mg / kg) for 5 days. Cisplatin was administered by intraperitoneal injection after 2 hours of oral administration of 2 mg / kg of normal hexane fraction. Experimental animals were fed with water only for 12 hours before treatment. After the completion of the experiment, the blood was collected and the serum was separated, and the uric acid and BUN of the serum were measured using the enzyme method (Youngdong, Korea). The creatine concentration was measured by the Jaffe modified direct method (Youngdong, Korea).

FIG. 6 is a graph showing the results of measurement of changes in blood urea nitrogen (BUN), serum creatine, and uric acid according to the dose of the normal hexane fraction. As shown in FIG. 6, the concentrations of blood urea nitrogen (BUN), serum creatine and uric acid in the serum were greatly increased by cisplatin. In addition, the concentration of blood urea nitrogen (BUN), creatine and uric acid in serum were decreased to 69.54, 50.78 and 61.26%, respectively, at the concentration of 100 mg / kg in the normal hexane fraction.

Example 7: Evaluation of kidney protection of female normal hexane fractions in an animal model of renal toxicity

Five-week-old ICR mice (males) were obtained from Raon Bio and purified for one week with free, regular solid feed and water. The environmental conditions of the animal room were set at a temperature of 23 ° C and 3 ° C, a relative humidity of 50 10%, an illumination time of 12 hours (8:00 am to 8:00 pm), a ventilation frequency of 10 to 20 times / . During the experiment, the temperature and humidity of the animal room were automatically controlled by constant temperature humidification.

The experimental animals were divided into three groups; (A) normal group; (B) cisplatin treated group; (C) cisplatin treated group and 50 mg / kg treated normal female fraction; (D) cisplatin treated group and 100 mg / kg normal female fraction Cisplatin, and 200 mg / kg of normal hexane fraction.

The hexane fraction was suspended in corn oil (50, 100, 200 mg / kg) for 5 days. Cisplatin was administered by intraperitoneal injection after 2 hours of oral administration of 2 mg / kg of normal hexane fraction. Experimental animals were fed with water only for 12 hours before treatment.

After the end of the experiment, the kidney tissue was removed, and the tissue was cut, fixed in 10% neutral formalin for 18 hours or more, dehydrated and embedded in paraffin to form a 4 μm section. Hematoxylin & eosin (H & E) staining was then performed and observed under a 200x optical microscope.

Fig. 7 shows photographs showing histopathological changes of kidney tissues extracted from (A) to (E) experimental animals. 7, when compared with the normal group (A), the cisplatin-treated group (B) showed severe denaturation of the glomeruli and tubules. In comparison with the cisplatin-treated group (B), the histopathological changes of the kidney were markedly decreased in the group treated with 100 mg / kg of the normal hexane fraction (D).

Claims (7)

A pharmaceutical composition for preventing, alleviating or treating renal toxicity caused by the administration of an anticancer agent, which comprises, as an active ingredient, a normal hexane fraction of a C _1-4 alcohol extract of a human.
The method according to claim 1,
The pharmaceutical composition for preventing, alleviating or treating renal toxicity caused by administration of an anticancer agent, wherein the anticancer agent is cisplatin.
The method according to claim 1,
Wherein the fraction is a liquid or lyophilized powder. The pharmaceutical composition for preventing, alleviating or treating renal toxicity caused by administration of an anticancer agent.
The method according to claim 1,
The pharmaceutical composition is prepared in any one form selected from the group consisting of tablets, capsules, powders, granules, pills, liquids and suspensions. The pharmaceutical composition for preventing, alleviating or treating renal toxicity .
A health food composition for the prevention of kidney damage from renal toxicity caused by administration of an anticancer agent, comprising a normal hexane fraction of a C _1-4 alcohol extract of a human.
6. The method of claim 5,
Wherein the fraction is in a liquid or lyophilized powder form.
6. The method of claim 5,
Wherein the health food composition is prepared in any one form selected from the group consisting of tablets, capsules, powders, granules, pills, liquid preparations and suspensions.

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