KR20140046565A - Composition for preventing or treating renal disease containing extract of amorpha fruticosa - Google Patents

Abstract

The present invention relates to a composition comprising an Amorpha fruticosa extract as an active ingredient for preventing or treating a renal disease. The composition according to the present invention has few side effects when applied to a human body and low possibility to generate resistance caused by long-term use by comprising natural products obtained from the nature as active ingredients. In particular, the composition of the present invention has superior efficacy for protecting kidney cells, thereby the composition can be valuably used for preventing or treating the renal disease. [Reference numerals] (AA) Cell survival rate; (BB) Concentration; (CC) Example (3-1) ug/ml; (DD) Example (4) uM; (EE) Example (3) uM

Description

Composition for the prevention or treatment of kidney disease containing weasel extract {COMPOSITION FOR PREVENTING OR TREATING RENAL DISEASE CONTAINING EXTRACT OF AMORPHA FRUTICOSA}

The present invention relates to a composition for preventing or treating kidney disease by including a weasel extract as an active ingredient.

The kidneys are the excretory organs of vertebrates that filter out waste products in the blood and send them out in the form of urine.

There are many different toxins that cause kidney toxicity. Chemicals known to cause renal toxicity include antisteroidal antipyretic analgesic anti-inflammatory agents such as phenacetin, aspirin and indomethacin, puromycin, daunomycin, cyclophosphamide, penicillamine, adriamycin, and cisplatin. , Immunosuppressive agents, aminoglycoside antibiotics such as amikacin, gentamycin, gentamycin, kanamycin, neomycin, sisomycin, streptomycin, tobramycin, cephalosporin antibiotics, carbapenem antibiotics, such as mefenenem, Heavy metals such as cadmium, lead, mercury, chromium, and inorganic and organic heavy metal compounds, chloroform, compounds such as D-serine, sulfonamide, 2-bromoethylene, hydrobromide, and mold toxins such as ocaratoxin and citrinin. In most cases, the exact mechanism by which a drug or drug causes kidney toxicity is not known. Many.

Kidneys are the target of toxic substances because the normal adult adult is likely to accumulate toxic substances in the blood because a large amount of blood flows through about 180 L of blood per day. In addition, in the production of urine, urine is temporarily stored in the kidneys, so toxic substances are concentrated and targeted.

Many synthetic preparations have been developed for the prevention or treatment of diseases caused by renal toxicity, but the importance of preparations derived from natural products has been highlighted due to stability issues such as the possibility of side effects occurring in the human body for a long time. .

The present inventors have found that a natural weasel has an effect in the prevention or treatment of kidney disease, and has completed the present invention.

Kumarappan et al., Ren. Fail., V. 30 (3), p. 307-322, 2008

It is an object of the present invention to provide a composition for the prevention or treatment of natural kidney diseases.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of kidney disease comprising a weasel extract as an active ingredient.

The composition according to the present invention includes a natural product obtained from nature as an active ingredient, as well as less side effects when applied to the human body, and is unlikely to develop resistance due to long-term use. In addition, the composition of the present invention can protect the oxidative damage of renal cells, and in particular, it is useful because it can protect the damage of renal cells caused by oxidative stress while maintaining the anticancer effect of the existing anticancer agent is not toxic. . In addition, the composition of the present invention can lower blood creatinine, and thus can be usefully used in the prevention or treatment of kidney disease.

Figure 1 relates to the renal cell damage protection effect on AAPH of each of the weasel extract and the compound isolated therefrom.
Figure 2 relates to the protective effect of renal cell damage on cisplatin of each of the weasel extract and the compound isolated therefrom.

The present invention relates to a composition for the prevention or treatment of kidney disease, comprising, in one aspect, Amorpha fruticosa extract or a fraction thereof as an active ingredient.

In the present specification, ' Amorpha fruticosa ' is a deciduous shrub of the dicotyledonous rosewood legume, also known as prunus vulgaris or dwarf saris , and is native to North America. The leaves are like a birch and the seeds are similar to baektae pods. It is about 3m high and has hairs on the branches, but gradually disappears as it grows. The leaves are shifted and the pinnate leaf is 1 time. The leaflets are 11-25 each, egg-shaped or oval, and the edges are flat. Flowers bloom in May-June and have a purplish blue color with a strong scent. Fruits are fruiting and fruiting in September. Fruit contains one seed and is kidney-shaped.

Weasel used in the present specification is not limited to the method of obtaining the cultivation, may be used by cultivating or commercially available. In addition, weasel is used herein may use part or all of the above ground or underground part of weasel herbaceous. In one embodiment of the present invention, but the ground portion (leaves, twigs, stems), roots and seeds of weasel, but not limited thereto.

As used herein, the term 'active ingredient' refers to a component that can exhibit activity alone or with a carrier that does not exhibit the desired activity.

In for the prevention and treatment of one aspect of the present invention prostrate composition, the ferret suffruticosa extract weasel fruticosa -C ₁ ~ C ₆ may contain an alcohol extract, specifically weasel fruticosa-methanol extract or ferret fruticosa -ethanol It may be an extract.

In the present specification, the 'weasel extract' may be a crude extract of water, C ₁ -C ₆ alcohol, and a solvent selected from the group consisting of a combination thereof. The C ₁ -C ₆ alcohol may specifically be methanol or ethanol. When the weasel is extracted with a solvent, it is preferable to extract by adding a solvent corresponding to about 5 to 15 times the weasel, and specifically, it is preferable to extract by adding about 10 times the solvent, but is not limited thereto. The extraction may be a heat extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction, and the like, there is no limitation if the extraction is obvious to those skilled in the art. The extraction may be performed at room temperature, but for more efficient extraction, the extraction may be performed under heating conditions, preferably at about 40 to 100 ° C., more preferably at about 80 ° C., but is not limited thereto. It is not. The extraction time is preferably about 2 to 4 hours, more preferably about 3 hours, but is not limited thereto, and may vary depending on conditions such as extraction solvent and extraction temperature. The extraction may be extracted one or more times several times in order to obtain a larger amount of the active ingredient, preferably one to five times, more preferably three times the continuous extraction can be used combined extract.

In this specification, weasel extract may include crude extract of weasel as described above, and may be included as a soluble fraction of the organic solvent obtained by further extracting the crude extract with a low polar organic solvent. Examples of the organic solvent include, but are not limited to, hexane, methylene chloride, ethyl acetate, n-butanol, and the like. The extract or the soluble fraction of the extract may be used as it is, or may be used as an extract form by concentration after filtration, and may be used as a form of a lyophilizate by concentration and freeze-drying.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention,

The weasel extract or fractions thereof includes one or more compounds, derivatives thereof or pharmaceutically acceptable salts thereof in the group consisting of Formula 1 and Formula 2,

[Chemical Formula 1]

(2)

In the above formulas,

R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, R31 or R32 are independently hydrogen, an alkyl group of C ₁ to C ₁₈ , C ₁ To C ₁₈ alkoxy group, C ₁ To alkenyl group of C ₁₈ or C ₁ To a C ₁₈ alkynyl group.

As used herein, the term "derivative" refers to all compounds that are changed to other substituents at substitutable positions of the compounds. The type of the substituent is not restricted, for example, C _{1 -10} which may be substituted each independently hydroxyl, phenoxy, thienyl, furyl, pyridyl, cyclohexyl, alkyl alcohol, alkyl di-alcohol or an optionally substituted phenyl Bicyclic hydrocarbon groups; C _{-6 5} cyclic hydrocarbon group which may be substituted with hydroxyl, hydroxymethyl, methyl or amino; Or a sugar moiety, but is not limited thereto. As used herein, the term 'sugar residue' refers to a group upon removal of one hydrogen atom from a polysaccharide molecule, and thus may mean a residue derived from, for example, a monosaccharide or an oligosaccharide.

As used herein, `` pharmaceutically acceptable '' means the approval of a government or equivalent regulatory body for use in animals, more specifically in humans, by avoiding significant toxic effects when used in conventional medicinal dosages. It can be received or approved, or listed in a pharmacopoeia or recognized as another general pharmacopeia.

As used herein, 'pharmaceutically acceptable salts' refers to salts according to one aspect of the invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. The salt is formed from (1) an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; (3-hydroxybenzoyl) benzoic acid, or an acetic acid, propionic acid, hexanoic acid, cyclopentenepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2- 4-methylbicyclo [2,2,2] -oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert Acid addition salts formed with organic acids such as butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid; Or (2) salts formed when the acidic proton present in the parent compound is substituted. In addition, the compounds according to the present disclosure may include not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that may be prepared by conventional methods.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the weasel extract or a fraction thereof is Amorphastilbol and 5,7-dihydroxy-6-geranyl flavanone (5, 7-dihydroxy-6-geranyl-flavanone) may include one or more compounds, derivatives thereof or pharmaceutically acceptable salts thereof.

[Formula 3] Amorphastilbol (Amorphastilbol)

5,7-dihydroxy-6-geranyl flavanone (5,7-dihydroxy-6-geranyl-flavanone)

 In the composition for the prevention or treatment of kidney disease, which is one aspect of the present invention, the composition may contain 0.001 to 10% by weight of weasel extract or a fraction thereof based on the total weight of the composition. When the weasel extract or fractions thereof is contained in an amount of less than 0.001% by weight based on the total weight of the composition, the effect of preventing or treating kidney disease is insignificant, and when it is contained in an amount of more than 10% by weight, the content of other components on the pharmaceutical is limited. do. In view of the above, weasel extract or fractions thereof, based on the total weight of the composition, 0.0011 to 9% by weight, 0.0012 to 8% by weight, 0.0013 to 7% by weight, 0.0014 to 6% by weight, 0.0015 to 5% by weight, 0.0016 It may be contained in 4 to 4% by weight, 0.0017 to 3% by weight, 0.0018 to 2% by weight or 0.0019 to 1% by weight, specifically may be contained in 0.002% by weight, but is not limited thereto.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the composition may contain 0.000025 to 0.25% by weight of the compound, its derivatives or pharmaceutically acceptable salts thereof, based on the total weight of the composition. Containing less than 0.000025% by weight of derivatives or pharmaceutically acceptable salts thereof, the effect of preventing or treating kidney disease is insignificant, and containing more than 0.25% by weight is appropriate for the content and formulation of other ingredients I can't. In view of the above, the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the compound, its derivatives or pharmaceutically acceptable salts thereof is 0.00005 ~ 0.2 weight, 0.000075 ~ 0.15 weight, 0.0001 with respect to the total weight of the composition It may be included in a concentration of ~ 0.1 weight or 0.000125 ~ 0.05 weight.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the weasel extract may include a C ₁ to C ₆ alcohol extract of weasel, and specifically, a methanol extract of weasel or ethanol of weasel. It may be an extract.

In the composition for preventing or treating kidney disease, which is an aspect of the present invention, the composition may include lowering creatinine in the blood.

In the present specification, creatinine is a substance generated from creatine present in muscle, and the daily output is constant in proportion to the amount of muscle. Urinary excretion of creatinine is proportional to glomerular filtration, and blood concentration of creatinine can be used as an indicator of renal function. The normal value is about 1 mg / dl. In kidney disease, the level of creatinine in the blood rises. In the present specification, lowering the creatinine level in blood means normally lowering the level higher than the normal creatinine level.

In the composition for preventing or treating kidney disease, which is an aspect of the present invention, the kidney disease may be a disease induced by renal toxicity due to oxidative stress. In the present specification, the oxidative stress may be due to oxidative damage caused by free radicals and / or deterioration of an antioxidant defense system in vivo. In the present specification, the kidney disease may be a disease according to kidney toxicity caused by destruction of kidney cells due to oxidative stress.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the kidney disease is nephritis, pyelonephritis, nephrotic syndrome, kidney cancer, acute pyelonephritis, chronic pyelonephritis, kidney tuberculosis, urinary tract infection, urolithiasis, ureteral stone , Acute renal failure, chronic renal failure, diabetic nephropathy, chronic glomerulonephritis, acute progressive nephritis, neproje syndrome, microglomerulosclerosis, mesothelial nephropathy, or membranous proliferative glomerulonephritis.

In the composition for preventing or treating kidney disease, which is an aspect of the present invention, the composition may prevent or treat kidney disease through antioxidant. The composition of the present invention can prevent the oxidation of kidney cells, thereby preventing the kidney cells from being destroyed.

In the composition for preventing or treating kidney disease, which is an aspect of the present invention, the composition may be a health food composition.

The composition may be processed into a drink, fermented milk, cheese, yogurt, juice, probiotic agent, and health supplement food or the like, and may be used in various other food additives.

In one embodiment, the composition may contain other ingredients and the like that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, additives such as perfume, coloring agent, bactericide, antioxidant, preservative, moisturizing agent, thickening agent, inorganic salt, emulsifier and synthetic polymer substance may be further added for improvement of physical properties. In addition, supplementary ingredients such as water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides and seaweed extract may be further included. The above components may be mixed and selected without difficulty by those skilled in the art depending on the purpose of formulation or use, and the amount thereof may be selected within a range that does not impair the objects and effects of the present invention.

The composition according to the present invention may be in various forms such as a solution, an emulsion, a viscous mixture, a tablet, a powder, etc., and may be administered by various methods such as simple drinking, injection administration, spraying or squeezing.

In the composition for the prevention or treatment of kidney disease, which is an aspect of the present invention, the composition may be a pharmaceutical composition.

In one embodiment, the pharmaceutical composition may be a pharmaceutical composition effective for kidney disease, and specifically, may be a pharmaceutical composition effective for kidney disease caused by oxidative stress. Specifically, the pharmaceutical composition is nephritis, pyelonephritis, nephrotic syndrome, kidney cancer, acute pyelonephritis, chronic pyelonephritis, kidney tuberculosis, urinary tract infections, urinary tract stones, ureteral stones, acute kidney failure, chronic kidney failure, diabetic nephropathy, chronic glomeruli It may be a pharmaceutical composition effective for the prevention, alleviation or treatment of symptoms of nephritis, including nephritis, acute progressive nephritis, nephropathy syndrome, microglomerular sclerosis, mesothelial nephropathy, or mesenchymal glomerulonephritis.

When the composition according to the present invention is applied to medicines, it may be formulated into an oral or parenteral dosage form in the form of solid, semi-solid or liquid by adding an inorganic or organic carrier to the composition as an active ingredient.

Examples of preparations for oral administration include tablets, pills, granules, soft or hard capsules, powders, fine granules, powders, emulsions, syrups, pellets and the like. Examples of the parenteral administration agent include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like. In order to formulate the active ingredient of the present invention, it can be easily formulated according to the conventional method. Surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffering agents, suspending agents and other adjuvants can be suitably used.

The pharmaceutical composition according to the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously and the like.

In addition, the dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art. Typical dosages are from 0.001 mg / kg / day to 2000 mg / kg / day, more specifically from 0.5 mg / kg / day to 1500 mg / kg / day.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Weasel sar used in the present invention used those native to Daegwallyeong highland.

[ Example  1] Preparation of Weasel Leaf Extract

[ Example  1-1] Preparation of Methanol Extracts from Weasel Ground

100 g of weasel tree roots including leaves, twigs and stems were immersed in 1 L of 100% methanol and refluxed for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 12.5 g of concentrate.

[ Example  1-2] Preparation of Water Extract from Weasel Ground

100 g of weasel tree ground including leaves, twigs, and stems were immersed in 1 L of water and refluxed for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 9.5 g of concentrate.

[ Example  1-3] Preparation of Ethanol Extracts from Weasel Leaf

100 g of the weasel ground part was soaked in 1 L of 100% ethanol and extracted under reflux for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 10.5 g of concentrate.

[ Example  2] Preparation of Weasel Root Extract

[ Example  2-1] Preparation of Methanol Extract of Weasel Root

100 g of Weasel root were soaked in 1 L of 100% methanol and extracted under reflux for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 11.2 g of concentrate.

[ Example  2-2] Preparation of Water Extract of Weasel Root

100 g of weasel roots were soaked in 1 L of water and refluxed for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 9.3 g of concentrate.

[ Example  2-3] Preparation of Ethanol Extract of Weasel Root

100 g of weasel roots were soaked in 1 L of 100% ethanol and extracted under reflux for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 10.0 g of concentrate.

[ Example  3] Preparation of Weasel Seed Extract

[ Example  3-1] Methanol Extract of Weasel Seed

100 g of ripe weasel seeds were soaked in 1 L of 100% methanol and extracted under reflux for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 14.7 g of concentrate.

[ Example  3-2] Preparation of Water Extract of Weasel Seed

100 g of ripe weasel seeds were soaked in 1 L of water and refluxed for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 10.7 g of concentrate.

[ Example  3-3] Preparation of Ethanol Extract of Weasel Fructus Seeds

100 g of ripe weasel seeds were soaked in 1 L of 100% ethanol and extracted under reflux for 3 hours. The extraction was repeated three times. The extract was dried under reduced pressure and concentrated to give 12.7 g of concentrate.

[ Example  4] Amorphous steel ball  detach

The methanol extract of the weasel fly seed prepared in [Example 3-1] was suspended in 500 ml of water, and then fractionated three times with 500 ml of ethyl acetate. The fractions were concentrated under reduced pressure to prepare 15 g of an ethyl acetate fraction. This fraction was subjected to silica gel chromatography using n-hexane-ethylacetate gradient system (10: 1-100% methanol) and divided into 14 small fractions (4E1-4E14). The fourth fraction was subdivided back into three subfractions by silica gel chromatography with chloroform-methanol (100: 1) solvent system, and preparative HPLC (Waters DeltaPrep 150 mL System, SunFire Silica 150 x 10) in the second subfraction. Amorphastyl ball (Formula 3) was separated (3 mg) using a mm column; mobile phase n-hexaneethylacetate (95: 5); flowrate 5 ml / min, UV detection at 254 nm. The structure of the amorphastyl ball was characterized using NMR and MS.

[Formula 3] Amorphastilbol (Amorphastilbol)

ESI-MS m / z 347 [M-H] < + >;

1 H NMR (500 MHz, CDCl 3) δ 7.48 (2H, d, J = 7.5 Hz), 7.35 (2H, t, J = 7.5 Hz), 7.25 (1H, br t, J = 7.5 Hz), 7.01 (1H , d, J = 16.5 Hz), 6.94 (1H, d, J = 16.5 Hz), 6.59 (2H, s), 5.22 (1H, m), 5.18 (1H, s), 5.06 (1H, m), 3.44 (2H, d, J = 7.0 Hz), 2.10 (4H, m), 1.83 (3H, s), 1.69 (3H, s), 1.60 (3H, s).

[ Example  5] 5,7-dihydroxy-6- Geranil Flavanese  detach

The methanol extract of the weasel fly seed prepared in [Example 3-1] was suspended in 500 ml of water, and then fractionated three times with 500 ml of ethyl acetate. The fractions were concentrated under reduced pressure to prepare 15 g of ethyl acetate. This fraction was subjected to silica gel chromatography using n-hexane-ethylacetate gradient system (10: 1-100% methanol) and divided into 14 small fractions (E1-E14). The fourth fraction was subdivided back into three subfractions by silica gel chromatography with a chloroform-methanol (100: 1) solvent system, and preparative HPLC (Waters DeltaPrep 150 mL System, SunFire Silica 150 x 10) in the first subfraction. 5,7-dihydroxy-6-geranyl flavanone (Formula 4) was separated (3 mg) using a mm column; mobile phase n-hexaneethylacetate (95: 5); flowrate 5 ml / min, UV detection at 254 nm. The structure of the 5,7-dihydroxy-6-geranyl flavanone was characterized using NMR and MS.

5,7-dihydroxy-6-geranyl flavanone (5,7-dihydroxy-6-geranyl-flavanone)

ESI-MS m / z 391 [M-H] < + >;

1 H NMR (500 MHz, CD3CN) δ 12.40 (1H, s), 7.46-7.40 (5H, m), 6.34 (1H, s), 6.02 (1H, s), 5.40 (1H, dd, J = 3.0, 13.0 Hz), 5.26 (1H, dt, J = 1.0, 7.0 Hz), 5.05 (1H, t, J = 7.0 Hz), 3.37 (2H, d, J = 7.0 Hz), 3.08 (1H, dd, J = 13.0, 17.0 Hz), 2.82 (1H, dd, J = 3.0, 17.0 Hz), 2.12-2.05 (4H, m), 1.81 (3H, s), 1.67 (3H, s), 1.59 (3H, s).

[ Experimental Example  One] Radically  Due to Oxidative  Kidney Cells for Injury Protection ability  evaluation

Renal cells like the LLC-PK1 cell line (CL-101, ATCC) are susceptible to oxidative damage, and the radical generator AAPH (2,2-Azobis (1-aminopropane) dihydrochloride) binds to oxygen molecules very rapidly. Produces peroxyl radical and lipid peroxidation, causing cytotoxicity (Miki et al., Arch. Biochem. Biophy., 258 (2), 373-380, 1987). Renal protective activity can be assessed using oxidative damage of LLC-PK1 cell lines using AAPH. In this example, the protective effect against kidney toxicity was evaluated using kidney cells (LLC-PK1) by the following experimental method reported in the literature (Yokozawa et al., Food Chem., 48, pp5068-5073, 2000). .

First, LLC-PK1 cells were treated with 5% fetal bovine serum (Gibco BRL Life Technologies), penicillin G 50 units / ml (Gibco BRL Life Technologies) and streptomycin (Grepcocin B. Incubated in a 95% air / 5% carbon dioxide incubator maintained at 37 ° C using DMEM / F12 (Gibco BRL Life Technologies, Grand Island, NY, USA) medium added. Cultured LLC-PK1 cells were introduced 10 ^{4 4} into 96-well culture plates and allowed to stabilize for 2 hours.

Subsequently, weasel fly methanol extract (1, 3, 10. 30 μg / m) prepared according to Example 3-1, amorphastilball (1, 3, 10, 30 uM) prepared according to Example 4 , 5,7-dihydroxy-6-geranyl flavanone (1, 3, 10, 30 uM) prepared according to Example 5 and the addition of a radical generating reagent (final concentration 10 mM AAPH) for 24 hours Then 10 μl of Ez-Cytox reagent (It's Bio, Korea) was added to each well, and then incubated at 37 ° C. After 2 hours, the cell viability was measured by measuring absorbance using a 450 nm detection wavelength in a PowerWave XS (Bio-Tek Instruments, Winnoski, VT, USA) microplate reader.

As a result (FIG. 1), 10 mM AAPH treatment reduced LLC-PK1 cell count by 23% of the AAPH untreated group, whereas weasel ethanol extract and compounds isolated therefrom inhibited such cell damage, 10 It was confirmed that the cell number reduced by AAPH at the concentration of µg / mL was improved to the normal level.

These results indicate that weasel extract or compounds isolated therefrom can protect kidney cells against renal cell damage resulting from oxidative stress.

[ Experimental Example  2] caused by anticancer drugs Oxidative  Kidney Cells for Injury Protection ability  evaluation

Cisplatin (CDDP), a representative anticancer agent, has been widely used as a treatment for solid cancers such as testicular cancer, ovarian cancer, bladder cancer, uterine cancer, and prostate cancer. It is restricted. These side effects are known to be caused by cispplanin production of free radicals (Atessahin et al., Basic Clin. Pharmacol. Exp., 213, pp 121-126, 2007). Renal cells, LLC-PK1 cell line (CL-101, ATCC), are susceptible to oxidative damage, and can be assessed for renal protective activity using oxidative damage of LLC-PK1 cell lines using cisplatin. In this example, the protective effect against kidney toxicity was evaluated using renal cells (LLC-PK1) by the following experimental method reported in the literature (Lee et al., Bioorg. Med. Chem. Lett., 22, pp5475). -5479, 2012).

First, LLC-PK1 cells were treated with 10% fetal bovine serum (Gibco BRL Life Technologies), penicillin G 50 units / ml (Gibco BRL Life Technologies) and streptomycin (Grepcocin B) Life Technologies 50 μg / ml Incubated in a 95% air / 5% carbon dioxide incubator maintained at 37 ° C using DMEM (Gibco BRL Life Technologies, Grand Island, NY, USA) medium added. Cultured LLC-PK1 cells were introduced 10 ^{4 4} into 96-well culture plates and allowed to stabilize for 2 hours.

Thereafter, weasel's methanol extract (1, 3, 10, 30 μg / m) prepared according to Example 3-1, prepared amorphastilball (1, 3, 10, 30, isolated by Example 4) uM), 5,7-dihydroxy-6-geranylflavanone (1, 3, 10, 30 uM) and cisplantin (final concentration 25 uM) isolated by Example 5 incubated for 24 hours 50 μl of MTT (1 mg / mL) reagent was added to each well, followed by incubation at 37 ° C. After 2 hours, the cell viability was measured by measuring absorbance using a 450 nm detection wavelength in a PowerWave XS (Bio-Tek Instruments, Winnoski, VT, USA) microplate reader.

As a result (Fig. 2), when 25 uM cisplantin treatment, LLC-PK1 cell number was reduced by 40% compared to the cisplantin untreated group, while weaselic methanol extract was found to inhibit such cell damage. . In particular, when the respective reagents according to Examples 3-1, 4 and 5 were treated at a concentration of 30 µg / mL, it was confirmed that the number of cells reduced by cisplantin was improved to the normal level. It was.

Therefore, weasel extract can protect renal cell damage from oxidative stress while maintaining the anticancer effect of cisplantin.

[ Experimental Example  3] Creatinine in Blood of Weasel Extract Descent  evaluation

After high-fat diet using 7-week-old C57BL / 6 mice (SPL Co., Ltd.), the effects of weasel extract and each drug used as a control on metabolic improvement efficacy were evaluated. Metformin was used as a positive control.

Mice were acclimated to the laboratory for 1 week and then divided into 3 groups. I) Non-treated group given normal diet (Purina Rat Feed, Central Experimental Animal Co., Ltd., Korea), ii) High-fat diet (AIN-76A, The group given 40% beef tallow) was given to the high fat diet group, i) 200 mg / Kg of weasel extract obtained in the above high fat diet and Example 3-1. Each drug was suspended orally in 0.8% methylcellulose solution and administered orally every day for 8 weeks.

Serum creatinine levels are widely used clinically as the most important indicator of renal function. After the end of administration according to Example 3-1, mice of each group fasted for 16 hours were anesthetized with ether and whole blood was obtained through the abdominal aorta, and then plasma creatinine was analyzed using plasma. Serum creatinine concentration was measured using the rate blank Jaffe Kinetic method, creatinine (Roche, USA) reagent and hitachi modular (Japan) instrument.

As a result (Table 1), the treatment group of weasel extract was found to lower blood creatinine levels by more than 37% compared to the high fat diet group.

Volume
(mg / kg body-weight / day) Creatinine (mg / dL) Untreated group - 0.09 0.03 It's a high-fat diet. - 0.19 ± 0.01 Example 3-1 200 0.12 + - 0.01

Examples of the formulation of the composition will be described below, but are not intended to limit the present invention but merely to be described in detail.

Formulation Example 1 Soft Capsule

80 mg, weasel extract, vitamin E 9 mg, vitamin C 9 mg, palm oil 2 mg, vegetable hardened oil 8 mg, lead 4 mg and lecithin 9 mg are mixed and mixed according to a conventional method to prepare a soft capsule fill solution. 400 mg per capsule is filled to prepare a soft capsule. In addition, a soft capsule sheet is prepared at a ratio of 66 parts by weight of gelatin, 24 parts by weight of glycerine, and 10 parts by weight of sorbitol solution and filled with the filler to prepare a soft capsule containing 400 mg of the composition according to the present invention.

[Formulation Example 2] Tablets

80 mg of weasel extract, 9 mg of vitamin E, 9 mg of vitamin C, 200 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose are mixed and granulated using a fluidized bed dryer, and then 6 mg of sugar ester is added. Tablets are prepared by tableting 500 mg of these compositions in a conventional manner.

[Formulation Example 3] Drinks

80 mg of weasel extract, 9 mg of vitamin E, 9 mg of vitamin C, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharides are mixed, and 300 ml of purified water is added to each bottle to make 200 ml. After filling the bottle sterilized for 4-5 seconds at 130 ℃ to prepare a drink.

[Formulation Example 4]

80 mg of weasel extract, 9 mg of vitamin E, 9 mg of vitamin C, 250 mg of anhydrous glucose, and 550 mg of starch were mixed, molded into granules using a fluidized bed granulator, and filled into sachets to prepare granules.

Formulation Example 5 Injection

Weasel extract ...... 20 mg

Sterile Distilled Water for Injection ...

pH adjuster ...

Injections are prepared in the above amounts per ampoule (2 ml) according to the conventional method for preparing injections.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Preparation Example 6 Preparation of Healthy Drinks

Weasel extract .................................... 1000 mg

Citric acid ................................................. ... 1000 mg

oligosaccharide................................................. .... 100 g

Plum concentrate ................................................ ..... 2 g

Taurine ................................................. ........... 1 g

Purified water was added to the mixture to complete 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

Weasel ( Amorpha) fruticosa ) A composition for the prevention or treatment of kidney disease, comprising an extract or a fraction thereof as an active ingredient. The method of claim 1,
The weasel extract or fractions thereof includes one or more compounds, derivatives thereof or pharmaceutically acceptable salts thereof in the group consisting of Formula 1 and Formula 2,
[Chemical Formula 1]

(2)

In the above formulas,
R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, R31 or R32 are independently hydrogen, an alkyl group of C ₁ to C ₁₈ , C ₁ To C ₁₈ alkoxy group, C ₁ To alkenyl group of C ₁₈ or C ₁ To a C ₁₈ alkynyl group, a composition for the prevention or treatment of kidney disease. The method of claim 1,
The weasel extract or fractions thereof is at least one compound in the group consisting of Amorphastilbol and 5,7-dihydroxy-6-geranyl flavanone (5,7-dihydroxy-6-geranyl-flavanone) A composition for the prophylaxis or treatment of kidney disease, comprising a derivative thereof or a pharmaceutically acceptable salt thereof. The method of claim 1,
The composition is a composition for the prevention or treatment of kidney disease, containing a weasel extract or a fraction thereof in an amount of 0.001 to 10% by weight based on the total weight of the composition. The method of claim 3,
The composition is a composition for preventing or treating kidney disease, containing 0.000025 to 0.025% by weight of the compound, its derivatives or pharmaceutically acceptable salts thereof relative to the total weight of the composition. The method of claim 1,
The weasel extract is a C ₁ ~ C ₆ alcohol extract of weasel, composition for the prevention or treatment of kidney disease. The method according to claim 6,
The alcohol is methanol or ethanol, the composition for the prevention or treatment of kidney disease. The method of claim 1,
The composition is for lowering creatinine in the blood, a composition for preventing or treating kidney disease. The method of claim 1,
The kidney disease is a disease induced by kidney toxicity caused by oxidative stress, a composition for preventing or treating kidney disease. 10. The method of claim 9,
The kidney disease is nephritis, pyelonephritis, nephrotic syndrome, kidney cancer, acute pyelonephritis, chronic pyelonephritis, kidney tuberculosis, urinary tract infection, urinary tract stones, ureteral stones, acute kidney failure, chronic kidney failure, diabetic nephropathy, chronic glomerulonephritis, acute progression A composition for the prophylaxis or treatment of kidney disease, including nephritis, neprogen syndrome, microglomerular sclerosis, mesothelial nephropathy, or membranous proliferative glomerulonephritis. The method of claim 1,
The composition is for preventing or treating kidney disease through antioxidant, composition for the prevention or treatment of kidney disease. 12. The method according to any one of claims 1 to 11,
The composition is a health food composition, a composition for preventing or treating kidney disease. 12. The method according to any one of claims 1 to 11,
The composition is a pharmaceutical composition, a composition for preventing or treating kidney disease.

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