KR102203926B1 - Composition for treating renal disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of kidney disease or renal fibrosis, comprising a stilt extract, and a health functional food for relieving kidney disease comprising a stilt extract.

Description

Composition for treating renal disease {Composition for treating renal disease}

The present invention relates to a pharmaceutical composition for the treatment of kidney disease, comprising the extract of twig.

The kidneys play an important role in maintaining the body's homeostasis. It regulates the volume of body fluid, the concentration and pH of ions in the blood, secretes waste products such as metabolic waste products, toxins and drugs, and performs blood pressure control, metabolic and endocrine functions. In addition, the kidneys activate vitamin D to help absorb calcium in the small intestine and are involved in the synthesis of various hormones.

Kidney disease refers to an abnormal state caused by a decrease in the overall function of the kidney or the kidney does not normally perform secretion, control, metabolic and endocrine functions, and is classified into chronic kidney disease and acute kidney disease.

Chronic kidney disease means a gradual loss of kidney function over months or years. Chronic kidney disease is caused by a variety of causes, but the final route is renal fibrosis. Chronic kidney disease can be caused by, for example, cardiovascular disease, hypertension, diabetes, glomerulonephritis, polycystic kidney disease and kidney transplant rejection.

Kidney damage is associated with the release of cytokines/growth factors such as TGF-β, epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Increased TGF-β expression is one of the most important pathogenesis mechanisms of renal fibrosis. TGF-β promotes fibroblast activity and induces matrix expression by interacting with TGF-β receptors.

Renal fibrosis, characterized by glomerulosclerosis and tubular interstitial fibrosis, is a common symptom of a variety of chronic kidney diseases. The occurrence of renal fibrosis is caused by excessive accumulation and deposition of extracellular matrix components. Renal fibrosis is a progressive process that ultimately leads to end-stage renal failure and is a disease that requires dialysis or kidney transplantation. However, there is no cure to slow the exacerbation of chronic kidney disease.

On the other hand, licorice extract or black corn extract has been suggested as a natural substance for treating renal fibrosis or kidney disease, but a technique for treating renal fibrosis or kidney disease by using a stilt extract has not been suggested.

Korean Patent Registration No. 10-1063524 Korean Patent Publication No. 10-2011-0060993

It is an object of the present invention to provide a pharmaceutical composition for treating kidney disease, or a pharmaceutical composition for inhibiting renal fibrosis, comprising a stilt extract.

1. A pharmaceutical composition for the treatment of kidney disease, comprising an extract of sty.

2. A pharmaceutical composition for the treatment of renal fibrosis, comprising an extract of sty.

3. The pharmaceutical composition according to item 1 or 2, wherein the snail extract reduces the expression of one or more proteins selected from the group consisting of αSMA, fibronectin, snail, phosphorylated p53, phosphorylated p38, and Bax.

4. The pharmaceutical composition according to item 1 or 2, wherein the agaric extract increases the expression of one or more proteins selected from the group consisting of Bcl2, and E-cadherin.

5. According to item 1, the kidney disease is acute kidney disease, end-stage kidney disease (ESKD), diabetic kidney disease (DN), IgA nephropathy (IgAN), HIV-related nephropathy, non-diabetic A pharmaceutical composition, which is one or more diseases selected from the group consisting of non-diabetic chronic kidney disease, focal segmental glomerulosclerosis (FSGS), microvariable nephrotic syndrome (MCD), and xanthine oxidase deficiency. .

6. A health functional food for relieving kidney disease, including the extract of sty.

7. A health functional food for relieving kidney fibrosis, including sagebrush extract.

8. The health functional food according to item 6 or 7, wherein the sorghum extract reduces the expression of one or more proteins selected from the group consisting of αSMA, fibronectin, snail, phosphorylated p53, phosphorylated p38, and Bax.

9. The health functional food according to item 6 or 7, wherein the agaric extract increases the expression of one or more proteins selected from the group consisting of Bcl2, and E-cadherin.

10. According to item 6 or 7, the kidney disease is acute kidney disease, end-stage kidney disease (ESKD), diabetic kidney disease (DN), IgA nephropathy (IgAN), HIV-related nephropathy, non At least one disease selected from the group consisting of non-diabetic chronic kidney disease, focal segmental glomerulosclerosis (FSGS), microvariable nephrotic syndrome (MCD), and xanthine oxidase deficiency, Health functional food.

A pharmaceutical composition or a health functional food comprising a stalk extract according to the present invention can treat renal fibrosis or relieve symptoms.

In addition, the pharmaceutical composition or health functional food comprising the styrofoam extract according to the present invention is a kidney disease, for example, acute kidney disease, end-stage kidney disease (ESKD), diabetes through the treatment or alleviation of renal fibrosis. Sexual kidney disease (DN), IgA nephropathy (IgAN), HIV-related nephropathy, non-diabetic chronic kidney disease, focal segmental glomerulosclerosis (FSGS), microvariant nephrotic syndrome (MCD), or xanthine oxidase deficiency.

1 is a result of confirming the decrease in renal fibrosis after administration of a stilt extract in a unilateral ureteral obstruction renal fibrosis mouse model by MT staining (low magnification, × 12.5). A. Normal control/distilled water; B. Normal control/Styria extract; C. Unilateral ureteral obstruction/distilled water; D. Unilateral ureteral obstruction/stit extract. In the normal control group, it was confirmed that no distinct pathological changes were observed when the administration of distilled water and stilt extract was observed (A, B). In a mouse model of unilateral ureteral obstruction, the ureter was enlarged at the medulla and cortical-medullary junctions of the kidney, and fibrosis of the interstitial region occurred. It was confirmed (C), and it was confirmed that the area of the fibrosis site of the kidney was reduced when administering the extract (D).
Figure 2 is a result of confirming the reduction of renal fibrosis after administration of styrofoam extract in a unilateral ureteral obstruction renal fibrosis mouse model by PAS staining (top) and MT staining (bottom) (high magnification, × 200). A. Normal control/distilled water; B. Normal control/Styria extract; C. Unilateral ureteral obstruction/distilled water; D. Unilateral ureteral obstruction/stit extract. In the normal control group, it was confirmed that no distinct pathological changes were observed when the administration of distilled water and stilt extract was observed (A, B). In a mouse model of unilateral ureteral obstruction, the ureter was enlarged at the medulla and cortical-medullary junctions of the kidney, and fibrosis of the interstitial region occurred. It was confirmed (C), and it was confirmed that the area of the fibrosis site of the kidney was reduced when administering the extract (D).
Figure 3 is a graph confirming the change in the fibrotic area according to the administration of a stilt extract in a unilateral ureteral obstruction renal fibrosis mouse model. Sham/DW: normal control/distilled water (n = 2); Sham/Styria extract: Normal control/Styria extract (n = 2); UUO/DW: unilateral ureteral obstruction/distilled water (n = 4); UUO/Styre extract: unilateral ureteral obstruction/Styre extract (n = 4). Sham control vs. UUO/DW P = 0.009, UUO/DW vs. UUO/Styria extract P = 0.029.
Figure 4 is a result of confirming the reduction of renal fibrosis after administration of a stilt extract in a unilateral ureteral obstruction renal fibrosis mouse model through immunohistochemical staining (low magnification, × 12.5). A. Normal control/distilled water; B. Normal control/Styria extract; C. Unilateral ureteral obstruction/distilled water; D. Unilateral ureteral obstruction/stit extract.
FIG. 5 is a result of confirming the reduction of renal fibrosis after administration of stilt extract in a unilateral ureteral obstruction renal fibrosis mouse model through immunohistochemical staining (high magnification, × 400). A. Unilateral ureteral obstruction/distilled water; B. Unilateral ureteral obstruction/stit extract.
6 is a result of confirming the change in the expression of renal fibrosis-related protein by Western blot after administration of a stilt extract in a unilateral ureteral obstruction renal fibrosis mouse model. Sham/DW: normal control/distilled water; Sham/Styria extract: Normal control/Styria extract; UUO/DW: unilateral ureteral obstruction/distilled water; UUO/Styria extract Unilateral ureteral obstruction/Styria extract.
7 is a result of confirming the change in fibrosis-related protein expression by Western blot after administration of the recombinant TGF-β and/or styrofoam extract in human renal proximal tubule primary cultured cells. CTL: control (distilled water); rTGFβ: Recombinant TGF-β.
8 is a result of confirming the change in the expression of fibrosis-related proteins in primary cultured cells of the human kidney proximal tubule after the administration of recombinant TGF-β and styrofolium extract by Western blot. A. αSMA protein expression; B. Phosphorylated p38 protein expression; C. Bcl-2 protein expression; D. E-cadherin protein expression.
9 is a result of confirming the increase in the protein expression of phosphorylated p38 and the decrease in the expression of phosphorylated p38 by the extract of chinensis in human proximal tubular cells when fibrosis is changed by recombinant TGF-β.

The invention will now be more fully described below with reference to the accompanying drawings, but only some, but not all, embodiments of the invention are illustrated. Indeed, these inventions can be embodied in many different forms and should not be construed as being limited to the embodiments presented herein. The singular forms used in this specification and the appended claims include plural objects unless clearly indicated otherwise.

The present invention provides a pharmaceutical composition for the treatment of kidney disease or a pharmaceutical composition for the treatment of kidney fibrosis, comprising the extract of sty.

The stalk may be a stalk (Actinidia arguta), a sputum (A. kolomikta), a snail (A. polygama), or a plant related to the genus. Unless otherwise stated, the term stalk as used herein refers to stilt fruit. The stool extract can be prepared by pulverizing stool, and then extracting the crushed stool with water or lower alcohol. The lower alcohol is a linear or branched alcohol having 1 to 5 carbons, and may be methanol, ethanol, propanol, propanol, butanol, or pentanol, or an isomer thereof.

The mouse unilateral ureteral obstruction (UUO) model provides a model of progressive renal fibrosis, and renal fibrosis is a representative feature of progressive renal disease. Surgically fabricated UUO can control the timing, severity and duration, and the recovery of obstruction is the basis for studying the treatment method of kidney disease (Robert L. Chevalier, et al., Ureteral obstruction as a model). of renal interstitial fibrosis and obstructive nephropathy, Kidney International, Vol. 75, Issue 11, pp. 1145-1152 (2009)).

UUO models can be constructed by methods well known in the art (Morrissey JJ, Klahr S, Differential effects of ACE and AT1 receptor inhibition on chemoattractant and adhesion molecule synthesis.Am J Physiol 1998; 274: F580-F586; Guo G, et al. Contribution of angiotensin II and tumor necrosis factor-a to the development of renal fibrosis.Am J Physiol Renal Physiol 2001, 280: F777-F785; Kaneto H et al., Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed rat kidney, Kidney Int 1994, 45: 1637-1647; Ishidoya S, et al. Angiotensin receptor antagonist ameliorates renal tubulointerstitial fibrosis caused by unilateral ureteral obstruction.Kidney Int 1995; 47: 1285-1294). For example, it can be manufactured by tying or cutting the ureter on one side of the mouse.

The kidney disease includes all kidney diseases in which fibrosis has occurred or is likely to occur in the future, for example, acute kidney disease, end-stage kidney disease (ESKD), diabetic kidney disease (DN), IgA nephropathy (IgAN), HIV-related nephropathy, non-diabetic chronic kidney disease, focal segmental glomerulosclerosis (FSGS), microvariant nephrotic syndrome (MCD), or xanthine It may be oxidase deficiency, but is not limited thereto.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier.

"Pharmaceutical acceptable carrier" is used as a diluent, adjuvant, excipient, or vehicle when administering the extract of the present invention, and is approved by the national management agency, or for use in animals, more specifically humans. It is listed in the generally known pharmacopoeia. The pharmaceutical carrier may be a liquid such as water or oil, and the oil includes oils derived from petroleum, animal, plant, or synthetic products, and oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutically acceptable carriers include saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, and urea. When administered to a patient, the stilt extract and pharmaceutically acceptable carrier of the present invention are preferably sterile. Salt solutions, liquid dextrose and glycerol solutions can also be used as liquid carriers, especially injectable solutions. Suitable pharmaceutical carriers may also include additives such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water and ethanol. The composition of the present invention may contain trace amounts of a wetting agent, an emulsifying agent, or a pH buffering agent, if necessary. The composition of the present invention may take a formulation suitable for solution, emulsion, sustained-release preparation or other use.

The pharmaceutical composition of the present invention can be administered in a variety of dosage forms. In one embodiment of the present invention, the pharmaceutical composition comprising the extract of the present invention may be formulated in a form suitable for oral, rectal, parenteral, nasal or transdermal administration, or administration by inhalation or suppository. .

The administration method is not particularly limited thereto, but any method of oral or parenteral administration may be used, and systemic administration or local administration may be possible, but systemic administration is more preferable, and oral administration is most preferable. Administration into the body of a patient is preferably oral administration, specifically, administration once to 10 times a day is basic, but it may be administered beyond that. In addition, the administration time may be short or sustained for a long time. Liquid dispersions for oral administration can be syrups, emulsions and suspensions.

The pharmaceutical composition of the present invention can be formulated by a known method by a person skilled in the art. For example, if necessary, it can be used parenterally in the form of an injection in a sterile solution or suspension of water or other pharmaceutically acceptable liquids. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc. are appropriately combined, and generally It can be formulated by blending in the unit dosage form required for the practice of pharmaceuticals recognized as. In the above formulation, the amount of the active ingredient is such that an appropriate dose within the indicated range can be obtained.

In addition, a sterile composition for injection can be formulated according to a conventional formulation using an excipient such as distilled water for injection. As the aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be mentioned. For example, alcohol, specifically ethanol, polyalcohol, such as propylene glycol, polyethylene glycol, nonionic surfactants, such as polysorbate 80(TM), and HCO-50 can be used in combination. Sesame oil and soybean oil are mentioned as the oily liquid, and it can be used together with benzyl benzoate and benzyl alcohol as a dissolution aid.

As an example of an injection formulation, for example, intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intraventricular injection, intracranial injection, intramedullary injection, etc. It is preferably intravenous injection.

In addition, buffers such as phosphate buffers, sodium acetate buffers, painless agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenols, and antioxidants may be combined. The prepared injection solution is usually filled in suitable ampoules.

Suspensions and emulsions may contain, as carriers, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection, together with styrofoam extract, can be prepared with a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and if necessary in suitable amounts. It may contain lidocaine hydrochloride.

The stilt extract of the present invention may exist in a solid or liquid form. Solvates may include non-aqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine and ethyl acetate, or they may include water as a solvent incorporated into the crystalline lattice. Solvates, which are solvents in which water is incorporated into the crystalline lattice, are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The present invention includes all such solvates.

When the composition or food of the present invention further provides for administration of one or more drugs, they may be administered simultaneously, sequentially or separately. They can be packaged together or separately. Also, they do not have to be administered simultaneously or they need to be in the same dosage form. Individual administrations can be administered on the same day or on different days, with the drug being administered as part of the same overall dosing treatment regimen. Concurrent means that drugs must be taken together or formulated as a single composition. Sequential means that the drugs are administered approximately simultaneously, preferably within about 1 hour of each other.

The therapeutically effective amount of the pharmaceutical composition is not particularly limited, but is preferably 5 mg/kg to 50 mg/kg, more preferably 10 mg/kg to 40 mg/kg, and 20 mg/kg to 30 mg Most preferred is /kg.

In one embodiment of the present invention, it provides a health functional food for relieving kidney disease or a health functional food for relieving kidney fibrosis, including a scythe extract.

The health functional food can be manufactured using conventional knowledge, and the health functional food according to the present invention may additionally contain commonly added ingredients such as excipients, sweeteners or extenders. The sweetener may be a natural sweetener such as taumatin and stevia extract, saccharin, or aspartame, but is not limited thereto.

The types of food are, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcohol It may be a beverage or a vitamin complex, but is not limited thereto.

The beverage may contain various flavoring agents or natural carbohydrates as an additional component, like ordinary beverages. As the natural carbohydrates described above, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In one embodiment of the present invention, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stable It may contain an agent, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may include flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food of the present invention.

αSMA (α-smooth muscle actin, α-smooth muscle actin) is known as α-actin-2, SMactin, or ASMA, and is a protein expressed by the ACTA2 gene (NM_001141945, NM_001613, NM_001320855), and is a protein expressed in all types of organ fibrosis. It is a molecular marker indicating the activation of myofibroblasts (Youhua Liu, Cellular and molecular mechanisms of renal fibrosis, Nat Rev Nephrol, 2011 Dec, 7(12): 684-696; Wynn TA.Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases.J Clin Invest. 2007, 117:524-529; Hinz B, et al. The myofibroblast: one function, multiple origins.Am J Pathol. 2007, 170:1807-1816; Nagamoto T, Eguchi G, Beebe DC (April 2000).Alpha-smooth muscle actin expression in cultured lens epithelial cells, Investigative Ophthalmology & Visual Science. 41 (5): 1122-9).

Fibronectin is an extracellular matrix glycoprotein that binds to integrin, a transmembrane receptor protein, and is known to be mainly expressed by the activity of fibroblasts (Youhua Liu, Cellular and molecular mechanisms of renal fibrosis, Nat Rev Nephrol, 2011 Dec, 7(12): 684-696), mainly expressed early in the fibrosis process of various organs (Eddy AA. Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol. 1996, 7, 2495-2508) ; Federica Genovese, et al., The extracellular matrix in the kidney: a source of novel non-invasive biomarkers of kidney fibrosis?, Fibrogenesis Tissue Repair, 2014, 7:4).

p53 is a tumor protein, also known as antigen NY-CO-13 or TRP53 (transformation-related protein 53), and is a protein expressed by the TP53 gene (NM_001276761, NM_000546, NM_001126112, NM_001126113, NM_001126114), and is phosphorylated in an occluded kidney. As it is known to increase the expression of p53, it is used as a representative molecular marker for fibrosis (Rohan Samarakoon, et al., TGF-β→SMAD/p53/USF2→PAI-1 transcriptional axis in ureteral obstruction-induced renal fibrosis, Cell Tissue Res (2012) 347:117-128).

Snail has four zinc finger motifs capable of specifically binding to the E-box motif at the C-terminus (Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002; 3: 155- 166), activated by the SMAD3-TGF-β pathway (Noeemie Simon-Tillaux, snail and kidney fibrosis, Nephrology Dialysis Transplantation, Volume 32, Issue 2, 1 February 2017, Pages 224-233), and the expression of SNAI1 in the kidney is It has been shown to induce spontaneous fibrosis (Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005, 132: 3151-3161; Boutet A, et al.. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney.EMBO J 2006, 25: 5603-5613).

Bax (Bcl-2-associated X protein) is a protein expressed by the BAX gene and belongs to the Bcl-2 gene family. In unilateral ureteral obstruction, Bcl2 protein expression initially increases, whereas Bax protein expression decreases and time is reduced. It is known that the expression of Bax protein increases as the expression of Bcl2 decreases over time (Zhang G, et al., Role of Apoptosis and Bcl-2/Bax in the Development of Tubulointerstitial Fibrosis during Experimental Obstructive Nephropathy, Experimental nephrology, 2001 , 9:71-80).

E-cadherin (epithelial-cadherin) is one of cell adhesion molecules, and plays an important role in forming an adhesive junction for adhesion between cells. It has a length of about 720-750 amino acids, consists of a small cytoplasmic portion, a transmembrane portion and a large extracellular portion, and is lost by TGFβ1, which induces fibrosis (Mangalakumar Veerasamy, et al., Differential regulation of E-cadherin and α). -smooth muscle actin by BMP 7 in human renal proximal tubule epithelial cells and its implication in renal fibrosis, AJP-Renal Physiol, VOL 297, F1238-F1248, 2009). Loss of E-cadherin has been known to cause fibrosis (Cho IJ, E-cadherin antagonizes transforming growth factor βgene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation, Hepatology. 2010 Dec;52(6):2053-64 ).

The p38 mitogen-activated protein kinase is a protein belonging to the mitogen-activated protein kinase (MAPK) family and responds to stress stimuli such as cytokines, ultraviolet rays, thermal shock and osmotic shock. p38 is activated by phosphorylation of threonine at positions 180 and 182 by MKK3 and SEK, and phosphorylated p38 phosphorylates and activates MAPKAP kinase 2 and phosphorylates transcription factors ATF2, Mac, and MEF2 (Tudor C, Marchese FP, Hitti E, Aubareda A, Rawlinson L, Gaestel M, Blackshear PJ, Clark AR, Saklatvala J, Dean JL (June 2009).The p38 MAPK pathway inhibits tristetraprolin-directed decay of interleukin-10 and pro-inflammatory mediator mRNAs in murine macrophages. FEBS Letters. 583 (12): 1933-8). On the other hand, it is known that the expression of phosphorylated p38 is increased in the kidney of unilateral ureteral obstruction model (Frank Y. Ma, et al., ASK1/p38 signaling in renal tubular epithelial cells promotes renal fibrosis in the mouse obstructed kidney, Am J. Physiol Renal Physiol 307: F1263-F1273, 2014).

TGFβ (transforming growth factor beta) is a multifunctional cytokine belonging to the transforming growth factor superfamily containing various isoforms, and is known to be a major factor inducing fibrosis in most chronic kidney diseases (Meng XM, et al., TGF-β the master regulator of fibrosis, Nat Rev Nephrol. 2016 Jun;12(6):325-38).

The following examples are intended for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

Example 1. Preparation of sputum extract

Dry stilts ( Actinia arguta ) were purchased and used in the experiment. Add 100 g of crushed scallionate to 800 ml of distilled water, stir well, and extract under reflux for 3 hours at an extraction temperature maintained at 85-95℃, and then the filtrate was separated, and the herbal medicine extract was concentrated under reduced pressure at 55-65℃. Freeze-drying was performed to obtain a powder of a snail extract in a yield of 35-40%.

Example 2. Histological changes in renal tissue fibrosis ( in vivo )

A 7-week-old male mouse was anesthetized by intraperitoneally administering zoletil (30mg/kg) and lumpun (Rompun 10m/kg), and an incision in the left flank to expose the kidneys to find the ureter, and then use 4-0 silk to find the ureter proximal. The unilateral ureteral obstruction (UUO) model, which is an animal experimental model for chronic kidney disease, was prepared by tying the cells at 1-2mm intervals, and then incision between them. To the mouse, the extract according to Example 1 was orally administered at a dose of 100 mg/kg/day for 10 days, and after the UUO procedure, the extract powder of the sputum extract was orally administered at a dose of 100 mg/kg/day every day, followed by UUO treatment. One week, the mice were sacrificed, the kidneys were removed, and the induction of fibrosis of the kidneys was observed.

Sham: Normal control (n=2); Sham/Styria extract: Normal control/Styria extract (n=2); UUO experimental group (UUO/DW, n=4); UUO/Styria extract: UUO experimental group/Styria extract administration (n=4) was divided into groups, the control group was orally administered with distilled water, and the experimental group was administered orally with a dose of 100 mg/kg/day.

The difference in the degree of renal fibrosis in the control group and the experimental group was confirmed histological changes by PAS (Periodic Acid-Schiff) and MT (Masson's trichrome) staining method (FIGS. 1 and 2). In addition, by quantifying the area of the degree of fibrosis through Image J software (NIH), it was confirmed that the fibrosis of the kidney tissue was significantly reduced after administration of the Darae extract (Fig. 3).

In addition, it was confirmed that renal fibrosis was reduced when the extract of chinensis was administered through immunohistochemical staining (FIGS. 4 and 5).

In addition, the difference in the expression of renal fibrotic proteins in each group was confirmed by Western blot method, and quantified using Image J software (NIH) to confirm the difference in protein expression in the group to which the Darae extract was administered (Fig. 6). As a result, in the UUO experimental group, the expression of αSMA, fibronectin, snail, phosphorylated p53, and Bax, which are known to increase as fibrosis progresses, was increased, but it was confirmed that the expression was suppressed by administering the extract, E-cadherin. It was found that the expression was increased by administering the serrata extract.

Example 3. Changes in expression level of renal tissue fibrosis-related factors (in vitro)

Human proximal tubular cells were subcultured and then recombinant TGF-β (PeproTech, Rocky Hill, NJ, USA) was administered at 2 ng/ml to induce fibrotic changes in tubular cells. Thereafter, the extract of serrata was administered at a concentration of 2.5, 5, 10 ㎍/ml, and changes in the expression levels of the fibrosis-related proteins αSMA (α-Smooth muscle actin), e-cadherin, and bcl2 were confirmed by Western blot method, Quantification was performed using Image J software (NIH).

The increase in αSMA was confirmed by administration of recombinant TGF-β, and after administration of the Darae extract, a significant decrease in αSMA and a significant increase in E-cadherin and bcl2 were confirmed (FIGS. 7 to 9 ). In addition, it was confirmed that the expression of phosphorylated p38 (pp38) decreased in a concentration-dependent manner upon administration of the Darae extract (FIG. 9).

For example, for the purposes of claim construction, the claims set forth below should not be construed in any way narrower than their literal language, and therefore exemplary embodiments from the specification should not be read as claims. Accordingly, it should be understood that the invention has been described as an example and is not a limitation on the scope of the claims. Accordingly, the invention is limited only by the following claims. All publications, issued patents, patent applications, books, and journal articles cited in this application are each incorporated herein by reference in their entirety.

Claims (10)

delete A pharmaceutical composition for the treatment of renal fibrosis, comprising an extract of sty. The pharmaceutical composition according to claim 2, wherein the agaric extract reduces the expression of one or more proteins selected from the group consisting of αSMA, fibronectin, snail, phosphorylated p53, phosphorylated p38, and Bax. The pharmaceutical composition according to claim 2, wherein the stalk extract increases the expression of one or more proteins selected from the group consisting of Bcl2, and E-cadherin. delete delete A health functional food for relieving kidney fibrosis, including a snail extract. The health functional food according to claim 7, wherein the stalk extract reduces the expression of one or more proteins selected from the group consisting of αSMA, fibronectin, snail, phosphorylated p53, phosphorylated p38, and Bax. The health functional food according to claim 7, wherein the agaric extract increases the expression of one or more proteins selected from the group consisting of Bcl2, and E-cadherin. delete

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