Composition for preventing, ameliorating, or treating kidney and / or liver diseases via antiinflammation, antioxidation, antifibrosis, and uremic toxin excretion, comprising complex (neprobin) of ganoderma lucidum extract and robinia pseudoacacia flower extract as active ingredient

Abstract

The present invention relates to a composition for preventing, ameliorating, or treating kidney and liver diseases, comprising a complex (NEPROBIN) of Ganoderma lucidum extract and Robinia pseudoacacia flower extract as an active ingredient. The complex provided from the present invention exhibits excellent effects in preventing kidney damage, maintaining kidney function, and regenerating kidney tissue by means of inhibition of NO production, reduction in cytokine (TNF-a) and Crp gene expression, increase in glutathione, reduction in ROS production, and increase in uremic toxin excretion, and exhibits excellent effects in preventing liver damage and improving liver function by means of inhibition of NO production, reduction in cytokine (IL6) and MCP1 and CXCL1 gene expression, increase in glutathione, suppression of expression of fibrosis markers (ACTA2, Vim, COL3A1) of hepatic stellate cells, restoration of GSH in liver tissue, increase in antioxidant enzyme activity, and suppression of expression of antiinflammatory- and antifibrotic-related genes and proteins, and thus, the complex is applicable as a pharmaceutical composition for preventing or treating kidney and / or liver diseases, and as a health functional food and food composition for preventing or ameliorating liver diseases.

Description

A composition for the prevention, improvement, or treatment of kidney and / or liver diseases through anti-inflammatory, antioxidant, anti-fibrotic, and uremic toxin elimination effects, comprising a complex of *Lysimachia vulgaris* extract and acacia flower extract (NEPROBIN) as an active ingredient.

[0001] The present application claims priority based on Korean application No. 10-2024-0166587 filed on November 20, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into the present application. The present invention relates to a composition for preventing, improving, or treating kidney and / or liver disease comprising a natural extract as an active ingredient, and more specifically, to a composition for preventing, improving, or treating kidney and / or liver disease comprising a complex of *Lysimachia vulgaris* extract and acacia flower extract (NEPROBIN) as an active ingredient.

[0002] The kidneys are organs located in the retroperitoneum, with one situated on each side. Their primary functions include excreting waste products generated as a result of nutrient intake and metabolism, as well as regulating blood pressure, hematopoietic function, and maintaining mineral balance. In particular, the excretion of metabolic waste through urine is a representative function of the kidneys; various waste products such as urea, creatinine, and uric acid, produced during the process of consuming food and generating energy, are eliminated through urine to prevent uremia caused by the accumulation of waste. A condition in which the kidneys, which perform these functions, are gradually damaged and unable to perform normal functions is called Chronic Kidney Disease (CKD). This condition progresses progressively over time and can eventually lead to Advanced Stage Renal Disease (ESRD). If kidney function deteriorates due to damage and the excretion of uremic toxins through the kidneys decreases, these substances accumulate in the body, further stimulating inflammation levels and oxidative stress. This can lead to various problems, including a decline in kidney function and the initiation of cardiovascular complications. Chronic kidney disease is difficult to diagnose and treat because it has no symptoms in the early stages; therefore, it is crucial to prevent the disease and delay its progression through regular nephrological checkups and care, lifestyle modifications, dietary therapy, and medication. Furthermore, when the disease has progressed significantly to the stage of end-stage renal failure, renal replacement therapies such as hemodialysis, peritoneal dialysis, and kidney transplantation are required.

[0003] Meanwhile, drug therapy for chronic kidney disease utilizes medications designed to treat the underlying disease, slow the progression to end-stage renal failure, and prevent or treat complications, thereby alleviating the symptoms caused by the disease. Types of medications used to treat chronic kidney disease include antihypertensives (imidapril, losartan, irbesartan, etc.), anemia treatments (erythropoietin, epoetin-alpha, darbepoetin-alpha, etc.), mineral regulation and prevention and treatment of bone diseases (hyperphosphatemia treatments, calcium supplements, active vitamin D, vitamin D analogs, etc.), nutritional supplements (vitamin B, vitamin C, folic acid, etc.), and uremia treatment (spherical activated carbon). Side effects of these drugs vary depending on the type; antihypertensives, for example, can cause side effects such as nausea, diarrhea, cough, fatigue, and edema, and should be administered with caution as they can worsen hyperkalemia. Anemia medications can increase blood pressure and therefore cannot be administered to patients with uncontrolled blood pressure; they also have side effects such as headache, joint pain, diarrhea, edema, blood clots, weakness, and chest pain. Among mineral regulators and medications for the prevention and treatment of bone diseases, hyperphosphatemia treatments may cause side effects such as hypocalcemia, headache, nausea, constipation, skin rash, intestinal obstruction, and intestinal perforation, while calcium supplements have side effects such as hypercalcemia, constipation, nausea, loss of appetite, and coma. Active vitamin D has side effects such as loss of appetite, constipation, headache, increased blood pressure, heart failure, and liver dysfunction, while vitamin D analogs have side effects such as hypercalcemia, headache, taste disturbances, agitation, upper respiratory tract infection, and conjunctivitis. Nutritional supplements have side effects such as nausea, diarrhea, and gastric distension, and uremia treatment agents have side effects such as constipation, loss of appetite, nausea, and skin rash.

[0004] As such, while various drugs are currently used to treat chronic kidney disease, they all have side effects. Furthermore, since these treatments are used to slow the progression to renal failure and treat complications, there are currently no drugs that can fundamentally restore kidney function. Therefore, there is a need to develop new natural product-based therapies that are safe and effective in improving and treating chronic kidney disease.

[0005]

[0006] Furthermore, the liver is a vital organ that plays essential roles in maintaining life, such as detoxification, nutrient metabolism, and bile production. However, the incidence of liver disease is continuously rising due to the increase in Westernized dietary habits, alcohol consumption, obesity, and metabolic syndrome. In particular, with the recent redefinition of the concept of Non-Alcoholic Fatty Liver Disease (NAFLD) as Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), it is being further emphasized that liver disease is a systemic condition closely linked to metabolic abnormalities, rather than simply alcohol consumption.

[0007] MASLD can progress from simple fatty liver to Metabolic Dysfunction-Associated Steatohepatitis (MASH), which is accompanied by inflammation and fibrosis, and is a major risk factor that can lead to cardiovascular disease, cirrhosis, and hepatocellular carcinoma. Currently, effective treatments for MASLD and various chronic liver diseases are limited, and prevention and lifestyle management are presented as key response strategies. Accordingly, there is a significantly increasing need for the development of functional foods and natural product-based materials capable of maintaining and improving liver health.

[0008] Recent studies have reported that naturally derived bioactive components can inhibit hepatocellular damage and contribute to the improvement of liver function through mechanisms such as antioxidant, anti-inflammatory, and lipid metabolism regulation; however, there have been no studies whatsoever regarding the therapeutic, mitigating, and preventive effects of a complex composition of *Chrysanthemum indicum* extract and acacia flower extract on liver diseases.

[0009] Against this backdrop, the present invention aims to contribute to the health functional food industry and the promotion of public health by discovering novel natural product-based therapeutic agents capable of being effective in the treatment and prevention of liver diseases and functional food ingredients capable of helping to promote liver health, and by scientifically verifying their efficacy.

[0010] The present invention aims to provide a composition based on natural products for improving or restoring kidney and / or liver function, or for preventing, improving, or treating kidney and / or liver diseases. In particular, the present invention aims to provide a composition for improving or restoring liver function, or for preventing, improving, or treating kidney and / or liver diseases, comprising a complex of *Lysimachia vulgaris* extract and *Acacia flower* extract (hereinafter referred to as NEPROBIN) as an active ingredient. The present invention aims to provide a pharmaceutical, health functional food, and / or food product comprising the above composition.

[0011] The present invention aims to prevent, improve, and / or treat tissue fibrosis.

[0012] Ganoderma licidum refers to the Reishi mushroom, which is widely distributed in North America, Asia, and Europe, and has been used as a medicinal mushroom in Korea, China, and Japan since ancient times. In traditional Korean medicine, Ganoderma licidum is used for neurasthenia, heart disease, hypertension, and various types of cancer; additionally, it is reported to possess various physiological activities such as anti-obesity, antioxidant, anti-diabetic, anti-tumor, and anticancer effects. In particular, Ganoderma licidum contains various substances including polysaccharides, polyphenols (gallic acid, quercetin, trans-cinnamic acid, kaempferol, hesperetin, naringenin), triterpenes, nucleosides, steroids, fatty acids, alkaloids, proteins, minerals, and vitamins.

[0013] The acacia tree (Robinia pseudoacacia L.) is a legume native to North America and widely distributed throughout Korea. Acacia flowers have traditionally been used for hemostasis, arthritis, and hypertension. They are used for both culinary and medicinal purposes and are known to have diuretic, sedative, antioxidant, antibacterial, anti-inflammatory, and DNA damage protective effects. Additionally, acacia flowers contain large amounts of vitamin C, phenolic compounds (gallic acid, chlorogenic acid, ferulic acid, caffeic acid, etc.), and flavonoids (robinin, apigenin, luteolin, kaempferol, myricetin, ruthoside, etc.).

[0014]

[0015] Accordingly, the inventors discovered that a complex of *Lysimachia vulgaris* extract and *Acacia flower* extract (NEPROBIN) can provide effects such as preventing kidney damage, maintaining kidney function, and regenerating kidney tissue through excellent inhibition of NO production, reduction of cytokine (TNF-α) and Crp gene expression, increase of glutathione, reduction of ROS production, and increased excretion of uremic toxins. Based on this discovery, the present invention was completed. Uremic toxins are proteins that accumulate in the body due to a decline in kidney function and cause damage. Chronic renal failure is a disease caused by a decrease in kidney function responsible for removing uremic toxins and persistent inflammation within the kidneys; it is known to be treated by excreting uremic toxins and reducing inflammation. When uremic toxins accumulate excessively, uremia occurs, which is a complication of end-stage renal failure and is known as a potential cause of cardiovascular disease progression. β2-microglobulin (β2-mg) is known as a representative uremic toxin.

[0016] In another embodiment, the inventors completed the present invention by discovering that a complex of *Lysimachia vulgaris* extract and *Acacia flower* extract (NEPROBIN) can provide excellent effects in improving liver function through anti-inflammation and anti-fibrosis, as it was proven that the complex protects liver function, reduces ROS in liver tissue, restores GSH, and increases antioxidant enzyme activity, and that it suppresses the expression of genes and proteins related to inflammation and fibrosis.

[0017] In another embodiment of the present invention, the inventors confirmed that a complex of *Lysimachia vulgaris* extract and *Acacia flower* extract (*NEPROBIN*) inhibits the expression of fibrosis-related genes and proteins and inhibits tissue fibrosis, thereby being effective in preventing, improving, and / or treating fibrosis, and thus completed the present invention.

[0018] In the present invention, the above-mentioned *Bullocho* extract and *Acacia* flower extract are characterized by being extracted with a solvent selected from the group consisting of water, organic solvents, or a mixture thereof.

[0019] In the present invention, the extract is preferably an ethanol extract, and the ethanol extract is preferably a 45 to 55% (V / V) extract.

[0020] In the present invention, the above-mentioned *Bullocho* extract and *Acacia* flower extract are characterized by being extracted at 50 to 120°C, more preferably at 70 to 100°C.

[0021] In the present invention, the complex of the acacia flower extract and the acacia flower extract is characterized by being mixed with the acacia flower extract in a weight ratio of 0.5:1 to 6:1 (acacia flower extract: acacia flower extract) relative to the acacia flower extract.

[0022] In the present invention, the complex of the *Bullocho* extract and the acacia flower extract is characterized by having the effects of preventing kidney damage, maintaining kidney function, and regenerating kidney tissue through inhibition of NO production, reduction of cytokine (TNF-α) and Crp gene expression, increase of glutathione, reduction of ROS production, and increased excretion of uremic toxins.

[0023] In the present invention, the kidney disease is characterized by comprising one or more selected from the group consisting of proteinuria, glomerulonephritis, renal failure, uremia, foamy urine, nephrotic syndrome, oliguria, chronic renal failure, acute renal failure, metabolic acidosis, nephritis, kidney injury, renal hypofunction, renal fibrosis, and nephrosclerosis. In particular, the composition of the present invention may be used for the treatment, prevention, or improvement of uremia, renal hypofunction, nephritis, and chronic renal failure.

[0024] The above complex of the *Bullocho* extract and acacia flower extract can provide effects of preventing liver damage and treating liver disease by inhibiting NO production, reducing the expression of cytokine (Il6) and Mcp1 and Cxcl1 genes, increasing glutathione, and suppressing the expression of fibrosis indicators (Acta2, Vim, Col3a1) in hepatic stellate cells.

[0025] The above liver disease may include one or more selected from the group consisting of non-alcoholic steatohepatitis (MASH), metabolism-associated steatohepatitis (NASH), alcoholic liver disease (ALD), chronic hepatitis B, liver fibrosis, and liver cirrhosis.

[0026] In the present invention, the composition is characterized as being a pharmaceutical composition for the prevention or treatment of kidney and / or liver disease.

[0027] In the present invention, the composition is characterized as being a food composition for the prevention or improvement of kidney and / or liver disease.

[0028] In addition, the present invention provides a pharmaceutical, a health functional food, and a food comprising the above composition.

[0029]

[0030] The extract provided by the present invention exhibits excellent effects in preventing kidney damage, maintaining kidney function, and regenerating kidney tissue through inhibition of NO production, reduction of cytokine (TNF-α) and Crp gene expression, increase of glutathione, reduction of ROS production, and increased excretion of uremic toxins; therefore, it can be utilized as a pharmaceutical composition for the prevention or treatment of kidney disease, as well as as a health functional food and food composition for the prevention or improvement of kidney disease.

[0031] The extract provided by the present invention has been proven to protect liver function, reduce ROS in liver tissue, restore GSH, and increase antioxidant enzyme activity, and has been confirmed to suppress the expression of genes and proteins related to inflammation and fibrosis. Since it has excellent effects in improving liver function through anti-inflammation and anti-fibrosis, it can be utilized as a health functional food and food composition for improving liver function.

[0032] The extract of the present invention can inhibit, prevent, improve, or treat fibrosis of tissues including the liver and kidneys.

[0033] Figure 1 is a graph showing the nitric oxide (NO) production inhibitory activity of Examples 1 to 3 and Comparative Examples 1 to 6 in mouse macrophages (RAW 264.7).

[0034] Figure 2 is a graph showing the TNF-α gene expression rates of Example 1 and Comparative Examples 5 to 6 in kidney epithelial cells (NRK-52E).

[0035] Figure 3 is a graph showing the intracellular glutathione levels of Example 1 and Comparative Examples 5 to 6 in kidney epithelial cells (NRK-52E).

[0036] Figure 4 is a graph showing the ROS level in the kidney tissue of Example 1 in a kidney disease model induced by an adenine diet.

[0037] Figure 5 is a graph showing the expression rate of the Crp gene of Example 1 in a kidney disease model induced by an adenine diet.

[0038] Figure 6 is a graph showing the TNF-α gene expression rate of Example 1 in a kidney disease model induced by an adenine diet.

[0039] Figure 7 shows the relative expression levels of mRNA for fibrosis-related genes (Col1a2, Vim, Acta2) in the kidney of Example 1. ### p < 0.001: Significance compared to the untreated control group. * p < 0.05, ** p < 0.005, *** p < 0.001: Significance compared to the AD diet group.

[0040] Figure 8 shows the protein expression of COL1, Vimentin, and α-SMA in the kidneys of Example 1. α-tubulin was used as an intrinsic control. Relative protein expression was quantified by densitometry. ### p < 0.001: Significance compared to the untreated control. * p < 0.05, ** p < 0.01: Significance compared to the AD diet group.

[0041] Figure 9 shows representative images of renal Sirius Red staining. The positive staining area was quantified. ### p < 0.001: Significance compared to the untreated control group. * p < 0.05, ** p < 0.01, *** p < 0.001: Significance compared to the AD diet group.

[0042] Figure 10a is a graph showing the gene expression levels of Il-6 of Example 1 and Comparative Examples 5 to 6 in AC2F hepatocytes.

[0043] Figure 10b is a graph showing the gene expression levels of Mcp1 of Example 1 and Comparative Examples 5 to 6 in AC2F hepatocytes.

[0044] FIG. 10c is a graph showing the gene expression levels of Cxcl1 of Example 1 and Comparative Examples 5 to 6 in AC2F hepatocytes.

[0045] Figure 11a is a graph showing the gene expression levels of Col3a1 in Example 1 and Comparative Examples 5 to 6 in LX-2 cells.

[0046] Figure 11b is a graph showing the gene expression levels of Asgr1 in Example 1 and Comparative Examples 5 to 6 in LX-2 cells.

[0047] Figure 12a is a graph showing blood AST levels when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0048] Figure 12b is a graph showing blood ALT levels when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0049] Figure 12c is a graph showing the blood CK-18 fragmentation index when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0050] Figure 13 is a graph showing liver GSH levels when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0051] Figure 14a is a graph showing the expression level of Il1b, a liver inflammation-related gene cytokines, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0052] Figure 14b is a graph showing the expression level of Mcp1, a liver inflammation-related gene chemokine, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0053] Figure 14c is a graph showing the expression level of Vcam1, an adhesion molecule and a liver inflammation-related gene, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0054] Figure 15a is a graph showing the expression level of Col1a2, a liver tissue fibrosis-related gene, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0055] Figure 15b is a graph showing the expression level of Col3a1, a liver tissue fibrosis-related gene, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0056] Figure 15c is a graph showing the expression level of Vim, a liver tissue fibrosis-related gene, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0057] Figure 15d is a graph showing the expression level of Acta2, a liver tissue fibrosis-related gene, when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0058] Figure 16a is a photograph showing the degree of fibrosis at the liver histologic level when treated with Example 1 in a mouse model of liver fibrosis induced by TAA.

[0059] Figure 16b is a graph showing the degree of fibrosis quantitatively.

[0060]

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0062] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0063]

[0064] According to one embodiment of the present invention, the present invention provides a composition for the prevention, improvement, or treatment of liver and / or kidney disease comprising a complex of Ganoderma lucidum extract and acacia flower (Robinia pseudoacacia L.) extract as an active ingredient.

[0065] The extract according to the present invention can be obtained through various extraction methods known in the field to which the present invention belongs, and specifically, it may be extracted with a solvent selected from the group consisting of water, organic solvents, or a mixture thereof.

[0066] The above water may be alkaline water, and the above organic solvent may include one or more of the following: polar organic solvents having 1 to 4 carbon atoms, such as anhydrous or hydrated lower alcohols, for example, methanol, ethanol, propanol, n-butanol, acetone; non-polar organic solvents such as ether, hexane, benzene, chloroform, ethyl acetate; vegetable organic solvents such as soybean oil, sesame oil; and mixtures thereof. The above organic solvent may be used alone or in a mixture of two or more types.

[0067] As a solvent used in the above extraction, one or more organic solvents selected from methanol, ethanol, propanol, n-butanol, and acetone, or a mixed solvent comprising water in these solvents may be used. Among these, when a mixed solvent of water and ethanol is used, the extraction efficiency of the active ingredient may be further improved. In the above mixed solvent of water and ethanol, the concentration of ethanol may be 10 to 90 weight%, preferably 30 to 90 weight%, more preferably 40 to 80 weight%, and most preferably 45 to 65 weight%.

[0068] Specifically, the content of the extraction solvent may be 1 to 100 times the weight of the solid content of the extract, more preferably 5 to 50 times, and most preferably 10 to 30 times.

[0069] In addition, the above extract may be extracted at room temperature or by heating under conditions where the active ingredients of the extract are not destroyed or are minimized. The extraction method is not particularly limited and, depending on the type of extract and the extraction method, methods such as reflux cooling extraction, water bath extraction, ultrasonic extraction, or cold maceration extraction may be used.

[0070] In the above extraction step, the number of extractions may be specifically repeated 1 to 5 times. In this case, the extraction efficiency of the active ingredient may be further improved.

[0071] For example, the extraction step may involve introducing an extractant and an extraction solvent into an extractor equipped with a reflux cooler, and then performing reflux cooling extraction at 50 to 120°C, more preferably 70 to 100°C, for 2 to 12 hours. In this case, the reflux cooling extraction method is a method of extracting by refluxing while heating and then cooling; specifically, since solvent loss may occur due to vaporization caused by heating, it refers to an extraction method that minimizes solvent loss by performing a cooling process together.

[0072] Alternatively, the extraction step described above can be performed by adding an extraction solvent to the extractable material and then extracting by heating in a water bath at 50 to 120°C for 0.5 to 72 hours.

[0073] Alternatively, the extraction step described above can be performed by adding an extraction solvent to the extractable material and then using an ultrasonic extractor for 1 to 20 hours.

[0074] Alternatively, the extraction step described above can be performed by adding an extraction solvent to the extractable material and then cold-macering it at 5 to 37°C for 1 to 15 days.

[0075] Alternatively, the extraction step may be performed by adding an extraction solvent in an amount of 10 to 30 times the weight of the extractant based on the dry solid of the extractant, extracting at 20 to 80°C for 1 to 72 hours, and then filtering and concentrating to obtain an extractant in a concentrated state.

[0076] Furthermore, the extractable material of the present invention includes not only the extractable material extracted by the extraction solvent described above, but also the extractable material extracted through a conventional purification process. For example, the active fraction obtained through various additional purification methods, such as a fraction obtained by passing through an ultrafiltration membrane having a specific molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or hydrophilicity), is also included in the extractable material of the present invention.

[0077] Filtration is a process of removing suspended solid particles from an extract. Particles may be filtered using cotton, nylon, paper, etc., or ultrafiltration, cryofiltration, centrifugation, etc., may be used, but is not limited thereto.

[0078] The step of concentrating and then drying the filtrate includes, but is not limited to, freeze-drying, vacuum drying, hot-air drying, spray drying, reduced-pressure drying, foam drying, high-frequency drying, infrared drying, etc. Depending on the case, a process of grinding the final dried extract may be added.

[0079] According to one embodiment of the present invention, the extract may be processed and used in various forms, such as an extract solution, a powder obtained by drying the extract solution, or a concentrate obtained by filtering and concentrating the extract solution. In addition, the extract is not particularly limited, but may be used in the form of, for example, a tincture containing a hydrochloric alcohol as an leaching solvent, a concentrate, an extract, a liquid extract, etc.

[0080] According to another embodiment of the present invention, the *Bullocho* extract prepared as described above may contain 0.50 to 5.73 mg / g of ganoderic acid A. Additionally, the acacia flower extract prepared as described above may contain 10 to 32.3 mg / g of robinin.

[0081] According to another embodiment of the present invention, a complex of *Bullocho* extract and acacia flower extract is preferably composed of *Bullocho* extract and acacia flower extract mixed in a weight ratio of 0.5:1 to 6:1 (*Bullocho* extract: acacia flower extract), more preferably in a weight ratio of 0.8:1 to 5:1 (*Bullocho* extract: acacia flower extract), and even more preferably in a weight ratio of 1:1 (*Bullocho* extract: acacia flower extract). When mixed in the above ratios, it is possible to enhance the effects of preventing kidney damage, maintaining kidney function, and regenerating kidney tissue by inhibiting NO production, reducing cytokine (TNF-α) and Crp gene expression, increasing glutathione, reducing ROS production, and increasing the excretion of uremic toxins. Furthermore, when mixed in the above ratios, it is most preferable in that it is possible to enhance the effects of improving liver function through anti-inflammatory and anti-fibrotic processes, such as protecting liver function, restoring GSH and increasing antioxidant enzyme activity, and suppressing the expression of genes and proteins related to inflammation and fibrosis.

[0082] As described above, the complex of *Lysimachia vulgaris* extract and *Acacia flower* extract exhibits excellent effects in preventing kidney damage, maintaining kidney function, and regenerating kidney tissue through inhibition of NO production, reduction of cytokine (TNF-α) and CRP gene expression, increase of glutathione, reduction of ROS production, and increased excretion of uremic toxins; therefore, it can be utilized as a pharmaceutical composition for the prevention or treatment of kidney disease, as well as as a health functional food and food composition for the prevention or improvement of kidney disease. Furthermore, because it exhibits excellent effects in improving liver function through anti-inflammatory and anti-fibrotic processes—such as liver protection, reduction of ROS in liver tissue, restoration of GSH and increased antioxidant enzyme activity, and inhibition of the expression of genes and proteins related to inflammation and fibrosis—it can be utilized as a pharmaceutical composition for the improvement or treatment of liver function, as well as as a health functional food and food composition for improving liver function.

[0083] According to one embodiment of the present invention, the pharmaceutical composition may further comprise, in addition to the extract of *Euonymus japonicus* and the extract of *Acacia* flowers as active ingredients, a suitable carrier, excipient, and diluent commonly used in the preparation of pharmaceutical compositions. Furthermore, solid or liquid formulation additives may be used in the preparation of the pharmaceutical composition, but are not limited thereto.

[0084] As excipients, lactose, sucrose, white sugar, glucose, oligosaccharide, corn starch, starch, talc, sorbitol, crystalline cellulose, dextrin, calcium carbonate, and silicon dioxide may be used. As binders, polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, dextrin, and pectin may be used. As lubricants, magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil may be used. As coloring agents, any that are generally permitted for addition to pharmaceuticals may be used. These tablets and granules may be appropriately coated as needed, such as by sugar coating or gelatin coating. Additionally, preservatives and antioxidants may be added as needed.

[0085] In addition, the pharmaceutical composition of the present invention can be prepared in any formulation commonly manufactured in the art, and the form of the formulation is not particularly limited, but can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and patches, external formulations, suppositories, and sterile injectable solutions according to ordinary methods, but is not limited thereto.

[0086] The preferred dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and body weight, the severity of the disease, the type of wound, the site of application, the frequency of administration, the time of administration, the formulation, and the type of adjuvant. Although the dosage is not particularly limited, the pharmaceutical composition of the present invention may typically be 0.1 to 2,000 mg per day. Administration may be performed once a day, divided into 2 to 3 doses per day at appropriate intervals, or intermittently at intervals of several days.

[0087] In addition, the dosage of the pharmaceutical composition of the present invention can be appropriately adjusted according to various relevant factors such as the route of administration, the patient's age, gender, body weight, severity of the patient, type of wound, application site, frequency of administration, time of administration, formulation, patient's condition, and type of adjuvant, and can be usefully used as a pharmaceutical composition for the prevention of kidney disease and / or improvement or treatment of liver function.

[0088] The composition obtained from the above method can provide a medicine for the prevention, treatment, or improvement of kidney disease, or for the improvement or treatment of liver function.

[0089] According to one embodiment of the present invention, the health functional food and food composition may be used as food, food additive, beverage, beverage additive, fermented milk, health functional food, etc. When used as food, food additive, beverage, beverage additive, or health functional food, it may be various types of food, fermented milk, meat, beverage, chocolate, snacks, confectionery, pizza, ramen, other noodles, chewing gum, ice cream, alcoholic beverage, vitamin complex, alcoholic beverage, and other health functional foods, but is not limited thereto.

[0090] When the complex of the *Bullocho* extract and the acacia flower extract of the present invention is prepared as a health functional food and a food composition, it may further include, in addition to the *Bullocho* extract and the acacia flower extract as active ingredients, ingredients that are typically added during food manufacturing.

[0091] The above-mentioned additive ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. The carbohydrates include conventional sugars such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, etc.), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings, natural flavorings (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) may be used.

[0092] For example, when the health functional food and food composition of the present invention are manufactured as a drink, in addition to the active ingredients of the present invention, namely the extract of *Euonymus japonicus* and the extract of *Acacia* flowers, pectin, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, and fruit juice may be additionally included.

[0093] The health functional foods and food compositions of the present invention include processed forms of all natural materials, such as foods, functional foods, nutritional supplements, health foods, and food additives. Health functional foods and food compositions of the above types may be manufactured in various forms according to conventional methods known in the art.

[0094] For example, as a health food, the above-mentioned *Bullocho* extract and acacia flower extract may be prepared in the form of tea, juice, or drink for consumption, or consumed in granulated, encapsulated, or powdered form. In addition, as a food, the natural extract of the present invention may be added to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and their processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). Furthermore, to use the extract of the present invention as a food additive, it may be prepared and used in the form of a powder or a concentrate.

[0095] The amount of the health functional food and food composition for the prevention of kidney disease or improvement of liver function according to the present invention can be appropriately adjusted according to individual differences such as age and degree of health status, or formulation and form, and can be usefully used as a health functional food and food composition for the prevention of kidney disease or improvement of liver function.

[0096] The composition obtained from the above method can achieve inhibition of NO production, reduction of cytokine (TNF-α) and Crp gene expression, protection of liver function, reduction of ROS in liver tissue, increase of glutathione, restoration of GSH and increased antioxidant enzyme activity, reduction of ROS production, and increased excretion of uremic toxins. Furthermore, by exhibiting effects of maintaining kidney and liver function and regenerating kidney tissue through anti-inflammatory and anti-fibrotic processes, such as the inhibition of fibrosis-related gene and protein expression, it can provide a health functional food and food product with excellent properties for the prevention of kidney disease or improvement of liver function.

[0097] In one embodiment of the present invention, the kidney disease may include one or more diseases selected from the group consisting of proteinuria, glomerulonephritis, renal failure, uremia, foamy urine, nephrotic syndrome, oliguria, chronic renal failure, acute renal failure, metabolic acidosis, nephritis, kidney damage, renal dysfunction, renal fibrosis, and renal sclerosis, and preferably may include one or more diseases selected from the group consisting of renal fibrosis and renal sclerosis.

[0098] In one embodiment of the present invention, the liver disease may include one or more selected from the group consisting of non-alcoholic steatohepatitis (MASH), metabolism-associated steatohepatitis (NASH), alcoholic liver disease (ALD), chronic hepatitis B, liver fibrosis, and liver cirrhosis, and in particular, may alleviate, mitigate, or treat the progression of liver fibrosis and / or liver cirrhosis in MASH / NASH, ALD, and chronic viral hepatitis (especially hepatitis B).

[0099] One embodiment of the present invention may provide a method for improving or treating liver and / or kidney disease by administering an effective amount of a complex of *Lysimachia vulgaris* extract and acacia flower extract to an individual in need thereof. Another embodiment of the present invention may provide a method for treating tissue fibrosis and / or sclerosis by administering an effective amount of a complex of *Lysimachia vulgaris* extract and acacia flower extract to an individual in need thereof.

[0100] One embodiment of the present invention may provide a use of a complex of *Bullocho* extract and acacia flower extract for improving, preventing, alleviating, or treating liver and / or kidney disease. Another embodiment may provide a use of *Bullocho* extract and acacia flower extract for use in the manufacture of pharmaceuticals or health functional foods for improving, preventing, alleviating, or treating liver and / or kidney disease.

[0101] One embodiment of the present invention may provide a use of the *Bullocho* extract and the *Acacia* flower extract for improving, preventing, alleviating, or treating tissue inflammation, fibrosis, and / or sclerosis. Another embodiment may provide a use of the *Bullocho* extract and the *Acacia* flower extract for use in the manufacture of pharmaceuticals or health functional foods for improving, preventing, alleviating, or treating tissue fibrosis and / or sclerosis.

[0102] One embodiment of the present invention may provide a use of a complex of *Bullocho* extract and acacia flower extract for improving, preventing, alleviating, or treating liver and / or kidney disease. Another embodiment may provide a use of *Bullocho* extract and acacia flower extract for use in the manufacture of pharmaceuticals or health functional foods for improving, preventing, alleviating, or treating liver and / or kidney disease.

[0103] All descriptions mentioned in the present specification regarding compositions for the prevention, improvement, or treatment of kidney disease or compositions and / or treatment methods for the prevention, improvement, or treatment of liver disease comprising a complex of Ganoderma lucidum extract and Robinia pseudoacacia L. extract as active ingredients may all apply to the said uses.

[0104]

[0105] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0106]

[0107] I. Assessment of the Degree of Improvement in Kidney Function

[0108] <Experimental Example 1> Comparison of NO production inhibitory effects of *Chrysanthemum indicum* extract and *Acacia flower* extract according to extraction solvent

[0109] 20g of dried *Bullocho* was weighed and washed with water. A solvent was added at 10 times the weight of the raw material, and the extract was extracted twice using a reflux cooling extraction method at 80°C for 3 hours. Water and 30 to 100% ethanol were used as the extraction solvents, respectively. The obtained extract was filtered through a 300 mesh filter and concentrated to 25 brix (±5 brix) in a rotary vacuum concentrator to obtain a concentrate, which was then dried. The dried powder was homogenized to prepare *Bullocho* extracts for five different solvents.

[0110] 20g of dried acacia flowers were weighed, and acacia flower extracts for each solvent were prepared using the same method as the method used to extract the above-mentioned acacia flower.

[0111] Each prepared extract was treated at a concentration of 100 μg / mL on RAW 264.7 mouse macrophage cells, in which NO production was induced with LPS (Lipopolysaccharide), to compare whether it inhibited NO production. The NO production inhibitory effects of the extracts of *Polygonum multiflorum* and *Acacia* flowers according to the extraction solvent are shown in Table 1 below.

[0112]

[0113] Sample NO Production Inhibitory Activity (%, mean±SD) Euonymus japonicus water extract 11.92±0.96 Euonymus japonicus 30%(v / v) ethanol extract 23.43±1.21 Euonymus japonicus 50%(v / v) ethanol extract 43.51±0.43 Euonymus japonicus 80%(v / v) ethanol extract 41.92±1.28 Euonymus japonicus 100%(v / v) ethanol extract 18.42±1.13 Acacia flower water extract 8.56±2.72 Acacia flower 30%(v / v) ethanol extract 19.75±0.54 Acacia flower 50%(v / v) ethanol extract 21.94±0.94 Acacia flower 80%(v / v) ethanol extract 19.18±1.09 Acacia flower 100%(v / v) ethanol extract 0±1.89

[0114] As shown in Table 1 above, in the case of the *Bullocho* extract, the 50% to 80% ethanol extracts showed excellent NO production inhibition effects, and among them, the 50% ethanol extract showed the best NO production inhibition effect. In addition, in the case of the *Acacia* flower extract, the 30% to 80% alcohol extracts showed excellent NO production inhibition effects, and among them, the 50% ethanol extract showed the best NO production inhibition effect. Therefore, it was confirmed that *Bullocho* extracts and *Acacia* flower extracts with excellent NO production inhibition effects can be obtained by using 50% to 80% ethanol as the extraction solvent, respectively. Among them, the NO production inhibition effect was best when 50% ethanol was used as the extraction solvent, so in the following experiments, extracts were prepared using 50% ethanol as the solvent.

[0115]

[0116] <Examples 1 to 3> Preparation of a complex of *Chrysanthemum indicum* extract and *Acacia flower* extract

[0117] 20g of dried *Bullocho* was weighed and washed, and 50% ethanol was added at 10 times the weight of the raw material. The mixture was extracted twice using a reflux cooling extraction method at 80°C for 3 hours. The obtained extract was filtered through a 300-mesh filter and concentrated to 25 brix (±5 brix) in a rotary vacuum concentrator to obtain a concentrate, which was then dried. The dried powder was homogenized to prepare *Bullocho* extract. Additionally, 20g of dried acacia flowers were weighed and washed, and 50% ethanol was added at 10 times the weight of the raw material. The mixture was extracted twice using a reflux cooling extraction method at 80°C for 3 hours. The obtained extract was filtered through a 300-mesh filter and concentrated to 25 brix (±5 brix) in a rotary vacuum concentrator to obtain a concentrate, which was then dried. The dried powder was homogenized to prepare acacia flower extract. The powdered extract of the *Bullocho* plant and the acacia flower extract prepared as described above were combined in the weight ratio shown in Table 2 below to prepare a final complex of the *Bullocho* extract and the acacia flower extract.

[0118]

[0119] Classification Bullocho : Acacia Flower Complex Ratio Bullocho Extract (Weight%) Acacia Flower Extract (Weight%) Example 11:15050 Example 23:17525 Example 35:183.3316.67

[0120] <Comparative Examples 1 to 6> Preparation of a complex of *Chenopodium album* extract and *Acacia flower* extract

[0121] Each extract was prepared in the same manner as in the above example, and the final complex of the red ginseng extract and acacia flower extract was prepared by mixing them in the weight ratios shown in Table 3 below.

[0122]

[0123] Classification Bullocho : Acacia Flower Complex Ratio Bullocho Extract (Weight%) Acacia Flower Extract (Weight%) Comparative Example 17: 187.5 12.5 Comparative Example 21: 32575 Comparative Example 31: 516.6 783.33 Comparative Example 41: 712.5 87.5 Comparative Example 51: 01000 Comparative Example 60: 10100

[0124] <Experimental Example 2> Confirmation of Components of Immortality Herb Extract and Acacia Flower Extract

[0125] Comparative Example 5 (Epipactis extract) and Comparative Example 6 (Acacia flower extract) were used as samples for the analysis of the main component content of the Epipactis extract and the Acacia flower extract and for the standardization of raw materials.

[0126] The major component of the above *Bullocho* extract was analyzed using high-speed liquid chromatography, and the major component was identified as 'ganoderic acid A'. To prepare the standard solution, ganoderic acid A was dissolved in 100% ethanol to obtain a concentration of 0.5 mg / mL, thereby preparing a standard stock solution. Subsequently, the standard stock solution was diluted with 100% ethanol and used as the standard solution. To prepare the test solution, the *Bullocho* extract was precisely weighed, 100% ethanol was added, and ultrasonic extraction was performed. The solution was then filtered through a 0.22–0.45 µm PTFE or PVDF membrane filter to be used as the test solution. For the analysis column, a stainless steel tube with an inner diameter of 4–6 mm and a length of 15–25 cm was packed with 5–10 µm octadecylsilyl silica gel for liquid chromatography. The column temperature was a constant temperature around 25°C, and the analysis was performed using a gradient method with (A) a 2% aqueous acetic acid solution and (B) acetonitrile as the mobile phase. A UV detector was used to detect ganoderic acid A at a wavelength of 254 nm.

[0127] As a result, as shown in Table 4 below, the content of ganoderic acid A in the extract of *Bullocho* prepared according to Comparative Example 5 was found to be in the range of 0.50 to 5.73 mg / g.

[0128]

[0129] Classification Ganoderic acid A content (mg / g) 15.73 21.07 32.74 41.25 50.50

[0130] The major component of the above acacia flower extract was analyzed using high-speed liquid chromatography, and the major component was identified as 'robinin'. To prepare the standard solution, robinin was dissolved in 100% methanol to obtain a concentration of 0.5 mg / mL, thereby preparing a standard stock solution. Subsequently, the standard stock solution was diluted with 100% methanol and used as the standard solution. To prepare the test solution, the acacia flower extract was accurately weighed, 100% methanol was added, and ultrasonic extraction was performed. The solution was then filtered through a 0.22–0.45 µm PTFE or PVDF membrane filter and used as the test solution. For the analysis column, a stainless steel tube with an inner diameter of 4–6 mm and a length of 15–25 cm was packed with 5–10 µm octadecylsilyl silica gel for liquid chromatography. The column temperature was a constant temperature around 25°C, and the analysis was performed using a gradient method with (A) 0.1% aqueous acetic acid solution and (B) acetonitrile as the mobile phase. A UV detector was used to detect robinin at a wavelength of 280 nm.

[0131] As a result, as shown in Table 5 below, the robinin content in the acacia flower extract prepared according to Comparative Example 6 was found to be in the range of 11.0 to 32.3 mg / g.

[0132]

[0133] Classification Robinin Content (mg / g) 116.1 211.0 332.3 430.7 521.0

[0134] <Experimental Example 3> Cytotoxicity Test: WST (Water-soluble tetrazolium salt) assay

[0135] To determine whether Examples 1 to 3 and Comparative Examples 1 to 6 exhibit toxicity to cells, cytotoxicity experiments were conducted using NRK-52E cells, a kidney cell line, and RAW 264.7 cells, a macrophage cell line.

[0136] To determine whether it exerts toxicity on the renal epithelial cell line NRK-52E and the macrophage cell line RAW 264.7, 3 x 10⁶ cells were placed in a 96-well plate using DMEM (Dulbecco's Modified Eagle Medium). 4 cell / well and 2X10 4 Cells were dispensed at a concentration of cell / well and cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture, Examples 1 to 3 and Comparative Examples 1 to 6 were mixed with the medium at different concentrations and added to each well of the cells. After culturing for 24 hours at 37°C and 5% CO2, the culture medium was removed, and WST-1 assay solution was added to each well and reacted in an incubator for 2 hours. Subsequently, the absorbance was measured at 450 nm using a micro-plate reader, and the cell viability was calculated according to Equation 1 below.

[0137] [Mathematical Formula 1]

[0138] Cell viability (%) = [(Absorbance of sample-treated group / Absorbance of untreated group) X 100]

[0139]

[0140] Sample (Combination Ratio) Treatment Concentration NRK-52E Cell Viability (%, mean±SD) RAW 264.7 Cell Viability (%, mean±SD) Control 100±1.65 100±4.54 Example 1 (1:1) 100 µg / mL 151.73±2.96 125.13±1.42 Example 2 (3:1) 100 µg / mL 146.86±1.45 122.51±1.13 Example 3 (5:1) 100 µg / mL 143.40±5.67 119.19±1.17 Comparative Example 1 (7:1) 100 µg / mL 128.89±1.55 114.82±1.78 Comparative Example 2 (1:3) 100 µg / mL 136.17±6.41 126.71±1.29 Comparative Example 3 (1:5) 100 µg / mL 126.28±4.23 127.39±1.31 Comparative Example 4 (1:7) 100 µg / mL 123.42±5.95 124.14±1.88 Comparative Example 5 (1:0) 100 µg / mL 124.76±1.84 12.74±0.17 Comparative Example 6 (0:1) 100 µg / mL 111.43±5.45 118.10±0.82

[0141] As a result, as shown in Table 6 above, no cytotoxicity was observed in Examples 1 to 3 and Comparative Examples 1 to 6, confirming that they can be used as safe materials for food, and all experiments were conducted at concentrations where no cytotoxicity was observed.

[0142]

[0143] <Experimental Example 4> Anti-inflammatory effect: NO production inhibition effect

[0144] To confirm the anti-inflammatory effect using Examples 1 to 3 and Comparative Examples 1 to 6, an experiment was conducted to inhibit NO production by inducing inflammation with LPS (Lipopolysaccharide) using mouse macrophage cell line RAW 264.7 cells.

[0145] To determine whether it inhibits NO production in RAW 264.7 mouse macrophage cells, 1 x 10⁶ cells were placed in a 24-well plate using DMEM (Dulbecco's Modified Eagle Medium). 5After seeding cells at a concentration of cell / well, they were cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture, Examples 1 to 3 and Comparative Examples 1 to 6 were mixed with the medium at various concentrations and added to each well. Additionally, LPS, an inflammation-inducing agent that promotes NO production, was added at a concentration of 1 μg / mL, and the cells were cultured for 24 hours at 37°C and 5% CO2. Only the supernatant from each well was collected, and the amount of NO produced in each well was measured at 540 nm using a micro-plate reader with a Nitric Oxide detection kit. The NO production inhibition rate was calculated as shown in Equation 2 below.

[0146] [Mathematical Formula 2]

[0147] NO production inhibition rate (%) = [1 - (Absorbance of sample-treated group / Absorbance of untreated group)] X 100

[0148]

[0149] As a result, when the NO production inhibitory effects of Examples 1 to 3 and Comparative Examples 1 to 6 were confirmed as shown in Figure 1, it was confirmed that Examples 1 to 3 had significantly superior anti-inflammatory effects compared to Comparative Examples 1 to 6, and among them, Example 1 showed the most superior NO production inhibitory effect. Through this, it was confirmed that the complex of *Bullocho* extract and acacia flower extract exhibited excellent anti-inflammatory activity at a weight ratio of 1:1 to 5:1 (*Bullocho* extract: acacia flower extract).

[0150]

[0151] <Experimental Example 5> Anti-inflammatory effect: Cytokine (TNF-α gene expression)

[0152] To confirm the cytokine expression inhibitory ability of renal epithelial cells using Example 1 and Comparative Examples 5 to 6, the gene expression level of TNF-α, a representative factor, was confirmed through qPCR.

[0153] To determine whether it inhibits TNF-α gene expression in the renal epithelial cell line NRK-52E, 1 x 10⁶ cells were placed in a 60 mm dish using DMEM (Dulbecco's Modified Eagle Medium). 6 After seeding cells at the cell concentration, they were cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture, the cells were pretreated with Example 1 and Comparative Examples 5 to 6 at a concentration of 100 μg / mL for 30 minutes, followed by treatment with 10 μg / mL of LPS (Lipopolysaccharide) for 1 hour to induce an inflammatory response. After 1 hour, the cells were lysed to isolate RNA, cDNA was synthesized from the isolated RNA, and qPCR was performed. To analyze the qPCR data, the 2-ΔΔCT method was used as a relative quantification approach, and the relative gene expression rate was calculated as shown in Equation 3 below. N-acetylcysteine ​​(NAC) was used as a positive control.

[0154] [Mathematical Formula 3]

[0155] Relative expression(%)=(TNF-α gene expression rate of sample-treated group / TNF-α gene expression rate of untreated group)

[0156]

[0157] As a result, as can be seen in Figure 2, when the TNF-α gene expression rates of Example 1 and Comparative Examples 5 to 6 were checked, Example 1 (2.52±0.85) showed a significantly superior TNF-α gene expression inhibitory effect compared to Comparative Examples 5 (3.27±0.50) to 6 (2.84±0.89), confirming that it has an anti-inflammatory effect in the kidney.

[0158]

[0159] <Experimental Example 6> Antioxidant Activity: Glutathione assay

[0160] In order to determine the level of glutathione (GSH), an endogenous substance with antioxidant capacity in kidney epithelial cells, using Example 1 and Comparative Examples 5 to 6, the intracellular GSH content was measured using NRK-52E cells, a kidney cell line.

[0161] To determine whether increasing the GSH content in the renal epithelial cell line NRK-52E cells exhibits an antioxidant effect, 2 x 10⁶ cells were placed in a 12-well plate using DMEM (Dulbecco's Modified Eagle Medium). 5 After seeding cells at a concentration of cell / well, they were cultured for 24 hours at 37°C and 5% CO2. After 24 hours of culture, the cells were pretreated with Example 1 and Comparative Examples 5 to 6 at a concentration of 100 μg / mL for 30 minutes, followed by treatment with 100 μM H2O2 for 1 hour to induce oxidative stress. After 1 hour, the cells were lysed, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. 1 mM EDTA-50 mM phosphate buffer was added to the supernatant, followed by the addition of o-phthalaldehyde. The mixture was reacted at room temperature for 20 minutes, and fluorescence was measured at excitation 360 nm and emission 460 nm. GSH concentration was calculated based on the quantitative curve of the standard solution, and the intracellular GSH content was calculated using Equation 4 below and expressed as nmole / μg protein. At this time, the study was conducted using N-acetylcysteine ​​(NAC) as a positive control.

[0162] [Mathematical Formula 4]

[0163] Intracellular GSH content (nmole / µg protein) = {[(Fluorescence value of sample treatment group / Fluorescence value of glutathione 1 nmole) / Reaction sample volume (μL)] X Protein amount of sample treatment group (µg / μL)}

[0164]

[0165] As a result, as can be seen in Figure 3, when the glutathione content of Example 1 and Comparative Examples 5 to 6 was measured, Example 1 (6.95±0.52 nmole / µg protein) showed a significantly superior effect in increasing glutathione content compared to Comparative Examples 5 (5.88±0.92 nmole / µg protein) to 6 (6.16±0.49 nmole / µg protein), confirming that it has an antioxidant effect against oxidative stress in the kidney.

[0166]

[0167] <Experimental Example 7> Experiment on the effect of preventing kidney damage

[0168] (1) Experiment to confirm the induction of kidney disease and uremic toxin excretion through a high-dose adenine diet

[0169] To confirm the reduction of renal damage in Example 1, renal disease was induced in 7-week-old male C57BL / 6J mice by administering a 0.25% high-dose adenine diet for 2 weeks. Along with the adenine diet, Example 1 was orally administered daily at doses of 100 mg / kg, 200 mg / kg, and 400 mg / kg, and dapagliflozin 10 mg / kg, a sglt2 inhibitor, was orally administered as a positive control. Urine from the mice orally administered for 2 weeks was collected using a metabolic cage, and the absorbance of β2-microglobulin (β2-MG) in the urine was measured at 450 nm using a mouse β2-MG ELISA kit. Although β2-MG is normally present in very small amounts in urine, its concentration in urine increases due to reduced reabsorption when the renal tubules are damaged or diseased; therefore, it serves as an indicator to confirm the kidney's function of uremic toxin excretion. The concentration of β2-MG was calculated based on the quantitative curve of the standard solution in the kit.

[0170] In addition, blood creatinine and blood urea nitrogen (BUN), which are known to accumulate in the blood as they are difficult to excrete through the kidneys when kidney disease occurs and are used as kidney function markers, were analyzed using a creatinine and BUN measurement kit after collecting mouse serum.

[0171]

[0172] Classification β2-MG(mg / mL, mean±SD) Creatinine(mg / dL, mean±SD) BUN(mg / dL, mean±SD) Negative control 0.108±0.026 2.92±0.622 6.32±2.60 Adenine diet Control 0.365±0.077 5.73±1.22 103.82±30.55 Dapagliflozin 10 mg / kg 0.205±0.059 4.02±0.747 4.89±18.89 Example 1 100 mg / kg 0.369±0.075 4.19±0.907 6.94±10.20 Example 1 200 mg / kg 0.274±0.044 3.47±0.946 8.96±15.47 Example 1 400 mg / kg0.273±0.0413.43±1.0061.02±17.14

[0173] As a result, as shown in Table 7 above, urinary β2-MG, blood creatinine, and BUN levels, which were elevated due to kidney disease caused by an adenine diet, decreased in a concentration-dependent manner in the oral administration group of the complex of *Bullocho* extract and acacia flower extract of Example 1, confirming that Example 1 maintains kidney function and increases the excretion of uremic toxins. Therefore, it was confirmed that Example 1 has the effect of reducing the occurrence of kidney disease by increasing the excretion of uremic toxins from the kidneys, preventing kidney damage, and maintaining kidney function.

[0174]

[0175] (2) Confirmation of oxidative stress indicators in kidney tissue: ROS

[0176] Oxidative stress in kidney tissue was measured using the DCFDA fluorescent dye. Kidney tissue was homogenized in PBS buffer, and the supernatant was reacted with the DCFDA fluorescent reagent (50 μM) for 10 minutes. Fluorescence changes were measured at excitation 485 nm and emission 530 nm, and the degree of ROS generation in the kidney tissue was quantified and analyzed.

[0177]

[0178] As shown in Figure 4, the ROS levels in kidney tissue were measured, and it was confirmed that the ROS level increased with the adenine diet, while the ROS level decreased in a concentration-dependent manner in the oral administration group of Example 1. Through this, it was confirmed that Example 1 has the effect of reducing the incidence of kidney disease by preventing kidney damage through reducing oxidative stress in the kidney.

[0179]

[0180] (3) Confirmation of kidney damage indicators and immune markers in kidney tissue: Crp, TNF-α

[0181] The gene expression levels of Crp and TNF-α, which are indicators of kidney tissue damage and immunity, were confirmed using qPCR. The Crp gene is a kidney damage indicator known to increase when inflammation occurs in the kidney, and the TNF-α gene is an inflammatory cytokine associated with inflammation. Kidney tissue was homogenized in Trizol to isolate RNA, and after synthesizing cDNA from the RNA, qPCR was performed. To analyze the qPCR data, the 2-ΔΔCT method was used as a relative quantification approach, and the relative gene expression rate was calculated as shown in Equation 5 below.

[0182] [Mathematical Formula 5]

[0183] Relative expression(%) = (Gene expression rate of sample administration group / Gene expression rate of control group)

[0184]

[0185] As shown in Figures 5 and 6, the expression levels of Crp and TNF-α genes in kidney tissue were examined, and it was confirmed that the increased expression levels of Crp and TNF-α genes in the adenine diet model were reduced in the oral administration group of Example 1. Through this, it was confirmed that Example 1 has the effect of reducing the occurrence of kidney disease by alleviating kidney damage and inflammation.

[0186]

[0187] (4) Height and weight measurement

[0188] To confirm the safety of Example 1, the change in kidney weight of experimental animals following administration was measured.

[0189]

[0190] Classification Kidney weight (mg, mean±SD) Negative control 3 13±38.55 Adenine diet Control 3 16.55±51.54 Dapagliflozin 10 mg / kg 3 28.82±57.03 Example 1 100 mg / kg 3 05.55±46.06 Example 1 200 mg / kg 295.64±30.05 Example 1 400 mg / kg 3 13.36±47.14

[0191] As a result, as shown in Table 8 above, it was found that administering Example 1 did not affect kidney weight, and the adenine diet also did not affect kidney weight. Through this, it was confirmed that Example 1 can be used as a safe material for pharmaceuticals and food.

[0192]

[0193] <Experimental Example 8> Confirmation of Inhibitory Effect on Renal Fibrosis

[0194] (1) Prepare mouse model

[0195] Animal experiments were conducted in accordance with the animal experiment guidelines of Pusan ​​National University (PNU) and were approved by the Institutional Animal Care Committee (IACUC) of Pusan ​​National University (IACUC Approval No.: PNU-2024-0412). Male C57BL / 6J mice were purchased from Hyochang Science in Daegu, South Korea. All mice were housed under conditions of 23±2°C, 60±5% relative humidity, and a 12-hour light-dark cycle, and were allowed free access to water and food. The experimental animals were randomly divided into four groups (n = 9–10). To induce chronic kidney disease (CKD), mice (9–10 weeks of age) were fed a 0.25% adenine diet (AD; JA BIO, Suwon, South Korea) for 2 weeks. To evaluate the renoprotective effects of natural compounds, each example was dissolved in 10% DMSO in water and administered orally once daily at doses of 100, 200, and 400 mg / kg. As a positive control, dapagliflozin (HY-10450, MedChemExpress, Monmouth Junction, NJ, USA) was administered orally at a dose of 10 mg / kg in the same solvent. Both the control group and the AD diet group received only the same 10% dimethyl sulfoxide aqueous solution. After the fasting period for each experimental group, mice were euthanized by CO2 inhalation and sacrificed for further analysis. Subsequently, serum and kidney tissue samples were collected and used for biochemical analysis.

[0196]

[0197] (2) Confirmation of anti-fibrotic effect

[0198] To evaluate the antifibrotic effects of the examples, the degree of fibrosis was analyzed in the same animal model. The gene expression of extracellular matrix proteins (Col1a2, Vim, and Acta2) in the kidneys of the example treatment groups was significantly reduced compared to the control group fed the AD diet (Fig. 7). The decrease in gene expression was particularly pronounced in the Example 1 200 mg (Col1a2: 57.1%, Vim: 53.7%, Acta2: 39.2%) and 400 mg (Col1a2: 37.8%, Vim: 26.1%, Acta2: 24.6%) treatment groups, which is a result compared to the kidneys of the control group fed the AD diet. Protein expression also showed a similar trend to gene expression. Treatment with Example 1 significantly reduced the expression of COL1, Vimentin, and α-SMA proteins compared to the control group fed the AD diet (Fig. 8). The decrease in protein expression was particularly pronounced in the Example 1 200 mg (COL1: 53.3%, Vimentin: 43.4%, and α-SMA: 59.8%) and 400 mg treatment groups (COL1: 48.7%, Vimentin: 32.1%, and α-SMA: 54.3%), compared to the control kidneys fed the AD diet. The degree of fibrosis was further evaluated histologically using Sirius Red (SR) staining. As a result of SR staining, the treatment with Example 1 significantly reduced fibrotic areas in the interstitial region (Fig. 9, left). Quantification of the positive SR staining areas revealed reductions of 34.7%, 55%, and 44.9% in the Example 1 100 mg, Example 1 200 mg, and Example 1 400 mg treatment groups, respectively (Fig. 9, right). Collectively, these results demonstrate that Example 1 effectively alleviates the development of fibrosis in the kidneys of a mouse model fed the AD diet.

[0199]

[0200] <Experimental Example 9> Anti-inflammatory effect: Cytokine (Il-6) and chemokine (Mcp1, Cxcl1)

[0201] The following experiment was conducted to confirm the protective efficacy of the test substance against the inflammatory response of hepatocytes. Using Example 1 and Comparative Examples 5 to 6, the gene expression levels of representative factors Il-6, Mcp1, and Cxcl1 were confirmed via qPCR to confirm the ability to inhibit cytokine and chemokine expression in hepatocytes.

[0202] To confirm gene expression inhibition in AC2F hepatocytes, 1 x 10⁶ AC2F cells were placed in a 60 mm dish. 6 After seeding, Example 1 and Comparative Examples 5 to 6 were treated at a concentration of 200 μg / mL. After 2 hours, 10 μg / mL of LPS (Lipopolysaccharide) was treated to induce an inflammatory response. After 1 hour, cells were lysed to isolate RNA, cDNA was synthesized from the isolated RNA, and qPCR was performed. To analyze the qPCR data, the 2-ΔΔCT method was calculated using a relative quantification approach, and silymarin was used as a positive control.

[0203] As a result, as can be seen in Figure 10, when the gene expression levels of Il-6, Mcp1, and Cxcl1 of Example 1 and Comparative Examples 5 to 6 were checked, it was found that Example 1 had a significantly superior inhibitory effect on the gene expression of Il-6, Mcp1, and Cxcl1 compared to Comparative Examples 5 to 6, confirming that it has an anti-inflammatory effect in the liver.

[0204]

[0205] <Experimental Example 10> Antifibrotic effect: Liver fibrosis genes (Col3a1 and Asgr1)

[0206] The following experiment was conducted to confirm the protective efficacy of the test substance against the fibrosis response of hepatic stellate cells. To confirm the ability to inhibit the TGFβ-induced fibrosis response of LX-2 cells using Example 1 and Comparative Examples 5 to 6, the gene expression levels of Col3a1, a gene that typically increases in the fibrosis response, and Asgr1, a gene that decreases, were confirmed via qPCR.

[0207] To induce a fibrotic response in LX-2 cells, 1 x 10⁶ LX-2 cells were placed in a 60 mm dish. 6 After seeding, the cells were pretreated with Example 1 and Comparative Examples 5 to 6 at a concentration of 200 μg / mL for 2 hours. After treating with 10 ng / ml of TGFβ for 24 hours to induce a fibrosis reaction, RNA was isolated from the cells and qPCR was performed. Silymarin was used as a positive control.

[0208] As a result, as can be seen in Figure 11, when the gene expression levels of Col3a1 in Example 1 and Comparative Examples 5 to 6 were checked, it was found that Example 1 significantly inhibited the gene expression of Col3a1 compared to Comparative Examples 5 to 6, and the gene expression of Asgr1 also increased more significantly, confirming that it has an antifibrotic effect in the liver.

[0209]

[0210] <Experimental Example 11> Experiment on Hepatoprotective and Liver Function Improvement Effects

[0211] (1) Prevention and treatment of liver disease using a mouse model of TAA-induced liver fibrosis

[0212] To confirm the hepatoprotective and liver function-improving effects of Example 1, liver disease was induced in 7-week-old male C57BL / 6J mice by intraperitoneal administration of Thioacetamide (TAA) for 4 weeks. Example 1 was orally administered daily along with TAA at 100 mg / kg, 200 mg / kg, and 400 mg / kg, and silymarin was orally administered as a positive control at 20 mg / kg. The body weight, feed intake, and water intake of the mice were measured daily. After measuring the final body weight at 4 weeks, the mice were sacrificed to sample blood, and the livers were dissected to measure and analyze the weight of the livers.

[0213] The concentrations of blood alanine aminotransferase (ALT) and blood aspartate aminotransferase (AST) were measured as markers to confirm functional changes in the liver. Blood AST levels were measured using the Asan set GOT Assay kit, and blood ALT levels were measured using the Asan set PT Assay kit. After reacting the substrates inside the kits with the blood, the absorbance was measured at 510 nm using a spectrophotometer. The results were calculated by converting the blood AST and ALT concentrations using the standard calibration curves inside the kits.

[0214] As a result, as can be seen in Figure 12, a significant increase in both ALT and AST in the blood of the TAA-treated group was observed, while a concentration-dependent decrease in ALT and AST in the oral administration group of Example 1 was observed. In addition, CK-18 fragments, which are known to increase with hepatocellular damage, were measured in the blood. A significant increase in CK-18 fragmentation was observed in the blood of the TAA-treated group, while a decrease was observed in the oral administration group of Example 1. Through this, it was confirmed that Example 1 has hepatoprotective efficacy.

[0215]

[0216] (2) Checking indicators of liver oxidative stress: GSH

[0217] Liver GSH levels were measured to identify indicators related to liver oxidative stress using a TAA-induced liver fibrosis mouse model. To measure tissue GSH levels, a quantification method using o-phthalaldehyde was employed. For the analysis to measure glutathione levels, 1 mM EDTA-50 mM phosphate buffer solution was added to the supernatant of a trichloroacetic acid (TCA)-treated homogenate, followed by the addition of o-phthalaldehyde, and the mixture was incubated at room temperature for 25 minutes. Fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. To determine the accurate concentration of GSH, a calibration curve was plotted and compared using GSH samples of known concentrations.

[0218] As a result, as can be seen in Figure 13, liver GSH levels decreased in the TAA treatment group and significantly increased in the oral administration group of Example 1. Through this, it was confirmed that Example 1 reduces oxidative stress.

[0219]

[0220] (3) Confirmation of expression levels of liver inflammation-related genes and proteins: Il1b, Mcp1 and Vcam1

[0221] We confirmed the expression levels of genes and proteins known to increase due to liver inflammation using a TAA-induced liver fibrosis mouse model. We performed qPCR to determine the gene expression levels of Il1b, a cytokine that typically increases in inflammatory responses, Mcp1, a chemokine, and Vcam1, an adhesion molecule.

[0222] As a result, as can be seen in Figure 14, it was confirmed that all indicators were significantly increased by TAA, and in the oral administration group of Example 1, it was confirmed that the expression levels of all inflammation-related genes were significantly reduced. Through this, it was confirmed that the TAA-induced hepatitis response was alleviated by Example 1.

[0223]

[0224] (4) Confirmation of expression levels of genes and proteins related to liver tissue fibrosis: Col1a2, Col3a1, Vim and Acta2

[0225] Using a TAA-induced liver fibrosis mouse model, we examined the expression levels of Col1a2, Col3a1, Vim, and Acta2, which are representative genes that increase during fibrosis.

[0226] As a result, as can be seen in Figure 15, the gene expression levels of Col1a2, Col3a1, Vim, and Acta2 were found to increase significantly in the TAA treatment model, and fibrosis was inhibited in the oral administration group of Example 1. Through this, it was confirmed that Example 1 is effective against TAA-induced liver tissue fibrosis.

[0227]

[0228] (5) Check the level of fibrosis at the histologic level of the liver

[0229] The following methods were used to confirm histological changes using a TAA-induced liver fibrosis mouse model. Liver sections (5 μm thick) embedded in paraffin were prepared according to standard procedures and stained with Sirius red. Images were captured using a microscope. In situ hybridization (ISH) experiments were performed using tissue samples fixed in formalin and embedded in paraffin. The RNAscope 2.5 HD Duplex Detection kit was used, employing specific probes that target RNA molecules within the tissue. Collagen and macrophages were stained using Mm-Col1a1 and Mm-Emr1, respectively, and the liver tissue samples underwent hybridization with these probes to specifically bind to complementary RNA sequences within the tissue sections. Following hybridization, the sections were processed according to the detection system included in the RNAscope assay kit. Images of the stained sections were captured using a microscope.

[0230] As a result, as can be seen in Figure 16, the positive region increased in the TAA group, similar to gene and protein expression levels, while a decreasing trend was observed in the oral administration group of Example 1. A significant effect was confirmed when calculated quantitatively.

[0231]

[0232] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0233] The present invention may provide a pharmaceutical composition for the prevention or treatment of kidney and / or liver disease. The present invention may provide a food composition for the prevention or improvement of kidney and / or liver disease. The present invention may provide a pharmaceutical product, a health functional food, and a food product comprising the above composition.

Claims

1. A composition for the prevention, improvement, or treatment of kidney disease comprising a complex of Ganoderma lucidum extract and Robinia pseudoacacia L. extract as an active ingredient.

2. A composition according to claim 1, characterized in that the above-mentioned *Bullocho* extract and acacia flower extract are extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

3. A composition according to claim 2, characterized in that the above-mentioned *Bullocho* extract and the acacia flower extract are each 45 to 55% (W / W) ethanol extracts.

4. A composition according to claim 1, characterized in that the above-mentioned *Bullocho* extract and acacia flower extract are extracted at 50 to 120℃.

5. A composition according to claim 1, wherein the complex of the *Bullocho* extract and the acacia flower extract is mixed with the acacia flower extract in a weight ratio of 0.5:1 to 6:1 (*Bullocho* extract: *Acacia flower extract*) relative to the *Bullocho* extract.

6. A composition according to claim 1, characterized in that the kidney disease comprises one or more selected from the group consisting of proteinuria, glomerulonephritis, renal failure, uremia, foamy urine, nephrotic syndrome, oliguria, chronic renal failure, acute renal failure, metabolic acidosis, nephritis, kidney damage, renal hypofunction, renal fibrosis, and nephrosclerosis.

7. A composition according to claim 1, characterized in that the composition is a pharmaceutical composition for the prevention or treatment of kidney disease.

8. A composition according to claim 1, characterized in that the composition is a food composition for the prevention or improvement of kidney disease.

9. A composition for the prevention or treatment of liver disease improvement comprising a complex of Ganoderma licidum extract and Robinia pseudoacacia L. extract as an active ingredient.

10. A composition according to claim 9, wherein the above-mentioned *immortality herb* extract and acacia flower extract are extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

11. A composition according to claim 9, characterized in that the above-mentioned *Bullocho* extract and *Acacia* flower extract are extracted at 50 to 120°C.

12. A composition according to claim 9, wherein the complex of the herb extract and the acacia flower extract is mixed with the acacia flower extract in a weight ratio of 0.5:1 to 6:1 (herb extract: acacia flower extract) relative to the herb extract.

13. A composition according to claim 9, characterized in that the complex of the above-mentioned *Bullocho* extract and acacia flower extract prevents liver damage and treats liver disease by inhibiting NO production, reducing cytokine (Il6) and Mcp1 and Cxcl1 gene expression, increasing glutathione, and suppressing the expression of fibrosis indicators (Acta2, Vim, Col3a1) in hepatic stellate cells.

14. A composition according to claim 9, characterized in that the liver disease comprises one or more selected from the group consisting of non-alcoholic steatohepatitis (MASH), metabolism-associated steatohepatitis (NASH), alcoholic liver disease (ALD), chronic hepatitis B, liver fibrosis, and liver cirrhosis.

15. A composition according to claim 9, characterized in that the composition is a pharmaceutical composition for the prevention or treatment of liver disease.

16. A composition according to claim 9, characterized in that the composition is a food composition for the prevention or improvement of liver disease.

17. A pharmaceutical product comprising the composition of any one of paragraphs 1 and 3 through 16.

18. A health functional food comprising the composition of any one of paragraphs 1 and 3 through 16.

19. A food comprising the composition of any one of paragraphs 1 and 3 through 16.

Images