Mulberry bark extract for the treatment of chronic kidney disease, its preparation method and uses

Abstract

This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a mulberry bark extract for treating chronic kidney disease, its preparation method, and its uses. The mulberry bark extract of this invention is obtained by extracting mulberry bark with a 75-100% (v / v) ethanol aqueous solution, concentrating the extract, and drying it. This invention is the first to experimentally discover that the ethanol extract of mulberry bark has significant TGF-β / Smad signaling inhibitory activity. Based on this, a new pharmaceutical use for mulberry bark and its ethanol extract is provided for inhibiting renal TGF-β signaling, suppressing renal fibrosis, and improving renal function, showing great application potential.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a mulberry bark extract for treating chronic kidney disease, its preparation method, and its uses. Background Technology

[0002] Chronic kidney disease (CKD) refers to a group of kidney diseases characterized by abnormal kidney structure or function for three consecutive months. This includes common conditions such as diabetic nephropathy, hypertensive nephropathy, CKD caused by various primary glomerular diseases, and CKD resulting from acute kidney injury. CKD can progress to kidney failure, causing fatal cardiovascular complications. In the adult population of my country, the prevalence of CKD is approximately 10.8%, making it one of the major chronic diseases in the country. Fibrosis is a pathological process caused by an imbalance between the synthesis and catabolism of extracellular collagen matrix, leading to excessive formation of extracellular matrix, which compresses and destroys parenchymal cells, resulting in organ dysfunction. Although various etiologies can lead to CKD, fibrosis is a common pathological feature of all types of CKD and is positively correlated with the deterioration of kidney function. Therefore, anti-fibrotic therapy is one of the key approaches to overcoming the bottleneck in CKD treatment. However, clinically, CKD treatment mainly focuses on addressing the underlying cause (eliminating or alleviating disease-inducing factors), and effective drugs targeting renal fibrosis are still lacking.

[0003] TGF-β / Smad signaling is a key signaling pathway promoting renal fibrosis. Extracellular TGF-β signaling molecules activate (phosphorylate) intracellular effector molecules Smad2 and Smad3 by binding to cell membrane receptors TGFBR1 and TGFBR2. Activated Smad2 / 3 then enter the nucleus to regulate fibrosis-related cellular phenotypes and gene expression. In fibrotic renal tissue, TGF-β signaling is highly activated. Knocking out or inhibiting TGF-β signaling in genetically modified mice significantly improves renal fibrosis and impaired renal function in CKD patients. Therefore, TGF-β signaling is an important target for the development of anti-renal fibrosis drugs.

[0004] Mulberry bark (Sangbaipi) refers to the dried root bark of the mulberry tree after the cork is removed. It is a common traditional Chinese medicine with effects such as purging the lungs and relieving asthma, promoting diuresis and reducing swelling, generating fluids and quenching thirst, and relieving constipation. It is often used to treat edema, diabetes, bronchitis, pleurisy, and pertussis. Meanwhile, existing research indicates that some traditional Chinese medicine compound formulas containing mulberry bark have a certain adjuvant therapeutic effect on CKD or organ fibrosis. However, there are currently no reports on the therapeutic effects of mulberry bark alone on CKD or organ fibrosis. Furthermore, based on traditional practice, traditional Chinese medicine compound formulas containing mulberry bark for the treatment of CKD or organ fibrosis are traditionally prepared by decocting in water (e.g., CN201610105305.8 Traditional Chinese medicine compound composition with anti-pulmonary fibrosis effect and its preparation method and application). There is currently no research or exploration in this field regarding the optimal method of administration of mulberry bark for CKD or organ fibrosis.

[0005] In summary, research on the therapeutic effects and material basis of mulberry bark in treating CKD or organ fibrosis is still insufficient. This situation makes it difficult to achieve more scientific, precise, and efficient application strategies when utilizing this traditional Chinese medicine, thus limiting the full utilization of its potentially effective active ingredients and restricting its widespread clinical application potential. Therefore, there is an urgent need in this field to further optimize the application of mulberry bark in the treatment of CKD or organ fibrosis. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a mulberry bark extract for treating chronic kidney disease, its preparation method, and its uses.

[0007] Use of mulberry bark or its extract as the sole active ingredient in the preparation of medicines for the treatment and / or prevention of chronic kidney disease and / or organ fibrosis.

[0008] Preferably, the drug is used to improve renal function impairment;

[0009] And / or, the drug is used to treat and / or prevent diseases of renal fibrosis, liver fibrosis, or myocardial fibrosis.

[0010] Preferably, the drug is used to inhibit TGF-β signaling activity;

[0011] And / or, the drug is used to inhibit the expression of serpine-1, Col1a1, fibronectin and α-SMA;

[0012] And / or, the drug is used to inhibit the activation of Smad2 / 3.

[0013] Preferably, the extract is the effective fraction of an alcoholic extract.

[0014] The present invention also provides a mulberry bark extract for the treatment and / or prevention of chronic kidney disease and / or organ fibrosis. The extract is obtained by extracting mulberry bark with a 75-100% (v / v) ethanol aqueous solution, concentrating the extract, and drying it.

[0015] Preferably, the concentration of the ethanol aqueous solution is 95% by volume.

[0016] Preferably, the amount of the ethanol-water solution used is 5-20 times the volume;

[0017] And / or, the extraction conditions are: soaking for 2-4 hours, followed by reflux for 10 minutes; or, soaking for 2-4 hours, followed by percolation extraction at a flow rate of 1-5 mL / min;

[0018] And / or, the concentration conditions are vacuum concentration at 55-75°C.

[0019] Preferably, the method includes the following steps: extracting mulberry bark with a 75-100% (v / v) ethanol aqueous solution, concentrating the extract, and drying it to obtain the extract.

[0020] The present invention also provides a medicament for treating and / or preventing chronic kidney disease, which is a preparation made by adding pharmaceutically acceptable excipients to the above-mentioned mulberry bark extract as the active ingredient.

[0021] To explore traditional Chinese medicines with significant anti-renal fibrosis effects, this invention conducted extensive drug activity screening studies, discovering that mulberry bark has significant anti-TGF-β signaling activity, and that the active ingredient in its ethanol extract has higher activity than its water extract. Furthermore, using different CKD animal models, it was confirmed that the active ingredient in the ethanol extract of mulberry bark can significantly inhibit CKD renal fibrosis and improve damaged kidney function. These data indicate that this traditional Chinese medicine or its ethanol extract, as a single active ingredient, has significant application potential in the clinical treatment of CKD characterized by renal fibrosis.

[0022] The technical solution proposed in this invention can optimize the efficacy of the traditional Chinese medicine mulberry bark, thereby significantly improving its clinical application effect and more effectively exploring and utilizing the intrinsic value of this traditional Chinese medicine, demonstrating broad application potential and development prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 The ethanol extract of mulberry bark inhibits TGF-β signaling activity in renal tubular epithelial TCMK1 cells. A. Dual-luciferase reporter gene assay to analyze the inhibition of TGF-β signaling reporter gene (pCAGA-Luc) expression by mulberry bark water / ethanol extract. B. RT-PCR detection of the inhibitory effect of mulberry bark ethanol extract (EECM) on TGF-β signaling target gene expression. C. TCMK1 cells were co-treated with EECM and TGF-β1 for 24 hours, and Smad2 / 3 phosphorylation was detected by Western blot. D. TCMK1 cells were pretreated with EECM for different times, then stimulated with TGF-β1 for 30 minutes, and Smad2 / 3 phosphorylation was detected by Western blot. ***p<0.001 vs GL3.basic or NC.#p<0.05,###p<0.001 vs pCAGA-luc or TGF-β1.

[0026] Figure 2 ECM improves renal fibrosis in UUO mice. A. HE and Masson staining show renal structure and collagen (blue) distribution. B. Western blot analysis of the expression of renal fibrosis proteins fibronectin, Col1a1, α-SMA, Smad3, and p-Smad3.

[0027] Figure 3 EECM improves adenine-induced (Ade) renal fibrosis and impaired renal function in mice. A. Weekly body weight curves of mice in each group. B. Serum creatinine. C. Blood urea nitrogen. D. Actual image of kidney morphology. E. HE and Masson staining showing kidney structure and fibrous collagen distribution. F. Western blot analysis of the expression of renal fibrosis proteins fibronectin, Col1a1, α-SMA, Smad3, and p-Smad3. ***p<0.001 vs NC. #p<0.05, ##p<0.01 and ###p<0.001 vs Ade. Detailed Implementation

[0028] In the following examples and experimental cases, reagents and materials not specifically described are all commercially available products.

[0029] Example 1: Mulberry bark alcohol extract (EECM) and its preparation method

[0030] The mulberry bark extract provided in this embodiment was prepared according to the following method:

[0031] Take 10g of mechanically crushed raw mulberry bark, add 100mL of 95% v / v ethanol aqueous solution and soak for 2 hours. In a cooling reflux device, heat and boil for 10 minutes to extract. Filter the liquid with filter paper, concentrate and dry under reduced pressure at 65℃, and weigh to obtain the ethanol extract of mulberry bark.

[0032] Example 2: Mulberry bark alcohol extract and its preparation method

[0033] The mulberry bark extract provided in this embodiment was prepared according to the following method:

[0034] Take 1 kg of mechanically pulverized raw mulberry bark, add 2 L of 95% ethanol aqueous solution and soak for 2 hours. Then, extract using 8 L of 95% ethanol aqueous solution in a percolation apparatus (total extraction solvent volume: 10 L) at a flow rate of 1 mL / min. Filter the extract through filter paper, concentrate by rotary evaporation under reduced pressure at 65℃, dry, and weigh. For animal administration, EECM is redissolved in physiological saline and sonicated to prepare a resuspension.

[0035] Comparative Example 1: Water extract of mulberry bark

[0036] The preparation method of the mulberry bark aqueous extract in this comparative example is the same as that in Example 1, except that the 95% v / v ethanol aqueous solution is replaced with ultrapure water.

[0037] The technical solution of the present invention will be further illustrated by the following experiments. The UUO mice and adenine nephropathy mice used in the following experiments are commonly used animal models in the prior art for studying chronic kidney disease (CKD).

[0038] Experimental Example 1: Mulberry bark alcohol extract inhibits TGF-β signaling activity in renal tubular epithelial cells

[0039] I. Experimental Methods:

[0040] The alcohol extract used in this experimental example was prepared according to the method of Example 1, and the water extract was prepared according to the method of Comparative Example 1. In subsequent experiments, the alcohol extract was redissolved in DMSO to an appropriate concentration, and the water extract was redissolved in ultrapure water to an appropriate concentration.

[0041] Luciferase assay to detect TGF-β signaling activity: Mouse proximal tubular epithelial cell line TCMK1 was seeded into 24-well plates and transfected with 500 ng of the TGF-β signaling reporter gene pCAGA-Luc plasmid or its control plasmid pGL3.basic for 24 hours using the PEI method (10 ng of Renilla luciferase vector was simultaneously transfected into each well as an internal control). The cells were then treated with the maximum non-cytotoxic dose of mulberry bark water / alcohol extract at 0.5% serum concentration for another 24 hours. To activate TGF-β signaling, 5 ng / mL TGF-β1 was added simultaneously. After treatment, pCAGA-Luc luciferase activity was analyzed using a dual-luciferase activity assay kit (Promega, Cat#E1910, USA).

[0042] Western blot and RT-PCR were used to detect the effect of mulberry bark extract on TGF-β signaling activity: TCMK1 cells were seeded in 6-well plates. When the cell density was close to 80%, the cells were starved in 0.5% serum medium for 6 hours. 5 ng / mL TGF-β1 and a specified concentration of mulberry bark extract were added and the cells were treated for a specific time. RNA and protein samples were extracted, and the expression levels of the corresponding genes / proteins were detected by RT-PCR and Western blot, respectively.

[0043] II. Experimental Results

[0044] In mouse proximal renal tubular epithelial cells (TCMK1), experiments showed that the maximum non-toxic dose of mulberry bark extract significantly inhibited the expression of the TGF-β reporter gene pCAGA-Luc. Figure 1 A), and the maximum non-toxic dose of the ethanol extract of mulberry bark was 32 μg / mL, equivalent to 0.76 mg of crude mulberry bark, while the maximum non-toxic dose of the aqueous extract of mulberry bark was 120 μg / mL, equivalent to 3.8 mg of crude mulberry bark. This indicates that compared with aqueous solution, 95% ethanol can more effectively extract the anti-TGF-β signaling active components from mulberry bark, with an extraction efficiency approximately 5 times that of the aqueous extract. In TCMK1 cells, treatment with the ethanol extract of mulberry bark (EECM) for 24 hours significantly inhibited the expression of downstream signaling target genes induced by TGF-β1, including serpine-1 and the extracellular matrix genes Col1a1 and fibronectin (…). Figure 1 B). Simultaneously, EECM can dose-dependently inhibit the activation (phosphorylation) of Smad2 / 3 without affecting its protein levels. Figure 1 C). More importantly, EECM pretreatment for 3 hours can inhibit TGF-β1-induced Smad2 / 3 phosphorylation (C). Figure 1 D).

[0045] The above experimental results indicate that extracts from different parts of mulberry bark have different inhibitory effects on TGF-β signaling active components, with the ethanol extract showing better efficacy than the water extract. The ethanol extract of mulberry bark can significantly inhibit the expression of downstream signaling target genes induced by TGF-β1, achieving inhibition of TGF-β1-induced Smad2 / 3 phosphorylation, thus possessing the potential for treating and preventing CKD.

[0046] Experimental Example 2: Mulberry bark extract inhibits TGF-β signaling and improves renal fibrosis in UUO mice.

[0047] I. Experimental Methods

[0048] In this experimental example, the alcohol extract prepared in Example 2 was used. When administering the drug to animals, the EECM was re-dissolved in physiological saline and then sonicated to prepare a resuspension.

[0049] Animal experiments using ureteroscopic ligation (UUO): 8-10 week old male C57BL / 6 mice were divided into 5 groups (n=10 per group): a normal control group (NC), a UUO model group (UUO), and low (L), medium (M), and high (H) dose EECM groups. A renal fibrosis model was established in the UUO and treatment groups using unilateral ureteroscopic ligation (UUO). After ligation, the UUO mice in the treatment groups were administered different doses of EECM by gavage once daily for 7 consecutive days. The high dose was 1.1 g EECM / kg body weight (equivalent to 30.3 g raw mulberry bark / kg body weight, corresponding to a human dose of 200 g / 60 kg body weight), the medium dose was 0.55 g EECM / kg body weight, and the low dose was 0.275 g EECM / kg body weight. The control model group and healthy mice were administered physiological saline by gavage. On day 7, the mice were sacrificed, and the ligated kidney tissue was collected for histopathological analysis, protein extraction, and Western blot detection of relevant protein expression.

[0050] II. Experimental Results

[0051] In UUO mice, low, medium, and high doses of EECM were administered by gavage for 7 consecutive days. HE staining showed that EECM could improve renal structural damage and reduce tubular dilation in UUO mice. Masson staining showed that EECM could reduce blue collagen matrix deposition in UUO kidneys. Figure 2 A). Correspondingly, Western blot analysis revealed that EECM could reduce the expression of fibronectin, Col1a1, and α-SMA in UUO kidneys (neutralized and high-dose EECM showed more significant inhibition of α-SMA). Simultaneously, neutralized and high-dose EECM significantly inhibited the expression and phosphorylation activation of Smad3. Figure 2 B).

[0052] The above experimental results in UUO mice demonstrate that the mulberry bark extract of the present invention can significantly inhibit the renal TGF-β signaling activity of UUO model animals, improve ureteral obstruction-induced renal fibrosis, and thus treat CKD.

[0053] Experimental Example 3: Mulberry bark alcohol extract inhibited TGF-β signaling activity and alleviated renal fibrosis and renal function impairment in adenine nephropathy mice.

[0054] I. Experimental Methods

[0055] In this experimental example, the alcohol extract prepared in Example 2 was used. When administering the drug to animals, the EECM was re-dissolved in physiological saline and then sonicated to prepare a resuspension.

[0056] Male C57BL / 6 mice aged 8-10 weeks were divided into 5 groups of 10 mice each: a normal control group (NC), an adenine model group (Ade), and low (L), medium (M), and high (H) dose EECM groups. Mice in the model and drug-treated groups were fed a diet containing 0.25% adenine for 3 weeks. Mice in the EECM groups were administered the same dose of adenine as in Experiment 2, once daily for 3 weeks. Mice in the control and model groups were administered physiological saline by gavage. Animal body weight was measured weekly during the experiment. At the end of the experiment, animals were sacrificed, serum was collected to measure renal function, kidney tissue was collected for routine pathological analysis, proteins were extracted, and the expression of relevant gene proteins was detected by Western blot.

[0057] II. Experimental Results

[0058] Compared to normal mice (NC), adenine-fed mice (Ade) experienced a sustained decrease in body weight, and different doses of EECM administration could improve the weight loss in Ade mice. Figure 3 A). Serum biochemical analysis showed that low, medium, and high doses of EECM intervention could reduce serum creatinine and blood urea nitrogen (BUN) levels in Ade mice to varying degrees and improve renal function. Figure 3 (B and C). In terms of appearance, the isolated kidneys of normal mice are reddish-brown, while the kidneys of Ade mice lose their reddish luster and appear pale yellow. EECM intervention can partially restore the reddish luster of the kidneys of Ade mice. Figure 3 D). HE staining showed that EECM significantly improved renal structural damage in Ade mice and reduced the area of ​​Masson collagen staining (D). Figure 3 E). Meanwhile, Western blot results showed that EECM intervention could inhibit the expression of fibronectin, Col1a1, and α-SMA, and the phosphorylation activation of Smad3 to varying degrees. Figure 3 F).

[0059] The above experimental results in mice with adenine nephropathy demonstrate that the mulberry bark extract of the present invention can significantly inhibit the activation of TGF-β signaling, inhibit fibrosis induced by toxic substances (adenine), and improve renal function damage, thereby treating CKD.

[0060] As can be seen from the above embodiments and experimental examples, the present invention provides an extract of mulberry bark, which is an alcoholic extract that can significantly inhibit TGF-β signaling, and the drug activity of the effective fraction of the alcoholic extract is higher than that of the water extract. This makes the mulberry bark extract provided by the present invention more effective in improving renal fibrosis in CKD, reducing renal function damage, and treating CKD. Therefore, the present invention has excellent application prospects.

Claims

1. Use of mulberry white bark extract as the only active ingredient in the preparation of a medicament for the treatment and / or prevention of renal fibrosis diseases, characterized in that: The extract is obtained by extracting mulberry bark with 75-100% ethanol aqueous solution, and concentrating and drying the extract; The amount of the ethanol aqueous solution is 5-20 times; The extraction conditions are as follows: soaking for 2-4 hours, heating and refluxing for 10 minutes; or, soaking for 2-4 hours, percolation extraction, and flow rate of 1-5 mL / min; The concentration conditions are as follows: concentration at 55-75 ℃ under reduced pressure.

2. Use according to claim 1, characterized in that: The medicine is used for inhibiting TGF-β signal activity; And / or, the medicine is used for inhibiting the expression of serpine-1, Col1a1, fibronectin and α-SMA; And / or, the medicine is used for inhibiting the activation of Smad2 / 3.

3. Use according to claim 1, characterized in that: The concentration of the ethanol aqueous solution is 95% by volume.

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