Rehmannia glutinosa polysaccharide, a preparation method thereof and application thereof in preparing a medicine for preventing and treating cisplatin-induced kidney injury

Abstract

The application discloses a rehmannia polysaccharide, a preparation method thereof and application of the rehmannia polysaccharide in preparation of a medicine for preventing and treating kidney injury caused by cisplatin. The rehmannia polysaccharide contains the following monosaccharides in percentage by mass: rhamnose (Rha) 10.265%-14.963%, glucuronic acid (GlcA) 0.514%-0.904%, galacturonic acid (GalA) 35.784%-40.596%, glucose (Glc) 3.128%-5.048%, galactose (Gal) 37.892%-41.438% and arabinose (Ara) 3.869%-4.865%. The structure of the rehmannia polysaccharide is clear, and the rehmannia polysaccharide shows a significant improvement effect on cisplatin nephrotoxicity through cell experiments and animal experiments, and has important value for developing a new and effective prevention and treatment product of cisplatin-induced acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a Rehmannia glutinosa polysaccharide, its preparation method, and its application in the preparation of drugs for preventing and treating cisplatin-induced kidney damage. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a sharp decline in glomerular filtration rate, often accompanied by elevated serum creatinine and decreased urine output. AKI is characterized by high morbidity and mortality, particularly among hospitalized and critically ill patients, and has become a major global public health challenge. Statistics show that approximately 13.3 million new AKI patients are diagnosed globally each year, with up to 1.7 million related deaths. More seriously, AKI often leads to poor long-term prognosis, including the development and progression of chronic kidney disease (CKD), and even end-stage renal disease (ESRD). Currently, there are still no effective drugs or methods to prevent or treat AKI clinically. Treatment mainly focuses on supportive care and renal replacement therapy, which is insufficient to fundamentally improve patient survival, reduce kidney tissue damage, or promote repair. Among the many causes, cisplatin-induced acute kidney injury (Cis-AKI) is a serious clinical problem in the field of oncology treatment. Cisplatin, as a first-line broad-spectrum chemotherapy drug, is widely used to treat various solid tumors such as head and neck cancer, lung cancer, ovarian cancer, and bladder cancer, with definite efficacy. However, its significant nephrotoxicity severely restricts its further clinical application and improvement of patient prognosis. Approximately one-third of chemotherapy patients experience varying degrees of renal dysfunction due to cisplatin, and currently, there are no safe and effective prevention and treatment strategies. Renal tubular epithelial cell (RTEC) damage is the main pathological basis of acute kidney injury (AKI). Due to their cellular and anatomical characteristics, functionally active RTECs are more susceptible to damage caused by nephrotoxins or irritant / respiratory nephrotoxic agents (I / R) than other cells in the kidney, leading to acute tubular necrosis and ultimately AKI. Therefore, protecting renal tubular epithelial cells from damage in the pathological state of AKI is an important direction for developing effective treatments.

[0003] Traditional Chinese medicine (TCM), with its multi-component, multi-target, and multi-pathway holistic regulatory characteristics, demonstrates unique potential in the prevention and treatment of complex diseases, providing new ideas and research directions for the prevention and treatment of Acinetobacterial Injection (AKI). Polysaccharides, as an important class of bioactive substances, are widely found in various TCM herbs and are one of the material bases for their efficacy. The bioactivity of polysaccharides is highly dependent on their detailed chemical structure (such as monosaccharide composition, glycosidic bond type, branching degree, and molecular weight). There is an urgent need to discover structurally well-defined active ingredients with specific structure-activity relationships from natural products, in order to provide new and effective candidate drugs for the clinical prevention and treatment of Cis-AKI. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a Rehmannia glutinosa polysaccharide, its preparation method, and its application in the preparation of drugs for preventing and treating cisplatin-induced kidney injury. This polysaccharide aims to target and improve cisplatin-induced acute kidney injury through a well-defined chemical structure, providing a new solution to overcome the shortcomings of existing prevention and treatment strategies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In its first part, this invention provides a Rehmannia glutinosa polysaccharide, which, by weight percentage, comprises the following monosaccharides: Rhamnose (Rha) 10.265%–14.963%, glucuronic acid (GlcA) 0.514%–0.904%, galacturonic acid (GalA) 35.784%–40.596%, glucose (Glc) 3.128%–5.048%, galactose (Gal) 37.892%–41.438%, and arabinose (Ara) 3.869%–4.865%.

[0007] In a specific embodiment of the present invention, the Rehmannia glutinosa polysaccharide comprises the following monosaccharide composition by mass percentage: rhamnose (Rha) 12.373%, glucuronic acid (GlcA) 0.779%, galacturonic acid (GalA) 37.623%, glucose (Glc) 4.277%, galactose (Gal) 39.692%, and arabinose (Ara) 4.083%.

[0008] According to an embodiment of the present invention, the weight-average molecular weight of the Rehmannia glutinosa polysaccharide is 3000-10000 Da, such as 5402 Da.

[0009] According to an embodiment of the present invention, the Rehmannia glutinosa polysaccharide comprises the following glycosidic bonds in molar percentage: t-Ara f 0.895%~0.997%, t-Xyl p 0.593%~0.673%, t-Rha p 0.886%~1.048%, 1,5-Ara f 0.968%~1.170%, 1,2-Rha p 8.002%~9.062%, t-Glc p 0.624%~0.772%, t-Glc p A 0.787%~0.853%, t-Gal p 17.917%~18.845%, t-Gal pA 4.132%~4.584%, 1,3,5-Ara f 0.713%–0.855%, 1,2,4-Rha p 5.062%~6.120%, 1,4-Gal p 2.108%~2.818%, 1,4-Gal p A 33.894%~38.318%, 1,4-Glc p 0.487%~0.559%, 1,3-Gal p 1.785%–1.915%, 1,6-Glc p 0.827%–1.495%, 1,6-Gal p 10.368%–12.656%, 1,3,4-Gal p A 1.541%~1.695%, 1,2,4-Gal p A 0.572%~0.764%, 1,4,6-Gal p A 0.709%~0.891%, 1,3,6-Gal p 0.485%~0.551%.

[0010] In a specific embodiment of the present invention, the Rehmannia glutinosa polysaccharide comprises the following glycosidic bonds, in molar percentage: t-Ara f 0.946%, t-Xyl p 0.633%, t-Rha p 0.967%, 1,5-Ara f 1.069%, 1,2-Rha p 8.532%, t-Glc p 0.698%, t-Glc p A 0.820%, t-Gal p 18.381%, t-Gal p A 4.358%, 1,3,5-Ara f 0.784%, 1,2,4-Rha p 5.591%, 1,4-Gal p 2.463%, 1,4-Gal p A36.106%, 1,4-Glc p 0.523%, 1,3-Gal p 1.850%, 1,6-Glc p 1.161%, 1,6-Gal p 11.512%, 1,3,4-Galp A 1.618%, 1,2,4-Gal p A0.668%, 1,4,6-Gal p A 0.800%, 1,3,6-Gal p 0.518%.

[0011] According to embodiments of the present invention, the Rehmannia glutinosa polysaccharide comprises at least one of the following: 1) Pectin containing HG domains; Preferably, the HG structural domain is composed as follows:

[0012] Where R represents H or -COOCH3, and R1 represents α-D-Gal p A-(1→or α-D-△) 4，5 -Gal p A-(1→; More preferably, other residues are connected to the C-2 and / or C-3 hydroxyl groups in the HG domain via glycosidic bonds; 2) Pectin containing RG-I domains with side chains; Preferably, the RG-I structural domain with side chains has the following composition:

[0013] Here, side chains represent side chains, and the side chains are β-D-Gal p -(1→4)-β-D-Gal p -(1→or→6)-α-D-Gal p -(1→Composition; 3) Short chains of α-galactan.

[0014] According to an embodiment of the present invention, the Rehmannia polysaccharide is derived from processed Rehmannia root.

[0015] Part Two, the present invention provides a method for preparing the Rehmannia glutinosa polysaccharide described in any of the above claims, comprising the following steps: S1. Add water to Rehmannia glutinosa and extract to obtain Rehmannia glutinosa aqueous extract; S2. Ethyl acetate is added to the aqueous extract of Rehmannia glutinosa obtained in step S1 for extraction, and the aqueous phase is collected. S3. Ethanol is added to the aqueous phase obtained in step S2 for alcohol precipitation, and the precipitate is collected to obtain crude polysaccharide of Rehmannia glutinosa. S4. The crude polysaccharide of Rehmannia glutinosa obtained in step S3 is mixed with hydrogen peroxide for decolorization to obtain decolorized crude polysaccharide; S5. The decolorized crude polysaccharide obtained in step S4 is purified sequentially using diethylaminoethyl cellulose (DEAE cellulose) and dextran gel (Sephadex) to obtain the Rehmannia glutinosa polysaccharide.

[0016] According to an embodiment of the present invention, in step S1, the ratio of prepared Rehmannia glutinosa to water is 1g:(10-20)mL, the extraction temperature is 90-100℃, the number of extractions is 2-3 times, and each extraction lasts 2-3 hours; for example, distilled water is added to prepared Rehmannia glutinosa at a ratio of 1g:20mL and boiled for 3.0 hours, and the extractions are combined after two extractions.

[0017] According to an embodiment of the present invention, in step S2, the volume ratio of the prepared Rehmannia glutinosa aqueous extract to the ethyl acetate is 1:(1-2), and the number of extractions is 2-4 times; for example, the prepared Rehmannia glutinosa aqueous extract is extracted with ethyl acetate at a volume ratio of 1:1 3 times.

[0018] According to an embodiment of the present invention, in step S3, the volume of ethanol is controlled to achieve a final concentration of 75% to 80%, such as 75%. Optionally, the method may further include a step of concentrating the aqueous phase under reduced pressure to one-third to one-half of the original solution, such as one-third, before the alcohol precipitation treatment. Specifically, the ethanol may be anhydrous ethanol, and the amount of ethanol added is based on the final concentration of ethanol in the solution, such as adding three times the volume of anhydrous ethanol. It is understood that the alcohol precipitation treatment step also includes a settling step, such as settling at 4°C for 8 to 12 hours. The precipitate can be further obtained by centrifugation, specifically at 3500 to 4500 r / min, such as centrifuging at 3500 rpm for 10 min. Further, after collecting the precipitate, the method may further include a step of dissolving the precipitate in water for dialysis, and then freeze-drying it after dialysis to obtain crude Rehmannia glutinosa polysaccharide. Specifically, the dialysis step includes: dialysis with running water in a 3500 Da dialysis bag for 48 hours, followed by dialysis with distilled water for 24 hours, and taking the solution from the bag.

[0019] According to an embodiment of the present invention, in step S4, the hydrogen peroxide is added in the form of a solution with a concentration of 120-180 mmol / L, such as 150 mmol / L. The ratio of the crude polysaccharide of Rehmannia glutinosa to the hydrogen peroxide solution is 1 mg:0.8-1.2 mL, such as 1 mg:1 mL. The decolorization conditions are as follows: pH 7.5-8.5, temperature 55-65℃, time 0.8-1.2 h, such as pH 8, temperature 60℃, time 1 h. Further, the method further includes the steps of dialysis and freeze-drying the decolorized system after decolorization. Specifically, the dialysis step includes: dialysis with running water in a 3500 Da dialysis bag for 48 hours, followed by dialysis with distilled water for 24 hours, and taking the solution from the bag.

[0020] According to an embodiment of the present invention, in step S5, the diethylaminoethyl cellulose purification step includes adding an aqueous solution (e.g., 10 mL) of the decolorized crude polysaccharide to a DEAE-52 anion exchange column, and eluting it sequentially with ultrapure water, and 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions at a rate of 0.5 mL / min and an elution time of 10 min / tube. Tubes 103-108 are collected, dialyzed, and then lyophilized. Specifically, the concentration of the aqueous solution of Rehmannia glutinosa crude polysaccharide can be 20 mg / mL. To further remove impurities, the method may further include filtering the aqueous solution of Rehmannia glutinosa crude polysaccharide using a 0.45 µm aqueous filter membrane before column chromatography.

[0021] According to an embodiment of the present invention, in step S5, the dextran gel purification step includes adding an aqueous solution (e.g., 10 mL) of the polysaccharide component purified by diethylaminoethyl cellulose to a Sephadex G-100 column, eluting with deionized water at a rate of 0.2 mL / min, collecting 5 mL per tube, collecting 14-30 tubes of polysaccharide components, dialyzing, and then lyophilizing. Specifically, the concentration of the aqueous solution of the polysaccharide component purified by diethylaminoethyl cellulose can be 5 mg / mL. To further remove impurities, the method may further include filtering the aqueous solution of the polysaccharide component purified by diethylaminoethyl cellulose using a 0.45 µm aqueous filter membrane before column chromatography.

[0022] Part Three, the present invention provides the use of any of the above-mentioned Rehmannia glutinosa polysaccharides, compositions containing said Rehmannia glutinosa polysaccharides, Rehmannia glutinosa polysaccharides prepared by any of the above-mentioned methods, or compositions containing Rehmannia glutinosa polysaccharides prepared by any of the above-mentioned methods in any of the following: A1) Prepare products that protect kidney function; A2) Prepare products for the prevention and / or treatment of kidney injury; A3) Prepare products for the prevention and / or treatment of nephrotoxicity; A4) Prepare products that protect renal tubular epithelial cells; A5) Preparation of antioxidant products; A6) To prepare products for the prevention and / or treatment of kidney damage caused by platinum compounds; A7) To prepare products for the prevention and / or treatment of nephrotoxicity caused by platinum compounds; A8) Prepare products that improve platinum-based compound-induced renal tubular epithelial cell damage; A9) Prepare products that enhance the cell viability or cell migration ability of renal tubular epithelial cells induced by platinum compounds; A10) Prepare products for the prevention and / or treatment of lipopolysaccharide-induced kidney damage; A11) Prepare products for the prevention and / or treatment of lipopolysaccharide-induced nephrotoxicity; A12) Prepare products that improve lipopolysaccharide-induced renal tubular epithelial cell damage; A13) Prepare products that enhance the cell viability or cell migration ability of lipopolysaccharide-induced renal tubular epithelial cells; A14) Prepare products for the prevention and / or treatment of oxidant-induced kidney damage; A15) Prepare products for the prevention and / or treatment of nephrotoxicity caused by oxidants; A16) Prepare products that improve oxidant-induced renal tubular epithelial cell damage; (A17) Prepare products that enhance the cell viability or cell migration ability of oxidant-induced renal tubular epithelial cells.

[0023] According to an embodiment of the present invention, the kidney injury is either acute kidney injury or chronic kidney injury; And / or, the platinum compound is cisplatin; And / or, the oxidant is hydrogen peroxide; And / or, the treatment includes reducing inflammatory cytokine levels, alleviating oxidative stress, and / or reducing mitochondrial dysfunction; And / or, the product is a drug or health food, or an intermediate used in the preparation of the drug or health food.

[0024] Part Four: This invention provides a product having the following functions, comprising Rehmannia polysaccharide as described in any one of the preceding claims or Rehmannia polysaccharide prepared by the method described in any one of the preceding claims; B1) Preparation to protect kidney function; B2) Prevention and / or treatment of kidney injury; B3) Prevention and / or treatment of nephrotoxicity; B4) Protects renal tubular epithelial cells; B5) Antioxidant; B6) Prevention and / or treatment of platinum-based kidney damage; B7) Prevention and / or treatment of platinum-based nephrotoxicity; B8) Improves platinum-based compound-induced renal tubular epithelial cell damage; B9) Enhances the cell viability or cell migration ability of renal tubular epithelial cells induced by platinum compounds; B10) Prevention and / or treatment of lipopolysaccharide-induced kidney damage; B11) Prevention and / or treatment of lipopolysaccharide-induced nephrotoxicity; B12) improves lipopolysaccharide-induced renal tubular epithelial cell damage; B13) Enhances the cell viability or cell migration ability of lipopolysaccharide-induced renal tubular epithelial cells; B14) Prevention and / or treatment of oxidative kidney damage; B15) Prevention and / or treatment of oxidant-induced nephrotoxicity; B16) improves oxidant-induced renal tubular epithelial cell damage; B17) enhances the cell viability or cell migration ability of oxidant-induced renal tubular epithelial cells.

[0025] According to an embodiment of the present invention, the product is a medicine or a health food; And / or, the product is a combination therapy for treating tumors, and also includes platinum compounds; And / or, the kidney injury is acute kidney injury or chronic kidney injury; And / or, the platinum compound is cisplatin; And / or, the oxidant is hydrogen peroxide; And / or, the treatment includes reducing inflammatory cytokine levels, alleviating oxidative stress, and / or reducing mitochondrial dysfunction.

[0026] The present invention has the following beneficial effects: (1) The material basis for high activity was clarified: For the first time, an active polysaccharide with a specific fine structure was successfully isolated and characterized from Rehmannia glutinosa. The structure of the polysaccharide was clearly defined (including its monosaccharide composition, glycosidic bond linkage mode, molecular weight range and spatial conformation, etc.), which laid a solid chemical foundation for subsequent quality control, structure-activity relationship studies and product development.

[0027] (2) Demonstrates significant improvement in cisplatin nephrotoxicity: In vitro and in vivo experiments showed that Rehmannia glutinosa polysaccharide (RRP) significantly enhanced cell viability and migration in a cisplatin-induced HK-2 cell injury model. It exerts a multi-pathway nephroprotective effect by enhancing antioxidant enzyme activity, reducing reactive oxygen species accumulation, improving mitochondrial energy metabolism, and inhibiting the release of pro-inflammatory factors. Furthermore, this polysaccharide is also effective against LPS and H2O2-induced cell damage, demonstrating broad-spectrum cytoprotective potential. In a cisplatin-induced acute kidney injury (Cis-AKI) animal model, this Rehmannia glutinosa polysaccharide effectively reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels, prevented cisplatin-induced weight loss and increased kidney / body mass index, and significantly improved the survival rate of experimental animals, confirming its clear nephroprotective effect.

[0028] (3) The advantage of this invention lies in the combination of "clear structure" and "definite efficacy": Compared with traditional crude extracts of Chinese medicine or polysaccharide components with unclear structures, this invention provides a single polysaccharide entity with clear structure and definite efficacy, effectively solving the key bottlenecks in existing research, such as unclear active ingredients, difficulty in quality control, and difficulty in elucidating the mechanism of action. It not only provides a candidate polysaccharide drug with a clear structure that has clinical application potential, but also provides the possibility for in-depth analysis of its mechanism of action against cisplatin nephrotoxicity at the molecular level, which is of great value for developing novel and effective Cis-AKI prevention and treatment strategies. Attached Figure Description

[0029] Figure 1A In the decolorization effect determination of Example 1 of the present invention, the cell viability of HK-2 cells was measured after 24 hours of exposure to culture media containing different concentrations of polysaccharides, both decolorized and undecolorized.

[0030] Figure 1B In the decolorization effect determination of Example 1 of the present invention, the cell viability of HK-2 cells exposed to culture media containing different concentrations of cisplatin for 24 hours was measured.

[0031] Figure 1C In the decolorization effect determination of Example 1 of the present invention, the cell viability of HK-2 cells was measured after 24 h of exposure in a culture medium containing 5 µM cisplatin and different concentrations of undecolorized polysaccharide (RRP-crude sugar).

[0032] Figure 1D In the decolorization effect determination of Example 1 of the present invention, the cell viability of HK-2 cells was measured after 24 h of exposure in a culture medium containing 5 µM cisplatin and different concentrations of decolorizing polysaccharide (RRP-decolorization).

[0033] Figure 2 The absorbance values ​​at 490 nm and the corresponding sodium chloride concentrations of the Rehmannia glutinosa polysaccharide solutions with different numbers of tubes obtained by column chromatography elution in Example 1 of this invention.

[0034] Figure 3 The absorbance of pure sugar RRP3 in different numbers of tubes after purification with Sephadex G-100 in Example 1 of this invention is measured at 490 nm.

[0035] Figure 4A The cell viability of HK-2 cells in Example 1 of this invention after being exposed for 24 hours in a culture medium containing 5 µM cisplatin and different concentrations of Rehmannia glutinosa polysaccharide RRP-0.2 purified and separated by DEAE-52 fiber column.

[0036] Figure 4BThe cell viability of HK-2 cells in Example 1 of this invention after being exposed for 24 hours in a culture medium containing 5 µM cisplatin and different concentrations of Rehmannia glutinosa polysaccharide RRP-0.3 purified and separated by DEAE-52 fiber column.

[0037] Figure 4C The cell viability of HK-2 cells in Example 1 of this invention after being exposed for 24 hours in a medium containing 5 µM cisplatin and different concentrations of Rehmannia glutinosa polysaccharide RRP-0.4 purified and separated by DEAE-52 fiber column.

[0038] Figure 5A The figure represents the hydroxyl radical scavenging rate of RRP-0.3 in Rehmannia glutinosa polysaccharide after purification and separation by DEAE-52 fiber column in Example 1 of this invention at different concentrations.

[0039] Figure 5B The figure represents the DPPH free radical scavenging rate of RRP-0.3 in Rehmannia glutinosa polysaccharide after purification and separation by DEAE-52 fiber column in Example 1 of this invention at different concentrations.

[0040] Figure 5C The ABTS at different concentrations of RRP-0.3 in Rehmannia glutinosa polysaccharide purified and separated by DEAE-52 fiber column in Example 1 of this invention. + Free radical scavenging rate.

[0041] Figure 5D The reducing power of RRP-0.3 in Rehmannia glutinosa polysaccharide purified and separated by DEAE-52 fiber column in Example 1 of this invention at different concentrations.

[0042] Figure 6A The cell viability of HK-2 cells in Example 1 of this invention after being exposed to culture medium containing different concentrations of pure sugar RRP3 for 24 hours is shown.

[0043] Figure 6B The cell viability of HK-2 cells in Example 1 of this invention after being exposed to a culture medium containing 5 µM cisplatin and different concentrations of pure sugar RRP3 for 24 h is shown.

[0044] Figure 7 This is a chromatogram of the composition determination of pure sugar RRP3 monosaccharide in Example 1 of the present invention.

[0045] Figure 8 is a scanning electron microscope image of pure sugar RRP3 in Example 1 of the present invention, with magnifications of 200 (A), 500 (B) and 1000 (C).

[0046] Figure 9 This is the infrared spectrum of pure sugar RRP3 in Example 1 of the present invention.

[0047] Figure 10This is the thermal analysis curve of pure sugar RRP3 in Example 1 of the present invention.

[0048] Figure 11 The results of cell scratch experiments in different experimental groups in Example 2 of this invention are shown.

[0049] Figure 12 The oxidative stress levels of HK-2 cells in different experimental groups in Example 2 of this invention are shown.

[0050] Figure 13 The ATP content of different experimental groups in Example 2 of the present invention.

[0051] Figure 14 shows the levels of pro-inflammatory cytokines in different experimental groups in Example 2 of the present invention: A-TNF-α; B-IL-6; C-IL-1β.

[0052] Figure 15 The intracellular ROS levels in different experimental groups in Example 2 of this invention.

[0053] Figure 16 These are JC-1 stained fluorescence images from different experimental groups in Example 2 of this invention.

[0054] Figure 17 The cell viability of HK-2 cells in Example 3 of this invention after being exposed to culture media containing different concentrations of LPS and H2O2 for 24 hours is shown.

[0055] Figure 18 The cell viability of HK-2 cells in Example 3 of this invention after being exposed to culture media containing different concentrations of RRP3 and LPS or H2O2 for 24 hours is shown.

[0056] Figure 19A The intracellular SOD levels in different experimental groups in Example 3 of this invention are shown.

[0057] Figure 19B The intracellular MDA levels in different experimental groups in Example 3 of this invention are shown.

[0058] Figure 19C The intracellular GSH levels in different experimental groups in Example 3 of this invention are shown.

[0059] Figure 20 The ATP content in the cells of different experimental groups in Example 3 of this invention is shown.

[0060] Figure 21 shows the levels of pro-inflammatory cytokines in different experimental groups in Example 3 of the present invention: A-TNF-α; B-IL-6; C-IL-1β.

[0061] Figure 22 The relative body weights of animals in different experimental groups in Example 4 of this invention.

[0062] Figure 23 Images of animal kidneys from different experimental groups in Example 4 of this invention.

[0063] Figure 24 These are the kidney organ indices of animals in different experimental groups in Example 4 of the present invention.

[0064] Figure 22 and 24 In the experimental group, relative body weight and kidney organ index during the experimental period were significantly different (p < 0.001) compared with the control group. p < 0.05, compared with the model group; p < 0.01, compared with the model group; p < 0.001, compared with the model group.

[0065] Figure 25 shows the blood creatinine (A) and urea nitrogen (B) levels of animals in different experimental groups in Example 4 of the present invention. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0068] Example 1: Preparation of Rehmannia glutinosa polysaccharides I. Preparation Method Prepare Rehmannia polysaccharide according to the following steps: (1) Preparation of crude polysaccharide from Rehmannia glutinosa Extract by boiling in 20 mL of distilled water at a ratio of 1 g to 20 mL for 3.0 h, repeating the extraction twice. Combine the extracts and extract three times with ethyl acetate at a 1:1 ratio. Concentrate the aqueous phase under reduced pressure to one-third of its volume, then slowly add three times the volume of anhydrous ethanol (final ethanol concentration 75%). Incubate overnight at 4°C, centrifuge at 3500 rpm for 10 min, redissolve the precipitate in distilled water, dialyze through a 3500 Da dialysis bag for 48 hours, then dialyze through distilled water for 24 hours. Freeze-dry the solution from the dialysis bag to obtain crude polysaccharide from Rehmannia glutinosa.

[0069] (2) Decolorization The crude polysaccharide of Rehmannia glutinosa was dissolved in 150 mmol / L H2O2 solution at a ratio of 1:1 (M / V, mg / mL). The pH was adjusted to 8 with NaOH, and the solution was decolorized in a water bath at 60℃ for 1 hour. Then, it was dialyzed with running water in a 3500 Da dialysis bag for 48 hours, followed by dialyzed with distilled water for 24 hours. The solution in the bag was collected and finally freeze-dried to obtain the decolorized crude polysaccharide.

[0070] Decolorization effect determination: Using glucose as a standard, the total carbohydrate content in the sample was determined using the phenol-sulfuric acid method; using bovine serum albumin as a standard, the protein content was determined using the Coomassie brilliant blue method; the absorbance of the solution before and after polysaccharide decolorization was measured at 450 nm, and the decolorization rate was calculated using the polysaccharide before decolorization as the standard; polysaccharide retention rate = sugar content × yield; yield = M H2O2脱色多糖 / M 未脱色多糖 The experimental results are shown in Table 1.

[0071] Table 1. Evaluation Results of Decolorization Effect

[0072] Effect of destaining on cell viability: HK-2 (human renal tubular epithelial cells) were cultured in Gibco DMEM / F-12 (1:1) (L-glutamine, 15 mM HEPES) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (15, 140, 122; Gibco) in a humid environment of 37°C and 5% CO2. In the experiment, HK-2 cells were exposed for 24 h to media containing polysaccharides (20, 40, 80, 160, 320, 640 µg / mL) and cisplatin (2.5, 5, 10, 20, 30, 40, 50 µM) (the medium with 0% cisplatin was the control group). Cells were then collected, and cell viability was assessed. The experimental results are shown in Figure 1.

[0073] As shown in Figure 1(A), the cell viability-promoting effects of both decolorized and undecolorized polysaccharides showed a trend of first increasing and then decreasing; and compared with undecolorized polysaccharides, the concentration of the decolorized polysaccharide with the strongest cell viability-promoting effect decreased from 320 μg / mL to 40 μg / mL. As shown in Figure 1(B), the cell viability of the 5 μM-50 μM cisplatin group was significantly lower than that of the control group, therefore 5 μM was selected as the modeling concentration of cisplatin.

[0074] HK-2 cells were exposed to a medium containing polysaccharides (0, 10, 20, 40, 80, 640 µg / mL before and after destaining) and cisplatin (5 µM) for 24 h. Cells were then collected and cell viability was assessed. The experimental results are shown in Figures 1(C)-(D).

[0075] As shown in Figures 1(C) and 1(D), the cell viability of the drug-treated group first increased and then decreased with the increase of polysaccharide concentration, and the optimal polysaccharide concentration was 20 μg / mL; decolorization further improved cell viability.

[0076] (3) Separation and purification Weigh 200 mg of crude Rehmannia glutinosa polysaccharide and dissolve it in 10 mL of ultrapure water. Centrifuge at 4000 r / min for 5 min. Filter the supernatant through a 0.45 µm aqueous filter membrane. Slowly add the polysaccharide solution to a pre-packed DEAE-52 anion exchange column. Elute sequentially with ultrapure water, and then with 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions at a rate of 0.5 mL / min for 10 min per column. The eluents from tubes 74-76, 103-108, and 133-135 were collected and combined. The eluents were dialyzed with running water in a 3500 Da dialysis bag for 48 hours, followed by dialyzed with distilled water for 24 hours. The eluents were then freeze-dried to obtain RRP-0.2, RRP-0.3, and RRP-0.4, respectively. The main polysaccharide component, RRP-0.3, was collected and prepared into a 5 mg / mL polysaccharide solution of 10 mL. After filtration through a 0.45 μm filter membrane, the solution was slowly added to a prepared Sephadex G-100 column and eluted with deionized water at a rate of 0.2 mL / min. 5 mL of the elution solution was collected from each tube. The polysaccharide components from tubes 14-30 were collected and dialyzed with running water in a 3500 Da dialysis bag for 48 hours, followed by dialyzed with distilled water for 24 hours. The eluents were then freeze-dried to obtain pure Rehmannia glutinosa sugar. In this study, α-glucose was used as a standard, and the sugar content of the eluent was determined using the phenol-sulfuric acid method. Elution curves were then plotted. Figure 2 Tubes 103-108 contained the main polysaccharide components, therefore they were combined and freeze-dried for further purification. Figure 3 As shown, the RRP pure sugar fraction in tube 14-30 after purification by Sephadex G-100 had the highest absorbance at 490 nm. Therefore, tubes 14-30 were combined as pure sugar and denoted as RRP3.

[0077] II. Assay of Rehmannia glutinosa polysaccharide cell viability and in vitro antioxidant activity (1) Cell culture and treatment The cell viability of Rehmannia glutinosa polysaccharides RRP-0.2, RRP-0.3, and RRP-0.4 purified and separated by DEAE-52 fiber column was detected to test their ameliorative effect on cisplatin-induced acute kidney injury.

[0078] The effect of RRP3 on improving cisplatin-induced acute kidney injury was tested by detecting the viability of RRP3 cells purified by dextran gel fiber column.

[0079] HK-2 (human renal tubular epithelial cells) were cultured in Gibco DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (15, 140, 122; Gibco) in a humid environment of 37°C and 5% CO2. In the experiments, HK-2 cells were exposed to a medium containing polysaccharides (20, 40, 80, 160 µg / mL) and cisplatin (5 µM) for 24 h. Cells were then collected, and cell viability was assessed.

[0080] (2) In vitro antioxidant activity assay The in vitro antioxidant activity of Rehmannia glutinosa polysaccharide RRP-0.3 was tested by measuring its reducing power, hydroxyl radical scavenging rate, DPPH radical scavenging activity, and ABTS+ radical scavenging activity after purification and separation by DEAE-52 fiber column.

[0081] Reducing power determination: 200 μL of sample solutions of different concentrations (0.05-10 mg / mL) were pipetted and added to phosphate buffer and potassium ferricyanide solution. The mixture was incubated in a water bath for 20 min and then cooled. Trichloroacetic acid and ferric chloride solution were then added and mixed thoroughly. Using vitamin C as a positive control, the absorbance at 700 nm was measured.

[0082] Hydroxyl radical scavenging rate determination: 50 μL of samples of different concentrations were mixed with an equal volume of FeSO4 and salicylic acid ethanol solution. After adding H2O2, the mixture was reacted at 37°C for 30 min. Using Vc as a control, the absorbance at 510 nm was measured, and the hydroxyl radical scavenging rate of the polysaccharide sample was calculated. The formula is as follows: Hydroxyl radical scavenging rate (%) = [1 (A X A X0 [) / A0]×100% (A0: absorbance excluding sample mixture, A) X : Absorbance of the sample solution group, A X0 (Absorbance of the mixture of solutions without H2O2).

[0083] DPPH free radical scavenging activity assay: Sample solutions of different concentrations were mixed with DPPH solution, and the absorbance at 517 nm was measured after the dark reaction, with vitamin C as a positive control. The DPPH free radical scavenging rate was calculated as follows: DPPH free radical scavenging rate (%) = [1 (A X A X0 [A0] × 100% (A0: absorbance excluding sample mixture, A0: absorbance) X : Absorbance of the sample solution group, A X0 (Absorbance of the mixture without DPPH solution).

[0084] ABTS + Free radical scavenging activity assay: ABTS reagent was mixed with K₂S₂O₈ solution and incubated in the dark for 12-16 h. The solution was then diluted with PBS to an absorbance of 0.70 ± 0.05. The sample was added and mixed with the ABTS solution, using VC as a positive control. ABTS was calculated using the formula. + Free radical scavenging rate. ABTS + Free radical scavenging rate (%) = [1 (A X A X0 [A0] × 100% (A0: absorbance of distilled water + ABTS mixture, Ax: absorbance of polysaccharide sample solution + ABTS mixture) x0 (Absorbance of polysaccharide sample solution + PBS buffer).

[0085] Experimental results are as follows Figure 4A As shown in Figure C, RRP-0.3 in Rehmannia glutinosa polysaccharide purified and separated by DEAE-52 fiber column showed the best effect in improving cisplatin-induced acute kidney injury in cells. Its in vitro antioxidant activity experiments indicated that it had the best scavenging effect on DPPH and ABTS+ free radicals, and this effect was dose-dependent within a certain range. Figure 5A -D). After purification by dextran gel fiber column, RRP-0.3 showed no toxic side effects in the range of 10-80 μg / mL. Figure 6A ), and it achieved the best improvement effect at 20 μg / mL ( Figure 6A -B).

[0086] III. Rehmannia glutinosa pure sugar structure (1) Molecular weight determination Accurately weigh 5 mg of sample, add 1 ml of 0.05 M NaCl solution to the sample to prepare a 5 mg / ml test sample solution, centrifuge at 8000 rpm for 10 min, collect the supernatant and filter it through a 0.22 μm microporous membrane, then transfer the sample to a 2 ml injection bottle for later use. Detection is performed using the HPGPC method with a high-performance gel permeation chromatography tandem column.

[0087] (2) Determination of monosaccharide composition Preparation of Standards: Weigh out 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and glucosamine galactose hydrochloride monosaccharides, and 10 mg of fucose. Dissolve them and bring the volume to 10 ml in a volumetric flask to prepare a standard stock solution. Then, dilute to the following serial dilutions, filter through a 0.22 μm microporous membrane, and transfer to sample vials.

[0088] Sample solution preparation: Take a clean chromatographic vial, accurately weigh 5 mg (±0.05 mg) of polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121 °C for 2 hours. Purge with nitrogen and dry. Add 3 mL of methanol to wash, then dry again, repeating the methanol washing 2-3 times. Dissolve in 1 mL of sterile water, transfer to a chromatographic vial for analysis.

[0089] PMP derivatization: Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolysate into a stoppered conical centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution, vortex to mix, and react in a 70 ℃ water bath for 1 h. After the reaction is complete, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, and take 0.3 mL and add water to make up to 1 mL.

[0090] Chromatographic method: Thermo U3000 high-performance liquid chromatography system, Agilent ZORBAX Eclipse XDB-C18 column (4.6). The column was 250 nm long and 5 μm wide. The mobile phase was acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, pH adjusted to 6.8 with 2 M NaOH) with isocratic elution. The volume ratio of acetonitrile to phosphate buffer was 17:83. The flow rate was 0.8 ml / min. The column temperature was 30 ºC. The detection wavelength was 250 nm. The injection volume was 10 μl.

[0091] (3) Scanning electron microscope: Take an appropriate amount of polysaccharide sample on conductive carbon tape, spray it with gold, and then place it in a scanning electron microscope for observation and photography. Set the working voltage to 10.0kV, observe the solid morphology of the sample at different magnifications, and adjust the clarity accordingly until the ideal field of view is obtained. Select an appropriate field of view to take pictures and record.

[0092] (4) Infrared spectroscopy: Weigh 1-2 mg of each dried polysaccharide sample into a mortar, add 200 mg of KBr powder, grind evenly, compress into tablets, and scan the samples using a Fourier transform infrared microscopy instrument with a wavelength range of 4000-400 cm⁻¹. -1 Record the infrared spectrum.

[0093] (5) Thermal analysis: Approximately 3 mg of polysaccharide sample was placed in an alumina crucible and then placed in a preheated thermogravimetric analyzer. The experiment was started. The programmed heating rate was 10 °C / min, and the scanning temperature range was 50–800 °C under static air atmosphere.

[0094] Experimental results are as follows Figure 7-10 As shown. Figure 7As shown, the mass percentages of the monosaccharide components in RRP are Rha:GlcA:GalA:Glc:Gal:Ara = 12.373:0.779:37.623:4.277:39.692:4.083, with a molecular weight of 5402 Da. Figure 8 shows the surface morphology of the sample at magnifications of 500, 1000, and 2000. As shown, under low magnification, the sample mainly exhibits a flaky or flaky distribution, with a smooth and tightly packed surface. Under high magnification, the tightly packed surface is still visible, with fine granular protrusions. Figure 9 Infrared spectroscopy analysis showed that the polysaccharide sample had a characteristic absorption peak at 3408.25 cm⁻¹. -1 A strong and broad absorption peak exists at 2932.51 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity spikes are absorption peaks of the CH stretching vibrations of methyl (-CH3) and methine (-CH2) groups; 1612.78 cm⁻¹ -1 The peak at 1417.21 cm⁻¹ is a characteristic absorption peak of the asymmetric stretching vibration of the free carboxyl group COO- in uronic acid, indicating that the polysaccharide sample is an acidic polysaccharide. -1， 1242.58 cm -1 The absorption peaks are due to the angular vibration of CH and the stretching vibration of CH, which together constitute the characteristic absorption of the sugar ring; 1147.04 cm⁻¹ -1 The stretching vibration of pyranose COC; 1076.77 cm⁻¹ -1 1027.50 cm -1 The presence of pyranoside was further confirmed at this location, indicating the bending vibration of the CO bond in the COC or COC structure; 918.28 cm -1 The peak represents the asymmetric ring stretching vibration of the pyran ring; 892.55 cm⁻¹ -1 This is a characteristic region of a β-pyranoside bond, suggesting that the polysaccharide contains β-pyranose; 764.78 cm -1 This displays the symmetrical ring stretching vibration peaks of the pyran ring. (Example:) Figure 10 As shown, the main changes in the sample occurred in three stages. In the first stage, from 30.14℃ to 230℃, the mass decreased by 10.08%, mainly due to the breaking of hydrogen bonds and the gradual loss of bound water. In the second stage, from approximately 230℃ to 700℃, the mass decreased by 54.06%, mainly due to the decomposition of polysaccharides and the disruption of chemical bonds. In the third stage, from 700℃ to 800℃, this can be attributed to the carbothermic decomposition of polysaccharides, with most of the sample transforming into ash and inorganic salts. The DTG curve shows a significant exothermic peak at 280℃ in the second stage.

[0095] (6) Methylation analysis Sample pretreatment: Weigh 5 mg of sample, dissolve in 1 ml of primary water, add 200 µl of 0.2 M MES, then add 500 µl of 200 mg / ml carbodiimide, and react at room temperature for 2 h. Add 1 ml of 2 M imidazole and 1 ml of 70 mg / ml NaBD4, and react for 3 h. Terminate the reaction with 300 μl of glacial acetic acid. Dialyze the sample for 48 h, and after dialysis, freeze-dry the sample for methylation. Dissolve 1 mg of the freeze-dried sample in 1 ml of DMSO. Add 30 mg of NaOH and incubate for 30 min. Add 250 μl of iodomethane solution, purge with nitrogen, and react in the dark for 1 h. Add another 250 μl of iodomethane solution and react for 1 h. Add 1 ml of water and 2 ml of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Pipette the lower dichloromethane phase and dry under nitrogen. Add 1 ml of 2M TFA and react at 121°C for 120 min. Dry under nitrogen at 30°C. Add 1 ml of freshly prepared 1 M NaBD4 (ammonia solution). Incubate with magnetic stirring at room temperature for 2.5 h. Terminate the reaction by adding 300 μL of acetic acid and dry under nitrogen. Dry twice with 2 ml of 5% (vol / vol) acetic acid in methanol at 40°C under nitrogen, then twice with 2 ml of methanol under nitrogen at 40°C. Add 1.5 mL of acetic anhydride, vortex to mix, and react at 100°C for 2.5 h. Add 2 ml of water and let stand for 10 min. Add 1 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat washing with water 3 times. Take the lower dichloromethane phase for analysis.

[0096] Chromatographic parameters: The chromatographic system used was an Agilent gas chromatograph (Agilent 7890A; Agilent Technologies, USA), with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The carrier gas was high-purity helium (purity not less than 99.999%), the flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, split injection was used, the split ratio was 10:1, and the solvent delay was 2.2 min.

[0097] Temperature program: 50℃ for 1.0 min, increase to 130℃ at 50℃ / min, increase to 230℃ at 3℃ / min, and hold for 2 min.

[0098] Mass spectrometry parameters: The mass spectrometry system used is an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source has an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode is full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0099] Table 2. Glycosidic bond content

[0100] (7) Nuclear magnetic resonance analysis The lyophilized sample was dissolved in 0.5 ml of D2O and measured using a 600 MHz Bruker NMR spectrometer. One-dimensional NMR (1H-NMR, 13C-NMR) and two-dimensional NMR (COSY, HSQC, HSQC-TOCSY, HMBC, NOESY) were performed. Calibration: HDO hydrogen δH = δ4.70 ppm, TMS carbon δC = δ0.00 ppm.

[0101] Table 3. Sugar residues in polysaccharide samples 1 H and 13 C chemical shift assignment

[0102] "--" indicates undetermined or not detected. Based on the monosaccharide composition, methylation results, and one-dimensional and two-dimensional NMR information analysis of the polysaccharide sample, it is indicated that the polysaccharide sample is a complex polysaccharide. It can be largely inferred that it is mainly composed of HG domains, pectin with side-chain RG-I domains, and a small amount of short α-galactan chains. In the HG domain, other residues are connected to the C-2 and C-3 hydroxyl groups by glycosidic bonds. The side chains of the RG-I domain are composed of →4)-β-D-Galp-(1→ or →6)-α-D-Galp-(1→). The possible structural elements of the polysaccharide are as follows:

[0103] Example 2, Cell Experiments and Mechanisms 1. Cell scratch assay Cells were distributed at a rate of 1 × 10⁶ cells per well. 6Cells were seeded at a density in 6-well plates and cultured for 24 hours. Then, the cells were scratched with a 200 μL pipette tip to ensure uniform damage. After washing three times with PBS to remove scratched cells, the cells were incubated for 24 hours with the prepared Rehmannia glutinosa pure saccharide RRP3 (20 μg / mL) and cisplatin (5 µM) culture medium. Untreated culture medium served as a blank control. Samples were collected, and the number of migrations of cells at the same location was recorded. Experimental results are as follows: Figure 11 As shown.

[0104] 2. Intracellular SOD and MDA levels To assess the oxidative stress level of HK-2 cells, commercial kits (Beyotime, Shanghai, China) were used to measure SOD and MDA levels. The specific procedures were as follows: HK-2 cells were homogenized in frozen phosphate-buffered saline (0.1 M, pH 7.4), the homogenate was filtered, and centrifuged at 4°C. SOD enzyme activity and lipid peroxidation levels were assessed by measuring the MDA content in the supernatant. SOD enzyme activity was expressed as activity units per milligram of protein, while MDA content was expressed as nanomoles per milligram of protein. Experimental results are shown below. Figure 12 As shown (Rehmannia glutinosa pure sugar RRP3 20μg / mL, cisplatin 5µM, no drug administration is blank).

[0105] 3. Measurement of intracellular ATP HK-2 cells were cultured in 6-well plates and then treated with a specific drug. ATP levels were determined using an ATP assay kit (Beyotime Biotechnology Co., Ltd., China). The procedure was performed according to the manufacturer's protocol. ATP values ​​were normalized to cellular protein concentration. Experimental results are shown below. Figure 13 As shown (Rehmannia glutinosa pure sugar RRP3 20μg / mL, cisplatin 5µM, no drug administration is blank).

[0106] 4. Cytokine assay Following the manufacturer's instructions, the levels of certain pro-inflammatory cytokines were assessed using an ELISA kit (BioLegend, San Diego, CA, USA). The results are shown in Figure 14 (Rehmannia glutinosa pure sugar RRP3 20 μg / mL, cisplatin 5 µM, no drug administration as blank).

[0107] 5. Intracellular ROS measurement Intracellular ROS levels were determined using a ROS detection kit (Beyotime Biotechnology, China) according to the manufacturer's protocol. Simply put, HK-2 cells were treated with the drug and incubated in serum-free medium containing DCFH-DA (10 μM) at 37°C for 20 min. Fluorescence intensity was assessed using a biphasic barrier system (Becton, Dickinson and Company, USA) with excitation at 488 nm and emission at 525 nm. Experimental results are as follows: Figure 15 As shown (Rehmannia glutinosa pure sugar RRP3 20μg / mL, cisplatin 5µM, no drug administration is blank).

[0108] 6. JC-1 staining Mitochondrial membrane potential was assessed using the JC-1 kit according to the manufacturer's instructions. Simply put, after treatment with the reagent, HK-2 cells were incubated in serum-free medium containing JC-1 in the dark at 37°C for 20 min. The plates were then examined using a fluorescence microscope, and images were randomly acquired. Experimental results are as follows: Figure 16 As shown (Rehmannia glutinosa pure sugar RRP3 20μg / mL, cisplatin 5µM, no drug administration is blank).

[0109] like Figure 11 Compared with the control group, the cell migration rate in the Cisplatin group was significantly decreased, and this trend was significantly improved after RRP3 administration. Figure 12 As shown, RRP3 reversed the increase in MDA levels induced by cisplatin in HK-2 cells. Simultaneously, RRP3 significantly increased superoxide dismutase (SOD) activity, indicating an opposite effect on RRP3-induced oxidative stress. Compared to the model group, RRP3 treatment significantly alleviated mitochondrial dysfunction in the kidneys, reflected in increased adenosine triphosphate (ATP) production. Figure 13 Cisplatin induced an increase in TNF-α, IL-6, and IL-1β, while RRP3 decreased these levels (Figure 14). Intracellular ROS were significantly generated in the cisplatin-treated group. ROS accumulation was eliminated after co-incubation with cisplatin and RRP3. Cells in the control and polysaccharide groups showed strong pale red fluorescence and relatively weak green fluorescence. After 24 h of cisplatin treatment, green fluorescence intensity increased while red fluorescence intensity decreased. However, with combined treatment of polysaccharide and cisplatin, red fluorescence gradually brightened while green fluorescence relatively weakened. Figure 15-16 These results indicate that polysaccharides have a mitigating effect on cisplatin-induced mitochondrial dysfunction.

[0110] Example 3: Constructing an Extended Model 1. Cell culture and processing HK-2 (human renal tubular epithelial cells) were cultured in Gibco DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (15, 140, 122; Gibco) at 37°C under humid conditions with 5% CO2. In the experiments, HK-2 cells were exposed for 24 h to medium containing pure polysaccharide RRP3 (20, 40, 80, 160 µg / mL) and LPS (10, 20, 30 µg / mL) or H2O2 (400, 800, 1600 µM). Cells were then collected, and cell viability was assessed.

[0111] HK-2 was used to screen for modeling concentrations of LPS and H2O2, which were 10 μg / mL and 400 μM, respectively. Figure 17 Subsequently, the optimal concentration of RRP3 in LPS- and H2O2-induced acute kidney injury was determined to be 40 μg / mL. Figure 18 ).

[0112] 2. Intracellular SOD and MDA levels To assess the oxidative stress level of HK-2 cells, commercial kits (Beyotime, Shanghai, China) were used to measure SOD and MDA levels. The specific procedures were as follows: HK-2 cells were homogenized in frozen phosphate-buffered saline (0.1 M, pH 7.4), the homogenate was filtered, and centrifuged at 4°C. SOD enzyme activity and lipid peroxidation levels were assessed by measuring the MDA content in the supernatant. SOD enzyme activity was expressed as activity units per milligram of protein, while MDA content was expressed as nanomoles per milligram of protein. Experimental results are shown below. Figure 19A -C (RRP3 40 μg / mL, LPS 10 μg / mL, H2O2 400 μM, with no drug administration as the blank).

[0113] like Figure 19A As shown in Figure C, RRP3 reversed the increase in MDA levels induced by LPS and H2O2 in HK-2 cells. At the same time, RRP3 significantly increased the activity of superoxide dismutase (SOD), indicating that it has the opposite effect on RRP oxidative stress.

[0114] 3. Measurement of intracellular ATP HK-2 cells were cultured in 6-well plates and then treated with a specific drug. ATP levels were determined using an ATP assay kit (Beyotime Biotechnology Co., Ltd., China). The procedure was performed according to the manufacturer's protocol. ATP values ​​were normalized to cellular protein concentration. Experimental results are shown below. Figure 20 The results are shown below (RRP3 40 μg / mL, LPS 10 μg / mL, H2O2 400 μM, with no drug administration as the blank).

[0115] like Figure 20 As shown, compared with the model group, RRP3 treatment significantly reduced mitochondrial dysfunction in the kidneys, which was reflected in increased production of adenosine triphosphate (ATP).

[0116] 4. Cytokine assay Following the manufacturer's instructions, the levels of certain pro-inflammatory cytokines were assessed using an ELISA kit (BioLegend, San Diego, CA, USA). The experimental results are shown in Figure 21 (RRP3 40 μg / mL, LPS 10 μg / mL, with no drug administration as a blank).

[0117] As shown in Figure 21, LPS induced an increase in TNF-α, IL-6, and IL-1β, while RRP3 decreased TNF-α, IL-6, and IL-1β.

[0118] Example 4, Animal Model 1. Animals Mice (male C57BL / 6, 6-8 weeks old, weighing 20-25g) were kept under a 12-hour light / dark cycle with free access to food and water, relative humidity of 60±10%, and 23±2℃ for one week for acclimatization.

[0119] 2. Research Design Mice were randomly divided into three groups (n = 8): (1) control group (normal mice), (2) cisplatin (20 mg / kg, ip), and (3) RRP3 + cisplatin (20 mg / kg, ip). Mice in each treatment group were orally administered RRP3 for 10 consecutive days. Normal mice were treated with saline. On day 7, the cisplatin group and all treatment groups received a single dose of cisplatin (20 mg / kg, ig) 30 min after administration to induce kidney injury. Three days after cisplatin injection, blood was collected by enucleation, and the mice were euthanized. Serum was separated by centrifugation at 4°C (2000 g, 15 min) and stored at -20°C. Kidneys were rapidly collected for subsequent measurements. Mouse body weight was recorded daily during the study period.

[0120] 3. Assess weight and kidney / body mass index. The mice were weighed before euthanasia; then, the renal system was surgically separated and weighed, and the renal organ index was measured as follows: kidney weight (mg) / body weight (g).

[0121] like Figure 22 As shown, RRP3 prevented weight loss in mice with cisplatin-induced acute kidney injury; Figure 23 As shown, the kidneys in the model group were yellowish-brown, while the kidneys in the RRP3 group were reddish-brown; Figure 24As shown, RRP3 prevented the increase in renal organ index in mice with cisplatin-induced acute kidney injury.

[0122] 4. Kidney function tests Serum BUN and Scr were measured using the BUN (CN: C013-2-1, Nanjing Jiancheng) and Scr detection kits (CN:AKNM008M, boxbio) as per the manufacturer's instructions.

[0123] As shown in Figure 25, cisplatin can upregulate serum BUN and Scr levels.

[0124] In summary, this study isolated and purified the polysaccharide RRP3 from Rehmannia glutinosa. In a cisplatin-induced human renal tubular epithelial cell (HK-2) injury model, this polysaccharide exhibited significant protective effects at concentrations of 20-40 μg / mL: it effectively enhanced cell viability and promoted cell migration and repair after injury; it strongly alleviated oxidative stress by enhancing superoxide dismutase (SOD) activity and reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels; simultaneously, it increased adenosine triphosphate (ATP) production, stabilized mitochondrial membrane potential, and improved mitochondrial function; and it significantly inhibited the release of key pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), exerting an anti-inflammatory effect. Furthermore, this polysaccharide also showed protective effects against lipopolysaccharide (LPS) and hydrogen peroxide (H2O2)-induced cell damage. Simultaneously, RRP3 prevented weight loss and increased renal organ indices in mice with cisplatin-induced acute kidney injury and significantly reduced serum BUN and Scr levels. These results indicate that Rehmannia glutinosa polysaccharide RRP provides a clear and effective candidate drug for the prevention and treatment of acute kidney injury induced by drugs such as cisplatin through synergistic effects via multiple pathways.

[0125] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A Rehmannia glutinosa polysaccharide, characterized in that, By weight percentage, it contains the following monosaccharides: Rhamnose (Rha) 10.265%–14.963%, glucuronic acid (GlcA) 0.514%–0.904%, galacturonic acid (GalA) 35.784%–40.596%, glucose (Glc) 3.128%–5.048%, galactose (Gal) 37.892%–41.438%, and arabinose (Ara) 3.869%–4.865%.

2. The Rehmannia glutinosa polysaccharide according to claim 1, characterized in that: The weight-average molecular weight of the Rehmannia glutinosa polysaccharide is 3000-10000 Da.

3. The Rehmannia polysaccharide according to any one of claims 1-2, characterized in that: Based on molar percentage, the Rehmannia glutinosa polysaccharide contains the following glycosidic bonds: t-Ara f 0.895%~0.997%, t-Xyl p 0.593%~0.673%,t-Rha p 0.886%~1.048%, 1,5-Ara f 0.968%~1.170%,1,2-Rha p 8.002%~9.062%,t-Glc p 0.624%~0.772%,t-Glc p A 0.787%~0.853%,t-Gal p 17.917%~18.845%,t-Gal p A 4.132%~4.584%,1,3,5-Ara f 0.713%~0.855%,1,2,4-Rha p 5.062%~6.120%,1,4-Gal p 2.108%~2.818%, 1,4-Gal p A33.894%~38.318%,1,4-Glc p 0.487%~0.559%,1,3-Gal p 1.785%~1.915%, 1,6-Glc p 0.827%~1.495%,1,6-Gal p 10.368%~12.656%,1,3,4-Gal p A 1.541%~1.695%, 1,2,4-Gal p A0.572%~0.764%, 1,4,6-Gal p A 0.709%~0.891%, 1,3,6-Gal p 0.485%~0.551%。 4. The Rehmannia polysaccharide according to any one of claims 1-3, characterized in that: The Rehmannia polysaccharide comprises at least one of the following: 1) Pectin containing HG domains; Preferably, the HG structural domain is composed as follows: Where R represents H or -COOCH3, and R1 represents α-D-Gal p A-(1→or α-D-△) 4，5 -Gal p A-(1→; More preferably, other residues are connected to the C-2 and / or C-3 hydroxyl groups in the HG domain via glycosidic bonds; 2) Pectin containing RG-I domains with side chains; Preferably, the RG-I structural domain with side chains has the following composition: Here, side chains represent side chains, and the side chains are β-D-Gal p -(1→4)-β-D-Galp-(1→or→6)-α-D-Galp-(1→composes of; 3) Short chains of α-galactan.

5. The method for preparing Rehmannia glutinosa polysaccharide according to any one of claims 1-4, comprising the following steps: S1. Add water to Rehmannia glutinosa and extract to obtain Rehmannia glutinosa aqueous extract; S2. Ethyl acetate is added to the aqueous extract of Rehmannia glutinosa obtained in step S1 for extraction, and the aqueous phase is collected. S3. Add ethanol to the aqueous phase obtained in step S2 for alcohol precipitation, collect the precipitate, and obtain crude polysaccharide of Rehmannia glutinosa. S4. The crude polysaccharide of Rehmannia glutinosa obtained in step S3 is mixed with hydrogen peroxide for decolorization to obtain decolorized crude polysaccharide; S5. The decolorized crude polysaccharide obtained in step S4 is purified sequentially using diethylaminoethyl cellulose and dextran gel to obtain the Rehmannia polysaccharide.

6. The preparation method according to claim 5, characterized in that: In step S1, the ratio of prepared Rehmannia root to water is 1g:(10-20)mL, the extraction temperature is 90-100℃, the extraction is performed 2-3 times, and each extraction lasts 2-3 hours. And / or, in step S2, the volume ratio of the prepared Rehmannia glutinosa aqueous extract to the ethyl acetate is 1:(1-2), and the extraction is performed 2-4 times; And / or, in step S3, the amount of ethanol used is controlled so that the final concentration of ethanol is 75% to 80%; And / or, in step S4, the hydrogen peroxide is added in the form of a solution with a concentration of 120-180 mmol / L, the ratio of the crude polysaccharide of Rehmannia glutinosa to the hydrogen peroxide solution is 1 mg: 0.8-1.2 mL, and the decolorization conditions are as follows: pH 7.5-8.5, temperature 55-65℃, and time 0.8-1.2 h; And / or, in step S5, the diethylaminoethyl cellulose purification step includes adding the aqueous solution of the decolorized crude polysaccharide to a DEAE-52 anion exchange column, and eluting it sequentially with ultrapure water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions at a rate of 0.5 mL / min and an elution time of 10 min / tube. Tubes 103-108 are collected, dialyzed, and then lyophilized. And / or, in step S5, the dextran gel purification step includes adding an aqueous solution of the polysaccharide component purified by diethylaminoethyl cellulose to a Sephadex G-100 column, eluting with deionized water at a rate of 0.2 mL / min, collecting 5 mL per tube, collecting 14-30 tubes of polysaccharide components, dialysis, and then lyophilizing.

7. The use of any one of the following: Rehmannia glutinosa polysaccharides according to claims 1-4, compositions comprising Rehmannia glutinosa polysaccharides according to claims 1-4, Rehmannia glutinosa polysaccharides prepared by the method according to any one of claims 5-7, or compositions comprising Rehmannia glutinosa polysaccharides prepared by the method according to any one of claims 5-7: A1) Prepare products that protect kidney function; A2) Prepare products for the prevention and / or treatment of kidney injury; A3) Prepare products for the prevention and / or treatment of nephrotoxicity; A4) Prepare products that protect renal tubular epithelial cells; A5) Preparation of antioxidant products; A6) To prepare products for the prevention and / or treatment of kidney damage caused by platinum compounds; A7) To prepare products for the prevention and / or treatment of nephrotoxicity caused by platinum compounds; A8) Prepare products that improve platinum-based compound-induced renal tubular epithelial cell damage; A9) Prepare products that enhance the cell viability or cell migration ability of renal tubular epithelial cells induced by platinum compounds; A10) Prepare products for the prevention and / or treatment of lipopolysaccharide-induced kidney damage; A11) Prepare products for the prevention and / or treatment of lipopolysaccharide-induced nephrotoxicity; A12) Prepare products that improve lipopolysaccharide-induced renal tubular epithelial cell damage; A13) Prepare products that enhance the cell viability or cell migration ability of lipopolysaccharide-induced renal tubular epithelial cells; A14) Prepare products for the prevention and / or treatment of oxidant-induced kidney damage; A15) Prepare products for the prevention and / or treatment of nephrotoxicity caused by oxidants; A16) Prepare products that improve oxidant-induced renal tubular epithelial cell damage; (A17) Prepare products that enhance the cell viability or cell migration ability of oxidant-induced renal tubular epithelial cells.

8. The application according to claim 7, characterized in that: The kidney injury is either acute kidney injury or chronic kidney injury; And / or, the platinum compound is cisplatin; And / or, the oxidant is hydrogen peroxide; And / or, the treatment includes reducing inflammatory cytokine levels, alleviating oxidative stress, and / or reducing mitochondrial dysfunction.

9. A product having the following functions, comprising Rehmannia glutinosa polysaccharide according to any one of claims 1-4 or Rehmannia glutinosa polysaccharide prepared by the method according to any one of claims 5-7; B1) Preparation to protect kidney function; B2) Prevention and / or treatment of kidney injury; B3) Prevention and / or treatment of nephrotoxicity; B4) Protects renal tubular epithelial cells; B5) Antioxidant; B6) Prevention and / or treatment of platinum-based kidney damage; B7) Prevention and / or treatment of platinum-based nephrotoxicity; B8) Improves platinum-based compound-induced renal tubular epithelial cell damage; B9) Enhances the cell viability or cell migration ability of renal tubular epithelial cells induced by platinum compounds; B10) Prevention and / or treatment of lipopolysaccharide-induced kidney damage; B11) Prevention and / or treatment of lipopolysaccharide-induced nephrotoxicity; B12) improves lipopolysaccharide-induced renal tubular epithelial cell damage; B13) Enhances the cell viability or cell migration ability of lipopolysaccharide-induced renal tubular epithelial cells; B14) Prevention and / or treatment of oxidative kidney damage; B15) Prevention and / or treatment of oxidant-induced nephrotoxicity; B16) improves oxidant-induced renal tubular epithelial cell damage; B17) enhances the cell viability or cell migration ability of oxidant-induced renal tubular epithelial cells.

10. The product according to claim 9, characterized in that: The product is a medicine or health food; And / or, the product is a combination therapy for treating tumors, and also includes platinum compounds; And / or, the kidney injury is acute kidney injury or chronic kidney injury; And / or, the platinum compound is cisplatin; And / or, the oxidant is hydrogen peroxide; And / or, the treatment includes reducing inflammatory cytokine levels, alleviating oxidative stress, and / or reducing mitochondrial dysfunction.

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