Eucommia ulmoides leaf extract and application thereof in prevention and treatment of adriamycin nephropathy

Abstract

The invention discloses a folium cortex eucommiae extract and application of the folium cortex eucommiae extract in prevention and treatment of adriamycin nephropathy, and relates to the technical field of medicines, the folium cortex eucommiae extract is prepared from dried folium cortex eucommiae through water extraction, heating at 65 DEG C, ultrasonic assistance and vacuum freeze drying, LC-MS / MS analysis shows that the folium cortex eucommiae extract contains no less than 200 components, the components mainly comprise flavonoids, terpenes and phenylpropanoids, and the content of the flavonoids, the terpenes and the phenylpropanoids is no less than 100%. The folium cortex eucommiae extract has no obvious cytotoxicity to HK-2 and HBZY-1 cells under the concentration of 0-1500mu g / mL, can be used for preparing medicines for preventing and treating adriamycin nephropathy, inhibits kidney cell apoptosis by down-regulating Bax and up-regulating Bcl-2 and obviously reduces the levels of IL-6, IL-1beta and TNF-alpha in serum, and in an animal model, 150-600mg / kg of the folium cortex eucommiae extract is continuously administrated for 21-30 days in an intragastric manner, so that the effect of preventing and treating adriamycin nephropathy is achieved. The traditional Chinese medicine composition can effectively relieve weight loss, reduce urine protein, serum creatinine and urea nitrogen, increase albumin and improve kidney pathological injury, and is suitable for relieving kidney toxicity in a prophylactic or therapeutic mode in subjects receiving adriamycin chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an extract of Eucommia ulmoides leaves and its application in the prevention and treatment of doxorubicin nephropathy. Background Technology

[0002] Doxorubicin nephropathy is a typical drug-induced chronic glomerular disease characterized by massive proteinuria, hypoalbuminemia, hyperlipidemia, and progressive renal function decline. Pathologically, it manifests as podocyte damage, glomerulosclerosis, and tubulointerstitial inflammatory infiltration. Its pathogenesis involves oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, and abnormal activation of apoptosis pathways. In recent years, research has increasingly focused on the application of natural products in the field of renal protection. For example, flavonoids, polyphenols, and saponins have been reported to have antioxidant, anti-inflammatory, and anti-apoptotic activities. Eucommia ulmoides, as a traditional Chinese medicine, has bark and leaves rich in bioactive components such as chlorogenic acid, genipin, and quercetin. Modern pharmacological studies have shown that Eucommia ulmoides extract has antihypertensive, anti-osteoporosis, and neuroprotective effects. However, its application in the prevention and treatment of doxorubicin-induced renal injury has not been systematically studied, especially lacking a complete technical solution based on a clear extraction process, mechanism of action, and animal model validation.

[0003] Currently, there are no specific effective drugs for treating doxorubicin nephropathy in clinical practice. Conventional interventions mainly rely on angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists to reduce proteinuria, but their blocking effect on fundamental cell damage is limited. Existing research on natural products mostly focuses on the in vitro effects of single components, which suffers from poor water solubility, low bioavailability, and insufficient in vivo stability. Research on crude extracts often fails to meet the requirements for new drug development due to non-standard preparation processes, unclear components, and lack of safety data. Current technologies generally lack integrated validation of multidimensional mechanisms of anti-apoptosis, anti-inflammation, and renal function improvement, and a complete pharmacodynamic evaluation system from cell models to animal models of chronic kidney disease has not been established. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides an extract of Eucommia ulmoides leaves and its application in the prevention and treatment of doxorubicin nephropathy. This addresses the problem that existing research on natural products often focuses on the in vitro effects of single components, resulting in poor water solubility, low bioavailability, and insufficient in vivo stability. Furthermore, research on crude extracts often suffers from non-standard preparation processes, unclear composition, and a lack of safety data, making it difficult to meet the requirements for new drug development. More importantly, existing technologies generally lack integrated verification of the multidimensional mechanisms of anti-apoptosis, anti-inflammation, and renal function improvement.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides an extract of Eucommia ulmoides leaves, obtained by the following steps:

[0008] After pulverizing the dried Eucommia ulmoides leaves, add distilled water at a mass-to-volume ratio of 1:10 and soak at room temperature for 24 hours.

[0009] The resulting mixture was heated in a water bath at 65°C for 60 minutes, followed by sonication for 30 minutes.

[0010] Filter the solution, collect the filtrate, and repeat the above extraction steps three times in total, then combine all the filtrates.

[0011] The combined filtrates were concentrated by rotary evaporation at 55°C to obtain a fluid extract.

[0012] The fluid extract was freeze-dried under vacuum to obtain a brownish-yellow powdered Eucommia ulmoides leaf extract.

[0013] As a preferred embodiment of the Eucommia ulmoides leaf extract of the present invention, wherein: the extract has no significant cytotoxicity to HK-2 human renal tubular epithelial cells or HBZY-1 rat glomerular mesangial cells in the concentration range of 0–1500 μg / mL.

[0014] Furthermore, the absence of obvious cytotoxicity means that, in the MTT assay, the cell viability is not less than 90% of the control group, and the cell morphology shows no obvious shrinkage, shedding, or fragmentation.

[0015] As a preferred embodiment of the Eucommia ulmoides leaf extract of the present invention, it is used in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy.

[0016] Furthermore, the term "medicine" here refers to a pharmaceutical preparation that uses the Eucommia ulmoides leaf extract as its active ingredient and is administered orally, by gavage, or by intravenous injection. Its dosage forms include, but are not limited to, granules, capsules, tablets, oral liquids, or lyophilized powder for injection.

[0017] As a preferred embodiment of the Eucommia ulmoides leaf extract of the present invention, the drug can reduce the levels of inflammatory factors IL-6, IL-1β and TNF-α in serum.

[0018] Furthermore, the reduction refers to the fact that, as detected by ELISA, the concentrations of IL-6, IL-1β, and TNF-α in the treated group animals were significantly lower than those in the model group (p<0.05), and this reduction showed a dose-dependent trend.

[0019] As a preferred embodiment of the pharmaceutical composition of the Eucommia ulmoides leaf extract of the present invention, the composition comprises a therapeutically effective amount of the Eucommia ulmoides leaf extract and pharmaceutically acceptable excipients.

[0020] Furthermore, the pharmaceutically acceptable excipients include fillers, disintegrants, lubricants, or stabilizers, the types and amounts of which comply with the relevant provisions of the Chinese Pharmacopoeia.

[0021] As a preferred embodiment of the pharmaceutical composition of Eucommia ulmoides leaf extract of the present invention, the pharmaceutical composition is administered by gavage, and the effective dose in animal models is 150 mg / kg, 300 mg / kg or 600 mg / kg.

[0022] Furthermore, the oral administration involves preparing a uniform suspension of Eucommia ulmoides leaf extract using physiological saline, with an administration volume of 10 mL / kg, once daily for 30 consecutive days.

[0023] In a second aspect, the present invention provides the use of Eucommia ulmoides leaf extract in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy, comprising:

[0024] As a preferred embodiment of the use of the Eucommia ulmoides leaf extract of the present invention in the preparation of a drug for the prevention and treatment of doxorubicin nephropathy, wherein: the doxorubicin-induced chronic kidney disease is characterized by urinary protein content >50mg / kg, elevated serum creatinine and urea nitrogen levels, decreased serum albumin, and interstitial inflammatory cell infiltration and hyaline casts in the renal tissue.

[0025] Furthermore, the urinary protein content was measured by collecting 24-hour urine in a metabolic cage using the Coomassie brilliant blue method, and the renal tissue pathological changes were observed by HE staining.

[0026] As a preferred embodiment of the use of the Eucommia ulmoides leaf extract of the present invention in the preparation of a drug for the prevention and treatment of doxorubicin nephropathy, wherein: the drug exerts a therapeutic effect by inhibiting renal cell apoptosis, specifically by downregulating the expression of the pro-apoptotic protein Bax and upregulating the expression of the anti-apoptotic protein Bcl-2, thereby reducing the apoptosis rate of HK-2 or HBZY-1 cells.

[0027] Furthermore, the expression levels of Bax and Bcl-2 were detected by Western blot, with β-actin as an internal control. The apoptosis rate was quantified by Annexin V-FITC / PI double staining combined with flow cytometry, and the sum of early and late apoptotic cells was used as the total apoptosis rate.

[0028] As a preferred embodiment of the use of the Eucommia ulmoides leaf extract of the present invention in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy, the present invention includes continuous administration for 21–30 days to alleviate weight loss, reduce proteinuria, and improve serum Scr, BUN, and ALB levels.

[0029] Furthermore, the continuous administration began on the day of doxorubicin modeling, with administration via gavage at a fixed time each day. Weight was recorded twice a week during this period. After the last administration, abdominal aortic blood and urine were collected after a 12-hour fast for indicator testing.

[0030] As a preferred embodiment of the use of the Eucommia ulmoides leaf extract of the present invention in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy, wherein the extract is administered in a preventive or therapeutic manner to subjects receiving doxorubicin chemotherapy to reduce their nephrotoxic side effects.

[0031] Furthermore, the prophylactic administration refers to the administration of Eucommia ulmoides leaf extract 1–3 days before the first dose of doxorubicin, and the therapeutic administration refers to intervention when proteinuria or renal dysfunction occurs after doxorubicin administration, both of which continue until the end of the chemotherapy cycle.

[0032] The beneficial effects of this invention are as follows: the preparation process of the Eucommia ulmoides leaf extract is stable, the components are clearly defined, and the safety is high. It can effectively prevent and treat doxorubicin-induced chronic kidney disease. It exerts a kidney-protective effect through a multi-target mechanism, including improving renal function, inhibiting apoptosis of renal tubular epithelial cells and mesangial cells, and reducing inflammatory response. The Eucommia ulmoides leaf extract can be made into oral or injectable preparations. In animal models, gavage administration can alleviate weight loss and renal pathological damage. It is also suitable for preventive intervention before doxorubicin chemotherapy or therapeutic application after chemotherapy, providing a natural, safe, and effective kidney protection strategy for clinical use. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram illustrating the establishment of the animal experimental model using Eucommia ulmoides leaf extract in Example 1.

[0035] Figure 2 This is a schematic diagram illustrating the detection of the effect of Eucommia ulmoides leaf extract on doxorubicin-induced apoptosis in Example 2.

[0036] Figure 3 This is a schematic diagram illustrating how Eucommia ulmoides leaf extract reduces kidney damage in Example 3. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] Example 1, referring to Figure 1 , Figure 2 and Figure 3 This is the first embodiment of the present invention, which provides a standardized preparation and component analysis of Eucommia ulmoides leaf extract. This embodiment aims to provide a reproducible, stable method for preparing Eucommia ulmoides leaf extract suitable for industrial production, and to conduct preliminary qualitative and quantitative analysis of its chemical components.

[0041] include:

[0042] 1.1 Raw Materials and Reagents

[0043] Dried Eucommia ulmoides leaves: Harvested from the authentic producing areas of Guizhou Province, identified by botanical taxonomists, with a moisture content of ≤8%, and crushed through a 20-mesh sieve;

[0044] Distilled water: Meets the purified water standards of the Chinese Pharmacopoeia;

[0045] Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-52AA);

[0046] Ultrasonic cleaning machine (Kunshan Ultrasonic Instruments Co., Ltd., KQ-500DE), frequency 40kHz;

[0047] Freeze dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd., LGJ-10);

[0048] LC-MS / MS system: ThermoFisher QExactive HF-X high-resolution mass spectrometry coupled with UHPLC.

[0049] 1.2 Extraction Process Steps

[0050] Soaking: Take 100g of dried Eucommia ulmoides leaf powder, add 1000mL of distilled water (solid-to-liquid ratio 1:10, w / v), and soak in a sealed container at room temperature (25±2℃) for 24 hours;

[0051] Heating extraction: Transfer the mixture to a 65°C constant temperature water bath and heat under reflux for 60 minutes;

[0052] Ultrasonic assistance: The device was then placed in an ultrasonic cleaner and ultrasonically treated for 30 minutes at 40kHz and 300W power.

[0053] Filtration and repeated extraction: Filter while hot (0.45μm filter membrane), collect the filtrate, and extract the residue twice more under the same conditions. Combine the three filtrates.

[0054] Concentration: The combined filtrates were rotary evaporated under reduced pressure at 55°C to a relative density of approximately 1.10 (measured at 60°C), yielding approximately 120 mL of a brownish-red fluid extract;

[0055] Freeze-drying: The fluid extract was pre-frozen to −80℃ and then freeze-dried under vacuum at −50℃ and 10Pa for 48 hours to obtain a brownish-yellow loose powder with a yield of about 20.5% (i.e. 20.5g / 100g raw material).

[0056] 1.3 Component Analysis

[0057] Untargeted metabolomics analysis of the extract was performed using UHPLC-Q Exactive HF-X high-resolution mass spectrometry.

[0058] The results showed that Eucommia ulmoides leaf extract contains a variety of chemical components, mainly including flavonoids, phenylpropanoids and iridoids, which are known to have antioxidant, anti-inflammatory and anti-apoptotic activities.

[0059] The above-mentioned components have good water solubility and stability, and most of them have been reported in the literature to have antioxidant, anti-inflammatory and anti-apoptotic activities, providing a material basis for subsequent pharmacodynamic studies.

[0060] Example 2 is the second embodiment of the present invention. This embodiment provides an evaluation of the in vitro safety and anti-renal cell apoptosis effect of Eucommia ulmoides leaf extract. In this embodiment, the safety of the extract on human renal tubular epithelial cells (HK-2) and rat glomerular mesangial cells (HBZY-1) was evaluated by the MTT assay, and its anti-apoptotic mechanism was investigated by using an doxorubicin-induced cell damage model.

[0061] include:

[0062] 2.1 Cell Culture

[0063] HK-2 cells were purchased from ATCC, and HBZY-1 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0064] Culture medium: DMEM / F12 + 10% FBS + 1% penicillin and streptomycin, cultured at 37℃ and 5% CO2.

[0065] 2.2 Cytotoxicity test (MTT assay)

[0066] Cells were seeded in 96-well plates (5×10³ cells / well). After 24 hours of cell adhesion, 0, 100, 300, 600, 1000, and 1500 μg / mL of Eucommia ulmoides leaf extract were added to each well, and the cells were cultured for another 48 hours. 20 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader.

[0067] Results: Within the concentration range of 0–1500 μg / mL, the survival rate of HK-2 and HBZY-1 cells was ≥92.5% (the control group was set at 100%), and no cell shrinkage, shedding or fragmentation was observed under the microscope, indicating that the Eucommia ulmoides leaf extract had no obvious cytotoxicity in this concentration range, which meets the characteristics described in claim 2.

[0068] 2.3 Doxorubicin-induced cell damage model and intervention

[0069] Model establishment: HK-2 cells were treated with 1 μM doxorubicin for 24 hours to simulate renal tubular epithelial injury;

[0070] Intervention group: Eucommia ulmoides leaf extract (final concentration 1000 μg / mL) was added during doxorubicin treatment.

[0071] Control group: Only an equal volume of PBS was added.

[0072] 2.4 Apoptosis Detection

[0073] Western Blot analysis of Bax / Bcl-2 protein expression:

[0074] Total protein was extracted and quantified using the BCA method;

[0075] SDS-PAGE electrophoresis, membrane transfer, incubation with primary antibodies (Bax, Bcl-2, β-actin) overnight at 4°C;

[0076] Secondary antibody incubation at room temperature for 1 hour, followed by ECL development.

[0077] Results: In the doxorubicin group, Bax expression was upregulated (p<0.01) and Bcl-2 was downregulated (p<0.01), while in the extract intervention group, Bax expression decreased by about 42%, Bcl-2 increased by about 58%, and the Bcl-2 / Bax ratio returned to near normal levels.

[0078] Annexin V-FITC / PI double staining flow cytometry:

[0079] Collect cells, wash with PBS, and resuspend in Binding Buffer;

[0080] Add Annexin V-FITC and PI, and stain for 15 minutes in the dark.

[0081] Apoptosis rate was detected by flow cytometry.

[0082] Results: The total apoptosis rate (early + late) in the doxorubicin group was 38.7±3.2%, while that in the extract intervention group it decreased to 16.4±2.1% (p<0.001), confirming that it inhibited renal cell apoptosis.

[0083] In summary, this embodiment verifies that Eucommia ulmoides leaf extract can effectively inhibit doxorubicin-induced renal cell apoptosis by regulating the Bax / Bcl-2 pathway at a safe concentration, supporting the use described in claim 5.

[0084] Example 3 is the third embodiment of the present invention. This embodiment provides a pharmacodynamic evaluation of Eucommia ulmoides leaf extract in a rat model of doxorubicin nephropathy. In this embodiment, a classic rat model of doxorubicin nephropathy is established, and the improvement effect of Eucommia ulmoides leaf extract on renal function, inflammatory factors and renal pathology is systematically evaluated.

[0085] include:

[0086] 3.1 Animals and Grouping

[0087] SPF-grade male SD rats, weighing 180–200g, were purchased from Zhiqinshuo (Chongqing) Biotechnology Co., Ltd.

[0088] After one week of acclimatization, the animals were randomly divided into 5 groups (n=10 / group):

[0089] Normal control group (Con): saline tail vein injection + saline gavage;

[0090] Model group (ADR): Doxorubicin via tail vein injection (3.5 mg / kg for the first time, 3.0 mg / kg for the second time) + CMC-Na by gavage;

[0091] Low-dose group (ADR+LE): Doxorubicin modeling + Eucommia ulmoides leaf extract 150mg / kg / d by gavage;

[0092] Medium-dose group (ADR+ME): Doxorubicin modeling + 300 mg / kg / day;

[0093] High-dose group (ADR+HE): Doxorubicin modeling + 600mg / kg / d.

[0094] 3.2 Dosing regimen

[0095] Doxorubicin was dissolved in normal saline and administered via tail vein twice: 3.5 mg / kg on day 1 and 3.0 mg / kg on day 14.

[0096] Starting from the day of modeling, the drug was administered by gavage at 9:00 AM every day. The extract was prepared into a suspension with physiological saline, and 2.5 mL was fixed for each rat. The required dosage was calculated based on body weight and dosage, and this was continued for 30 consecutive days.

[0097] Record your weight twice a week;

[0098] On day 30, the patient fasted for 12 hours, blood was drawn from the abdominal aorta, and urine was collected over 24 hours using a metabolic cage.

[0099] 3.3 Indicator Testing

[0100] Kidney function indicators:

[0101] Urine protein: determined using the Coomassie Brilliant Blue (CBB) method;

[0102] Serum creatinine (Scr): Detected using a sarcosine oxidase method kit;

[0103] Blood urea nitrogen (BUN): Detected using a urease method kit;

[0104] Serum albumin (ALB): Detected using a bromocresol green assay kit.

[0105] Inflammatory factors: Serum IL-6, IL-1β, and TNF-α were detected by ELISA.

[0106] Kidney pathology: The left kidney was fixed in 4% paraformaldehyde, paraffin sections were prepared, and hematoxylin and eosin (HE) stained. Microscopic observation showed that in the model group, there was tubular dilation, extensive interstitial inflammatory cell infiltration, and hyaline cast formation. In contrast, the high-dose treatment group showed a significant reduction in the above-mentioned pathological damage, and the glomerular structure remained basically intact.

[0107] Pathological results: In the model group, there was renal tubular dilation, extensive interstitial inflammatory cell infiltration, and hyaline cast formation; in the high-dose group, the pathological effect was reduced, and the glomerular structure remained basically intact.

[0108] 3.4 Conclusion

[0109] Eucommia ulmoides leaf extract, administered orally at doses of 150–600 mg / kg for 30 consecutive days, showed the following effects:

[0110] Relieves weight loss caused by doxorubicin;

[0111] Reduce urinary protein, serum creatinine (Scr), and urine protein (BUN);

[0112] Increase serum albumin;

[0113] Inhibits systemic inflammation (decreases IL-6, IL-1β, and TNF-α);

[0114] Improves pathological damage to kidney tissue.

[0115] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An extract of Eucommia ulmoides leaves, characterized in that: Obtained through the following steps: After pulverizing the dried Eucommia ulmoides leaves, add distilled water at a mass-to-volume ratio of 1:10 and soak at room temperature for 24 hours. The resulting mixture was heated in a water bath at 65°C for 60 minutes, followed by sonication for 30 minutes. Filter the solution, collect the filtrate, and repeat the extraction steps above three times. Combine all the filtrates. The combined filtrates were concentrated by rotary evaporation at 55°C to obtain a fluid extract. The fluid extract was freeze-dried under vacuum to obtain a brownish-yellow powdered Eucommia ulmoides leaf extract.

2. The Eucommia ulmoides leaf extract as described in claim 1, characterized in that: Within the concentration range of 0–1500 μg / mL, it showed no significant cytotoxicity to HK-2 human renal tubular epithelial cells or HBZY-1 rat glomerular mesangial cells.

3. Use of the Eucommia ulmoides leaf extract as described in claim 1 or 2 in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy.

4. The use as described in claim 3, characterized in that: The doxorubicin nephropathy model was established by tail vein injection of doxorubicin. The initial injection dose was 3.5 mg / kg, and an additional injection of 3.0 mg / kg was given on day 14. The model was considered successful when the 24-hour urinary protein content was greater than 50 mg / kg on day 21.

5. The use as described in claim 3 or 4, characterized in that: The drug exerts its therapeutic effect by inhibiting renal cell apoptosis, specifically by downregulating the expression of the pro-apoptotic protein Bax and upregulating the expression of the anti-apoptotic protein Bcl-2, thereby reducing the apoptosis rate of HK-2 or HBZY-1 cells.

6. The Eucommia ulmoides leaf extract as described in claim 1, characterized in that: The drug can reduce the levels of inflammatory factors IL-6, IL-1β and TNF-α in serum.

7. A pharmaceutical composition of Eucommia ulmoides leaf extract, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of Eucommia ulmoides leaf extract and physiological saline as a pharmaceutically acceptable solvent, and is an oral suspension.

8. The pharmaceutical composition according to claim 7, characterized in that: The oral suspension is administered by gavage, with a volume of 2.5 mL per rat. The rats are weighed every three days before administration, and the required extract mass is calculated based on the rat weight and the target dose of 150 mg / kg, 300 mg / kg, or 600 mg / kg. The suspension is then prepared with physiological saline to the corresponding concentration and administered once daily for 30 consecutive days.

9. The use of the Eucommia ulmoides leaf extract as described in claim 3 in the preparation of a medicament for the prevention and treatment of doxorubicin nephropathy, characterized in that: This includes continuous administration for 30 days to alleviate weight loss, reduce urinary protein excretion, and improve serum creatinine, blood urea nitrogen, and albumin levels.

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