Chemicals and materials
Eucommia folium was purchased from Huayu Agricultural Development Co., Ltd. (Yantai, Shandong Province, China). The Uric Acid (UA) Content Assay Kit, Urea Nitrogen (BUN) Content Assay Kit, Creatinine (Cr) Content Assay Kit, and Xanthine Oxidase (XOD) Activity Assay Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Potassium sulfonate (purity ≥ 99.0%) and hypoxanthine (purity ≥ 99.0%) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Nontargeted metabolomics detection-related reagents were obtained from Beijing Solarbio Technology Co., Ltd. Untargeted metabolomics detection-related reagents were obtained from the Shenzhen Weike Meng Technology Group Co., Ltd. (Shenzhen, China).
Preparation of DZ
The preparation process for DZ is as follows: ① Picking: The suitable time for picking Eucommia folium leaves is approximately June 20th. Leaves that are mature, have full flesh and a dark green colour are chosen. When picking, the petioles are cut off, stacked neatly, and processed on site. Do not store them for too long. ② Purification: After entering the workshop, Eucommia folium should first be cleaned with clean water to remove the dust attached to the surface of the leaves. The cleaning requires rinsing twice with running tap water, without rubbing, to prevent damage to the leaves. ③ Air drying: The cleaned Eucommia folium leaves were dried with a fan. ④ Cut the pages: The cleaned Eucommia folium leaves were placed evenly into a leaf cutter and were cut into thin 0.5 cm strips. ⑤ Finish filming: The Eucommia folium leaves were cut into strips and sent to a dryer for withering. The withering temperature should be controlled at approximately 510 degrees Celsius. After withering, the leaves should be immediately cooled to the ambient temperature in a tracked air cooler to prevent residual heat from ripening and affecting the withering effect. ⑥ Returning tide: After withering and cooling, the Eucommia folium leaves should be placed in an open tea basket and left to settle for 4 hours to naturally regain moisture. ⑦ Drying. The rehydrated Eucommia folium leaves were placed into a dryer for drying, with a drying temperature controlled of approximately 180 degrees and not too high. This process was repeated 1–2 times. ⑧ Titian: Place the Eucommia folium leaves that have been dried twice into a tea stir fry machine and stir fry until they are semicharred. It is advisable to stir fry until there is an aroma overflow. ⑨ Sealing: After stir frying the Eucommia folium leaves, they were cooled thoroughly and stored in a sealed bag. ⑩ Chen Hua: Eucommia folium was placed in breathable cotton bags, which were then placed in an ageing workshop for natural temperature and humidity ageing treatment. The humidity should not exceed 60%. When the humidity is too high, attention should be given to dehumidification to prevent mould growth. ⑪ Fermentation: The aged Eucommia folium leaves are placed into a fermentation chamber for fermentation. ⑫ Compression moulding: A cake press is used to press the fermented Eucommia folium leaves into the desired shape to obtain the DZ.
Preparation of the DZ water extract
A total of 100 g of DZ was added to 5 L of distilled water for reflux extraction, and the mixture was extracted for 1 hour each time; this process was repeated twice. After extraction, the water extracts were combined and filtered with a 200-mesh filter cloth. The water extract was concentrated to 100 mL using a rotary evaporator. The concentrated solution was freeze dried into a powder and weighed. Finally, 15.37 g of powder was obtained, which was the DZ water extract, with an extraction rate of 15.73%.
Animals and ethics statement
A total of 50 adult male C57BL/6 mice (4 weeks old) were used in this study. All animals were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Licence number: SCXK(jing)2019-0010. All the animals were kept in steel rodent cages, and the room temperature for the animals was controlled at 22 ± 2℃. The humidity was controlled at 60–80%, with a 12 h light:dark cycle (light period: 07:00–19:00; dark period: 19:00–07:00). The animal use protocol listed below was reviewed and approved by the Binzhou Medical University Institutional Animal Care and Use Committee (2023 − 198).
Mouse HUA model
A mouse HUA model was constructed by using a gavage of potassium oxonate (300 mg/kg) + hypoxanthine (500 mg/kg). Potassium oxonate was administered orally at a volume of 0.1 mL for 4 weeks, and a potassium oxonate solution was prepared by adding 3 g of potassium oxonate and 40 mL of 0.5% CMC-Na. Then, 0.1 mL of hypoxanthine was added by gavage for 4 weeks, and the hypoxanthine solution was prepared as follows: 5 g of hypoxanthine + 40 mL of 0.5% CMC-Na.
Design of the animal experimental groups
Fifty adult male C57BL/6 mice were randomly divided into 5 groups: the control group, HUA model group, 0.1 mg/kg DZ water extract group (DZ-L), 1 mg/kg DZ water extract group (DZ-M), and 10 mg/kg DZ water extract group (DZ-H). The treatment for each experimental group was as follows: in the control group, 0.25 mL of distilled water was orally administered for 4 weeks. For the HUA model group, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution was administered by gavage. After 1 week, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution + 0.05 mL of distilled water were added by gavage for 3 weeks. DZ-L group: gavage of 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution. After 1 week, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution + 0.1 mg/kg DZ water extract were gavaged for 3 weeks. In the DZ-M group, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution were gavaged. After 1 week, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution + 1 mg/kg DZ water extract were gavaged for 3 weeks. In the DZ-H group, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution were gavaged. After 1 week, 0.1 mL of potassium oxonate solution + 0.1 mL of hypoxanthine solution + 10 mg/kg DZ water extract were gavaged for 3 weeks.
Biochemical index detection
After the model was established, 1 mL of blood was taken from the mouse eyeball, left at room temperature for 2 h, centrifuged (3500 rpm) for 10 min, collected, packaged, and stored at -20℃ for future use. By strictly following the instructions of the UA, Cr, BUN, and XOD test kits, the UA, Cr, BUN, and XOD levels in the serum were determined.
Detecting pathological damage to the kidneys and glomeruli
After blood collection, the C57BL/6 mice were euthanized via the cervical dislocation method, and their livers were removed. The livers were washed with precooled physiological saline and dried with filter paper. The livers were then fixed with 4% neutral formaldehyde. HE-stained sections were used to observe pathological damage in renal tissue, whereas PAS-stained sections were used to observe pathological damage in glomeruli.
Untargeted serum metabolomics detection
Untargeted metabolomics analysis was performed on the serum of each group of mice using liquid chromatography‒mass spectrometry. After the model was established, 1 mL of blood was collected from the mouse eyeball, left at room temperature for 2 h, and centrifuged at 3500 rpm for 10 min, after which 150 µL of serum was collected. The serum was then sent to the Weike Meng Technology Company for untargeted metabolomics analysis.
Case collection
Patient source: Eighty patients with hyperuricaemia, who were admitted to the Affiliated Hospital of Binzhou Medical College from March 2023 to October 2023, including 40 males and 40 females aged between 40 and 65 years, were selected as the research group.
Patient selection criteria: These criteria were developed on the basis of the American College of Rheumatology (ACR) gout classification criteria of 1977 and the ACR/EULAR gout classification criteria of 2015.
The inclusion criteria for hyperuricaemia were as follows: (1) met the diagnostic criteria for hyperuricaemia, which means that the diagnostic criterion for hyperuricaemia was that the fasting blood uric acid levels were measured twice on different days, with levels > 420 µmol/L in males and levels > 360 µmol/L in females; (2) ages between 40 and 65 years, the sex of which is not limited; (3) acute onset, with a course of disease within 14 days; and (4) no gout symptoms have yet appeared. (5) All participants provided informed consent and signed an informed consent form.
The exclusion criteria were as follows: not taking diuretics or medications to lower uric acid levels within 3 months; acute or chronic infections; hypertension; coronary heart disease; malignant tumours; liver and kidney diseases; diabetes; blood system diseases; abnormal liver and kidney function or gout; and pregnancy or lactation.
The case dropout criteria were as follows: ① patients who experienced serious adverse events or complications and were not suitable for continued treatment; ② patients who had poor compliance; who used medication that did not reach 80% of the prescribed amount or exceeded 120% of the prescribed amount; ③ patients who voluntarily withdrew or were lost to follow-up during the treatment process; ④ patients who had not completed the entire course of treatment, which affected the efficacy or safety assessment; and ⑤ patients whose incomplete information affected the validity and safety of the judgement.
Patient dropout treatment: ① After the subject falls off, the researcher should try to contact the subject as much as possible, inquire about the reasons, and complete the evaluation items as much as possible. ② The dropout cases are all required for the set of statistics to provide a full analysis, and dropout patients do not need to be supplemented separately.
The termination test criteria were as follows: ① Those who experienced serious adverse events; ② during the course of the disease, the condition worsened, and ineffective cases were treated; ③ serious deviations occurred during the implementation of clinical trial protocols; and ④ the subjects requested to withdraw during the clinical trial process.
DZ efficacy testing
A total of 30 HUA volunteers, including 20 males and 10 females, were selected for this study. Volunteers drank DZ powder each day for 4 weeks, with a daily intake of 10 g. Weekly testing of serum UA levels in HUA volunteers was performed and serum UA levels were retested after discontinuing DZ for one month.
Safety evaluation criteria
According to the "Guidelines for Clinical Research of Traditional Chinese Medicine New Drugs (Trial)" published in 2002, the main focus of interest is the digestive disorders and abdominal pain of patients before and after treatment, as well as other adverse reactions, such as gastrointestinal discomfort, bloating, vomiting, diarrhoea, bloody stools, and black stools, and safety indicators, such as liver and kidney function. The degrees of symptoms were classified as follows: ① no adverse reactions; ② mild reactions were mild, short-lived, and tolerable; ③ moderate reactions were severe, stopped, and could be relieved without treatment; and ④ severe reactions, terminated, were symptomatic.
Statistical analysis
The data are presented as the means ± standard deviations from at least six independent experiments. Statistical differences were determined by using Student’s t-tests, where P < 0.05 was considered to indicate statistical significance. The analyses were performed via the Statistical Program for Social Sciences Software (International Business Machines Corporation, New York, USA).