Application of total flavonoids of chrysanthemum morifolium and metabolites thereof in preparation of uric acid-lowering or liver and kidney protecting drugs or health products

Abstract

The application discloses application of total flavones of chrysanthemum morifolium ramat and metabolites thereof in preparation of uric acid-lowering or liver and kidney protecting drugs or health care products. The total flavones of chrysanthemum morifolium ramat contain various flavone compounds. In a hyperuricemia model mouse, the total flavones of chrysanthemum morifolium ramat can significantly reduce blood uric acid level, and has a significant protective effect on liver and kidney, and the effect is achieved by inhibiting the activity and expression of xanthine oxidase and adenosine deaminase in the liver, and regulating the expression of uric acid related transporters in the kidney. Meanwhile, metabolites of the total flavones of chrysanthemum morifolium ramat, luteolin-7-O-glucuronide and apigenin-7-O-glucuronide, can also play a pharmacological role in the mouse body. In vitro experiments and mouse primary hepatocytes further prove that luteolin-7-O-glucuronide and apigenin-7-O-glucuronide have an inhibiting effect on the activity and expression level of xanthine oxidase. The application widens the application range of traditional Chinese medicinal materials, and provides a new direction for development of efficient and low-toxic uric acid-lowering and anti-gout drugs or health care products.

Description

Technical Field

[0001] This invention pertains to pharmaceutical applications, primarily involving the use of total flavonoids from Hangzhou white chrysanthemum and their metabolites in the preparation of drugs or health products that lower uric acid or protect the liver and kidneys. Background Technology

[0002] With improved living standards and changes in dietary structure, the incidence of metabolic diseases in the population is on the rise. Among them, hyperuricemia (HUA) has received increasing attention. Hyperuricemia can be diagnosed when fasting serum uric acid levels exceed 420 μmol / L (approximately 7 mg / dL) in two separate tests on different days. Hyperuricemia may be caused by excessive uric acid production during purine metabolism or by impaired uric acid clearance from the body.

[0003] This trend is gradually affecting younger populations, with some patients with hyperuricemia and gout also experiencing obesity and hypertension, subsequently developing complications such as hyperlipidemia, chronic kidney disease, diabetes, cardiovascular and cerebrovascular diseases, and metabolic syndrome, severely impacting their quality of life. Hyperuricemia is also an early stage of gout and gouty arthritis, and most patients are unaware of their condition before experiencing symptoms. Therefore, strengthening prevention education and patient intervention measures is crucial. Currently, the main drug for treating hyperuricemia is allopurinol. For gout patients, first-line drugs include allopurinol, febuxostat, and benzbromarone. Allopurinol plays a key role in lowering uric acid levels by inhibiting xanthine oxidase (XO / XDH) in the uric acid production pathway; while benzbromarone mainly blocks uric acid transporter 1 (URAT1) in the kidneys, promoting uric acid excretion. However, these drugs are often accompanied by various adverse reactions, and most patients need to take medication for more than a year. Therefore, there is an urgent need to explore plant-derived alternatives with higher safety and better efficacy to treat hyperuricemia through a multi-pronged strategy.

[0004] According to the Chinese Pharmacopoeia, chrysanthemum is the dried capitulum of Chrysanthemum morifolium Ramat, a plant in the Asteraceae family. Medicinal materials are classified into "Haoju," "Chuju," "Gongju," "Hangju," and "Huaiju" based on their origin and processing methods. Hangbai chrysanthemum, as a historically significant edible plant, is widely used in the food and pharmaceutical fields globally, especially in China. Studies show that Hangbai chrysanthemum has a long history of alleviating various discomforts and diseases related to oxidative stress and metabolism, with its main active ingredient being flavonoids. However, no application of total flavonoids from Hangbai chrysanthemum in the prevention, improvement, and / or treatment of hyperuricemia has been found. Nevertheless, some studies indicate that after entering the body, through a series of metabolic absorption processes, the flavonoids in Hangbai chrysanthemum mainly exist in the form of glycosides (metabolites) rather than aglycones (prototypes) (CHEN Z, ZHENG S, LI L, et al. Metabolism of flavonoids in human: a comprehensive review[J]. Curr Drug Metab, 2014, 15(1):48-61.). Therefore, the potential pharmacological benefits of Hangzhou white chrysanthemum extract and the specific physiological effects of its components have become an important research area. Most studies focus only on the active molecules existing in the form of aglycones, and most are in vitro experiments; therefore, the exact mechanism of action of chrysanthemum extract remains unclear. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of total flavonoids of Hangzhou white chrysanthemum and its metabolites in the preparation of drugs or health products that lower uric acid or protect the liver and kidneys. The total flavonoids of Hangzhou white chrysanthemum and its metabolites all have anti-hyperuricemia activity and can exert liver and kidney protective effects.

[0006] This study validated the ameliorative effect of total flavonoids from Hangzhou white chrysanthemum on hyperuricemia in an animal model. Further, it demonstrated its ameliorative effect on purine metabolism disorders through multiple target pathways (uric acid production pathway: XO / XDH, ADA; uric acid excretion pathway: URAT1, ABCG2, OAT1, OAT3).

[0007] This study further explored the pharmacological effects of various metabolites of flavonoids, confirming that flavonoid metabolites (luteolin-7-O-glucuronide and apigenin-7-O-glucuronide) also have the effect of lowering uric acid levels by inhibiting xanthine oxidase and transport proteins. The in vivo metabolism of luteolin and apigenin from total flavonoids of Hangzhou white chrysanthemum to luteolin-7-O-glucuronide and apigenin-7-O-glucuronide, respectively, is as follows:

[0008]

[0009] This provides a novel and more practical dietary therapy for the treatment of hyperuricemia, taking into account its safety and efficacy. It also makes it possible to validate the therapeutic properties of the extract in vivo, providing new insights and guidance on how to utilize the extract to treat hyperuricemia.

[0010] Furthermore, in order to achieve the objectives of this invention, the following technical solution is adopted:

[0011] Preferably, the preparation method of the total flavonoids of Hangzhou white chrysanthemum includes the following steps: Extracting Hangzhou white chrysanthemum with 70% ethanol, typically three to five times, and then concentrating the extract at below 65°C to remove the solvent and obtain a concentrate. The concentrate is loaded onto a resin column until the column is completely saturated. After saturation, the resin is washed with deionized water to remove unbound substances, and then eluted with ethanol. The eluent is collected, concentrated to dryness, ground, and spray-dried to obtain the total flavonoids of Hangzhou white chrysanthemum, also known as the total flavonoid extract of Hangzhou white chrysanthemum, wherein the content of apigenin-7-O-glucoside is not less than 60 mg / g, the content of luteolin-7-O-glucoside is not less than 90 mg / g, the content of luteolin is not less than 5 mg / g, the content of apigenin is not less than 0.5 mg / g, and the content of chlorogenic acid is not less than 15 mg / g.

[0012] Preferably, the total flavonoids of Hangzhou white chrysanthemum are used in the preparation of drugs, health products and foods that lower plasma uric acid levels.

[0013] Preferably, the total flavonoids of Hangzhou white chrysanthemum are used in the preparation of drugs, health products and foods that inhibit xanthine oxidase and / or adenosine deaminase.

[0014] Preferably, the total flavonoids of Hangzhou white chrysanthemum are used in the preparation of drugs or health products that improve renal function damage caused by hyperuricemia.

[0015] Preferably, the total flavonoids of Hangzhou white chrysanthemum are used in the preparation of drugs or health products for repairing liver function damage caused by hyperuricemia.

[0016] Preferably, the application of luteolin-7-O-glucuronide, a metabolite of total flavonoids from Hangzhou white chrysanthemum, in the preparation of xanthine oxidase inhibitors, and the application of apigenin-7-O-glucuronide, a metabolite of total flavonoids from Hangzhou white chrysanthemum, in the preparation of xanthine oxidase inhibitors.

[0017] Preferably, the drug or health product is prepared from an effective amount of total flavonoids of Hangzhou white chrysanthemum, plus pharmaceutically acceptable excipients or auxiliary ingredients, and is in the form of a liquid preparation, solid preparation or semi-solid preparation, and is administered orally.

[0018] Preferably, for an adult weighing 60 kg, the adult dosage of the total flavonoids of Hangzhou white chrysanthemum is 0.3–1.2 g / person / day.

[0019] The advantages of this invention are:

[0020] (1) Currently, there is still no consensus on the first-line drug for hyperuricemia, and the drugs used clinically all have significant side effects. This invention has found that total flavonoids of Hangzhou white chrysanthemum have significant uric acid-lowering activity without significant side effects. As an edible plant that is both food and medicine, Hangzhou white chrysanthemum has definite curative effects, is safe and controllable, and has broad development potential. It can help expand the clinical application of drugs for hyperuricemia, improve the quality of life of patients, and play a beneficial role in the renal and liver function damage caused by uric acid.

[0021] (2) This invention has determined the main mechanism by which total flavonoids of Hangzhou white chrysanthemum exert their pharmacological effects. They can inhibit the activity and expression of xanthine oxidase in the body, regulate the expression of renal transporters, and the inhibitory effect is dose-dependent. This is a promising dietary aid for individuals with elevated uric acid levels.

[0022] (3) This invention identified and evaluated the pharmacological effects of the metabolites in mice after administration of total flavonoids of Hangzhou white chrysanthemum. In vitro experiments determined that luteolin-7-O-glucuronide and apigenin-7-O-glucuronide inhibited the expression and activity of xanthine oxidase, providing a pharmacological basis for the clinical application of flavonoid drugs. Attached Figure Description

[0023] Figure 1 HPLC chromatogram of total flavonoids in Hangzhou white chrysanthemum.

[0024] Figure 2 A schematic diagram of mouse plasma uric acid concentration in Example 1. Data in the figure are expressed as mean ± SEM, n = 8-10; compared with the control group (CON), **P < 0.01; compared with the model group (HUA), # P<0.05, ## P<0.01, ### P<0.001.

[0025] Figure 3 Example 1: Mouse plasma urea nitrogen (BUN) Figure 3 a) Creatinine (CRE) Figure 3 b) Concentration diagram. Data in the figure are expressed as mean ± SEM, n = 8; compared with the control group (CON), *P < 0.05, **P < 0.01; compared with the model group (HUA), # P<0.05, ## P<0.01, ### P<0.001.

[0026] Figure 4 Example 1: Mouse plasma alanine aminotransferase (ALT) Figure 4 a) Aspartate aminotransferase (AST) Figure 4 b) Total cholesterol in the liver (TC) Figure 4 c) Liver triglycerides (TG) Figure 4 d) Horizontal diagram. Data in the diagram are expressed as mean ± SEM, n = 8; compared with the blank group (CON), **P < 0.01, ***P < 0.001; compared with the model group (HUA), # P<0.05, ## P<0.01, ### P<0.001.

[0027] Figure 5 A representative diagram of xanthine oxidase (XOD) protein expression in mouse liver in Example 2. Figure 5 a) Representative diagram of adenosine deaminase protein expression ( Figure 5 b) and protein expression quantification statistics ( Figure 5 c), plasma activity results ( Figure 5 d, Figure 5 e) Schematic diagram. Data in the figure are expressed as mean ± SEM, n = 8; compared with the blank group (CON), *P < 0.05, **P < 0.01, ***P < 0.001; compared with the model group (HUA), # P<0.05, ## P<0.01, ### P<0.001.

[0028] Figure 6 Example 3: Mouse kidney ABCG2 ( Figure 6 a) and its protein expression quantitative statistical graph ( Figure 6 b), URAT1( Figure 6 c) and its protein expression quantitative statistical graph ( Figure 6 d). Data in the figure are expressed as mean ± SEM, n = 4–6; compared with the blank group (CON), *P < 0.05; compared with the model group (HUA), # P<0.05, ## P<0.01.

[0029] Figure 7 In Example 4, the XO / XDH ratio and protein content of primary mouse hepatocytes were measured. Figure 7 a) Schematic diagram of expression changes and statistical graph of protein expression quantification ( Figure 7 b). Data in the figure are expressed as mean ± SEM, n = 3; compared with the blank group (CON), ***P < 0.001; compared with the model group (HUA), ### P<0.001.

[0030] Figure 8 In Example 5, the XO / XDH protein in mouse primary liver cells treated with luteolin-7-O-glucuronide and apigenin-7-O-glucuronide was... Figure 8 a) Schematic diagram of expression changes and statistical graph of protein expression quantification ( Figure 8 b) Uric acid concentration ( Figure 8 c), Statistical graph of the inhibition rate of XO / XDH by in vitro experimental drugs ( Figure 8 d). Data in the figure are expressed as mean ± SEM, n = 6; compared with the blank group (CON), *P < 0.05, ***P < 0.001; compared with the model group (HUA), # P<0.05, ### P<0.001. Detailed Implementation

[0031] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0032] Example 1: Component analysis of total flavonoids in Hangzhou white chrysanthemum

[0033] 1.1 Preparation of solution

[0034] Using a 70% methanol solution, in triplicate, a 1 mg / mL solution of total flavonoids powder extracted from Hangzhou white chrysanthemum was prepared, sonicated for 30 min, diluted 100 times, and then injected.

[0035] 1.2 Chromatographic and Mass Spectrometry Conditions

[0036] Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QQUITY, Agilent Technologies, USA) was used with a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column. Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Flow rate: 0.3 mL / min; Gradient elution program: 0-5 minutes, 12%-18% B; 5-20 minutes, 18%-30% B; 20-25 minutes, 30%-50% B; 25-26 minutes, 50%-90% B; 26-27 minutes, 90% B; 27-27.5 minutes, 90%-12% B; 27.5-30 min, 12% B. Mass spectrometry: Electrospray ionization source, negative ion mode.

[0037] 1.3 Results Analysis

[0038] like Figure 1As shown, CME is mostly composed of flavonoid glycosides, with the highest content components being luteolin-7-O-glucoside (peak 7 in the figure) and apigenin-7-O-glucoside (peak 13 in the figure). The content of apigenin-7-O-glucoside is 63 mg / g, the content of luteolin-7-O-glucoside is 110 mg / g, the content of luteolin is 6 mg / g, the content of apigenin is 0.5 mg / g, and the content of chlorogenic acid is 20 mg / g.

[0039] Example 2: Uric acid-lowering effect of total flavonoids from Hangzhou white chrysanthemum

[0040] 2.1 Animal grouping and administration

[0041] Forty-eight male C57BL / 6 mice (18-22g) were housed under SPF conditions, with a temperature controlled at 23-25℃, humidity at 45%-55%, and a 12-hour light-dark cycle. Throughout the study, the mice had free access to food and water. After a 7-day acclimatization period, the 48 mice were randomly assigned to six groups: CON (normal control), HUA (hyperuricemia model), ALL (allopurinol), and CME (total flavonoids extract of Chrysanthemum morifolium), at doses of 50, 100, and 200 mg / kg / day, respectively. Except for CON, the mice in all other groups were administered PO (250 mg / kg / day) and hypoxanthine (250 mg / kg / day) solutions daily by gavage to induce the hyperuricemia model (all drugs were uniformly suspended in 0.5% CMC-Na carrier solution).

[0042] 2.2 Sample Collection

[0043] On day 14, two hours after the last administration, mice were anesthetized, and blood was collected from the orbital vein. The plasma was collected and centrifuged (3000 rpm, 10 min). After blood collection, the mice were euthanized, and the liver and kidney tissues were rapidly separated, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0044] 2.3 Determination of Biochemical Indicators

[0045] Serum uric acid (UA) concentration, serum creatinine (CRE), blood urea nitrogen (BUN) levels, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice were determined using liquid chromatography-mass spectrometry (LC-MS). Liver triglycerides (TG) and total cholesterol (TC) were determined according to kit methods. Results are shown below. Figure 2 , Figure 3 a, b, Figure 4 ad.

[0046] 2.4 Results Analysis

[0047] As shown in the figure, in the HUA model, treatment with total flavonoids from Hangzhou white chrysanthemum reduced plasma uric acid levels, thereby achieving a therapeutic effect on hyperuricemia. Compared with the CON group, the plasma uric acid level in the HUA group was significantly increased by 108% (P < 0.01). Notably, CME treatment reversed the observed trend; compared with the HUA group, plasma uric acid levels in the CME 50, CME 100, and CME 200 groups decreased by 42.8% (P < 0.05), 51.7% (P < 0.01), and 57.2% (P < 0.01), respectively. Figure 2 Allopurinol treatment reduced plasma uric acid levels in mice by 75% (P < 0.001). In conclusion, CME effectively reduces plasma uric acid concentration in a dose-dependent manner, thereby alleviating hyperuricemia in affected animals.

[0048] Excessive uric acid can cause renal dysfunction and impair renal filtration efficiency. CRE and BUN are important indicators for evaluating renal function. Elevated plasma CRE and BUN levels in HUA mice indicate renal impairment compared to the control group. All tested doses of CME (50, 100, and 200 mg / kg) effectively reduced CRE and BUN levels, with the highest dose (200 mg / kg) significantly improving biochemical indicators reflecting normal renal function, and significantly reducing CRE and BUN levels. Specifically, unlike allopurinol, CME reduces plasma CRE and BUN levels in a dose-dependent manner. Figure 3 a) and b) thus illustrate the dose-dependent nephroprotective effect.

[0049] Excess uric acid plays a detrimental role in hepatocellular damage, promoting the progression of non-alcoholic fatty liver disease (NAFLD) through multiple pathways, including insulin resistance, oxidative stress, and inflammation. ALT and AST are classic indicators for evaluating liver function. Compared to control mice, HUA mice showed increased liver volume, elevated liver-to-body weight ratio, significantly elevated serum liver injury markers (AST and ALT), and disrupted liver lipid levels (TC and TG). Increasing doses of CME effectively reversed these abnormalities, particularly at 200 mg / kg CME treatment, which significantly reduced plasma and liver tissue AST and ALT levels compared to untreated HUA mice. Figure 4 ad).

[0050] Example 3: Total flavonoids from Hangzhou white chrysanthemum exert their uric acid-lowering effect by inhibiting the function and expression of hepatic xanthine oxidase (XO / XDH) and adenosine deaminase (ADA).

[0051] 3.1 Total flavonoids from Hangzhou white chrysanthemum inhibit the activity of xanthine oxidase and adenosine deaminase.

[0052] The activities of xanthine oxidase and adenosine deaminase in mouse plasma were determined according to the xanthine oxidase kit and adenosine deaminase assay method. Results are shown below. Figure 5 .

[0053] 3.2 Total flavonoids from Hangzhou white chrysanthemum reduce xanthine oxidase expression.

[0054] Mouse liver proteins were extracted using RIPA, and changes in XO / XDH and ADA protein expression were measured by Western blotting. (See results below.) Figure 5 ae.

[0055] 3.3 Results Analysis

[0056] To further explore the molecular pathways by which CME lowers uric acid, this study examined key enzymes involved in purine catabolism. Compared to the CON group, the HUA group showed significantly upregulated XO / XDH protein expression and increased activity. This abnormal expression / activity was effectively counteracted by treatment with all tested doses of CME and allopurinol. Furthermore, compared to control mice, HUA mice showed significantly increased protein expression of ADA, a key enzyme in purine metabolism, in the liver. However, different doses of CME inhibited ADA protein expression and correspondingly reduced plasma ADA activity. Figure 5 (ae). In summary, the results suggest that total flavonoids from Hangzhou white chrysanthemum may exert their uric acid-lowering effect by simultaneously inhibiting the activity and expression of xanthine oxidase and adenosine deaminase.

[0057] Example 4: Total flavonoids from Hangzhou white chrysanthemum exert uric acid-lowering effects by regulating URAT1 and ABCG2 expression. 4.1 Total flavonoids from Hangzhou white chrysanthemum regulate the expression of URAT1 and ABCG2.

[0058] Proteins were extracted from mouse kidneys using RIPA, and the expression changes of URAT1 and ABCG2 proteins were measured by Western blotting. (See results below.) Figure 6 ad.

[0059] 4.2 Results Analysis

[0060] Uric acid metabolism largely depends on the functional kinetics of renal transporters, which coordinate renal and intestinal excretion pathways. Approximately two-thirds of total uric acid is cleared by the kidneys, while the remainder is excreted through the intestines. In the HUA group, this study observed a pattern of transporter expression dysregulation characterized by downregulation of ABCG2 expression. Figure 6 a, b), while URAT1 expression is upregulated ( Figure 6 c, d). Different doses of CME treatment, especially the maximum of 200 mg / kg, significantly improved and effectively restored the expression of transport proteins.

[0061] Example 5: Verification of the inhibitory effect of total flavonoids from Hangzhou white chrysanthemum on XO / XDH expression in primary mouse liver cells. 5.1 Isolation of primary liver cells.

[0062] Mice were anesthetized, their abdominal cavities were opened, and an indwelling needle was inserted into the inferior vena cava for perfusion with buffer and 0.05% collagenase. After completion, the liver was removed. After rinsing, the cells were placed in DMEM complete medium. The cell membrane was torn open with forceps, and the cells were shaken to disperse in the medium. The cells were filtered through a cell strainer and centrifuged, the supernatant was discarded, and the cells were resuspended in 10 ml of a 1:1 mixture of 90% Percoll and DMEM complete medium. After centrifugation, the supernatant of dead cells and culture medium was discarded, and the cells were resuspended in complete medium. Finally, the cells were incubated at 37°C and 5% CO2 at 2×10⁻⁶. 5 Seeds were produced at a density of cells / ml.

[0063] 5.2 Effects of total flavonoids from Hangzhou white chrysanthemum on protein expression of XO / XDH in primary liver cells

[0064] The total flavonoid extract of *Chrysanthemum indicum* was dissolved in ethanol. Primary liver cells were seeded in 24-well plates and cultured for 24 h. A hyperuricemia model was established, and *Chrysanthemum indicum* flavonoids were administered. The culture medium was discarded, and DMEM complete medium containing DMEM + ethanol, 2.5 mM adenosine (a uric acid precursor) + ethanol, 2.5 mM adenosine + 100 and 200 μg / mL of *Chrysanthemum indicum* flavonoids was added respectively. Cells were cultured for another 24 h, and xanthine oxidase 0.05 U / mL was added. Cell proteins were collected, and Western blotting was used to determine changes in XO / XDH protein expression. Results are shown below. Figure 7 a, b.

[0065] 5.3 Results Analysis

[0066] As shown in the figure, stimulation of cells with adenosine and xanthine oxidase resulted in a significant increase in xanthine oxidase levels in primary mouse liver cells. However, the addition of total flavonoids from Hangzhou white chrysanthemum significantly reduced xanthine oxidase protein levels in primary mouse liver cells. Figure 7 a) and b) thus concluded that the total flavonoids of Hangzhou white chrysanthemum have an inhibitory effect on the expression of XO / XDH in primary liver cells.

[0067] Example 6: Inhibitory effect of major metabolites in mouse plasma on XO / XDH expression and activity

[0068] 6.1 Determination of major metabolites in mouse plasma

[0069] The main metabolites present in mouse plasma after CME administration were detected using liquid chromatography-mass spectrometry (LC-MS).

[0070] 6.2 In vitro xanthine oxidase inhibition experiment

[0071] Xanthine is one of the precursors of uric acid and can be converted into uric acid under the action of xanthine oxidase. Therefore, in vitro, using xanthine as a substrate, xanthine oxidase and different concentrations of luteolin-7-O-glucuronide and apigenin-7-O-glucuronide (10, 50, 250 μM) were added, with allopurinol as a positive control. The inhibition rate of the drug against XO / XDH was calculated by measuring the concentration of the generated uric acid. The optimal XO / XDH reaction concentration was 20 mU / L, the system was phosphate buffer, the XO / XDH enzyme pre-incubation time was 15 min, and after precise co-incubation of the substrate and inhibitor for 5 min, the enzyme reaction was terminated with 4 volumes of ice-cold acetonitrile. After shaking and centrifugation, the supernatant was used for uric acid concentration determination. The in vitro XO / XDH inhibition rate results are shown below. Figure 8 d.

[0072] XO / XDH inhibition rate % = (Uric acid concentration at 5 min in the group without inhibitor - Uric acid concentration at 5 min in the group with inhibitor) / (Uric acid concentration at 5 min in the group without inhibitor - Uric acid concentration at 0 min in the group without inhibitor) * 100%

[0073] 6.3 Effects of flavonoid glucuronide on protein expression of XO / XDH in primary hepatocytes

[0074] Primary mouse liver cells were isolated as described in 4.2. Luteolin-7-O-glucuronide and apigenin-7-O-glucuronide were dissolved in DMSO. Primary liver cells were seeded in 24-well plates and cultured for 24 h. A hyperuricemia model was established, and the cells were then drugged. The culture medium was discarded, and DMEM complete medium containing DMSO, 2.5 mM adenosine (a uric acid precursor) + DMSO, 2.5 mM adenosine + 50 μM luteolin-7-O-glucuronide, and apigenin-7-O-glucuronide were added respectively. Cells were cultured for another 24 h, and xanthine oxidase 0.05 U / mL was added. Cells were collected, and protein was extracted. Western blotting was used to determine changes in XO / XDH protein expression. Results are shown below. Figure 8 a.

[0075] 6.4 Analysis

[0076] Previous studies have highlighted the extensive metabolism of flavonoids in CMEs in vivo; most flavonoids do not exist in vivo as aglycones but are primarily metabolized to glucuronides by glucuronidase. CMEs mainly consist of glucosides, which undergo a single-phase metabolism in mice to become aglycones, followed by a two-phase metabolism to forms such as glucuronidation, sulfation, or methylation. Considering the central role of flavonoid metabolites after CME administration, this study demonstrates the presence of these metabolites, particularly luteolin-7-O-glucuronic acid (L7GA) and apigenin-7-O-glucuronic acid (A7GA), which are present in high concentrations in plasma. Notably, LC-MS / MS analysis showed that the plasma glucuronide conjugates (0.5 h after administration of 200 mg / kg CME: luteolin-7-O-glucuronide, 23.9 μM; apigenin-7-O-glucuronide, 59.8 μM; luteolin and apigenin were almost undetectable) were consistent with previous findings. LC-MS / MS analysis of mouse plasma after CME administration showed that the concentration of flavonoid glucuronide metabolites was much higher than that of the flavonoid parent compounds.

[0077] Under hyperuricemic conditions, XO protein expression increased in mouse primary hepatocytes. Treatment with luteolin-7-O-glucuronide and apigenin-7-O-glucuronide effectively reduced XO / XDH protein expression. Figure 8 a, b), which lowers the uric acid level in the culture medium ( Figure 8 c). In vitro experiments also showed that luteolin-7-O-glucuronide and apigenin-7-O-glucuronide had inhibitory activity against XO / XDH, with inhibition rates of 62% and 51%, respectively, at a concentration of 250 μM. Figure 8 d). Therefore, the flavonoid metabolite glucuronide of CME can exert its pharmacological effect by inhibiting the expression and activity of XO / XDH in mice, thereby lowering uric acid levels.

Claims

1. Application of apigenin-7-O-glucuronide in the preparation of uric acid-lowering drugs.

Images