Application of rosmarinic acid in preparation of medicine or health food for preventing and treating hyperuricemia of patients with renal insufficiency

Abstract

The invention discloses application of rosmarinic acid in preparation of a medicine or health food for preventing and treating hyperuricemia of a patient with renal insufficiency. The medicine for preventing and treating hyperuricemia of the patient with renal insufficiency is an XOD inhibitor or an ABCG2 gene expression promoter. The rosmarinic acid can significantly reduce the serum uric acid level of HUA mice induced by high purine diet, promote ABCG2-mediated intestinal uric acid excretion, and relieve HUA-induced intestinal inflammation and barrier injury by adjusting intestinal flora. The rosmarinic acid is safe and free of toxic and side effects, and has very important application prospects.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical preparation technology, and in particular to the application of rosmarinic acid in the preparation of drugs or health foods for the prevention and treatment of hyperuricemia in patients with renal insufficiency. Background Technology

[0002] Hyperuricemia (HUA) is a common metabolic disease caused by abnormally high levels of uric acid in the bloodstream due to impaired uric acid metabolism. Clinically, HUA is defined as a serum uric acid level greater than or equal to 360 μmol / L in women and ≥420 μmol / L in men. Uric acid is a purine metabolite. In the human body, exogenous purines are mainly synthesized in the liver, intestines, and vascular endothelium, while endogenous purines originate from damaged and dead cells, ultimately being converted into uric acid through the action of adenosine deaminase, purine nucleoside phosphorylase, and xanthine oxidase. Approximately two-thirds of uric acid is excreted through the kidneys, and one-third through the intestines. Excessive uric acid synthesis or insufficient excretion can cause HUA. Long-term HUA can lead to the deposition of urate crystals in the joints, eventually inducing gout. In addition, HUA is also a high-risk factor for kidney disease, cardiovascular disease, and metabolic syndrome.

[0003] Currently, the treatment of hyperuricosuric effects (HUA) in patients with renal insufficiency remains controversial. Studies have shown that clinical pharmacological interventions do not benefit all patients with renal insufficiency, especially when using drugs that rely on renal excretion. In severe renal insufficiency, uricosuric drugs such as benzbromarone have limited efficacy and increase the risk of kidney damage. The risk of hypersensitivity syndrome is significantly increased in patients with renal insufficiency when using allopurinol. When the glomerular filtration rate (eGFR) is <30 ml / min / 1.73 m³ / min... 2 The dosage of this drug should be halved. Febuxostat is more tolerated in these patients because it is primarily metabolized in the liver, but long-term use still carries side effects such as liver damage. For patients with renal insufficiency, intervention with this type of drug not only has reduced efficacy but can also further aggravate kidney damage. Therefore, it is necessary to seek effective, safe, and cost-effective methods to alleviate HUA.

[0004] Xanthine oxidase (XOD) is a key enzyme in uric acid synthesis. It catalyzes the conversion of hypoxanthine to xanthine, ultimately producing uric acid, which is then excreted from the body by urate transporters. Adenosine triphosphate-binding cassette transporter G2 (ABCG2), a member of the ABC transporter superfamily, is highly expressed in the proximal tubules of the kidney and intestinal villi, and is the main transporter responsible for uric acid excretion by the kidneys and intestines. ABCG2-mediated intestinal uric acid excretion plays a crucial role in maintaining uric acid homeostasis. Especially in cases of renal insufficiency, the intestine can excrete up to 60% of uric acid. Therefore, XOD and ABCG2 are considered key targets for alleviating hyperuricemia (HUA). Furthermore, the role of gut microbiota in uric acid metabolism is increasingly recognized. Multiple studies have confirmed a close link between gut microbiota dysbiosis and HUA or gout, with these patients exhibiting reduced beneficial bacteria and enrichment of opportunistic pathogens, as well as impaired intestinal barrier function. Maintaining gut homeostasis is of great significance for the prevention and treatment of HUA. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides the application of rosmarinic acid in the preparation of drugs or health foods for preventing and treating hyperuricemia in patients with renal insufficiency. Rosmarinic acid can significantly reduce serum uric acid levels in HUA mice induced by a high-purine diet, promote ABCG2-mediated intestinal uric acid excretion, and alleviate HUA-induced intestinal inflammation and barrier damage by regulating the intestinal flora. Rosmarinic acid is safe and has no toxic side effects, demonstrating significant application potential.

[0006] The technical solution of the present invention is as follows: The purpose of this invention is to provide the application of rosmarinic acid in the preparation of drugs or health foods for the prevention and treatment of hyperuricemia in patients with renal insufficiency.

[0007] In one embodiment of the present invention, the drug for preventing and treating hyperuricemia in patients with renal insufficiency is an XOD inhibitor or an ABCG2 gene expression promoter.

[0008] In one embodiment of the present invention, the drug for preventing and treating hyperuricemia in patients with renal insufficiency further contains a drug carrier and / or pharmaceutical excipients.

[0009] In one embodiment of the present invention, the pharmaceutical excipient is one or more of the following: filler, binder, wetting agent, disintegrant, lubricant, and flavoring agent.

[0010] In one embodiment of the present invention, the drug carrier is a microcapsule, microsphere, nanoparticle, or liposome.

[0011] In one embodiment of the present invention, the dosage form of the drug for preventing and treating hyperuricemia in patients with renal insufficiency is granules, capsules, tablets, pills or oral liquid.

[0012] In one embodiment of the present invention, the drug has at least one of the following effects: (1) Lower serum uric acid levels; (2) Inhibits serum and liver XOD activity; (3) Promotes the expression of ABCG2 in the gut; (4) Promotes the excretion of uric acid from the intestines; (5) Inhibits the secretion of pro-inflammatory cytokines in the intestine; (6) Promotes the secretion of anti-inflammatory cytokines in the intestine; (7) Upregulates the levels of tight junction proteins and mucins in the gut, reducing intestinal leakage; (8) Regulates intestinal flora and enhances intestinal barrier.

[0013] Beneficial effects: Rosmarinic acid has low membrane permeability, resulting in an oral bioavailability of only 1.57%, with most of it being utilized by gut microbiota. Rosmarinic acid possesses various biological activities, including anti-inflammatory, antioxidant, antiviral, and antibacterial properties. However, no studies have been reported on relieving hypersensitivity to rosmarinic acid through the intestinal route.

[0014] Rosmarinic acid significantly reduced serum uric acid levels in HUA mice induced by a high-purine diet. The average serum uric acid concentration in mice treated with rosmarinic acid was 26.2% lower than that in the model group.

[0015] Rosmarinic acid intervention reduced serum and liver XOD activity in mice by 29.6% and 27.3% respectively compared to the model group.

[0016] Rosmarinic acid promotes uric acid excretion in the mouse intestine. The mRNA level of ABCG2 in the mouse ileum was upregulated to 3.66 times that of the model group. Fecal uric acid content increased by 89.3% compared to the model group.

[0017] Rosmarinic acid alleviates HUA-induced intestinal inflammation by inhibiting the secretion of pro-inflammatory cytokines IL-1β, IL-18, TNF-α, and IL-6 in the gut and promoting the secretion of the anti-inflammatory cytokine IL-10.

[0018] Rosmarinic acid alleviates HUA-induced intestinal barrier damage by increasing the expression of tight junction proteins Occludin, ZO-1, and mucin2.

[0019] Rosmarinic acid regulates gut microbiota and increases Dubosiella , Turicibacter and Bifidobacterium The relative abundance of opportunistic pathogens is reduced. Bacteroides The relative abundance of [something] promotes the maintenance of intestinal homeostasis.

[0020] In summary, drugs and health products derived from rosmarinic acid can prevent and treat kidney failure (HUA) in patients with renal insufficiency from various angles, and are safe and free of toxic side effects, thus having very important application prospects. Attached Figure Description

[0021] Figure 1 The effect of rosmarinic acid on serum uric acid levels in mice.

[0022] Figure 2 The effect of rosmarinic acid on XOD activity in mice. (A) Serum XOD activity; (B) Liver XOD activity.

[0023] Figure 3 The effect of rosmarinic acid on uric acid excretion in mice. (A) Urinary uric acid level; (B) Fecal uric acid level.

[0024] Figure 4 The mRNA transcription level of uric acid secretion protein ABCG2 in the ileum of mice in each group was detected by RT-qPCR.

[0025] Figure 5 This represents the cytokine levels in the ileum.

[0026] Figure 6 Histopathological staining of the ileum and colon.

[0027] Figure 7 The mRNA transcription levels of the ileal tight junction proteins Occludin, ZO-1, and the mucin 2 were measured.

[0028] Figure 8 To assess intestinal permeability by detecting serum concentrations of fluorescein isothiocyanate-dextran (FITC-dextran) in mice after oral administration.

[0029] Figure 9 To analyze the regulatory effect of rosmarinic acid on the intestinal flora of mice based on 16S rRNA gene sequencing.

[0030] In the above figure, experimental data are expressed as mean ± standard error (mean ± SEM); those marked with , , Both represent statistical differences between the two groups, respectively indicating p <0.05、 p <0.01、 p <0.001. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following experiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0032] Example 1: Rosmarinic acid reduces uric acid levels in HUA mice by promoting intestinal uric acid excretion. The specific steps are as follows: (1) Six-week-old male ICR mice were used as experimental subjects. Feeding environment: temperature 22-26℃, humidity 40%-60%, and the animal room environment was based on a 12h light and 12h dark cycle.

[0033] (2) After one week of adaptation to the environment, the mice were randomly divided into five groups: blank group, model group, benzbromarone group, low-dose rosmarinic acid group and high-dose rosmarinic acid group. All drugs were prepared into suspensions using sodium carboxymethyl cellulose (CMC-Na) and administered orally to the mice.

[0034] The control group was fed a standard diet plus CMC-Na; the model group was fed a high-purine diet (standard diet plus 400 mg / kg potassium oxonate plus 400 mg / kg inosine plus 400 mg / kg hypoxanthine); the benzbromarone group was fed a high-purine diet plus 10 mg / kg benzbromarone (positive control); and the rosmarinic acid group was fed a high-purine diet plus rosmarinic acid, including low doses (20 mg / kg) and high doses (40 mg / kg). Mice were fed continuously for 4 weeks. Urine and feces were collected from each group the day before the end of the experiment. After modeling, mice were sacrificed, and serum, liver, and intestinal tissue were collected. A portion of all collected tissues was flash-frozen in liquid nitrogen and stored at -80°C, while another portion was fixed with 4% paraformaldehyde. All animal experiments were approved by the Animal Ethics Committee of Dalian University of Technology and conducted in accordance with the guidelines of the National Institute of Animal Science. Uric acid levels and XOD activity were detected using commercial kits. Serum and urine samples were used directly for testing. Liver tissue was homogenized in ice-cold PBS, centrifuged, and the supernatant was used for XOD activity detection. Fecal samples were sonicated in PBS for 20 minutes, then incubated at 37°C for 1 hour, and the supernatant was centrifuged to detect uric acid content.

[0035] Total RNA was extracted from the ileum using Trizol reagent and reverse transcribed into cDNA using the Evo M-MLV RT Kit (AGBio, China). RT-qPCR analysis was performed using TB Green® Premix Ex Taq™ II (Takara, Japan) to detect the mRNA level of ABCG2 in the ileum, and Equation 2 was used. -ΔΔCtCalculate. The primer sequences are: ABCG2 F-5'-GAACTCCAGAGCCGTTAGGAC-3', R-5'-CAGAATAGCATTAAGGCCAGGTT-3'. β-actin F-5'-GAGACCTTCAACACCCCAGC-3', R-5'-ATGTCACGCACGATTTCCC-3'.

[0036] The results are as follows Figure 1-4 As shown, after 4 weeks of feeding with a high-purine diet, serum uric acid levels and XOD activity in the model group mice significantly increased, indicating that the HUA model had been established. After rosmarinic acid intervention, serum uric acid levels in HUA mice significantly decreased, with the high-dose group showing a 26.2% decrease compared to the model group. Furthermore, serum and liver XOD activity were significantly inhibited, decreasing by 29.6% and 27.3% respectively compared to the model group. In addition, fecal uric acid content in mice significantly increased under rosmarinic acid intervention, with the high-dose group showing an 89.3% increase compared to the model group. The mRNA level of uric acid secretion protein ABCG2 in the high-dose group was 3.66 times that of the model group, indicating a significantly enhanced intestinal uric acid excretion in mice.

[0037] Example 2: Rosmarinic acid reduces HUA-induced intestinal inflammation The specific steps are the same as steps (1) to (2) in Example 1.

[0038] The ileum tissue obtained in Example 1 was homogenized in ice-cold PBS, centrifuged, and the supernatant was collected. Cytokine levels in the sample were detected using a commercial kit. Results are as follows: Figure 5 Among them, (A) IL-1β; (B) IL-18; (C) TNF-α; (D) IL-6; (E) IL-10. It is evident that rosmarinic acid reversed the HUA-induced increase in pro-inflammatory cytokines (IL-1β, IL-18, TNF-α, IL-6) and decrease in anti-inflammatory cytokines (IL-10), thereby alleviating intestinal inflammation.

[0039] The ileum and colon tissues fixed in 4% paraformaldehyde obtained in Example 1 were prepared into paraffin sections. After dewaxing and rehydration, the sections were stained for histopathological analysis. Hematoxylin-eosin (H&E) staining was performed using standard methods to observe the intestinal villus structure, and Alcian blue (AB) staining was used to show the mucin content. Results are as follows... Figure 6 As shown in the figure, the model group mice exhibited ileal villi atrophy, damage, and shedding, along with reduced ileal and colonic mucin secretion, indicating impaired goblet cell function. Rosmarinic acid treatment alleviated these pathological changes.

[0040] Example 3: Rosmarinic acid reduces HUA-induced intestinal barrier damage The specific steps are the same as steps (1) to (2) in Example 1.

[0041] Total RNA was extracted from the ileum tissue obtained in Example 1 and analyzed by RT-qPCR to detect the mRNA levels of the tight junction proteins Occludin, ZO-1, and the mucin 2 in the ileum. Primer sequences were as follows: Occludin F-5'-ATGTCCGGCCGATGCTCTC, 3'-TTGGCTGCTCTTGGGTCTGTAT; ZO-1 F-5′-TTTTTGACAGGGGGAGTGG, 3′-TGCTGCAGAGGTCAAAGTTCAAG; Mucin2 F-5'-TTCGGCACGAGCAACTTTG, 3'-GGCAGGACACCTTGTCATTG.

[0042] The results are as follows Figure 7 As shown, the mRNA levels of Occludin, ZO-1, and Mucin2 were downregulated in the model group, while transcriptional levels were restored after rosmarinic acid intervention.

[0043] In the intestinal permeability assay, 150 μL of FITC-dextran (80 mg / mL, dissolved in PBS, molecular weight 4000) was orally administered to mice. Serum was collected 4 hours later, and fluorescence intensity was measured at excitation (485 nm) and emission (535 nm). Serum FITC-dextran levels were calculated using a standard curve. Results are as follows: Figure 8 As shown in the figure, the serum FITC-dextran level in the model group mice was significantly increased, indicating intestinal barrier damage and leakage. Intestinal damage was alleviated after rosmarinic acid intervention.

[0044] Example 4: Rosmarinic acid regulates gut microbiota The specific steps are the same as steps (1) to (2) in Example 1.

[0045] The mouse feces collected in Example 1 were subjected to 16S rRNA gene sequencing analysis. The results are as follows: Figure 9 As shown, (A) Venn diagram of OUT between groups; (B) hierarchical clustering tree of samples; (C) principal coordinate analysis (PCoA); (D) microbial distribution map at the genus level; (E, F) effect size of linear discriminant analysis between groups (LEfSe).

[0046] Venn diagrams and hierarchical clustering analyses at the OTU level showed significant differences in microbial composition among the groups. Figure 9A and B). β-diversity analysis based on PCoA showed a clear separation between the model group and the control group. The distribution of the rosmarinic acid intervention group partially overlapped with that of the model group, but the overall trend was segregated, indicating that RA has a regulatory effect on the gut microbiota. Figure 9 C). Furthermore, among the top 20 most abundant bacterial genera, rosmarinic acid intervention significantly increased the number of beneficial *Dunaliella* spp. ( Dubosiella) genus *Zurichella* ( Turicibacter) and Bifidobacterium spp. Bifidobacterium The relative abundance of Bacteroides spp. (a type of opportunistic pathogen) was reduced. Bacteroides) ( Figure 9 D). LEfSe analysis identified the main differentially expressed bacterial genera between the model group and the rosmarinic acid group as: Dubosiella and Bifidobacterium ( Figure 9 E).

[0047] In summary, rosmarinic acid reduces serum uric acid levels in mice by inhibiting XOD activity and promoting intestinal uric acid excretion. Furthermore, rosmarinic acid also inhibits HUA-induced intestinal inflammation and barrier damage, possibly through regulation of the gut microbiota. These data indicate that rosmarinic acid has significant uric acid-lowering effects and can be used in the preparation of anti-HUA drugs and health products, providing a new, safe, and effective means of preventing and treating HUA.

[0048] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. Application of rosmarinic acid in the preparation of drugs or health foods for the prevention and treatment of hyperuricemia in patients with renal insufficiency.

2. The application according to claim 1, characterized in that, Drugs used to prevent and treat hyperuricemia in patients with renal insufficiency include XOD inhibitors or ABCG2 gene expression promoters.

3. The application according to claim 1, characterized in that, Drugs for preventing and treating hyperuricemia in patients with renal insufficiency also contain drug carriers and / or pharmaceutical excipients.

4. The application according to claim 3, characterized in that, Pharmaceutical excipients are one or more of the following: fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

5. The application according to claim 3, characterized in that, The drug carrier is a microcapsule, microsphere, nanoparticle or liposome.

6. The application according to claim 1, characterized in that, The dosage forms of drugs for preventing and treating hyperuricemia in patients with renal insufficiency are granules, capsules, tablets, pills, or oral liquids.

7. The application according to claim 1, characterized in that, The drug has at least one of the following effects: (1) Lower serum uric acid levels; (2) Inhibits serum and liver XOD activity; (3) Promotes the expression of ABCG2 in the gut; (4) Promotes the excretion of uric acid from the intestines; (5) Inhibits the secretion of pro-inflammatory cytokines in the intestine; (6) Promotes the secretion of anti-inflammatory cytokines in the intestine; (7) Upregulates the levels of tight junction proteins and mucins in the gut, reducing intestinal leakage; (8) Regulates intestinal flora and enhances intestinal barrier.

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