Application of paederia scandens extract PSMF rich in flavonoid compounds in preparation of products for reducing uric acid

Abstract

The invention provides an application of a paederia scandens extract PSMF rich in flavonoid compounds in preparation of a product for reducing uric acid. Animal experiment research finds that the PSMF intervenes and relieves weight loss and food intake reduction of a mouse with hyperuricemia, and meanwhile, the decrease of liver and kidney indexes is reversed. In addition, the PSMF also significantly reduces the levels of serum uric acid, xanthine oxidase, adenosine deaminase, creatinine, blood urea nitrogen and the like of hyperuricemia mice, inhibits inflammatory response, and protects liver and kidney injury. And finally, the PSMF improves in-vivo uric acid homeostasis by regulating the expression of the uric acid synthesis gene and the uric acid transporter gene. Results show that PSMF intervention has an effect of regulating uric acid homeostasis, has a good treatment effect on hyperuricemia, and is expected to be used as a natural dietary supplement for treating and preventing hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the technical field of hyperuricemia treatment, and relates to application of PSMF, a flavonoid-rich extract of Parsley striata, in preparing a product for lowering uric acid. Background Art

[0002] Hyperuricemia (HUA), a widespread chronic metabolic disease, is characterized by uric acid levels in the blood that exceed the normal range (≥420 μmol / L). The main cause of the disease is uric acid metabolism disorder. According to surveys, about 80% of endogenous uric acid comes from the decomposition of nucleic acids and adenosine triphosphate (ATP), while the remaining 20% ​​comes from exogenous dietary purines. With the improvement of the quality of life, hyperuricemia has become the second largest potential life threat after diabetes. Long-term intake of high-purine foods has led to a continuous increase in the incidence of hyperuricemia, and the age of onset tends to be younger. According to recent survey reports, the overall prevalence of hyperuricemia in China is at least 13.3%. In addition, hyperuricemia is closely associated with a variety of chronic diseases such as gout, obesity, type 2 diabetes, kidney disease, hypertension, and cardiovascular disease.

[0003] At present, the treatment strategies for hyperuricemia mainly include drug therapy and dietary adjustment. The core of dietary adjustment is to reduce the intake of purine-rich foods. However, it is usually difficult to normalize the patient's serum uric acid level by adjusting the diet alone. Therefore, drug intervention of uric acid levels is necessary. In clinical practice, drugs such as allopurinol and benzbromarquinone are widely used to treat hyperuricemia. However, the side effects of these drugs, such as drug-induced hepatitis, hematological toxicity, renal damage, and allergic reactions, limit their application in clinical treatment. Therefore, the discovery of safer and more efficient active ingredients from natural plant resources has attracted widespread attention from scientific researchers in order to find alternative or auxiliary treatments for hyperuricemia. Summary of the invention

[0004] The present invention aims to provide an application of PSMF, an extract of Polygonum multiflorum rich in flavonoids, in the preparation of a product for lowering uric acid. The results of animal experiments in the present invention show that PSMF exhibits significant advantages in lowering blood uric acid levels and protecting the liver and kidneys, and is expected to be used as a natural dietary supplement for the treatment and prevention of hyperuricemia.

[0005] The above-mentioned object of the present invention is achieved through the following scheme:

[0006] Application of PSMF, a flavonoid-rich extract of Polygonum multiflorum, in the preparation of uric acid-lowering products.

[0007] Preferably, the product includes but is not limited to medicine.

[0008] Preferably, the PSMF achieves uric acid lowering by alleviating the weight loss and reduced food intake caused by potassium oxonate and adenine, while reversing the decrease in liver and kidney indexes.

[0009] Preferably, the PSMF achieves uric acid lowering by reducing blood urea nitrogen (BUN) and creatinine (CRE) levels, inhibiting serum adenosine deaminase (ADA) activity, and reducing xanthine oxidase (XOD) activity.

[0010] Preferably, the PSMF reduces uric acid by inhibiting the increase of serum IL-1β level and restoring serum IL-10 level, thereby inhibiting the inflammatory response induced by adenine and potassium oxonate.

[0011] Preferably, the PSMF reduces uric acid by alleviating liver and kidney damage caused by hyperuricemia, including improving degeneration, necrosis and cavitation of liver cells and alleviating renal tubular epithelial cell necrosis and interstitial fibrosis.

[0012] Preferably, the PSMF achieves uric acid lowering by down-regulating the mRNA expression of XDH and PRPS1.

[0013] Preferably, the PSMF achieves uric acid lowering by restoring the mRNA expression of ABCG2 and OAT1 and down-regulating the mRNA expression of GLUT9.

[0014] Preferably, the PSMF is prepared by the following method: first, the leaves of the Croton tiglium are freeze-dried, then crushed and passed through a 60-mesh sieve, then 60% ethanol is used as an extraction solvent, mixed at a solid-liquid ratio of 1:40, and then extracted using ultrasonic-assisted technology. Finally, the extract is separated by a vacuum filtration device to obtain a Croton tiglium extract.

[0015] More preferably, the method further comprises: concentrating the Cistanche deserticola extract under reduced pressure at 45°C to obtain an extract concentrate, followed by purification using AB-8 macroporous resin, washing with distilled water, desorbing with 95% ethanol, and freeze-drying the desorbed solution to obtain a Cistanche deserticola extract PSMF rich in flavonoids.

[0016] More preferably, the conditions for extraction by ultrasonic-assisted technology are: 300W, 45°C, 30min; and the conditions for freeze-drying are: -40°C, 20Pa, 48h.

[0017] The beneficial effects of the present invention are:

[0018] The present invention discloses for the first time the use of PSMF, a flavonoid-rich extract of Psoralea corylifolia, in the preparation of uric acid-lowering products. The present invention has found through animal experimental studies that PSMF intervention alleviates weight loss and reduced food intake in hyperuricemia mice, and reverses the decline in liver and kidney indexes. In addition, PSMF also significantly reduces the levels of serum uric acid, xanthine oxidase (XOD), adenosine deaminase (ADA), creatinine (CRE) and blood urea nitrogen (BUN) in hyperuricemia mice, inhibits inflammatory response, and protects liver and kidney damage. Finally, PSMF improves uric acid homeostasis in the body by regulating the expression of uric acid synthesis genes and uric acid transporter genes. The results show that PSMF intervention has the effect of regulating uric acid homeostasis, has a good therapeutic effect on hyperuricemia, and is expected to be used as a natural dietary supplement for the treatment and prevention of hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of PSMF intervention on physiological signs of mice with hyperuricemia. Flow chart of experimental treatment of mice (A), body weight (B), weight gain (C), water intake (D), food intake (E) and liver and kidney index (F). Lowercase letters indicate significant differences (p<0.05, the same below);

[0020] Figure 2 Effects of PSMF intervention on biochemical indices of hyperuricemia mice. Serum uric acid (A), BUN (B), CRE (C) and ADA (D) levels, liver XOD activity (E), serum IL-1β level (F), liver IL-1β level (G) and serum IL-10 level (H);

[0021] Figure 3 Effects of PSMF intervention on liver tissue of hyperuricemia mice (HE staining, 100× and 200×);

[0022] Figure 4 Effects of PSMF intervention on renal morphology in hyperuricemia mice;

[0023] Figure 5 Effects of PSMF intervention on renal tissue of hyperuricemia mice (Mason staining, 100× and 200×);

[0024] Figure 6 Effects of PSMF intervention on XDH (A) and PRPS1 (B) mRNA gene expression in hyperuricemia mice;

[0025] Figure 7 Effects of PSMF intervention on ABCG (A), OAT1 (B) and GLUT9 (C) mRNA gene expressions in hyperuricemia mice.

[0026] Figure 8It is a schematic diagram showing the main technical routes and results of the present invention. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] Example

[0029] 1. Preparation of PSMF and determination of flavonoid content

[0030] 1. Preparation of PSMF

[0031] Paederia scandens (Lour.) Merrill is the dried leaves of Paederia scandens (Lour.) Merrill, a plant of the Rubiaceae family.

[0032] The extraction and purification method of the extract of Polygonum multiflorum is as follows: the Polygonum multiflorum leaves are freeze-dried and then crushed with a powder grinder and sieved through a 60-mesh sieve. The treated powder is mixed with 60% ethanol at a solid-liquid ratio of 1:40, and extracted with the aid of ultrasonic-assisted extraction technology (300W, 45°C, 30min). After the extraction is completed, the filtrate is separated by a vacuum filtration device, and the extract concentrate is obtained by reduced pressure concentration at 45°C. Subsequently, the concentrate is purified by AB-8 macroporous resin, washed with distilled water, and desorbed with 95% ethanol (v / v). The desorbed solution is concentrated by a rotary evaporator, and the concentrated solution is freeze-dried (-40°C, 20Pa, 48h), and finally a Polygonum multiflorum extract (PSMF) rich in flavonoids is prepared.

[0033] 2. Determination of total flavonoids content in PSMF

[0034] The aluminum chloride colorimetric method was used. The specific method was as follows: first, 100 μL of appropriately diluted sample was added with 100 μL of distilled water and 50 μL of 5% sodium nitrite solution, and the reaction was continued for 6 min; then, 50 μL of 10% aluminum chloride solution was added, and the reaction was continued for 6 min; finally, 400 μL of 1 mol / L sodium hydroxide solution and 300 μL of distilled water were added, and the reaction was continued for 15 min. The absorbance was measured at 510 nm using rutin ethanol solution (0-1000 μg / mL) as the standard. The total flavonoid content of PSMF was 760.33 mg RE / gDW.

[0035] 2. Animal Experiment Design

[0036] Forty male C57BL / 6 mice (8 weeks old, body weight (20 ± 2g) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. Before the experiment, the mice were fed adaptively for one week. The mice were housed in an SPF-grade experimental animal room with a temperature of 20-24°C, a humidity of 55-70%, and a light / dark cycle of 12h. All experimental procedures were carried out in accordance with the standards of the "Regulations on the Management of Experimental Animals" and approved by the Animal Experiment Ethics Committee of Hainan University (NO.HNUAUCC-2023-00178). The 40 mice were evenly divided into 4 groups: control group, model group, allopurinol treatment group (10 mg / kg BW / d) and PSMF treatment group (200 mg / kg BW / d). All mice were fed a standard diet. Potassium oxonate (300 mg / kg BW / d) and adenine (100 mg / kg BW / d) were combined with 0.5% CMC-Na mixture was administered orally once a day for 4 weeks in the model group, allopurinol group, and PSMF group to induce HUA mice ( Figure 1 A). During the experiment, the treatment group was given allopurinol or PSMF by gavage once a day, and the control group and the model group were gavaged with an equal volume of 0.5% CMC-Na. The weight changes of mice were monitored weekly, and the food intake and water intake were recorded. After the experiment, the mice were fasted for 12 hours and euthanized. The blood samples of mice were centrifuged at 4000rpm for 20 minutes at 4℃ to collect serum. The liver and kidney tissues were washed, weighed, cut into several pieces, and fixed with 10% neutral formaldehyde fixative and liquid nitrogen, respectively. All samples were stored at -80℃ for subsequent analysis.

[0037] 3. Determination of biochemical indicators

[0038] Uric acid (UA), creatinine (CRE), urea nitrogen (BUN), adenosine deaminase (ADA), interleukin 1β (IL-1β), interleukin 10 (IL-10) in serum, xanthine oxidase (XOD) in liver, and interleukin 1β (IL-1β) in kidney were detected using kits purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.

[0039] 4. Tissue Sections

[0040] The kidney and liver tissues fixed with 10% neutral formaldehyde were embedded in paraffin and cut into 4 μm thick sections by a microtome, and then stained with hematoxylin-eosin (HE) and Masson (Masson). Finally, images of kidney and liver pathological sections were obtained using Pannoramic MIDI.

[0041] 5. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0042] Total RNA from liver and kidney tissues was extracted using a tissue RNA extraction kit (G3640-50T), and RNA was reverse transcribed into cDNA using a reverse transcription kit (G3337-50). Quantification was performed using a CFX-96 fluorescence quantitative PCR instrument. Gene detection was performed using SYBR Green Master Mix, and GAPDH was used as an internal reference gene. The primer sequences of the target gene and the internal reference gene are shown in Table 1. Gene expression was performed using 2 -ΔΔCT Method confirmed.

[0043] Table 1 Primer sequences of target genes and internal reference genes

[0044]

[0045] 6. Results and Analysis

[0046] 1. PSMF intervention can restore the physiological signs of hyperuricemia mice

[0047] Effects of PSMF on body weight of mice with hyperuricemia Figure 1 BC. During the experimental period, the body weight of control mice continued to increase, while that of HUA mice decreased significantly (p<0.05). Allopurinol treatment significantly attenuated the weight loss (p<0.05), and PSMF intervention showed a similar trend to allopurinol. Figure 1 The results of DE showed that the food intake and water intake of the control group mice were stable, while the food intake of the model mice was significantly reduced and the water intake was significantly increased (p<0.05). Compared with the model group, allopurinol and PSMF intervention increased the food intake of HUA mice (p<0.05), however, there was no significant difference in water intake compared with the model group (p>0.05, Figure 1 D and E). Figure 1 As shown in Figure F, the liver and kidney indexes of the model group were significantly higher than those of the control group (p<0.05), indicating that potassium oxonate and adenine may exhibit renal and liver toxicity in mice. Allopurinol and PSMF significantly reversed the increase in organ indexes of HUA mice (p<0.05). The results showed that PSMF intervention alleviated the weight loss and reduced food intake caused by potassium oxonate and adenine in HUA mice, and reversed the decrease in liver and kidney indexes.

[0048] 2. PSMF intervention regulates purine metabolism in hyperuricemia mice

[0049] The effect of PSMF on the uric acid level of HUA mice was investigated by measuring the levels of blood uric acid (UA), urea nitrogen (BUN), creatinine (CRE), and the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD). Compared with the control group mice (64.49 μmol / L), the serum uric acid level of the model group mice was significantly increased to 83.90 μmol / L (p<0.05). This result showed that the hyperuricemia model was successfully established. Compared with the model group, the serum uric acid levels of the allopurinol group and the PSMF group decreased by 33.44% and 22.04%, respectively, indicating that the intervention of PSMF and allopurinol can effectively reduce the uric acid level ( Figure 2 A). In the model group mice, serum urea nitrogen (BUN) and creatinine (CRE) levels increased significantly, and ADA activity was enhanced (p<0.05). PSMF intervention significantly reduced the BUN and CRE levels of HUA mice and inhibited serum ADA activity (p<0.05, Figure 2 BD). XOD catalyzes xanthine and hypoxanthine to produce uric acid, and its activity is closely related to the uric acid level in the body. Figure 2 As shown in E, the XOD activity in liver tissue of HUA mice (13.03U / g prot) was significantly higher than that of normal mice (9.06U / g prot, p<0.05). Allopurinol significantly reduced the activity of XOD in the liver (8.23U / g prot, p<0.05), while PSMF intervention had a similar effect to allopurinol, with an XOD activity of 10.67U / g prot. PSMF intervention can improve serum renal function indicators and regulate the activity of purine metabolism-related enzymes, thereby improving hyperuricemia in mice.

[0050] 3. PSMF intervention can reduce the inflammatory response in hyperuricemia mice

[0051] High uric acid levels in the body are often accompanied by inflammatory responses. The effects of PSMF intervention on the alleviation of inflammatory responses in HUA mice were evaluated by detecting pro-inflammatory factors (IL-1β) and anti-inflammatory factors (IL-10). Figure 2 As shown in FH, adenine and potassium oxonate stimulation significantly increased serum and liver IL-1β levels and significantly reduced serum IL-10 levels in HUA mice (p<0.05). As expected, allopurinol and PSMF intervention inhibited the increase in serum IL-1β levels and restored serum IL-10 levels (p<0.05). The results showed that PSMF intervention inhibited the inflammatory response induced by adenine and potassium oxonate.

[0052] 4. PSMF intervention can alleviate liver damage in hyperuricemia mice

[0053] HE staining was used to observe the effect of PSMF intervention on the liver tissue of hyperuricemia mice. Figure 3 As shown in the figure. The liver tissue cell structure of the mice in the control group remained normal, and no obvious cell degeneration or necrosis was observed. In contrast, the liver tissue structure of the mice in the model group showed significant deformation, necrosis and vacuolation. Compared with the model group, the degeneration, necrosis and vacuolation of the liver cells of mice were significantly improved after treatment with allopurinol and PSMF.

[0054] 5. PSMF intervention can alleviate renal damage in hyperuricemia mice

[0055] The appearance and shape of the kidneys Figure 4 The kidneys of normal mice were reddish brown, while the kidneys of mice in the model group were white, edematous, and had obvious lesions. Allopurinol and PSMF interventions improved the condition to varying degrees. Figure 5 As shown. The glomerular morphology of the kidneys of the control group mice remained intact, the proximal tubular brush border structure was clear, and there was no obvious pathological damage. The kidneys of the model group mice showed obvious renal damage, including loss of internal brush border structure, inflammatory infiltration of renal interstitial cells, renal interstitial fibrosis, and tubular epithelial cell necrosis. PSMF intervention alleviated tubular epithelial cell necrosis and interstitial fibrosis. Similar effects were observed in the allopurinol group. The results clearly demonstrated the alleviating effect of PSMF intervention on liver and kidney damage in HUA mice.

[0056] 6. PSMF intervention can inhibit uric acid synthesis and promote uric acid excretion in HUA mice

[0057] Uric acid is the end product of purine metabolism in the body and is excreted from the body through the urinary system and digestive system. Phosphoribosyl pyrophosphate synthase (PRPS) and xanthine dehydrogenase (XDH), encoded by PRPS1 and XDH, play an important role in purine metabolism. Compared with the control group, the mRNA expression of XDH and PRPS1 in the HUA group was significantly upregulated by 148.37% and 202.08%, respectively (p<0.05), and the gene transcription levels of the two enzymes in PSMF-treated mice were significantly decreased ( Figure 6 , p<0.05). The kidney maintains uric acid homeostasis mainly by inhibiting the renal tubular reabsorption of uric acid or increasing uric acid secretion. In the distal renal tubule, uric acid secretion is mediated by transporters ABCG2 and OAT1.

[0058] Compared with the control group, potassium oxonate and adenine led to a significant decrease in the mRNA expression of ABCG2 and OAT1 in the model group mice (p<0.05, Figure 7 ). After allopurinol and PSMF intervention, the mRNA expression of ABCG2 and OAT1 was restored. Studies have shown that GLUT9 plays a key role in the reabsorption of uric acid in the proximal tubule. In this study, the mRNA expression level of GLUT9 in HUA mice was 1.69 times that of normal mice ( Figure 7 , p<0.05). Compared with the model group, the mRNA expression of GLUT9 in the allopurinol group and PSMF group decreased by 27.22% and 47.34%, respectively (p<0.05). The results showed that PSMF intervention may control the uric acid level in HUA mice by regulating the expression of uric acid metabolism-related genes.

[0059] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of PSMF, a flavonoid-rich extract of Polygonum multiflorum, in the preparation of uric acid-lowering products.

2. The use according to claim 1, characterized in that: The PSMF reduces uric acid by alleviating the weight loss and reduced food intake induced by potassium oxonate and adenine, while reversing the decrease in liver and kidney indexes.

3. The use according to claim 1, characterized in that: The PSMF reduces uric acid by reducing urea nitrogen and creatinine levels, inhibiting serum adenosine deaminase activity, and reducing xanthine oxidase activity.

4. The use according to claim 1, characterized in that: The PSMF suppresses the increase of serum IL-1β level and restores serum IL-10 level, thereby suppressing the inflammatory response induced by adenine and potassium oxonate to achieve uric acid reduction.

5. The use according to claim 1, characterized in that: The PSMF achieves uric acid lowering by alleviating liver and kidney damage caused by hyperuricemia.

6. The use according to claim 1, characterized in that: The PSMF reduces uric acid by downregulating the mRNA expression of XDH and PRPS1.

7. The use according to claim 1, characterized in that: The PSMF reduces uric acid by restoring the mRNA expression of ABCG2 and OAT1 and downregulating the mRNA expression of GLUT9.

8. The use according to claim 1, characterized in that: The PSMF is prepared by the following method: first, the leaves of the Herba Lycopodii are freeze-dried, then crushed and passed through a 60-mesh sieve, then 60% ethanol is used as an extraction solvent, mixed at a solid-liquid ratio of 1:40, then extracted using ultrasonic-assisted technology, and finally, the extract is separated by a vacuum filtration device to obtain the Herba Lycopodii extract.

9. The use according to claim 8, characterized in that: The method also includes: concentrating the extract of Cnidium monnieri under reduced pressure at 45° C. to obtain an extract concentrate, then purifying it using AB-8 macroporous resin, washing it with distilled water, desorbing it with 95% ethanol, and freeze-drying the desorbed solution to obtain a Cnidium monnieri extract PSMF rich in flavonoids.

10. The use according to claim 9, characterized in that: The conditions for extraction using ultrasonic-assisted technology were: 300W, 45°C, 30min; the conditions for freeze-drying were: -40°C, 20Pa, 48h.

Images