Method for protecting in-vitro acute gout cell model based on polyphenol compound

Abstract

The invention is applicable to the technical field of bioengineering, and provides a method for protecting an in-vitro acute gout cell model based on a polyphenol compound. According to the method, the process for ultrasonic extraction of the polyphenol compounds in the inonotus obliquus is optimized, and the optimal conditions are that the material-liquid ratio is 1: 40 g / mL, the ultrasonic time is 30 min, the ultrasonic power is 280 W, and the predicted extraction rate value is 0.866% and is verified to be 0.888%. Polyphenolic compounds (protocatechuic acid, protocatechualdehyde and osmundone) have concentration-dependent inhibition on xanthine oxidase, the ability sequence is osmundone > protocatechualdehyde > protocatechuic acid, the inhibition is reversible, and the inhibition types are competitive, miscible and miscible. Under the safe concentration, ROS, NO and LDH in acute gout model cells induced by MSU can be reduced, the activity of SOD is improved, it is prompted that inonotus obliquus has the potential hyperuricemia resisting effect, and a basis is provided for industrial production of polyphenol of inonotus obliquus.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for protecting an in vitro acute gout cell model based on polyphenol compounds. Background Art

[0002] Inonotus obliquus Inonotus obliquus Inonotus obliquus, commonly known in my country as chaga, birch mushroom, or white chaga, primarily grows on the trunks of deciduous trees such as birch and elm. Betulinum, a member of the Basidiomycota, Hymeromycetes, Ostreophytes, and the genus Inonotus, is highly cold-resistant. It contains a variety of active ingredients, including polysaccharides, polyphenols, triterpenes, melanin, and lignin. It also contains inorganic elements such as calcium, silicon, iron, zinc, copper, aluminum, and phosphorus, as well as amino acids such as glycine, aspartic acid, and threonine. Modern pharmacological studies have shown that Inonotus obliquus exhibits anti-tumor, lipid-lowering, hypoglycemic, antioxidant, anti-inflammatory, and hepatoprotective activities. For example, the lanostane-type triterpenoid compound, inonotinol E (ITE), exhibits potent inhibitory activity against tumor cells. ITE significantly inhibits the growth and migration of breast cancer cells, with no significant toxicity in patients. Ye et al. found that Inonotus obliquus can regulate intestinal microbial abundance to maintain a dynamic balance, thereby controlling inflammation and alleviating symptoms associated with type 2 diabetes. It effectively reduced body weight and fasting blood glucose in diabetic mice, and alleviated the severity of intestinal, liver, kidney, and pancreatic lesions in diabetic mice. Lee et al. found that Inonotus obliquus alcohol inhibited the expression of mitogen-activated protein kinase and NADP oxidase 5, and effectively reduced the levels of NF-κB, as well as cytokines such as NO, ROS, COX-2, IL-1β, IL-6, and TNF-α. This, in turn, blocked the activation of the MAPK-NOX5 and NF-κB signaling pathways, protecting human dermal fibroblasts and attenuating the expression of aging genes. Wang Shiqi et al. determined that crude Inonotus obliquus polysaccharides and their graded alcohol-precipitated polysaccharides (e.g., IOP40 and IOP60) exhibited inhibitory effects on xanthine oxidase (XO) activity; both are mixed inhibitors of XO. Furthermore, Inonotus obliquus has anti-fatigue and protective effects against neurodegenerative diseases.

[0003] Hyperuricemia and gout are currently common diseases, and are becoming more common among younger people. XO, a key enzyme in purine metabolism in the body, catalyzes the conversion of xanthine and hypoxanthine to uric acid, accompanied by the production of superoxide anion free radicals and hydrogen peroxide. When uric acid levels are within the normal range, it can act as an antioxidant and neuroprotectant, but excessive or insufficient excretion can lead to hyperuricemia, which may be accompanied by complications such as hypertension, diabetes, and atherosclerosis. Long-term hyperuricemia can induce gouty arthritis. In addition, the excessive free radicals produced during uric acid production are also associated with heart disease, skin diseases, aging, and Alzheimer's disease. Currently, research on the treatment mechanisms of gout and hyperuricemia is mostly related to XO. Commonly used uric acid-lowering drugs in clinical practice are mostly synthetic, but they have toxic side effects such as liver damage and allergies. Therefore, screening XO inhibitors from natural compounds of plant origin has become a research hotspot. Edible and medicinal fungi rich in active ingredients such as polyphenols (such as Inonotus obliquus) are considered to have the potential to prevent and treat hyperuricemia and gout-related diseases because of their safety and greater compliance with modern health concepts.

[0004] However, in the existing technology, research on Inonotus obliquus has mostly focused on polysaccharide compounds and their anti-tumor activity, while research on other components (especially polyphenols) is relatively limited. Moreover, most of the research is still in the laboratory stage, and the industrial preparation process has not yet been perfected. At the same time, the research on the mechanism of action of the active ingredients of Inonotus obliquus in preventing and treating hyperuricemia and gout is still not in-depth, and lacks effective research model support. To this end, the present invention proposes a method based on polyphenol compounds to protect in vitro acute gout cell models, aiming to provide a powerful tool for in-depth exploration of the mechanism of action of Inonotus obliquus polyphenols in acute gout and promote the research and development of related natural medicines. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for protecting an in vitro acute gout cell model based on polyphenol compounds, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions: The method for protecting an in vitro acute gout cell model based on polyphenolic compounds comprises the following steps: RAW 264.7 cells were cultured in a medium containing 150 μg / mL sodium urate for 24 h to induce an acute gout cell model. Adding a polyphenol compound to the model cells, wherein the polyphenol compound is protocatechuic aldehyde, protocatechuic acid or porphyrone, and detecting at least one indicator of cell survival rate, ROS content, NO content, LDH release and SOD activity after culturing; The concentration of protocatechuic aldehyde is 2.5-10 μM, the concentration of protocatechuic acid is 12.5-50 μM, and the concentration of porphyrone is 2.5-10 μM.

[0007] Furthermore, the polyphenol compounds can significantly reduce the content of ROS and NO in model cells, reduce the release of LDH, and significantly increase the activity of SOD.

[0008] Furthermore, the half-maximal inhibitory concentrations of the polyphenolic compounds on xanthine oxidase were: protocatechuic aldehyde 5.92 ± 0.40 mM, protocatechuic acid 26.25 ± 0.52 mM, and porphyrone 4.91 ± 0.42 mM.

[0009] Furthermore, the polyphenolic compounds inhibit xanthine oxidase in a reversible manner, wherein protocatechuic aldehyde and protocatechuic acid exhibit mixed inhibition, and porphyrone exhibits competitive inhibition.

[0010] Furthermore, the polyphenolic compounds were extracted from Inonotus obliquus by ultrasound-assisted extraction, and the extraction conditions were a solid-liquid ratio of 1:40 g / mL, an ultrasound time of 30 min, and an ultrasound power of 280 W.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This study used total polyphenol content as an indicator and a response surface experiment designed using Design-Expert 13 software. The ultrasonic extraction process for polyphenols from Inonotus obliquus was optimized, with the solid-liquid ratio, ultrasonic time, and ultrasonic power as influencing factors. The optimal extraction process was determined to be a solid-liquid ratio of 1:40 g / mL, an ultrasonic time of 30 min, and an ultrasonic power of 280 W. Under these conditions, the predicted polyphenol extraction rate was 0.866%, and the experimental extraction rate reached 0.888%, which is close to the theoretical value. Furthermore, the inhibitory effect of the main polyphenolic compounds (protocatechuic acid, protocatechuic aldehyde, and porphyrone) from Inonotus obliquus on xanthine oxidase (XO) was evaluated using the uric acid content in the enzymatic reaction system as an indicator. The results showed that within the appropriate concentration range, the inhibition of XO activity by the three compounds was concentration-dependent, with the inhibitory ability ranking as porphyrinone > protocatechuic aldehyde > protocatechuic acid. Enzyme reaction kinetic analysis showed that the inhibition of XO by the three compounds was reversible, with protocatechuic aldehyde and protocatechuic acid exhibiting mixed inhibition, and porphyrinone exhibiting competitive inhibition. Furthermore, the present invention used MSU to induce RAW 264.7 cells to establish an in vitro acute gout cell model. It was found that 2.5-10 μM protocatechuic aldehyde, 12.5-50 μM protocatechuic acid, and 2.5-10 μM porphyrinone had no toxic side effects on the cells and significantly reduced ROS and NO levels, decreased LDH release, and increased SOD activity in the model cells, indicating that protocatechuic aldehyde, protocatechuic acid, and porphyrinone have potential protective effects on acute gout cells. In summary, the present invention not only optimizes the extraction process of Inonotus obliquus polyphenols, but also confirms that its main polyphenol compounds have the effects of inhibiting XO activity and protecting acute gout cells, suggesting that Inonotus obliquus has potential anti-hyperuricemia efficacy and provides theoretical support and basis for the industrial production of its polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The effect of solid-liquid ratio on the polyphenol extraction content.

[0013] Figure 2 The effect of ultrasonic time on the polyphenol extraction content.

[0014] Figure 3 The effect of ultrasonic power on the polyphenol extraction content.

[0015] Figure 4 is the material-liquid ratio ( A ) and ultrasound time ( B ) on the extraction of polyphenols from Inonotus obliquus; A is AB 3D graph of the influence of factors on Y; B is AB Contour plot of factor interactions.

[0016] Figure 5 is the material-liquid ratio ( A ) and ultrasonic power (C ) on the extraction of polyphenols from Inonotus obliquus; A is AC 3D graph of the influence of factors on Y; B is AC Contour plot of factor interactions.

[0017] Figure 6 is the ultrasound time ( B ) and ultrasonic power ( C ) on the extraction of polyphenols from Inonotus obliquus; A is BC 3D graph of the influence of factors on Y; B is BC Contour plot of factor interactions.

[0018] Figure 7 represents the inhibitory effects of PAH, PA and OS on XO activity; A represents the inhibitory effect of PAH on XO activity; B represents the inhibitory effect of PA on XO activity; C represents the inhibitory effect of OS on XO activity.

[0019] Figure 8 Figure 1 is the relationship between n and [XO] at different concentrations of PAH, PA and OS; A is the relationship between n and [XO] at different concentrations of PAH; B is the relationship between n and [XO] at different concentrations of PA; C is the relationship between n and [XO] at different concentrations of OS.

[0020] Figure 9 Figure 3 is the LB curve and slope and intercept change trend diagram of the inhibitory effect of PAH on XO; A is the LB curve of PAH; B is the effect of PAH on the slope of the LB curve; C is the effect of PAH on the intercept of the LB curve.

[0021] Figure 10 Figure 3 is the LB curve of PA's inhibitory effect on XO and its slope and intercept changing trend diagram; A is the LB curve of PA; B is the effect of PA on the slope of the LB curve; C is the effect of PA on the intercept of the LB curve.

[0022] Figure 11 Figure 2 is the LB curve and slope change trend diagram of the inhibitory effect of OS on XO; A is the LB curve of OS; B is the effect of OS on the slope of the LB curve.

[0023] Figure 12 is the relative survival rate of RAW 264.7 cells under the action of different concentrations of MSU (n = 5, ** P < 0.01).

[0024] Figure 13 The relative survival rates of RAW 264.7 cells under the action of different concentrations of allopurinol, PAH, PA and OS (n = 5, ** P < 0.01,* P<0.05); where A is the relative survival rate of RAW 264.7 cells treated with different concentrations of allopurinol; B is the relative survival rate of RAW 264.7 cells treated with different concentrations of PAH; C is the relative survival rate of RAW 264.7 cells treated with different concentrations of PA; D is the relative survival rate of RAW 264.7 cells treated with different concentrations of OS.

[0025] Figure 14 MTT assay was used to detect the relative survival rate of cells in each group after the addition of PAH, PA, and OS (n = 4, compared with the control group: ** P < 0.01; compared with the model group: ## P < 0.01); A is the relative survival rate of cells in each group after PAH addition; B is the relative survival rate of cells in each group after PA addition; C is the relative survival rate of cells in each group after OS addition.

[0026] Figure 15 The effect of drug addition on the relative intensity of ROS (n = 5, compared with the control group: ** P < 0.01; compared with the model group: # P <0.05, ## P < 0.01); where A is the effect of PAH on the relative intensity of ROS; B is the effect of PA on the relative intensity of ROS; C is the effect of OS on the relative intensity of ROS.

[0027] Figure 16 Effects of drug addition on the relative content of NO (n = 3, compared with the control group: ** P < 0.01; compared with the model group: # P <0.05, ## P < 0.01); where A is the effect of PAH addition on the relative content of NO; ​​B is the effect of PA addition on the relative content of NO; ​​C is the effect of OS addition on the relative content of NO.

[0028] Figure 17 is the amount of LDH released in each group after drug addition (n = 3, compared with the control group: ** P < 0.01; compared with the model group: # P <0.05, ## P < 0.01); where A is the amount of LDH released after PAH was added; B is the amount of LDH released after PA was added; and C is the amount of LDH released after OS was added.

[0029] Figure 18 Effects of drug addition on SOD activity (n = 3, compared with the control group: ** P < 0.01; compared with the model group: # P <0.05, ## P < 0.01); where A is the effect of PAH on SOD activity; B is the effect of PA on SOD activity; C is the effect of OS on SOD activity. DETAILED DESCRIPTION

[0030] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0031] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0032] Example 1: Optimization of polyphenolic compounds from Inonotus obliquus using response surface methodology; 1.1 Effect of solid-liquid ratio on polyphenol extraction; according to Figure 1 As can be seen, within the range of 1:10 to 1:40 g / mL, the polyphenol content in the crude extract gradually increased with increasing solid-liquid ratios. At a solid-liquid ratio of 1:40 g / mL, the polyphenol content in the crude extract reached a peak of 0.85%. However, at a solid-liquid ratio of 1:50 g / mL, the polyphenol content in the crude extract decreased. At a small solid-liquid ratio, the Inonotus obliquus powder cannot be fully immersed in the solvent, resulting in insufficient dissolution of the polyphenols in methanol and premature saturation of the solvent. As the solid-liquid ratio increases, the increased contact area increases the diffusion rate of the polyphenols into the solvent, increasing their solubility in methanol. When the solid-liquid ratio is too high, reaching 1:50 g / mL, the polyphenol content begins to decline. Due to the increased solvent volume, the solubility of other active substances also increases, affecting the purity of the polyphenol extraction and increasing solvent costs. In summary, the polyphenol extraction effect was optimal when the solid-liquid ratio was 1:40 g / mL, so the solid-liquid ratios of 1:30, 1:40, and 1:50 g / mL were selected as the three levels of response surface optimization.

[0033] 1.2 Effect of ultrasonic time on polyphenol extraction; Depend on Figure 2As shown, the polyphenol content in the crude extract of Inonotus obliquus showed a clear trend of first increasing and then decreasing with increasing ultrasonication time within the range of 10 to 50 minutes. The polyphenol content in the crude extract reached a maximum of 0.77% at an ultrasonication time of 30 minutes. When the ultrasonication time was less than 30 minutes, the polyphenol content was low, which may result from incomplete ultrasonication, with cavitation failing to fully incorporate the polyphenols in the powder into the solvent. When the ultrasonication time exceeded 30 minutes, the polyphenol concentration decreased. This phenomenon may be due to the destruction of the polyphenol structure and the breakage of the chemical bonds in the compounds, resulting in a reduced extraction yield. Considering that the polyphenol content in the crude extract of Inonotus obliquus was significantly higher at an ultrasonication time of 30 minutes than at other ultrasonication time levels, 20, 30, and 40 minutes were selected as the three levels for the response surface optimization of this factor.

[0034] 1.3 Effect of ultrasonic power on polyphenol extraction; Depend on Figure 3 The results show that polyphenol extraction is significantly affected by ultrasonic power. Within the 240-280 W range, the extraction rate increases with increasing ultrasonic power, reaching 0.81% at 280 W. Ultrasonic powers above 400 W may cause degradation and denaturation of phenolic compounds, resulting in a decrease in polyphenol extraction yield. Therefore, 240, 280, and 320 W were selected as the three levels for response surface optimization.

[0035] 1.4 Response surface experiment optimization results and variance analysis; Based on the results of the previous single-factor experiments, a three-factor, three-level experimental design was conducted for the selected extraction conditions using Design-Expert 13 software. The Box-Behnken response surface methodology within the software was used to design an experiment to optimize the polyphenol extraction rate of Inonotus obliquus. Seventeen experimental schemes with different ratios were obtained, and the results are shown in Table 1.

[0036] Table 1 Response surface design and results

[0037] The material-liquid ratio ( A ), ultrasound time ( B ), ultrasonic power ( C ) The regression equation of the polyphenol extraction rate (Y) of Inonotus obliquus was obtained by regression fitting of the three factors: Y = -1.57470 + 0.042810 A + 0.052887 B + 0.005212 C +0.000182 AB - 0.000021 AC+ 6.55616 × 10 -6 BC - 0.000516 A 2 - 0.001019 B 2 -7.79829 × 10 -6 C 2 .

[0038] The results of variance analysis of the response surface model are shown in Table 2. P < 0.0001, reaching an extremely significant level, indicating that the model can effectively reflect the differences in the extraction rate of polyphenols from Inonotus obliquus. P The value is 0.3944 ( P > 0.05), which is not significant, indicating that the model has a good fit and is of practical significance. Overall, the model is accurate and reliable. As shown in Table 3, the correlation coefficient of the model is R 2 = 0.9753, adjusted coefficient of determination R 2 adj = 0.9436, indicating that this model can explain 97.53% of the variability of the three factors and 94.36% of the variation in the polyphenol extraction rate of Inonotus obliquus can be explained by this model, further verifying the stability of the model. F Value size ( F The larger the value, the greater the influence of the single factor on the polyphenol extraction rate of Inonotus obliquus). It can be seen that the influence of the single factor is: A (Material to liquid ratio)> B (Ultrasound time)> C (ultrasound power).

[0039] Table 2 Variance analysis of polyphenol extraction yields using the Box-Behnken response surface model equation

[0040] Note: * indicates significant difference ( P <0.05); ** indicates extremely significant difference ( P <0.01).

[0041] Table 3 Analysis of variance of quadratic regression model

[0042] Through in-depth analysis of Design Expert 13 software, we can obtain the response surface 3D diagram and contour analysis diagram of the interaction between any two factors, such as Figure 4 A and B, Figure 5 A and B and Figure 6 As shown in A and B. Generally speaking, if the middle part of the contour map presents an elliptical shape and the steepness of the response surface is higher, it means that the change of the factor has a more significant impact on the response value, and it also indicates that the interaction between the two factors is stronger. Figure 4 (material-liquid ratio and ultrasonic time), Figure 5 (material-liquid ratio and ultrasonic power) and Figure 6 The ultrasound time and power graph demonstrates the specific effects of different factors on the extraction rate of polyphenols from Inonotus obliquus. Each interaction between two factors is elliptical, indicating a significant impact on the extraction rate. Among the interactions between the factors, the response surface corresponding to the interaction between ultrasound time and liquid-to-liquid ratio has the most pronounced curvature. This phenomenon clearly demonstrates that, among the many factors, the interaction between ultrasound extraction time and liquid-to-liquid ratio has the most significant impact on the extraction rate of polyphenols from Inonotus obliquus.

[0043] 1.5 Optimal extraction process; Using the polyphenol content in the crude extract of Inonotus obliquus as the response value, a Box-Behnken fitting and variance analysis were performed using Design-Expert 13 software. The optimal crude extraction conditions were a solid-liquid ratio of 1:40 g / mL, an ultrasonic time of 30 min, and an ultrasonic power of 280 W. The theoretical maximum polyphenol content in the crude extract of Inonotus obliquus was 0.866%. Verification analysis based on the predicted optimal conditions revealed a maximum polyphenol content of 0.888% in the crude extract of Inonotus obliquus, which is close to the theoretical value, further demonstrating the authenticity and reliability of the regression model after response surface optimization.

[0044] Example 2: The inhibitory effects of the main polyphenolic compounds of Inonotus obliquus on xanthine oxidase (XO); 2.1 Analysis of XO inhibitory activity in vitro; The results of the in vitro inhibitory activity of different concentrations of inhibitors (polyphenols, referred to as polyphenols) on XO are as follows Figure 7 As shown in Figures A to C, as the concentrations of PAH (protocatechuic aldehyde), PA (protocatechuic acid) and OS (ospermone) increased, the inhibition rates of the three on XO increased in a concentration-dependent manner. The half-maximal inhibitory concentration (IC 50 ) It can be seen that: PAH, PA, OS have an IC of XO 50They are 5.92 ± 0.40, 26.25 ± 0.52 mM, and 4.91 ± 0.42 mM, respectively. It can be seen that the three polyphenols all have good XO inhibition ability, among which OS has the best inhibitory effect, and the inhibitory ability of PAH is similar to OS. Although the inhibitory ability is weaker than that of more common polyphenols such as baicalein, quercetin, and galangin, the current research on the half-maximal inhibitory concentration of XO of these three polyphenols is very limited. It is worth noting that IC 50 The assay results are susceptible to methodological influences, and differences in reaction time, reaction conditions, sample concentration, and drug source can all lead to biased results. Furthermore, the ranking of PAH and OS in terms of their inhibitory ability against XO is opposite to their ranking in terms of their DPPH scavenging ability. This is presumably due to the stronger interaction between OS and amino acid residues in XO than between OS and PAH, resulting in OS's greater inhibitory ability against the enzyme.

[0045] 2.2 Analysis of inhibition reversibility; By measuring the change of reaction rate with enzyme concentration at different inhibitor concentrations, the reversibility of the inhibitory effect on XO can be analyzed. Figure 8 As shown in A to C, the substrate concentration is fixed at 250 μM, and the reaction rate (n) and XO concentration (200, 400, 600, 800 U·L) are plotted at different concentrations of PAH, PA, and OS. −1 ). The results showed a good linear relationship across all concentration groups, passing through the origin. The slopes of the fitted lines gradually decreased with increasing PAH, PA, and OS concentrations. This indicates that all three polyphenols can reduce the XO catalytic reaction rate but cannot completely inactivate XO, consistent with reversible inhibition. Therefore, it can be concluded that the inhibition of XO by PAH, PA, and OS is reversible.

[0046] 2.3 Analysis of inhibition types; The type of inhibition can be determined by the Lineweaver-Burk double reciprocal curve (LB curve). Reversible inhibition types include competitive inhibition, non-competitive inhibition, uncompetitive inhibition, and mixed inhibition. n The reaction starts with the addition of XO and the 1 / [ S ]-1 / n Fit the straight line to determine the type of inhibition of the compound on XO. Mixed inhibition is usually calculated according to formula 1. K m and V m For the secondary plot of the LB curve, according to formula 2 and formula 3, we can get K i and Kis For competitive inhibition, the kinetic equation is calculated by Equation 4 K m and V m .

[0047] Mixed inhibition: Formula 1: ; Formula 2: ; Formula 3: ; Competitive inhibition: Formula 4: ; Where: n and V m are the rate and maximum reaction rate of the enzymatic reaction, respectively; [ I ]and[ S ] are the concentrations of inhibitors (PAH / PA / OS) and substrate xanthine, respectively; K m and K i are the Michaelis constant and the inhibition constant, respectively; K is is the binding enzyme inhibition constant. K i Represents the ability of the inhibitor to bind to the free enzyme. K is It represents the binding ability of the inhibitor to the enzyme-substrate complex. The smaller the value, the easier and tighter the binding.

[0048] The results showed that when the PAH concentration was less than 5.79 mM, the LB fitting line intersected in the second quadrant ( Figure 9 A); As the PAH concentration increases, the slope of the fitting line gradually increases ( Figure 9 Middle B), y The axis intercept gradually increases ( Figure 9 C), indicating the maximum reaction rate ( V max ) gradually decreases, the Michaelis constant ( K m ) gradually increases, which is consistent with the performance of mixed inhibition. According to the LB equation quadratic plot calculation, K i and K is 0.54 mM and 8.53 mM respectively, K i < K isThis indicates that the affinity of PAH to XO is greater than the affinity of XO to the substrate. When the PA concentration is less than 6.49 mM, the obtained LB fitting line intersects in the first quadrant ( Figure 10 A); As the PA concentration increases, the slope of the fitting line gradually increases ( Figure 10 Middle B), y The axis intercept gradually decreases ( Figure 10 Middle C), description V max Increase, K m Increase, this phenomenon belongs to mixed inhibition. According to the LB equation quadratic plot calculation, K i is 1.84 mM, K is Negative values ​​cannot be used as a basis for judgment. When the OS concentration is less than 2.24 mM, the LB fitting line intersects at y axis( Figure 11 Middle A); As the OS concentration increases, the slope of the fitting line gradually increases ( Figure 11 In B), the y-intercept remains unchanged, indicating V max constant, K m increases, indicating competitive inhibition when the concentration is less than 2.24 mM.

[0049] Example 3: Protective effects of polyphenolic compounds on acutely ventilated cells; 3.1 Screening of acute gout cell models; Macrophage RAW264.7 cells were inoculated in high-glucose DMEM and cultured in a 37°C, 5% CO2 incubator for 24 h. Cells in the logarithmic growth phase were collected and cell suspensions were prepared. 100 μL of cell suspension (approximately 6 × 10 cells per ml) was added to each well of a 96-well plate. 4 cells), added 100 μg / mL sodium urate (MSU) solution (dissolved in DMEM medium), and cultured at 37°C for 24 h to establish an acute gout cell model. Figure 12 As shown in the figure, compared with the control group (MSU concentration of 0 μg / mL), the cell viability was approximately 85% after 24 hours of culture when 150 μg / mL MSU was added. When the MSU concentration reached 200 μg / mL, the cell viability was significantly reduced (approximately 60%). Therefore, a safe concentration of 150 μg / mL was selected for subsequent experiments.

[0050] 3.2 Determination of safe concentration of polyphenols; In order to determine the safe concentration of polyphenolic compounds, the relative survival rate of RAW 264.7 cells was used as an indicator to investigate the cytotoxic effects of allopurinol, PAH, PA, and OS. Figure 13 As shown in Figure A, compared with the control group (0 μg / mL), the relative cell survival rate decreased significantly after treatment with 25 μg / mL allopurinol; the survival rate of the 12.5 μg / mL group was not significantly different from that of the control group, so 12.5 μg / mL was selected as the safe concentration of allopurinol. Figure 13 As shown in Figure B, compared with the control group (0 μM), the relative survival rate of cells decreased significantly after treatment with 20 μM PAH; the survival rates of the 2.5, 5, and 10 μM groups were not significantly different from those of the control group, so 2.5, 5, and 10 μM were selected as the safe concentrations of PAH. Figure 13 As shown in Figure C, compared with the control group (0 μM), the relative survival rate of cells after treatment with 100 μM PA showed a downward trend; the survival rates of the 12.5, 25, and 50 μM groups were not significantly different from those of the control group, so 12.5, 25, and 50 μM were selected as the safe concentrations of PA. Figure 13 As shown in Figure D, compared with the control group (0 μM), the relative cell viability decreased significantly after treatment with 20 μM OS. The viability of the 2.5, 5, and 10 μM groups remained unchanged, so 2.5, 5, and 10 μM OS concentrations were selected as safe concentrations. These safe concentrations eliminate the potential for drug toxicity to interfere with cells and provide a suitable drug concentration basis for subsequent studies of inflammatory markers in cell models.

[0051] 3.3 Protective effects of polyphenols on acute gout cell model; To explore the protective effects of PAH, PA, and OS on acute gout cell models, the relative survival rate of RAW 264.7 cells was used as an indicator. Figure 14 As shown in Figures A-C, compared with the control group, the cell viability in the model group (MSU concentration 150 μg / mL) was significantly reduced to 59.93 ± 4.32%, successfully inducing an acute gout cell model. Compared with the model group, the addition of 2.5, 5, and 10 μM PAH, 12.5, 25, and 50 μM PA, and 2.5, 5, and 10 μM OS significantly increased cell viability. This indicates that PAH, PA, and OS can significantly enhance the survival rate of the MSU-induced acute gout cell model, exerting a protective effect against cell damage and laying the foundation for subsequent studies of anti-inflammatory mechanisms.

[0052] 3.4 Effects of polyphenols on ROS (reactive oxygen species) production in RAW264.7 cells; RAW 264.7 cells will cause oxidative stress due to MSU stimulation, produce excessive ROS, which may activate NLRP3 inflammasome and trigger gout attacks. Figure 15 As shown in Figures A-C, compared with the control group, the ROS content in the model group (MSU concentration of 150 μg / mL) was extremely significantly increased. Compared with the model group, the low, medium, and high concentrations of PAH (2.5, 5, and 10 μM), PA (12.5, 25, and 50 μM), and OS (2.5, 5, and 10 μM) in the treatment groups all significantly reduced ROS content, indicating that PAH, PA, and OS have the ability to resist oxidative stress and maintain body function balance.

[0053] 3.5 Effects of polyphenols on NO secretion in RAW 264.7 cells; NO is a biological signaling molecule that is widely present in cells and has important biological regulatory functions. It plays an important role in the process of inflammatory diseases. Figure 16 As shown in Figures A to C, compared with the control group, the model group (MSU concentration of 150 μg / mL) showed an extremely significant increase in NO content. Compared with the model group, low, medium, and high concentrations of PAH (2.5, 5, and 10 μM), PA (12.5, 25, and 50 μM), and OS (2.5, 5, and 10 μM) significantly or extremely significantly decreased NO content in the treatment groups, indicating that PAH, PA, and OS can inhibit NO release in the MSU-induced RAW 264.7 macrophage model.

[0054] 3.6 Effects of polyphenols on LDH (lactate dehydrogenase) release in RAW 264.7 cells; When cells are damaged or undergo apoptosis, the cell membrane permeability changes and LDH is released outside the cell and can be detected in the cell culture medium. Figure 17 As shown in Figures A to C, compared with the control group (Control), the LDH release of cells in the model group (MSU concentration 150 μg / mL) increased significantly ( P < 0.01), indicating that MSU can effectively damage the RAW 264.7 cell membrane and cause cell damage. Compared with the model group, LDH release was significantly reduced in the low, medium, and high concentrations of PAH (2.5, 5, and 10 μM), PA (12.5, 25, and 50 μM), and OS (2.5, 5, and 10 μM) treatment groups, indicating that PAH, PA, and OS can reduce cell damage in the acute gout model and improve cell survival.

[0055] 3.7 Effects of polyphenols on SOD (superoxide dismutase) activity in RAW 264.7 cells; As an important protective enzyme against oxidative damage, SOD can effectively remove superoxide anion free radicals, ensuring that cells are protected from oxidative damage and regulating the metabolic activity of organisms. Figure 18 As shown in Figures A-C, compared with the control group, the SOD activity in the model group (MSU concentration of 150 μg / mL) was extremely significantly decreased. Compared with the model group, the SOD activity in the low, medium, and high concentrations of PAH (2.5, 5, and 10 μM), PA (12.5, 25, and 50 μM), and OS (2.5, 5, and 10 μM) treatment groups was significantly or extremely significantly increased, indicating that PAH, PA, and OS can enhance SOD activity in the MSU-induced acute gout model in RAW 264.7 cells.

[0056] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for protecting an in vitro acute gout cell model based on polyphenolic compounds, characterized in that: The following steps are involved: RAW 264.7 cells were cultured in a medium containing 150 μg / mL sodium urate for 24 h to induce an acute gout cell model. Adding a polyphenol compound to the model cells, wherein the polyphenol compound is protocatechuic aldehyde, protocatechuic acid or porphyrone, and detecting at least one indicator of cell survival rate, ROS content, NO content, LDH release and SOD activity after culturing; The concentration of protocatechuic aldehyde is 2.5-10 μM, the concentration of protocatechuic acid is 12.5-50 μM, and the concentration of porphyrone is 2.5-10 μM.

2. The method according to claim 1, characterized in that The polyphenol compounds can significantly reduce the contents of ROS and NO in model cells, reduce the amount of LDH released, and significantly increase the activity of SOD.

3. The method according to claim 1, characterized in that The half-maximal inhibitory concentrations of the polyphenolic compounds on xanthine oxidase were: protocatechuic aldehyde 5.92 ± 0.40 mM, protocatechuic acid 26.25 ± 0.52 mM, and porphyrone 4.91 ± 0.42 mM.

4. The method according to claim 1, wherein The polyphenolic compounds inhibit xanthine oxidase in a reversible manner, wherein protocatechuic aldehyde and protocatechuic acid exhibit mixed inhibition, and porphyrone exhibits competitive inhibition.

5. The method according to claim 1, characterized in that The polyphenolic compounds are extracted from Inonotus obliquus by ultrasound-assisted extraction, and the extraction conditions are: solid-liquid ratio 1:40 g / mL, ultrasound time 30 min, and ultrasound power 280 W.

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