Pu'er tea polyphenol-polysaccharide composition and application thereof in improving insulin resistance

Abstract

The invention belongs to the technical field of medicines and health-care products, and particularly discloses a Pu'er tea polyphenol-polysaccharide composition and application thereof in improving insulin resistance. The compound consists of Pu'er tea polyphenol and polysaccharide, and can synergistically regulate and control glycolipid metabolism of HepG2 cells. According to the compound, by activating a PI3K-Akt signal channel, GSK3B kinase activity is inhibited, GYS1 expression is up-regulated, glycogen synthesis is promoted, and abnormal accumulation of glucose-6-phosphate is remarkably reduced; meanwhile, a glutamine metabolic axis is regulated and controlled, the overexpression of the GLS gene is inhibited, and the stable state of glutamine is recovered. According to the composition, the glucose consumption of insulin resistance model cells can be increased by 48.23%, the glycogen synthesis amount is increased by 27.62%, and lipid droplet deposition is reduced by 53.47%. The molecular mechanism of the polyphenol-polysaccharide composition for synergistically improving insulin resistance through a multi-target metabolism-gene network is disclosed for the first time, and a new scheme is provided for developing a natural product composition for treating diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and health care products, and particularly relates to a Pu'er tea polyphenol-polysaccharide composition and its application in improving insulin resistance and regulating glucose and lipid metabolism. Background Art

[0002] Insulin resistance (IR) is the core pathological basis of metabolic diseases such as type 2 diabetes mellitus (T2DM), obesity, and non-alcoholic fatty liver disease. It manifests as decreased insulin sensitivity in hepatocytes, leading to reduced glucose uptake, impaired glycogen synthesis, and abnormal lipid accumulation. According to the International Diabetes Federation, the number of people with diabetes worldwide reached 536.6 million in 2021, with over 140 million in China, and the prevalence is trending towards younger age. Targeted regulation of insulin resistance is a key scientific proposition for current treatment.

[0003] Pu'er tea is rich in bioactive ingredients such as polyphenols and polysaccharides. Studies have confirmed that individual components have the potential to regulate glucose and lipid metabolism and improve insulin resistance. For example, tea polyphenols can inhibit the activity of key gluconeogenic enzymes, while tea polysaccharides can promote insulin secretion. Pu'er tea has excellent metabolic regulatory abilities. Existing technologies have revealed the metabolic regulatory potential of individual active ingredients such as polyphenols and polysaccharides, but a complete understanding of the synergistic effects of multiple components within this natural complex system is lacking.

[0004] Current research on Pu'er tea has largely focused on evaluating the efficacy of individual components, such as tea polyphenols or tea polysaccharides, while ignoring the synergistic effects of components within a natural complex system. Furthermore, the molecular mechanisms of insulin resistance are complex, involving dynamic changes in signaling pathways (such as PI3K-Akt and AMPK), gene expression (such as SOCS3 and GYS1), and metabolites (such as glutamine and glucose-6-phosphate). Multi-omics technologies are urgently needed to uncover the underlying mechanisms of Pu'er tea polyphenol-polysaccharide combined intervention. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention aims to provide a Pu'er tea polyphenol-polysaccharide composition with a synergistic effect, clarify its optimal ratio and mechanism of action for improving insulin resistance, and provide a basis for the development of natural insulin resistance intervention drugs or health products.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one embodiment, the present invention provides a Pu'er tea polyphenol-polysaccharide composition, which comprises Pu'er tea polyphenols and Pu'er tea polysaccharides. Specifically, the concentration of Pu'er tea polyphenols is 12.5-240 μg / mL, the concentration of Pu'er tea polysaccharides is 12.5-120 μg / mL, and the composition is non-toxic to HepG2 cells.

[0008] Preferably, the ratio of Pu'er tea polyphenols to Pu'er tea polysaccharides is a combination of 180-220 μg / mL Pu'er tea polyphenols and 80-120 μg / mL Pu'er tea polysaccharides, and more preferably a combination of 200 μg / mL Pu'er tea polyphenols and 100 μg / mL Pu'er tea polysaccharides.

[0009] In one embodiment, the present invention provides a method for screening a Pu'er tea polyphenol-polysaccharide composition for improving insulin resistance, comprising the following steps:

[0010] (1) The toxicity of Pu'er tea polyphenols and Pu'er tea polysaccharides at different concentrations on HepG2 cells was detected by CCK-8 assay, and the safe concentration range was determined to be 12.5-200 μg / mL for Pu'er tea polyphenols and 12.5-100 μg / mL for Pu'er tea polysaccharides;

[0011] (2) Establishing an insulin-resistant HepG2 cell model using continuous stimulation with palmitic acid;

[0012] (3) Pu'er tea polyphenol-polysaccharide compositions with different concentrations are applied to the model cells, and the composition with the best ratio is screened out by detecting glucose consumption, glycogen synthesis and oil red O stained lipid droplet area.

[0013] In another embodiment, the present invention also includes the glucose consumption detection by measuring the glucose concentration in the cell culture medium using a glucose kit, the glycogen synthesis detection by measuring the glycogen content in the cell pellet using a glycogen determination kit, and the Oil Red O staining by quantifying the lipid droplet area using Image J software.

[0014] In another embodiment, the present invention further comprises that the palmitic acid concentration is 150 μM.

[0015] In one embodiment, the present invention provides a use of a Pu'er tea polyphenol-polysaccharide composition in the preparation of a medicine or health product for improving insulin resistance, wherein the composition can significantly increase glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells and reduce intracellular lipid accumulation.

[0016] In another embodiment, the present invention further comprises the composition regulating glycogen synthesis via the PI3K-Akt / GSK3B-GYS1 signaling axis.

[0017] In one embodiment, the present invention provides a method for detecting the effect of a Pu'er tea polyphenol-polysaccharide composition on improving insulin resistance, comprising detecting the following indicators:

[0018] (1) Transcriptomic indicators: differential expression of SOCS3, PCK1, PCK2, and GYS1 as differentially expressed genes;

[0019] (2) Metabolomics indicators: changes in the levels of glutamine, glucose-6-phosphate, fructose-6-phosphate, and uridine diphosphate-N-acetylglucosamine as differential metabolites;

[0020] The differentially expressed genes and differential metabolites were identified by RNA-seq sequencing and LC-MS / MS mass spectrometry analysis.

[0021] In one embodiment, the present invention provides a method for preparing a Pu'er tea polyphenol-polysaccharide composition, comprising:

[0022] (1) Pu'er tea is used as raw material, and high-purity Pu'er tea polyphenols are obtained through hot water extraction, ethyl acetate extraction, macroporous resin purification and dichloromethane decaffeination;

[0023] (2) subjecting the extracted aqueous phase to ethanol precipitation, washing, and vacuum drying to obtain Pu'er tea polysaccharides;

[0024] (3) The Pu'er tea polyphenols and polysaccharides are mixed in proportion to prepare a composition.

[0025] In another embodiment, the present invention also includes that the Pu'er tea is made from Yunnan large-leaf sun-dried green tea, the obtained Pu'er tea polyphenol purity is 85.93%, the Pu'er tea polysaccharide purity is 54.19%, and the Pu'er tea polyphenols and polysaccharides are mixed in a ratio of 12.5-200 μg / mL: 12.5-100 μg / mL to prepare a composition.

[0026] The beneficial effects achieved by the present invention are:

[0027] (1) Dual improvement of glucose and lipid metabolism: The optimal combination of Pu'er tea polyphenols (200 μg / mL) and polysaccharides (100 μg / mL) was verified by an in vitro HepG2 cell model to significantly increase glucose consumption (48.23% higher than the model group) and glycogen synthesis (27.62% higher) in insulin-resistant cells, and reduce lipid droplet deposition (the oil red O staining area decreased by 53.47%). The combination achieves multi-dimensional intervention on insulin resistance by regulating the insulin receptor signaling pathway, inhibiting gluconeogenesis (downregulating PCK1 / PCK2), promoting glycogen synthesis (upregulating GYS1) and improving lipid metabolism (reducing lipid droplet accumulation), thus making up for the limitations of a single component. The composite system has a significantly better regulatory effect on glucose and lipid metabolism than a single component, reflecting the synergistic effect of polyphenols and polysaccharides.

[0028] (2) The present invention clearly defines molecular targets and signal pathway regulation. 1,849 differentially expressed genes were screened by RNA-seq, enriched in the "insulin signaling pathway" and "insulin resistance pathway", including key genes such as SOCS3 (down-regulated, relieving insulin signal inhibition), GYS1 (up-regulated, promoting glycogen synthesis), PCK1 / PCK2 (down-regulated, inhibiting gluconeogenesis), etc., providing direct targets for drug development. In addition, 17 key differential metabolites were identified, which inhibit the activity of the hexosamine pathway and reduce the production of uridine diphosphate-N-acetylglucosamine by regulating the metabolism of amino acids such as glutamine and serine and the level of phosphate sugars (such as glucose-6-phosphate), thereby improving energy metabolism disorders associated with insulin resistance.

[0029] (3) The present invention has the safety and applicability of natural origin. CCK-8 detection determined that polyphenols (12.5-200 μg / mL) and polysaccharides (12.5-100 μg / mL) were non-cytotoxic when used alone or in combination, providing a safety basis for subsequent in vivo experiments and product development. Compared with chemically synthesized drugs, Pu'er tea polyphenol-polysaccharide compositions are derived from natural medicinal and edible resources, have controllable side effects, and have high biocompatibility. They are suitable for the development of functional foods or health products, and are particularly suitable for long-term intervention in insulin resistance and related metabolic diseases.

[0030] (4) This invention illustrates for the first time that Pu'er tea polyphenols and polysaccharides improve insulin resistance by synergistically regulating sugar metabolism and amino acid metabolism in parallel, providing a new natural product intervention strategy for the prevention and treatment of metabolic diseases such as type 2 diabetes and obesity.

[0031] (5) The present invention has multi-field application potential. The composition prepared by the present invention can be used to prepare drugs or functional foods that improve insulin resistance and reduce the risk of diabetes and complications by regulating hepatocyte glucose and lipid metabolism. A method for screening multi-component synergistic systems from natural products (such as CCK-8 toxicity detection and multi-omics analysis) is provided, which is suitable for the development of other medicinal and edible resources and promotes natural product research in the field of metabolic disease prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1a .Effects of Pu'er tea polyphenols and polysaccharides on the activity of HepG2 cells.

[0033] Figure 1b .Effects of Pu'er tea polyphenols and polysaccharides combined on the activity of HepG2 cells;

[0034] Figure 1c .Establishment and identification of the HepG2 cell insulin resistance model, where (A) shows the effect of different concentrations of PA treatment on HepG2 cell viability, and (B) is the comparison of glucose consumption between normal group cells and insulin resistance model cells.

[0035] Figure 2a .The effect of Pu'er tea polyphenols-polysaccharides on the lipid content in HepG2 cells was observed after Oil Red O staining.

[0036] Figure 2b Quantification of Oil Red O staining area.

[0037] Figure 3 Analysis of the number of differentially expressed genes among the Pu'er tea polyphenol-polysaccharide group, model group, and control group: (A) is a bar chart showing the number of differentially expressed genes between the Pu'er tea polyphenol-polysaccharide group and the model group, and between the model group and the control group; (B) is a Venn diagram showing the comparative analysis of differentially expressed genes between the model group / control group and the Pu'er tea polyphenol-polysaccharide group / model group; (C) is a cluster diagram showing the differentially expressed genes between the Pu'er tea polyphenol-polysaccharide group and the model group.

[0038] Figure 4a .GO Biological Process enrichment analysis results.

[0039] Figure 4b .GO Cellular Component enrichment analysis results.

[0040] Figure 4c .GO Molecular Function enrichment analysis results.

[0041] Figure 5 Bubble chart of KEGG enrichment analysis of differentially expressed genes in the Pu'er tea polyphenol-polysaccharide group and the model group.

[0042] Figure 6 PCA analysis chart of the control group, model group and Pu'er tea polyphenol-polysaccharide treatment group.

[0043] Figure 7 OPLS-DA score graphs: (A) is the OPLS-DA score graph of the control group and the model group; (B) is the OPLS-DA score graph of the model group and the Pu'er tea polyphenol-polysaccharide treatment group.

[0044] Figure 8 Heat map of differential metabolites between the model group and the Pu'er tea polyphenol-polysaccharide group.

[0045] Figure 9 KEGG enrichment analysis diagram of the model group and control group.

[0046] Figure 10 KEGG enrichment analysis diagram of Pu'er tea polyphenol-polysaccharide group and model group.

[0047] Figure 11 Correlation analysis of differentially expressed genes and metabolites in IR-HepG2 cells regulated by Pu'er tea polyphenols-polysaccharides. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to specific examples, but the present invention is not limited by the examples.

[0049] Unless otherwise specified, the experimental materials, reagents, instruments and methods used in the following examples are conventional experimental materials, reagents, instruments and methods in the art and can be purchased through commercial channels.

[0050] Example 1: Preparation of Pu'er Tea Polyphenol-Polysaccharide Composition and Determination of the Safe Range and Optimal Ratio of the Composition

[0051] 1.1 Extraction of Pu'er Tea Polyphenols and Pu'er Tea Polysaccharides

[0052] Raw material pretreatment: Using 2022 Yunnan Jingdong County Pu'er ripe tea as raw material, use 90℃ hot water at a material-liquid ratio of 1:10 to soak for 2.25 hours to fully extract the active ingredients in the tea.

[0053] Primary separation: The extract is filtered and passed through a 400-mesh sieve to remove tea residue and large particles. The filtrate is concentrated under reduced pressure at 50-60°C to retain the activity of heat-sensitive substances.

[0054] Polyphenol purification: The concentrate is extracted with ethyl acetate, and the organic phase is retained after separation. The organic phase is gradient eluted with a macroporous adsorption resin (HP20) to remove pigments and small molecular impurities. To further improve the purity of tea polyphenols, dichloromethane is used for decaffeination. The tea polyphenol sample is dissolved in an appropriate amount of water, and dichloromethane is added (volume ratio 1:1) and mixed thoroughly. The liquid is allowed to stand and separate, and the lower organic phase (containing caffeine) is discarded, and the upper aqueous phase is retained. Repeat the above steps twice until the caffeine is completely removed. The aqueous phase is vacuum dried to obtain tea polyphenols. The purity of tea polyphenols is determined by high-performance liquid chromatography (HPLC), and a Pu'er tea polyphenol sample with a purity of 85.93% is finally obtained.

[0055] Polysaccharide Extraction: Ethanol was added to the aqueous phase after extraction to precipitate the polysaccharides, and the precipitate was collected by centrifugation. The precipitate was washed twice with anhydrous ethanol, acetone, and ether, alternating to remove residual lipids and tannins. The washed precipitate was vacuum-dried to obtain tea polysaccharides. The purity of the tea polysaccharides was determined by UV-Vis spectrophotometry, resulting in a Pu'er tea polysaccharide sample with a purity of 54.19%.

[0056] 1.2 Establishment of a HepG2 insulin resistance cell model induced by palmitic acid (PA)

[0057] First, the solution was prepared, and the complete culture medium was DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0058] Palmitic acid solution: Add 0.065 g of palmitic acid to 2.5 mL of 0.1 M NaOH and dissolve thoroughly in a 70°C water bath. Then, add 1 mL dropwise to 10 mL of 10% BSA solution in a 37°C water bath to obtain a 10 nM palmitic acid solution. Sterilize by filtering through a 0.22 μm filter and store at 4°C until needed.

[0059] Metformin solution: Weigh 0.083 g of metformin hydrochloride powder into 10 mL of pure water, dissolve thoroughly, and mix to obtain a 50 mM metformin stock solution. Sterilize the solution by filtering with a 0.22 μm filter and store at 4°C until use.

[0060] Cell Thawing and Culture: Remove frozen HepG2 cells from liquid nitrogen and quickly place in a preheated 37°C water bath for approximately 1 minute to thaw. Centrifuge at 1000 rpm for 5 minutes. After transferring to a clean bench, discard the cryopreservation solution and add 1 mL of complete culture medium. Pipet the cells evenly with a pipette. Transfer to a fresh dish containing complete culture medium and shake horizontally in a cross-section. Place in a cell culture incubator for culture. HepG2 cells are cultured in a 37°C incubator with 5% CO2 until the logarithmic growth phase. Change the culture medium based on cell status. Passage or other procedures are performed when the cell density reaches 80-90%.

[0061] Cell passaging: When the cell density reaches 80-90%, discard the original culture medium, wash twice with PBS buffer, add 1mL of trypsin digestion solution, and digest for 2-3 minutes in a 37°C cell culture incubator (adjust the digestion time according to the cell digestion status). When the cells are observed to shrink and round under a microscope and fall off the cell dish, immediately add 1mL of complete culture medium to terminate the digestion. Use a pipette tip to pipette evenly and transfer the suspension to a centrifuge tube. Centrifuge at 1000rpm for 5 minutes. After centrifugation, discard the supernatant, add 2mL of complete culture medium and pipette evenly. Inoculate the cells into two new cell culture dishes at a passage ratio of 1:2, shake them horizontally and place them in the incubator.

[0062] Cell cryopreservation: After cells have grown to a density of 80-90%, wash them twice with PBS buffer, digest them according to the subculture process, centrifuge them, add serum-free freezing solution and evenly resuspend the cells. Transfer them to cryopreservation tubes, mark them with information, and place them in a -80℃ freezer overnight. The next day, transfer them to a liquid nitrogen tank for long-term storage.

[0063] CCK-8 assay for cytotoxicity of Pu'er tea polyphenols and polysaccharides against HepG2 cells: The CCK-8 kit can be used to monitor cell proliferation and cytotoxicity. It utilizes mitochondrial dehydrogenases to reduce WST-8 in the CCK-8 reagent to an orange-yellow formazan product. The color of the formazan product is proportional to the number of viable cells. The OD value, measured at a wavelength of 450 nm using a microplate reader, indirectly reflects cell viability and metabolic activity. After HepG2 cells reached the logarithmic growth phase, a cell suspension was prepared and seeded at a density of 5,000 cells / well in 96-well plates, with six replicates per group. After incubation at 37°C and 5% CO₂ until cells adhered, the culture medium was discarded and the cells were treated with varying concentrations of Pu'er tea polyphenols and polysaccharides. A control group received complete culture medium without Pu'er tea polyphenols and polysaccharides. After incubation for 24 h, the original culture medium was discarded, and 90 μL of new culture medium and 10 μL of CCK-8 solution were added to each well. The cells were cultured in a cell culture incubator in the dark for 2 h, and the absorbance at 450 nm was measured using a microplate reader.

[0064] Establishment and characterization of a HepG2 cell insulin resistance model: The pre-prepared palmitic acid solution was diluted with culture medium to 100, 150, 200, 250, and 300 μM. The effects of different concentrations of PA solution on HepG2 cell viability were tested according to the method in 2.1.2.5. After the HepG2 cells reached the logarithmic growth phase, a cell suspension was prepared and seeded into a 24-well plate. The cells were divided into a normal group (without insulin), a normal group (with insulin), a model group (without insulin), and a model group (with insulin), with six replicate wells per group. After culturing at 37°C and 5% CO2 until the cells adhered, serum-free medium was replaced and starved for 12 hours. The medium was then discarded, and the normal group cells were treated with complete medium for 24 hours, and the model group cells were treated with complete medium containing 150 μM PA for 24 hours. The original medium was then discarded, and the cells were washed twice with PBS. DMEM medium with / without insulin was added for 30 minutes. The culture medium of each well was taken, and the glucose consumption of each group of cells was determined using a glucose kit to determine whether the insulin resistance model was successfully established.

[0065] Effects of Pu'er Tea Polyphenols and Polysaccharides on Glucose Consumption in IR-HepG2 Cells: After HepG2 cells were cultured to the logarithmic growth phase, an IR-HepG2 cell model was established according to the above method. After setting up two groups, the control and model groups, the remaining groups were treated with metformin, 200μg / mL TP, 100μg / mL TPS, 100μg / mL TP + 100μg / mL TPS, 50μg / mL TP + 100μg / mL TPS, 33μg / mL TP + 100μg / mL TPS, 200μg / mL TP + 100μg / mL TPS, and 200μg / mL TP + 66μg / mL TPS, respectively. Six replicates were set up for each group. After 24 hours of intervention, the cell culture medium from each well was collected and 2μL was added to each well into a 96-well plate. A distilled water group and a standard group were also set up. 200 μL of glucose kit working solution was added to the above 96-well plate, incubated in a 37°C incubator for 10 min, and the absorbance at 505 nm was measured using a microplate reader.

[0066] Glucose concentration = (sample OD - blank OD) / (standard OD - blank OD) × standard concentration

[0067] Glucose consumption = DMEM medium glucose concentration - sample glucose concentration

[0068] Effects of Pu'er tea polyphenols and polysaccharides on glycogen synthesis in IR-HepG2 cells: After HepG2 cells were cultured to the logarithmic growth phase, an IR-HepG2 cell model was established according to the above method. Two groups, control and model, were set up, and the rest of the cells were treated with metformin, 200 μg / mL TP, 100 μg / mL TPS, 100 μg / mL TP+100 μg / mL TPS, 50 μg / mL TP+100 μg / mL TPS, 33 μg / mL TP+100 μg / mL TPS, 200 μg / mL TP+100 μg / mL TPS, and 200 μg / mL TP+66 μg / mL TPS, with 6 replicates per group. After 24 hours of intervention, cells were harvested by trypsin digestion to prepare a cell suspension. The suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cell pellet was washed with buffer and then re-centrifuged to collect the cell pellet. 0.225 mL of alkaline solution was added to each tube, and the suspension was in a boiling water bath for 20 minutes. After cooling with running water, 0.225 mL of double-distilled water was added to prepare the glycogen detection solution. After the sample addition reaction was performed according to Table 1, a blank tube was used for zero adjustment, and the absorbance of each tube was measured at 620 nm.

[0069] Glycogen content (mg) = (absorbance of the assay tube ÷ absorbance of the standard tube) × concentration of the standard × 10 ÷ 1.11 × total volume of the glycogen test solution

[0070] Table 1. Glycogen content detection sample addition table

[0071] Blank tube Standard tube Assay tube Double distilled water / mL 1.0 - 0.9 Standard solution / mL - 1.0 - Glycogen test solution / mL - - 0.1 Color development solution / mL 2 2 2

[0072] Oil Red O staining: After HepG2 cells reached the logarithmic growth phase, an IR-HepG2 cell model was established according to the method in 2.1.2.6. Two control and model groups were established. The remaining cells were treated with metformin, 200 μg / mL TP, 100 μg / mL TPS, or 200 μg / mL TP + 100 μg / mL TPS, respectively, with three replicates per group. After 24 hours of treatment, the culture medium was discarded, and each well was gently washed twice with PBS. Each well was then fixed with 4% paraformaldehyde for 30 minutes. Following fixation, the cells were gently washed twice with PBS, followed by a rinse in 60% isopropanol. After the rinse, the isopropanol was discarded, and Oil Red O staining solution was added to each well for 10 minutes. The Oil Red O was discarded, and the cells were rinsed again with 60% isopropanol. The isopropanol was discarded, PBS was added to each well, and the cells were observed under a microscope and photographed.

[0073] Data processing: All experimental data are expressed as mean ± standard deviation (x ± SD). Statistical analysis was performed using SPSS software, and graphs were constructed using GraphPad Prism software. Data between multiple groups were analyzed using one-way analysis of variance (ANOVA), and data between two groups were analyzed using the T-test. P < 0.05 indicated a statistically significant difference, and P < 0.01 indicated a statistically significant difference.

[0074] The results showed that Pu'er tea polyphenols (0, 12.5, 25, 50, 100, 200, 400, 600, 800 μg / mL) and Pu'er tea polysaccharides (0, 12.5, 25, 50, 100, 200, 400, 600, 800 μg / mL) were added to HepG2 cells that were cultured to stably adhere to the wall. After incubation for 24 hours, the OD value at 450 nm was measured using the CCK-8 method to determine the cell viability. Figure 1aAs shown in the figure, compared with the control group, in the Pu'er tea polyphenols group, within the range of 12.5-200 μg / mL, the relative cell viability did not change significantly with increasing concentrations; however, when the concentration reached 400 μg / mL, the HepG2 cell activity decreased significantly compared with the blank group; in the Pu'er tea polysaccharide group, within the range of 12.5-100 μg / mL, the relative cell viability did not change significantly with increasing concentrations; however, when the concentration reached 200 μg / mL, the HepG2 cell activity decreased significantly compared with the blank group. This shows that Pu'er tea polyphenols within the concentration range of 12.5-200 μg / mL and Pu'er tea polysaccharides within the concentration range of 12.5-100 μg / mL are non-toxic to HepG2 cells and can be used for subsequent experiments. To investigate the cytotoxicity of Pu'er tea polyphenols and polysaccharides when acting synergistically, we set low, medium, and high doses of Pu'er tea polyphenols and polysaccharides within their respective non-toxic concentration ranges and measured cell viability at ratios of 1:1, 1:2, 1:3, 2:1, and 3:1. Figure 1b As shown in the figure, compared with the control group, there was no significant change in the relative cell viability of the 15 different Pu'er tea polyphenols and polysaccharide synergistic groups, indicating that the Pu'er tea polyphenols and polysaccharide synergistic groups were non-toxic to HepG2 cells in the selected concentration combination. Therefore, high doses (100μg / mL TP+100μg / mL TPS, 50μg / mL TP+100μg / mL TPS, 33μg / mL TP+100μg / mL TPS, 200μg / mL TP+100μg / mL TPS, 200μg / mL TP+66μg / mL TPS) were selected from 5 different ratios as synergistic groups for subsequent experiments.

[0075] To further determine the optimal PA concentration for inducing insulin resistance in HepG2 cells in this study, the effects of different concentrations of PA on HepG2 cell viability were examined. Figure 1c As shown, after 24 hours of PA treatment (100-300 μM), cell viability decreased from 100.00±7.11% to 46.83±5.46%, with a dose-dependent inhibition of cell activity. Because 200-300 μM PA significantly inhibited cell viability, a 24-hour treatment with 150 μM PA was used to induce a cell insulin resistance model.

[0076] The glucose oxidase method was used to determine the glucose consumption of cells in the normal group and the model group. On this basis, insulin (Ins) was added to verify whether the HepG2 cell insulin resistance model was successfully established. Figure 1cAs shown in the results, after 24 hours of PA treatment, the glucose consumption of the model group cells was significantly decreased compared with the normal control group. After 30 minutes of Ins stimulation, the glucose consumption of the normal control group increased significantly, but there was no significant difference in glucose consumption in the model group after the addition of Ins, and the difference was significant compared with the normal cells added with Ins. These results indicate that the insulin resistance HepG2 cell model was successfully established.

[0077] A glucose kit was used to measure the effects of different Pu'er tea polyphenol-polysaccharide groups on glucose consumption in IR-HepG2 cells. The experimental results are shown in Table 2. Compared with the normal control group, the glucose consumption of the model group cells was significantly decreased, indicating that the cell model was successfully established. The glucose consumption of the metformin group cells was significantly increased compared with the model group cells, and there was no significant difference compared with the control group, indicating that metformin significantly increased the glucose consumption of IR-HepG2 cells. Compared with the model group, the glucose consumption of the Pu'er tea polyphenol-polysaccharide group cells was significantly increased, among which the glucose consumption of the 200μg / mL TP+100μg / mL TPS synergistic group changed most significantly, and there was no significant difference between the control group and the metformin group, indicating that this group has a similar effect as metformin in improving the glucose consumption of IR-HepG2 cells.

[0078] Table 2 Effects of Pu'er tea polyphenols and polysaccharides on glucose consumption in IR-HepG2 cells

[0079] Group Glucose consumption / mM Normal group <![CDATA[3.519±0.109 ## ]]> Model Group <![CDATA[2.351±0.037** ΔΔ ]]> Metformin group <![CDATA[3.597±0.185 ## ]]> 200 μg / mL tea polyphenols <![CDATA[3.116±0.124** ##ΔΔ ]]> 100 μg / mL tea polysaccharides <![CDATA[3.276±0.097* ##Δ ]]> 100μg / mL tea polyphenols + 100μg / mL tea polysaccharides <![CDATA[2.976±0.146** ##ΔΔ ]]> 50μg / mL tea polyphenols + 100μg / mL tea polysaccharides <![CDATA[2.962±0.063** ##ΔΔ ]]> 33μg / mL tea polyphenols + 100μg / mL tea polysaccharides <![CDATA[2.990±0.149** ##ΔΔ ]]> 200μg / mL tea polyphenols + 100μg / mL tea polysaccharides <![CDATA[3.485±0.108 ## ]]> 200μg / mL tea polyphenols + 66μg / mL tea polysaccharides <![CDATA[2.692±0.201** #ΔΔ ]]>

[0080] Note: Compared with the normal group, *P<0.05; compared with the normal group, **P<0.01; compared with the model group, #P<0.05; compared with the model group, ##P<0.01; compared with the metformin group, △P<0.05; compared with the metformin group, △△P<0.01, the same below.

[0081] A glycogen assay kit was used to measure the effects of different Pu'er tea polyphenol-polysaccharide groups on glycogen synthesis in IR-HepG2 cells. The experimental results are shown in Table 3. Compared with the control group, the glycogen synthesis of the model group cells was significantly decreased. Compared with the model group cells, the metformin group and the other Pu'er tea polyphenol-polysaccharide groups except 100μg / mL TPS and 200μg / mL TP + 66μg / mL TPS significantly increased the glycogen synthesis of IR-HepG2 cells. From a numerical point of view, the synergistic Pu'er tea polyphenol / polysaccharide group of 200μg / mL TP + 100μg / mL TPS improved the glycogen synthesis of IR-HepG2 cells most significantly, and was closest to the control group.

[0082] Table 3 Effects of Pu'er tea polyphenols and polysaccharides on glycogen synthesis in IR-HepG2 cells

[0083]

[0084] Oil red O staining was used to observe the lipid accumulation in cells. Figure 2a As shown in the figure, there are a few oil droplets gathered around the cells in the normal group, and the distribution is scattered and the number is small; the number of oil droplets in the cells of the model group induced by PA is significantly increased and densely distributed, indicating that PA induces fatty degeneration of the cells. Compared with the model group, the number and size of oil droplets in IR-HepG2 cells treated with metformin, 200μg / mL TP, 100μg / mL TPS, and 200μg / mL TP+100μg / mL TPS have a decreasing trend to varying degrees. The area of the stained area was quantified using Image J software. Figure 2b As can be seen, compared with the normal group, the staining area of the model group increased significantly, while the staining area of IR-HepG2 cells treated with metformin, 200 μg / mL TP, and 200 μg / mL TP+100 μg / mL TPS decreased significantly compared with the model group, and had no significant change compared with the normal group. The staining area of IR-HepG2 cells treated with 100 μg / mL TPS showed no obvious downward trend and had no significant change compared with the model group.

[0085] CCK-8 assays revealed that Pu'er tea polyphenols within the 12.5-200 μg / mL concentration range, and Pu'er tea polysaccharides within the 12.5-100 μg / mL concentration range, were non-toxic to HepG2 cells. Furthermore, the combination of 200 μg / mL Pu'er tea polyphenols and 100 μg / mL Pu'er tea polysaccharides was also non-toxic to HepG2 cells. Insulin resistance (IR-HepG2) was induced in HepG2 cells using a 150 μM PA concentration combined with 24-hour treatment. Treatment with different ratios of Pu'er tea polyphenols and polysaccharides significantly increased glucose consumption and glycogen synthesis, while Oil Red O staining area was significantly reduced in all treatment groups. The combination of 200 μg / mL TP and 100 μg / mL TPS had the most significant improvement: glucose consumption increased by 48.23%, glycogen synthesis increased by 27.62%, and Oil Red O staining area decreased by 53.47% compared to the model group. This study indicates that Pu'er tea polyphenols and polysaccharides can alleviate insulin resistance by improving glucose and lipid metabolism. This study clarifies the functional characteristics of the polyphenol-polysaccharide complex system in alleviating insulin resistance by synergistically regulating glucose and lipid metabolism homeostasis, providing key experimental evidence for subsequent analysis of the mechanism of action.

[0086] Example 2: Transcriptome analysis of the effect of Pu'er tea polyphenols and polysaccharides on improving insulin resistance in HepG2 cells

[0087] The human hepatocellular carcinoma cell line (HepG2) used in this experiment was purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences.

[0088] Total RNA extraction from cells: Cell samples from the control group, model group, and Pu'er tea polyphenol-polysaccharide treatment group (three biological replicates for each group) were placed in EP tubes. After collecting the cell pellet, 350 μL of Buffer RLT was added (10 μL of β-ME was added to every 1 mL of Buffer RLT before use). The tubes were pipetted and mixed to dissolve the cells. The tubes were allowed to stand at room temperature for 5 minutes. Add 1 times the volume of supernatant solution of 75% ethanol solution and mix thoroughly with a pipette tip; pass the entire sample into an RNeasy MinElute spin column with a 2 mL collection tube, centrifuge at 10,000 × g at room temperature for 15 seconds, and discard the waste liquid; add 350 μL RW1, centrifuge at 10,000 × g at room temperature for 15 seconds, and discard the waste liquid; add a mixture of 10 μL DNase I and 70 μL Buffer RDD and let it stand at room temperature for 15 minutes; add 350 μL RW1, centrifuge at 10,000 × g at room temperature for 15 seconds, and discard the waste liquid; add 500 μL RPE, centrifuge at 10,000 × g at room temperature for 15 seconds, and discard the waste liquid; add 500 μL RPE again, centrifuge at 10,000 × g at room temperature for 15 seconds, and discard the waste liquid; transfer the RNeasy MinElute spin column to a new collection tube and centrifuge at full speed for 5 minutes at room temperature; Transfer the column to a new 1.5 mL centrifuge tube, add 20 μL RNase-free water, and centrifuge at full speed at room temperature to elute RNA.

[0089] Transcriptome library construction and sequencing: The RNA sample is denatured at an appropriate temperature to open its secondary structure, and oligo (dT) magnetic beads are used to enrich mRNA; Frag / Primer Buffer is added and reacted at an appropriate temperature for a certain time to obtain fragmented mRNA; the first-strand / second-strand synthesis reaction system is prepared respectively, and the reaction program is set to synthesize the first-strand / second-strand cDNA; the double-stranded cDNA end is repaired and an A base is added to the 3' end; the adapter ligation reaction system is prepared and the reaction program is set to connect the adapter to the cDNA; the PCR reaction system is prepared, the product is amplified, and the library is constructed; after the library quality is tested, the DNA sequence is sequenced based on DNBSEQ. TM Sequencing platform.

[0090] Sequencing data quality control: After sequencing, raw data (Raw Reads) are obtained. Raw data from sequencing may contain reads of low quality, adapter contamination, and excessive N content of unknown bases. These reads must be removed before data analysis to ensure the reliability of the results. SOAPnuke software is used to perform quality control on raw data and generate clean reads.

[0091] Sequence genome alignment: Obtain the gene sequence to be aligned from the NCBI database, and use HISAT2 to align CleanReads with the reference genome sequence. For samples with good sample quality and sufficient sequencing data, most transcripts will be fully covered, and reads will be evenly distributed in various regions of the transcript.

[0092] Gene expression analysis: FPKM is a commonly used normalization method for normalizing gene expression levels in high-throughput sequencing data. After obtaining the number of reads for each gene (i.e., gene expression level) using HTSeq, FPKM is used to normalize it, making gene expression levels comparable across samples and providing a reliable data foundation for further differential expression analysis and functional studies.

[0093] Differentially expressed genes were screened using the DESeq2 method, with q value (adjusted p-value) < 0.05 and log2FC > 2 or log2FC < -1 as the threshold for screening differentially expressed genes.

[0094] GO enrichment analysis of differentially expressed genes: All differentially expressed genes were mapped to the entries of the Gene Ontology database (http: / / www.geneontology.org / ), the number of genes in each entry was calculated, and then the hypergeometric test was applied to find the significantly enriched GO entries. The basic function phyper in the R language package was used.

[0095] (https: / / stat.ethz.ch / R-manual / R-devel / library / stats / html / Hypergeometric.html) Calculate p-value. Perform multiple testing correction. The correction software package is q-value

[0096] (https: / / bioconductor.org / packages / release / bioc / html / qvalue.html). Finally, q-value (corrected p-value) ≤ 0.05 was used as the threshold, and GO terms that met this condition were defined as significantly enriched GO terms.

[0097] KEGG enrichment analysis of differentially expressed genes: Pathway significant enrichment analysis uses KEGG pathways as units and applies hypergeometric tests to identify significantly enriched pathways. The R language package phyper function was used.

[0098] (https: / / stat.ethz.ch / R-manual / R-devel / library / stats / html / Hypergeometric.html) Calculate p-value. Perform multiple testing correction. The correction software package is q-value

[0099] (https: / / bioconductor.org / packages / release / bioc / html / qvalue.html). Finally, a q-value (corrected p-value) ≤ 0.05 was used as the threshold, and pathways that met this condition were defined as pathways that were significantly enriched in differentially expressed genes.

[0100] To further investigate the effects of Pu'er tea polyphenols and polysaccharides on insulin resistance in HepG2 cells, RNA-seq was used to analyze the transcriptome of HepG2 cells in the control, model, and Pu'er tea polyphenols and polysaccharide-treated groups. To ensure sequencing data quality, preprocessing was performed, and quality analysis results were obtained. Details are shown in Table 4. Cleanbases refers to the total amount of sequencing data after quality control. This value is calculated by multiplying the number of clean reads by the read length. Base quality is an important indicator of sequencing quality. Q20 represents the percentage of bases with a sequencing quality of 99% or higher, generally exceeding 85%. Q30 represents the percentage of bases with a sequencing quality of 99.9% or higher, generally exceeding 80%. In this study, Q20 values for all samples exceeded 97%, and Q30 values exceeded 92%. GC content reflects the base distribution. In this study, the GC content of each sample remained around 50%, indicating a relatively uniform base distribution. Total mapping is a key metric in transcriptome analysis, representing the percentage of clean reads that can be mapped to the genome. Generally speaking, an alignment rate of 65% or higher is considered ideal. In this study, clean reads from each sample were aligned to the reference genome, and the results showed a Total mapping range of 97.78% to 97.99%. This result strongly demonstrates that the reference genome used for this sequencing was appropriate and that no contamination or other interference was present during the experiment. In addition to Total mapping, Unique mapping is also an important parameter for evaluating alignment results. Unique mapping indicates the percentage of clean reads with unique mapping positions on the reference sequence. In this study, Unique mapping ranged from 89.46% to 89.99% for each sample. These data demonstrate the high validity of the sequencing data and the good match between the selected reference genome and the sequencing data, providing a reliable foundation for further transcriptome analysis. Therefore, further analysis is feasible.

[0101] Table 4. Sequencing data quality analysis and sequence alignment results

[0102]

[0103] The differentially expressed genes between the groups were screened using q-value < 0.05 and |log2FoldChange| > 2 as the thresholds for differentially expressed genes. Figure 3As shown, a total of 1270 differentially expressed genes were screened out between the control group and the model group, of which 645 were upregulated and 625 were downregulated. At the same time, a total of 1849 differentially expressed genes were screened out between the model group and the Pu'er tea polyphenol-polysaccharide group, of which 1076 differentially expressed genes were upregulated and 773 differentially expressed genes were downregulated. Statistical analysis of the differentially expressed genes between the model / control group and the Pu'er tea polyphenol-polysaccharide / model group was performed, and 1034 differentially expressed genes overlapped ( Figure 3 ), accounting for 81.42% of the differentially expressed genes in the model / control group, indicating that Pu'er tea polyphenols-polysaccharides have a significant regulatory effect on the gene expression dynamics of HepG2 cells with insulin resistance. Cluster analysis was performed on the 1849 differentially expressed genes screened from the PTE and MC groups, such as Figure 3 As shown in the figure, differentially expressed genes showed consistent expression patterns within the groups, but formed a clear boundary between the groups, reflecting significant differences between the two groups. Among the differentially expressed genes in the PTE group, the number of up-regulated genes was predominant.

[0104] In order to further study the effects of Pu'er tea polyphenols and polysaccharides on biological processes, we conducted Gene Ontology (GO) enrichment analysis on the differentially expressed genes obtained. In this part of the study, we conducted enrichment analysis on the GO terms of biological process (BP), cellular component (CC) and molecular function (MF), respectively. The number of differentially expressed genes exceeded 2 and the Q value was less than 0.05. The results of BP enrichment were summarized and a bubble chart was drawn. Figure 4a As shown in Figure 2, the differentially expressed genes between the Pu'er tea polyphenol-polysaccharide treatment group and the model group were mainly enriched in the regulation of lipid metabolic process, cellular response to insulin stimulation, and insulin receptor signaling pathway. The CC enrichment results showed that Figure 4b As shown in Figure 2, the differentially expressed genes between the Pu'er tea polyphenol-polysaccharide treatment group and the model group were mainly enriched in mitochondria, cytoplasmic stress granules, lipid droplets and other items. The MF enrichment results showed that Figure 4cAs shown in the figure, the differentially expressed genes between the Pu'er tea polyphenol-polysaccharide treatment group and the model group were mainly enriched in protein binding, ATP binding, metal ion binding and other items.

[0105] KEGG enrichment analysis was performed on the differentially expressed genes in the Pu'er tea polyphenol-polysaccharide group and the model group. The 20 most significantly enriched signal pathways were screened out according to the Qvalue value, such as Figure 5 The results showed that pathways enriched for differentially expressed genes regulated by Pu'er tea polyphenols and polysaccharides included the insulin signaling pathway, cellular senescence, the p53 signaling pathway, thermogenesis, cholesterol metabolism, the FoxO signaling pathway, the AMPK signaling pathway, and insulin resistance. Insulin resistance and the insulin signaling pathway were particularly closely associated with insulin resistance in HepG2 cells. To further explore the underlying mechanisms, we carefully compared the enriched insulin resistance and insulin signaling pathways with the differentially expressed genes. The results revealed that genes such as SOCS3, PCK1, PCK2, and GYS1 had significantly significant q-values, suggesting that these genes may be differentially expressed genes with a significant impact on insulin resistance in HepG2 cells during the Pu'er tea polyphenol-polysaccharide-regulated process.

[0106] Compared with the model group, the Pu'er tea polyphenol-polysaccharide group had 1,849 differentially expressed genes, of which 1,076 were upregulated and 773 were downregulated. GO enrichment analysis of these differentially expressed genes revealed that within the biological process category, the differentially expressed genes were primarily enriched in lipid metabolic process regulation, cellular response to insulin stimulus, and the insulin receptor signaling pathway. Within the cellular component category, the differentially expressed genes were primarily enriched in mitochondrion, cytoplasmic stress granule, and lipid droplet. Within the molecular function category, the differentially expressed genes were primarily enriched in protein binding, ATP binding, and metal ion binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes revealed that insulin resistance and the insulin signaling pathway were significantly enriched and closely associated with insulin resistance in HepG2 cells. Further comparative analysis found that the q values of genes such as SOCS3, PCK1, PCK2 and GYS1 were significantly higher, which may be the regulatory targets of Pu'er tea polyphenols and polysaccharides in improving insulin resistance.

[0107] Example 3 Metabolomic analysis of Pu'er tea polyphenols-polysaccharides in improving insulin resistance in HepG2 cells

[0108] Cell samples from the control group, model group and Pu'er tea polyphenol-polysaccharide treatment group were placed in EP tubes and resuspended evenly with 100 μL ultrapure water extract; 50 μL of cell suspension was taken, 200 μL of pure methanol (-20°C pre-cooled) extract was added, and vortexed at 2500 r / min for 2 min; the cell samples were then quick-frozen in liquid nitrogen for 5 min, taken out and thawed on ice for 5 min, vortexed for 2 min to mix, and this step was repeated 3 times; then centrifuged at 4°C, 12000 r / min for 10 min, and 200 μL of the supernatant was aspirated into a new centrifuge tube; the tube was placed in a -20°C refrigerator for 30 min, and centrifuged at 4°C, 12000 r / min for 10 min. 180 μL of the supernatant was passed through a protein precipitation plate for analysis on the machine.

[0109] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (Waters ACQUITY H-Class D, https: / / www.waters.com / nextgen / cn / zh.html) and tandem mass spectrometry (MS / MS) ( 6500+, https: / / sciex.com.cn / ).

[0110] Ultra-performance liquid chromatography was performed using an ACQUITY UPLC BEH Amide column (1.7 μm, 100 mm × 2.1 mm id), the column temperature was 40°C, the injection volume was 2 μL, the mobile phase A was ultrapure water (10 mM ammonium acetate, 0.3% ammonia water), the mobile phase B was 90% acetonitrile / water (V / V), the flow rate was 0.40 mL / min, and the mobile phase gradient was set as follows: 0-1.2 min A / B was 5:95 (V:V), 8 min A / B was 30:70 (V:V), 9.0-11 min A / B was 50:50 (V:V), and 11.1-15 min A / B was 5:95 (V:V).

[0111] Mass spectrometry was performed using an electrospray ionization (ESI) source at 550°C, a mass spectrometer voltage of 5500 V in positive ion mode, a mass spectrometer voltage of -4500 V in negative ion mode, and a curtain gas (CUR) of 35 psi. Each ion transition was scanned and detected in a Q-Trap 6500+ using optimized declustering potential (DP) and collision energy (CE).

[0112] A MWDB (Metware Database) database was constructed based on the standard samples to perform qualitative analysis on the data from mass spectrometry detection. In the multiple reaction monitoring (MRM) mode of the triple quadrupole mass spectrometer, the quadrupole first screens the precursor ions of the target substance and excludes ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced to ionize by the collision chamber and then break to form multiple fragment ions. The fragment ions are then filtered by the triple quadrupole to select the required characteristic fragment ions, eliminating interference from non-target ions and obtaining accurate quantification. After obtaining the mass spectrometry analysis data of different samples, the chromatographic peaks of all target substances are integrated and quantitative analysis is performed using the standard curve.

[0113] Statistical analysis was performed using R software to obtain principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA). PCA is an unsupervised statistical analysis method for multidimensional data that aims to project high-dimensional data into a low-dimensional space while preserving the main variability in the data. It converts the original data into a set of linearly independent variables by finding the principal components of the data, that is, the directions of maximum variance in the data. This analysis method is often used to study how to reveal the internal structure among multiple variables through a small number of principal components. In other words, a small number of principal components are derived from the original variables, preserving as much information as possible from the original variables and being independent of each other. OPLS-DA is a supervised discriminant analysis statistical method that removes orthogonal variation (irrelevant to the classification) from the data by separating variation that is relevant and irrelevant to the classification, retaining only information relevant to the classification, further improving the interpretability and predictive power of the model.

[0114] Variable Importance in Projection (VIP) can be used to initially screen for metabolites that differ between groups. A VIP ≥ 1 is often used as a basic criterion for screening differential metabolites. A VIP value greater than 1 indicates that the metabolite has strong explanatory and predictive capabilities for the model. FC values can also be combined to further screen for differential metabolites. In this study, metabolite differences were considered significant when VIP ≥ 1 and FC ≥ 2 or FC ≤ 0.5.

[0115] The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a comprehensive database broadly divided into four categories: phylogenetic information, genomic information, chemical information, and health information. As the leading public database for pathways, KEGG provides pathway queries covering the metabolism of carbohydrates, nucleosides, amino acids, and the biodegradation of organic matter. It not only provides all possible metabolic pathways but also comprehensively annotates the enzymes catalyzing each step, including amino acid sequences and links to the PDB library. It is a powerful tool for in vivo metabolic analysis and metabolic network research. Metabolites are annotated and displayed using the KEGG database.

[0116] All experimental data are expressed as mean ± standard deviation (x ± SD) and statistically analyzed using SPSS software, and graphs were constructed using GraphPad Prism software. Data between multiple groups were analyzed using one-way analysis of variance (ANOVA), and data between two groups were analyzed using the T-test. P < 0.05 indicated a statistically significant difference, and P < 0.01 indicated a statistically significant difference.

[0117] The results can be seen, such as Figure 6 As shown, there is a clear separation between the metabolic profiles of the model group cells and the control group cells, indicating that the metabolic profile of the HepG2 cell insulin resistance model after establishment is significantly different from that of the control group cells. The Pu'er tea polyphenol-polysaccharide group cells and the model group cells have significant clustering distinctions, suggesting that Pu'er tea polyphenol-polysaccharide treatment has a significant improvement effect on the cell profile of the model group. The possible reasons for the partial overlap of the three groups of samples are: first, PCA is an unsupervised analysis method that focuses on maximizing the explanation of overall variance and may ignore subtle metabolic differences related to the phenotype; second, Pu'er tea polyphenol-polysaccharides may improve insulin resistance by regulating specific metabolic pathways rather than causing global metabolic profile changes. This targeted regulation may result in the differences in the overall metabolic pattern in PCA not being fully captured by the first two principal components. In order to overcome the above limitations, the following analysis was combined with supervised multivariate statistical analysis (OPLS-DA) and differential metabolite screening (VIP>1 and P<0.05).

[0118] Principal component analysis (PCA) provided a preliminary visualization of metabolite data between experimental groups. However, as an unsupervised dimensionality reduction method, PCA can only provide a relatively basic overview of inter-group differences. To further pinpoint the key metabolites underlying inter-group differences, statistical analysis based on orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Compared to PCA, the OPLS-DA model better separated the metabolic profiles of the different treatment groups, effectively distinguishing variants associated with inter-group differences from those not associated with them. This allowed for a sharper focus on metabolic changes associated with the improvement of insulin resistance in Pu'er tea polyphenol-polysaccharide treatment. The accuracy of the OPLS-DA model is determined by multiple metrics. The R2Y value reflects the model's ability to explain the training set Y (i.e., group classification). Values closer to 1 indicate a stronger explanatory power for the inter-group classification. The Q2 value, derived through methods such as cross-validation, represents the model's predictive power. It reflects the model's accuracy in predicting new samples. Similarly, higher Q2 values indicate a well-constructed model and a more reliable prediction of metabolic differences in unknown samples. The results of OPLS-DA analysis are as follows Figure 7 As shown, the metabolic profiles of the model and control cell samples were significantly different, with R2Y and Q2 values of 0.974 and 0.779, respectively, indicating that the model has high explanatory and predictive capabilities, suggesting that the metabolic profiles of the model and control groups were significantly different. The Pu'er tea polyphenol-polysaccharide treatment group was also significantly different from the model group, with R2Y and Q2 values of 0.938 and 0.684, respectively, suggesting that the Pu'er tea polyphenol-polysaccharide treatment group had a significant regulatory effect on the metabolic profile of IR-HepG2 cells.

[0119] Metabolite data from the Pu'er tea polyphenol-polysaccharide group and the model group were analyzed. Based on the screening criteria of VIP > 1 and P < 0.05, a total of 17 differential metabolites were identified (Table 5). Among these, 9 belonged to amino acids and their derivatives (tyrosine, glutamine, serine, alanine, cystine, L-citrulline, ornithine, arginine, and L-cysteic acid); 4 sugar phosphates (D-xylulose 5-phosphate, fructose-6-phosphate, glucose-6-phosphate, and sedoheptulose-7-phosphate); 2 nucleotides and their metabolites (uridine diphosphate-N-acetylglucosamine and deoxycytidylic acid); and 2 organic acids and their derivatives (pyruvate and succinate). Further analysis revealed that these differential metabolites exhibited significant differences between the Pu'er tea polyphenol-polysaccharide group and the model group, with 7 differential metabolites upregulated (e.g., glutamine, L-citrulline, ornithine) and 10 downregulated (e.g., alanine, tyrosine, and glucose-6-phosphate).

[0120] Table 5. Differential metabolites between the Pu'er tea polyphenol-polysaccharide group and the model group

[0121]

[0122]

[0123] This study analyzed the metabolite data of the Pu'er tea polyphenol-polysaccharide group and the model group based on the screening criteria of VIP>1 and P<0.05, and screened out differential metabolites. The overall distribution is as follows Figure 8 As shown in the heat map, there are significant differences in metabolite expression between the model group and the Pu'er tea polyphenols-polysaccharide group, indicating that Pu'er tea polyphenols-polysaccharides have a certain regulatory effect on the metabolic disorder of the model group.

[0124] KEGG enrichment analysis was performed on the differential metabolites between the model group and the control group. Figure 9 The enriched pathways were mainly insulin resistance, central carbon metabolism in cancer, protein digestion and absorption, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, etc.

[0125] The KEGG enrichment analysis of the differential metabolites between the Pu'er tea polyphenol-polysaccharide group and the model group showed that Figure 10 The enriched pathways were mainly insulin resistance, central carbon metabolism in cancer, protein digestion and absorption, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, etc.

[0126] Therefore, at the metabolic level, compared with the model group, 17 differential metabolites were found after Pu'er tea polyphenol-polysaccharide treatment, including 9 amino acids and their derivatives, including tyrosine, glutamine, serine, alanine, cystine, L-citrulline, ornithine, arginine, and L-cysteic acid; 4 sugar phosphates, including D-xylulose 5-phosphate, fructose-6-phosphate, glucose-6-phosphate, and sedoheptulose-7-phosphate; 2 nucleotides and their metabolites, including uridine diphosphate-N-acetylglucosamine and deoxycytidylic acid; and 2 organic acids and their derivatives, including pyruvate and succinate. Among them, 7 differential metabolites were upregulated (such as glutamine, L-citrulline, ornithine, etc.), and 10 differential metabolites were downregulated (such as alanine, tyrosine, serine, glucose-6-phosphate, etc.). After treatment with Pu'er tea polyphenols and polysaccharides, KEGG pathways were primarily enriched in insulin resistance, central carbon metabolism, protein digestion and absorption, alanine, aspartate and glutamate metabolism, and glucose, serine, and threonine metabolism. These pathways are associated with insulin resistance, energy metabolism, protein digestion and absorption, and the metabolism of various amino acids. Pu'er tea polyphenols and polysaccharides may act on the insulin resistance pathway by downregulating the levels of fructose-6-phosphate, glucose-6-phosphate, and uridine diphosphate-N-acetylglucosamine, thereby attenuating the activity of the hexosamine pathway and improving insulin resistance.

[0127] Example 4: Transcriptome and metabolome correlation analysis of Pu'er tea polyphenols-polysaccharides in improving insulin resistance in HepG2 cells

[0128] Based on metabolomics data, this study conducted a comparative analysis of the key metabolic pathways enriched by differentially expressed genes and metabolite markers in the transcriptome, and used the Pearson correlation coefficient to perform statistical analysis on the correlation between the screened metabolite markers and differentially expressed genes.

[0129] As shown in the results, in this study, we first used RNA sequencing (RNA-seq) technology to conduct a transcriptomic study on cells, aiming to preliminarily reveal the regulatory effects of Pu'er tea polyphenols and polysaccharides on IR-HepG2 cell transcripts. At the same time, we conducted a comprehensive metabolomic analysis of the intracellular metabolites of IR-HepG2 cells treated with Pu'er tea polyphenols and polysaccharides to systematically depict the changes in the intracellular metabolic profile.

[0130] To elucidate the mechanisms linking gene expression changes and metabolic profiles in IR-HepG2 cells treated with Pu'er tea polyphenols and polysaccharides, we conducted a comparative analysis by integrating metabolic pathways enriched by metabolomics-screened metabolic markers and transcriptomics-identified differentially expressed genes. Comprehensive analysis of the metabolomics and transcriptomics findings revealed several co-regulated pathways. As shown in Table 6, these co-regulated pathways primarily focus on amino acid metabolism, energy metabolism, metabolic disease mechanisms, and nutrient absorption and transport, suggesting that Pu'er tea polyphenols and polysaccharides may regulate the metabolism and function of IR-HepG2 cells through multiple pathways and targets. Further data analysis revealed that genes such as BCAT2, GPT2, ACY1, SDSL, CBS, GLS, GSK3B, GYS1, NFKB1, NOS3, PIK3CD, and PIK3R1 were significantly differentially expressed in multiple co-regulated pathways, suggesting that these genes and related pathways may be key targets for Pu'er tea polyphenols and polysaccharides in improving insulin resistance in IR-HepG2 cells.

[0131] Table 6. Pu'er tea polyphenols-polysaccharides improve the pathways co-enriched in the metabolome and transcriptome of IR-HepG2 cells

[0132]

[0133]

[0134] Through metabolomics analysis, we screened out differential metabolites related to Pu'er tea polyphenol-polysaccharide treatment, and combined with transcriptomics data, analyzed the Pearson correlation between differential metabolites and differentially expressed genes. Figure 11As shown, there was a significant correlation between differentially expressed metabolites screened from metabolomics and differentially expressed genes identified by transcriptomics (P < 0.05, |correlation coefficient| > 0.8). A total of 604 metabolite-gene pairs were found to be significantly correlated, of which 336 were positively correlated and 268 were negatively correlated. Among them, GYS1, a gene closely related to glucose metabolism, showed a significant negative correlation with glucose-6-phosphate (G6P) (correlation coefficient = -0.895); GSK-3B gene expression showed a significant positive correlation with G6P (correlation coefficient = 0.924); and glutamine showed a significant negative correlation with glutaminase (GLS) gene expression (correlation coefficient = -0.874).

[0135] Therefore, by integrating metabolomics and transcriptomics data, the molecular mechanism by which Pu'er tea polyphenols and polysaccharides improve insulin resistance was revealed. The study found that in terms of glucose metabolism, Pu'er tea polyphenols and polysaccharides significantly reduced GSK3B expression, while upregulating GYS1 expression and significantly reducing the accumulation of the metabolite G6P. This revealed that Pu'er tea polyphenols and polysaccharides may regulate glycogen synthesis through the PI3K-Akt / GSK3B-GYS1 axis and significantly reduce the abnormal accumulation of G6P, thereby improving liver cell glucose metabolism disorders. In terms of amino acid metabolism, Pu'er tea polyphenols and polysaccharides significantly inhibited GLS overactivation and reduced abnormal glutamine consumption, thereby restoring the homeostatic balance of the amino acid metabolic pathway and alleviating insulin resistance. Pu'er tea polyphenols and polysaccharides regulate glycogen synthesis through the PI3K-Akt / GSK3B-GYS1 axis and significantly reduce abnormal G6P accumulation, thereby improving hepatocyte glucose metabolism disorders. Furthermore, in the alanine, aspartate, and glutamate metabolic pathways, glutamine is catalyzed by the GLS enzyme to produce glutamate, which plays a key role in maintaining energy and redox homeostasis. Pu'er tea polyphenols and polysaccharides can restore glutamine levels and normalize GLS gene expression. This mechanism may be by inhibiting excessive GLS enzyme activation and reducing abnormal glutamine consumption, thereby maintaining metabolic pathway homeostasis. These results suggest that Pu'er tea polyphenols and polysaccharides can comprehensively improve insulin resistance in HepG2 cells by synergistically regulating glucose metabolism and amino acid metabolism in parallel.

[0136] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Pu'er tea polyphenol-polysaccharide composition, characterized in that: The composition comprises Pu'er tea polyphenols and Pu'er tea polysaccharides; specifically, the concentration of Pu'er tea polyphenols is 12.5-240 μg / mL, the concentration of Pu'er tea polysaccharides is 12.5-120 μg / mL, and the composition is non-toxic to HepG2 cells.

2. The composition according to claim 1, characterized in that The ratio of Pu'er tea polyphenols to Pu'er tea polysaccharides is a combination of 180-220 μg / mL Pu'er tea polyphenols and 80-120 μg / mL Pu'er tea polysaccharides, and preferably a combination of 200 μg / mL Pu'er tea polyphenols and 100 μg / mL Pu'er tea polysaccharides.

3. A method for screening a Pu'er tea polyphenol-polysaccharide composition for improving insulin resistance, characterized in that: The following steps are involved: (1) The toxicity of Pu'er tea polyphenols and Pu'er tea polysaccharides at different concentrations on HepG2 cells was detected by CCK-8 assay, and the safe concentration range was determined to be 12.5-200 μg / mL for Pu'er tea polyphenols and 12.5-100 μg / mL for Pu'er tea polysaccharides; (2) Establishing an insulin-resistant HepG2 cell model using continuous stimulation with palmitic acid; (3) Pu'er tea polyphenol-polysaccharide compositions with different concentrations were applied to the model cells, and the composition with the best ratio was screened out by detecting glucose consumption, glycogen synthesis and oil red O stained lipid droplet area.

4. The method according to claim 3, characterized in that The glucose consumption assay was performed by measuring the glucose concentration in the cell culture medium using a glucose kit, the glycogen synthesis assay was performed by measuring the glycogen content in the cell pellet using a glycogen assay kit, and the oil red O staining was performed by quantifying the lipid droplet area using Image J software.

5. The method according to claim 3 or 4, characterized in that The palmitic acid concentration was 150 μM.

6. Use of the Pu'er tea polyphenol-polysaccharide composition according to claim 1 or 2 in the preparation of a medicine or health product for improving insulin resistance; specifically, the composition can significantly increase glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells and reduce intracellular lipid accumulation.

7. The use according to claim 7, characterized in that The composition regulates glycogen synthesis through the PI3K-Akt / GSK3B-GYS1 signaling axis.

8. A method for detecting the effect of a Pu'er tea polyphenol-polysaccharide composition on improving insulin resistance, characterized in that: Including detection of the following indicators: (1) Transcriptomic indicators: SOCS3, PCK1, PCK2, and GYS1 were differentially expressed as differentially expressed genes; (2) Metabolomics indicators: changes in the levels of glutamine, glucose-6-phosphate, fructose-6-phosphate, and uridine diphosphate-N-acetylglucosamine as differential metabolites; The differentially expressed genes and differential metabolites were identified by RNA-seq sequencing and LC-MS / MS mass spectrometry analysis.

9. A method for preparing the Pu'er tea polyphenol-polysaccharide composition according to claim 1 or 2, characterized in that: include: (1) Pu'er tea was used as raw material, and high-purity Pu'er tea polyphenols were obtained through hot water extraction, ethyl acetate extraction, macroporous resin purification and dichloromethane decaffeination; (2) The extracted aqueous phase is subjected to ethanol precipitation, washing, and vacuum drying to obtain Pu'er tea polysaccharides; (3) The Pu'er tea polyphenols and polysaccharides are mixed in proportion to prepare a composition.

10. The preparation method according to claim 9, characterized in that The Pu'er tea is made from Yunnan large-leaf sun-dried green tea. The obtained Pu'er tea polyphenols have a purity of 85.93% and the Pu'er tea polysaccharides have a purity of 54.19%. The Pu'er tea polyphenols and polysaccharides are mixed in a ratio of 12.5-200 μg / mL: 12.5-100 μg / mL to prepare a composition.

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